1

UNIT 3

COLLECTION AND PROCESSING OF SAMPLE FOR FUNGAL INFECTION IN SKIN,

NAIL AND HAIR

LEARNING OBJECTIVE

To understand the concept of skin fungal infection.

To understand the concept of nail fungal infection.

To understand the concept of hair fungal infection.

To understand the concept of LCB.

To understand the concept of KOH.

To understand the concept of INDIA INK.

3.1 COLLECTION OF SKIN SCRAPPINGS

Dermatophytes such as Trichophyton, Microsporum and Epidermophyton causes skin

infection. It may be caused by skin infection.

3.1.1 PROCEDURE

First of all clean the lesion with 70% ethanol.

Scrap the material from the periphery of the lesion with a sterilized scalpel..

Place the material in a clean and sterilized petridish .

The scrapping specimen should be transported to the clinical lab in petridish..

If 70% ethanol causes burning sensation into the lesion then it should be cleaned with

distilled water.

3.2 COLLECTION OF NAIL SCRAPPINGS

Aspergillus, Candida and Dermatophytes like Trichophyton, Microsporum,

Epidermophyton causes nail infection.

3.2.1 PROCEDURE

First of all clean the nails with 70% ethanol.

Scrap the material from the distal end of the nail with a sterilized scalpel.

Discard the first 4-5 scrappings and collect the latter ones.

Place the material in a clean petridish.

The scrapped specimen should be transportd to the clinical laboratory.

3.3 COLLECTION OF HAIR SCRAPPINGS

Piedra, Trichosporon, Trichophyton, Microsporum and Epidermophyton causes

fungal infection in hair. Infected hair is identified by exposure to uv light.

3.3.1 PROCEDURE

Hair should be collected from areas of scaling or alopecia.

The infected hair are plucked by using sterilized forceps.

In young patients, removal of hair is difficultso place a strip of clear tape over the

infected area and then remove it.

2

Place the collected hair into a clean and sterilized petridish.

The specimen should be transported to the clinical laboratory in petridish.

3.4 POTASSIUM HYDROXIDE PREPARATION

Potassium hydroxide (KOH) preparation is used for the rapid detection of fungal elements in

clinical specimen, as it clears the specimen making fungal elements more visible during

direct microscopic examination. KOH is a strong alkali. When specimen such as skin, hair,

nails or sputum is mixed with 20% w/v KOH (preparation of KOH is posted at the end of this

post), it softens, digests and clears the tissues (e.g., keratin present in skins) surrounding the

fungi so that the hyphae and conidia (spores) of fungi can be seen under microscope.

3.4.1USES

In diagnostic laboratories, KOH mount is one of the main methods of investigating fungal

infections. It is used as a primary screening tool, it detects fungal elements present but may

not necessarily identify the species of the fungi. To identify the fungal isolate, specimen must

be cultured in either general purpose fungal culture media such as Sabouraud Dextrose Agar

(SDA) or specific media based on the type of anticipated isolate.) KOH preparation is

recommended in the following suspected conditions (this is not the exclusive lists);

3.4.2 PROCEDURE OF KOH PREPARATION

Place a drop of KOH solution on a slide.

Transfer the specimen (small pieces) to the drop of KOH, and cover with glass. Place the slide in a petri dish, or other container with a lid, together with a damp piece of

filter paper or cotton wool to prevent the preparation from drying out.

As soon as the specimen has cleared, examine it microscopically using the 10X and 40X objectives with the condenser iris diaphragm closed sufficiently to give a good

contrast.

If too intense a light source is used the contrast will not be adequate and the

unstained fungi will not be seen.

3.4.3 DISADVANTAGES OF KOH PREPARATION

Experience required since background artifacts are often confusing.

Clearing of some specimens may require an extended time

3.4.4PROCEDURE TO MAKE KOH PREPARATION

Weigh 20 g potassium hydroxide (KOH) pellets.

Transfer the chemical to a screw-cap bottle.

Add 50 ml distilled water, and mix until the chemical is completely dissolved, add remaining distilled water and make the volume 100 ml.

Label the bottle and mark it corrosive. Store it at room temperature. The reagent is

stable for up to 2 years.

3

3.5LACTOPHENOL COTTON BLUE

The lactophenol cotton blue (LPCB) wet mount preparation is the most widely used method

of staining and observing fungi and is simple to prepare. The preparation has three

components:

Phenol: kills any live organisms;

Lactic acid : It preserves fungal structures, and

Cotton blue : It stains the chitin in the fungal cell walls.

3.5.1PREPARATION

Place a drop of seventy percent alcohol on a clean microscope slide.

Material from cultures of filamentous fungi should be removed using a stiff

inoculating wire not the loop used for manipulations with bacteria or yeasts.

Flame the wire by holding it upright in the hottest part of the Bunsen flame, just

above the blue cone, until the whole length of the wire glows red hot.

You must ensure that the inoculating wire has cooled before placing it in a fungal

culture – it should have cooled sufficiently after approximately ten seconds.

Remove the cap from the tube but do not put it on the bench. Kill any contaminating microorganisms by flaming the neck of the tube.

Remove a small amount of the culture. For fungal cultures, it is often useful to take a little of the agar medium together with the fungus. In any case, the material should be

disturbed as little as possible when being transferred to the slide.

Flame the neck of the tube once more and replace the cap.

Immerse the fungal material in the drop of seventy percent alcohol. This drives out the air trapped between the hyphae.

Tease out the material very gently with mounted needles.

Do not forget to sterilise the inoculating wire and the needles after use by heating to red heat in a Bunsen flame

Fungal structures are readily visualised after staining with a lactophenol cotton blue dye preparation.

Before the alcohol dries out add one or at most two drops of the stain. A common fault is to add too much to the preparation. Holding the coverslip between your index

finger and thumb, touch one edge of the drop of stain with the edge of the coverslip.

Lower the coverslip gently onto the slide, trying to avoid air bubbles. Your preparation is now ready for examination.

Make the initial examination using a low power objective lens. The thinner parts of the preparation, generally around the edges of the mounted material, will yield the

best images.

Switch to a higher power 40X objective for more detailed examination of spores.

4

3.6 INDIA INK

The main purpose of Negative staining is to study the morphological shape,

size and arrangement of the bacteria cells that is difficult to stain.

eg: Spirilla. It can also be used to stain cells that are too delicate to be heat-

fixed.It is also used to prepare biological samples for electron microscopy.

It is used to view viruses, bacteria, bacterial flagella, biological membrane

structures and proteins or protein aggregates, which all have a low electron-

scattering power. It is also used for the study and identification of aqueous

lipid aggregates like lamellar liposomes (le), inverted spherical micelles (M)

and inverted hexagonal HII cylindrical (H) phases by Negative staining

transmission electron microscopy.The main purpose of Negative staining is to

study the morphological shape, size and arrangement of the bacteria cells that

is difficult to stain. eg: Spirilla. It can also be used to stain cells that are too

delicate to be heat-fixed. It is also used to prepare biological samples for

electron microscopy. It is used to view viruses, bacteria, bacterial flagella, biological membrane structures and proteins or protein aggregates, which all

have a low electron-scattering power. It is also used for the study and

identification of aqueous lipid aggregates like lamellar liposomes (le),

inverted spherical micelles (M) and inverted hexagonal HII cylindrical (H)

phases by Negative staining transmission electron microscopy.

VERY SHORT ANSWER TYPE QUESTIONS

Q1. Expand LCB.

Q2 Expand KOH.

Q3 Define negative staining.

Q4. Use of cotton blue.

Q5. Use of lactic acid.

SHORT ANSWER TYPE AND LONG ANSWER TYPE QUESTIONS

Q1.Discuss the principle and procedure of LCB.

Q2. Discuss the principle and procedure of KOH

. Q3. Discuss the principle and procedure of India ink.

Q4.Discuss collection and processing of skin specimen.

Q5. Discuss collection and processing of skin specimen

Q6. . Discuss collection and processing of skin specimen

5

UNIT4

FUNGAL CULTIVATION

LEARNING OBJECTIVE

To learn the concept of fungal cultivation.

To learn the concept of isolation of candida

To learn the concept of of isolation of Dermatophytes.

To learn the concept of of isolation of pencillium

To learn the concept of of isolation of Rhizopus

To learn the concept of of isolation of Mucor

To learn the concept of of isolation of Aspergillus.

4.1 TECHNIQUES OF FUNGAL CULTIVATION

4.1.1 INTRODUCTION

Aseptic Technique: In most microbiological procedures, it is necessary to protect

instruments, containers, media, and stock cultures from contamination by

microorganisms constantly present in the environment. Aseptic technique involves the

sterilization of tools, glassware, and media before use as well as measures to prevent

subsequent contamination by contact with nonsterile objects.

Equipment and Work Area: To culture bacteria or fungi, you need the

following materials

Disinfectant solution such as 70% ethanol, 4% household bleach solution.

Alcohol or gas (Bunsen) burner.

Inoculating loop for bacteria, yeasts, and fungi with abundant spores; scalpel

or half-spearpoint needle for other fungi Stock culture (the original culture

from which other cultures will be started)

Sterile medium in petri dishes or culture tubes

Soap for washing hands.

Lab coat or old, clean shirt, especially while you are staining cultures.

Before working with bacterial or fungal cultures, always wash your hands

with soap and water. Next, prepare a work area. Select an area that is as free

from drafts as possible. Turn off the air-conditioner and fans, and close all

windows and doors. Wipe the work area with 70% ethanol or a similar

disinfectant solution. Arrange your materials conveniently on the clean work

surface. Do not smoke, eat, or drink while working with cultures. Media

Preparation The first step in media preparation is to assemble the equipment

and ingredients. You will need a balance, spatula, weighing paper, 1-L

graduated cylinder, glass stirring rod, a large flask or beaker, and culture

tubes or petri dishes. You can either use a recipe to prepare a particular

medium from scratch or purchase any of the commercially available

dehydrated media. The media most commonly used are nutrient agar

(bacteria), potato dextrose agar (fungi), and Sabouraud dextrose agar (fungi).

After assembling the equipment and ingredients, weigh the dry ingredients

accurately. Place a sheet of weighing paper on the pan to protect the balance

and to facilitate transferring the material into a flask. Using the weighing

paper as a funnel, pour the dry ingredients into a large flask or beaker. Add

the proper amount of distilled water and swirl the flask to dissolve the dry

material. Agar-containing media must be heated slowly, just to boiling, to

dissolve the agar. Gently agitate the medium during the heating process by

either stirring or shaking the flask. Watch the flask carefully: agar burns

easily and boils over quickly. Pour the liquid agar or broth into bottles or

culture tubes and cap them loosely. Autoclave the medium in the bottles or

culture tubes to sterilize it. If the medium is to be used to pour dishes,

autoclave it in the plugged flask in which it was mixed. When sterilization is

complete, lay the tubes on a slant tray. Tighten the tops for storage only after

the agar solidifies. If plates are to be poured, disinfect the work area and stop

all air drafts. Let the flask cool until it is easy to handle (20 to 40 minutes).

Lay out sterile petri dishes and light a Bunsen burner. Remove the stopper

from the flask and flame the mouth. Lift the cover of the dish at just enough of

an angle to pour in the medium. Pour the agar slowly to avoid bubble

formation; if bubbles do form, pass the burner flame quickly over the surface

of the agar severa times, which should cause the bubbles to burst. Pour

enough agar to fill the dish about one-half full, replace the cover, and allow

the dish to stand undisturbed until the agar solidifies.

Sterilization Procedures Many microorganisms produce highly resistant

spores that remain viable even after exposure to dry heat or boiling water for

several hours. Steam under pressure is used to increase the temperature

enough to kill any contaminating microorganisms. Steam penetrates

wrappings and loosely capped articles, sterilizing the contents. The home

pressure cooker works on this principle. An autoclave is, in essence, a large,

self-contained pressure cooker that goes through the heating, sterilizing, and

cooling cycles automatically. If an autoclave is not available, you can use a

large pressure cooker on a stove as long as you follow a few rules:

Read the directions for your brand of cooker and follow them

carefully.

Make sure there is sufficient water in the cooker.

Don’t start timing until 15 pounds per square inch (psi) have been

reached. 4. At the end of 15 minutes, allow the pressure cooker to cool

slowly. Media should be sterilized for 15 minutes at a temperature of

121°C and a pressure of 15 psi. Glassware and contaminated articles

like old stock cultures should be autoclaved for 30 minutes at 121°C

and 15 psi.

4.2 GENERAL CHARACTERISTICS AND ISOLATION OF

DERMATOPHYTES

Superficial fungal infections of the skin is public health problem .

Dermatophytosis which involves keranized tissues such as superficial skin,

nails and hair, is the commonest condition that is encountered in the

dermatology clinics. These infections are usually ignored and patient presents

to outpatient department only if cosmetically disfiguring or due to

inconvenience caused by pruritus. This results in many not seeking medical

help immediately thus increasing the disease load in the community and

increased risk of transmission. The epidemiology of dermatophytosis has

evolved over the last many decades. Whether developed countries or

developing countries these changes have occurred due to changing life styles,

economy, increasing leisure activities and environmental factors. People who

are exposed to soil, and animals occupationally or as a leisure activity, are at

risk of acquiring dermatophyte infectionof nail, it is left for longer period till

the cells are dissolved. The slide was examined for the presence of fungal

elements.

Culture: Sabouraud’s dextrose agar (SDA) with gentamicin (8mg/litre)were

used for culturing the specimens initially. Later dermatophyte test

medium(DTM) was also included for culturing the specimens. All cultures

were incubated at 250 C in biochemical oxygen demand incuators/B.O.D

incubator for 4 weeks before issuing a negative report. Plates and tubes were

examined everyday during first week and every two days thereafter. This was

done to detect any bacterial or saprophytic fungal contamination and also to

re-inoculate in case contamination occurred. The day of the appearance of the

suspected dermatophyte colony was noted for assessing the rate of growth of

the isolates.

4.3 GENERAL CHARACTERISTICS AND ISOLATION OF

PENICILLIUM.

4.3.1MACROSCOPIC APPEARANCE

Surface: Texture velvety to powdery; Green, blue-green, gray-green, white,

yellow, or pinkish on the surface.

Reverse: Usually white to yellowish, sometimes red or brown.

Growth Rate: Moderately rapid to rapid.

Note: If the isolate produces a red reverse and diffuse pigment in the agar, P. marneffei must be

considered and the organism should be tested for thermal dimorphism; this is especially relevant if

the patient has recently visited southeast Asia.

4.3.2 DISTINGUISHING FEATURES

Penicillium is distinguished by its frequently greenish colonies and its branching or simple

conidiophores supporting phialides in brush-like clusters known as penicilli. It is

differentiated from Paecilomyces by its phialides lacking long, pointed apical extensions. In

contrast to Scopulariopsis , its conidia lack a truncate base. P. marneffei produces downy

gray-green colonies, often with a brownish or red tint caused by the presence of red or

yellow pigmented sterile hyphae in the colony. The colonies, when incubated at 37 degrees C.

on Sabouraud Dextrose Agar, characteristically lose this pigmentation and convert into

yeast-like cells multiplying by fission. The diagnosis of infection due to P. marneffeirests on

the histopathologic demonstration of cells multiplying by fission in the interior of leukocytes.

4.3.3 HABITAT

Penicillium are cosmopolitan, predominant in regions of temperate climate. Penicillia figure

among the most common types of fungi isolated form the environment. Of the approximately

150 recognized species, some are frequently implicated in the deterioration of food products

where they may produce mycotoxins. Other species are producers of penicillin. Infections

with P. marneffei are primarily acquired in mountainous provinces of Northern Thailand,

Laos, Myanmar, and Southeastern China.

4.3. 4 PATHOGENICITY

Normally considered nonpathogenic with the exception of Penicillium marneffei , a

dimorphic species capable of causing infection of the lymphatic system, the lungs, the liver,

the skin, the spleen, and the bones. It has been known to cause keratitis (inflammation of the

cornea), external ear, respiratory, and urinary tract infections, and endocarditis after

insertion of valve prostheses.

4.3.5 RECOMMENDED MEDIA

Corn Meal Agar

Inhibitory Mold Agar

Malt Extract Agar

Potato Dextrose Agar

Sabouraud Dextrose Agar

Wort Agar

Incubate at 25 degrees C. for 2-7 days.

4.4 GENERAL CHARACTERISTICS AND ISOLATION OF RHIZOPUS

4.4.1MICROSCOPIC APPEARANCE

Hyphae broad, not or scarcely septate; rhizoids and stolons present; sporangiophores brown,

solitary or in tufts on the stolons, diverging from the point at which the rhizoids form;

sporangia rather round; apophysis absent or scarcely apparent; sporangiophores ovoid.

4.4.2 MACROSCOPIC APPEARANCE

Surface: Texture deeply cottony; White becoming gray-brown on surface.

Reverse: Pale white.

Growth Rate: Very rapid growth.

4.4.3 DISTINGUISHING FEATURES

Rhizopus is recognized by the presence of well developed rhizoids situated at the point where

sporangiophores are attached to the stolons. In contrast to Mucor , Rhizomucor and Absidia ,

4.4.4 HABITAT

Rhizopus are cosmopolitan, frequently isolated from soil and agricultural products (cereal,

vegetables, etc.). Certain species are plant pathogens.

Pathogenic

Maximum

Growth

Temperature

Rhizoid

Length

(um)

Sporaniophore

Length

(um)

Sporangium

Length

(um) Columellae

Sporangio-

spores

Positive 50-52°C 100-

120

200-1000 40-130 Slightly

elongated;

distinct

apophysis

Equal size,

average length 4-

6um; smooth to

slightly striated;

almost round to

slightly elongated

Postive 40-46°C 150-

300

500-3500 50-250 Almost

round

Variable size,

average length 6-

8um; striated;

elongated to

lemon-shaped

Negative 30-32°C 300-

350

1500-4000 150-350 Almost

round

Variable in size,

average length 9-

11um; very

striated;polyhedric

4.4.5PATHOGENICITY

Rhizopus is the principal agent of mucormycosis (formally zygomycosis). This rapidly

progressing infection is characterized by the cerosis of tissues and the production of infarcts

in the brain, the lungs, and the intestines. Primarily, it is patients suffering from diabetic

ketoacidosis, malnutrition, severe burns, or immunocompromising conditions who are most

at risk to develop this type of infection.

4.4.6 RECOMMENDED MEDIA

Corn Meal Agar

Malt Extract Agar

Potato Dextrose Agar

Sabouraud Dextrose Agar

Wort Agar

INCUBATION

Temperature: 25 degrees C.

Time: 2-7 days.

4.5GENERAL CHARACTERISTICS AND ISOLATION OF MUCOR

Mucor is a filamentous fungus found in soil, plants, decaying fruits and vegetables. As well

as being ubiquitous in nature and a common laboratory contaminant, Mucor spp. may cause

infections in man, frogs, amphibians, cattle, and swine. Most of the Mucor spp. are unable to

grow at 37°C and the strains isolated from human infections are usually one of the few

thermotolerant Mucor spp. The genus Mucor contains several species. The most common

ones are Mucor amphibiorum, Mucor circinelloides, Mucor hiemalis, Mucor indicus, Mucor

racemosus, and Mucor ramos

4.5.1.PATHOFENICITY

Mucor spp. are among the fungi causing the group of infections referred to as zygomycosis.

Although the term mucormycosis has often been used for this syndrome, zygomycosis is now

the preferred term for this angio-invasive disease. Zygomycosis includes mucocutaneous and

rhinocerebral infections, as well as septic arthritis, dialysis-associated peritonitis, renal

infections, gastritis and pulmonary infections. Diabetic ketoacidosis and immunosuppression

are the most frequent predisposing factors. Desferoxamine treatment, renal failure, extensive

burns, and intravenous drug use may also predispose to development of zygomycosis.

Vascular invasion that causes necrosis of the infected tissue, and perineural invasion are the

most frustrating features of these infections. Of note, due to its relatively limited activity,

itraconazole prophylaxis in immunosuppressed patients may select the fungi in phylum

Zygomycota as the cause of infections .

Macroscopic Features

Colonies of Mucor grow rapidly at 25-30°C and quickly cover the surface of the agar. Its

fluffy appearance with a height of several cm resembles cotton candy. From the front, the

color is white initially and becomes grayish brown in time. From the reverse, it is

white. Mucor indicus is an aromatic species and may grow at temperatures as high as

40°C. Mucor racemosus and Mucor ramosissimus, on the other hand, grow poorly or do not

grow at all at 37°C.

Microscopic Features

Nonseptate or sparsely septate, broad (6-15 µm) hyphae, sporangiophores, sporangia, and

spores are visualized. Intercalary or terminal arthrospores (oidia) located through or at the

end of the hyphae and few chlamydospores may also be produced by some species.

Apophysis, rhizoid and stolon are absent. Sporangiophores are short, erect, taper towards

their apices and may form short sympodial branches. Columella are hyaline or dematiaceous

and are hardly visible if the sporangium has not been ruptured. Smaller sporangia may lack

columella. Sporangia are round, 50-300 µm in diameter, gray to black in color, and are filled

with sporangiospores. Following the rupture of the sporangia, sporangiospores are freely

spread. A collarette may sometimes be left at the base of the sporangium following its

rupture. The sporangiospores are round (4-8 µm in diameter) or slightly elongated.

Zygospores, if present, arise from the mycelium.

The branching of sporangiophores (branched or unbranched), the shape of the

sporangiospores (round or elongated), maximum temperature of growth, presence of

chlamydospores, assimilation of ethanol, and molecular analysis aid in differentiation

of Mucor spp. from each other .

.The features that help in differentiation of these genera are summarized in the table below

Genus

Best

grow

th

Sporangiop

hore

Apoph

ysis

Colum

ella

Sporangi

um

Rhizo

id

Stylosp

ore

Absidia 45°C Branched,

hyaline

Conical

, not

promin

ent

Dome-

shaped

Pear-

shaped

+, but usuall

y

indisti

nct

–

Apophysomyces

>42°C

Usually unbranched,

grayish-

brown

Bell-

shaped,

not

promin

ent

Usually dome-

shaped,

rarely

elongat

ed

Pear-shaped

+ –

Mortierella 40°C Branched,

hyaline – – Spherical + +/-

Mucor <37°

C

Branched or unbranched,

hyaline

– +, in

varying

shapes

Spherical – –

Rhizomucor

54°C Branched,

brown –

Spherical

Spherical + –

Rhizopus 45°C Unbranched and brown

mostly

Not promin

ent

Spheric

al or

elongat

ed

Spherical + –

VERY SHORT ANSWER TYPE QUESTIONS

Q1. Name fungal culture medium.

Q2. Expand SDA

Q3. Expand CMA

Q4.Uses of penicillium.

Q5.Define zygomycetes.

Q6. Define pathogenicity.

SHORT ANSWER TYPE AND LONG ANSWER TYPE QUESTIONS

Q1.How can we isolate the candida species from the specimen.

Q2. How can we isolate the penicillium species from the specimen

Q3.How can we isolate the rhizopus species from the specimen

Q4.How can we isolate the mucor species from the specimen

Q5.How can we isolate the dermatophytes species from the specimen

1

UNIT 5

INTRODUCTION TO IMMUNOLOGY IMMUNITY

LEARNING OBJECTIVE

To learn the concept of immune system

To learn the concept of innate immunity

To learn the concept of acquired immunity

5.1 INTRODUCTION

Immunity Humans have three types of immunity — innate, adaptive, and passive:

5.1.1INNATE IMMUNITY

Everyone is born with innate (or natural) immunity, a type of general protection. Many of

the germs that affect other species don't harm us. For example, the viruses that cause

leukemia in cats or distemper in dogs don't affect humans. Innate immunity works both ways

because some viruses that make humans ill — such as the virus that causes HIV/AIDS —

don't make cats or dogs sick. Innate immunity also includes the external barriers of the body,

like the skin and mucous membranes (like those that line the nose, throat, and

gastrointestinal tract), which are the first line of defense in preventing diseases from entering

the body. If this outer defensive wall is broken (as through a cut), the skin attempts to heal the

break quickly and special immune cells on the skin attack invading germs. Adaptive Immunity

The second kind of protection is adaptive (or active) immunity, which develops throughout

our lives. Adaptive immunity involves the lymphocytes and develops as people are exposed to

diseases or immunized against diseases through vaccination.

5.2 PASSIVE IMMUNITY

Passive immunity is "borrowed" from another source and it lasts for a short time. For

example, antibodies in a mother's breast milk provide a baby with temporary immunity to

diseases the mother has been exposed to. This can help protect the baby against infection

during the early years of childhood. Everyone's immune system is different. Some people

never seem to get infections, whereas others seem to be sick all the time. As people get older,

they usually become immune to more germs as the immune system comes into contact with

more and more of them. That's why adults and teens tend to get fewer colds than kids — their

bodies have learned to recognize and immediately attack many of the viruses that cause

colds.

The first part of the immune system that meets invaders such as bacteria is a group of

proteins called the complement system. These proteins flow freely in the blood and can

quickly reach the site of an invasion where they can react directly with antigens - molecules

that the body recognizes as foreign substances. When activated, the complement proteins can

- trigger inflammation - attract eater cells such as macrophages to the area .

Phagocytes This is a group of immune cells specialized in finding and "eating" bacteria,

viruses, and dead or injured body cells. There are three main types, the granulocyte, the

macrophage, and the dendritic cell. The granulocytes often take the first stand during an

infection. They attack any invaders in large numbers, and "eat" until they die. The pus in an

2

infected wound consists chiefly of dead granulocytes. A small part of the granulocyte

community is specialized in attacking larger parasites such as worms. The macrophages

("big eaters") are slower to respond to invaders than the granulocytes, but they are larger,

live longer, and have far greater capacities. Macrophages also play a key part in alerting the

rest of the immune system of invaders. Macrophages start out as white blood cells called

monocytes. Monocytes that leave the blood stream turn into macrophages. The dendritic

cells are "eater" cells and devour intruders, like the granulocytes and the macrophages. And

like the macrophages, the dendritic cells help with the activation of the rest of the immune

system. They are also capable of filtering body fluids to clear them of foreign organisms.

5.2.1. LYMPHOCYTES: B-CELLS AND T-CELLS

The lymphatic system The receptors match only one specific antigen. White blood cells

called lymphocytes originate in the bone marrow but migrate to parts of the lymphatic system

such as the lymph nodes, spleen, and thymus. There are two main types of lymphatic cells, T

cells and B cells. The lymphatic system also involves a transportation system - lymph vessels

- for transportation and storage of lymphocyte cells within the body. The lymphatic system

feeds cells into the body and filters out dead cells and invading organisms such as bacteria.

On the surface of each lymphatic cell are receptors that enable them to recognize foreign

substances. These receptors are very specialized - each can match only one specific antigen.

To understand the receptors, think of a hand that can only grab one specific item. Imagine

that your hands could only pick up apples. You would be a true apple-picking champion - but

you wouldn't be able to pick up anything else. In your body, each single receptor equals a

hand in search of its "apple." The lymphocyte cells travel through your body until they find

an antigen of the right size and shape to match their specific receptors. It might seem limiting

that the receptors of each lymphocyte cell can only match one specific type of antigen, but the

body makes up for this by producing so many different lymphocyte cells that the immune

system can recognize nearly all invaders.

5.2.2 T - CELLS

T cells come in two different types, helper cells and killer cells. They are named T cells after

the thymus, an organ situated under the breastbone. T cells are produced in the bone marrow

and later move to the thymus where they mature. Helper T cells are the major driving force

and the main regulators of the immune defense. Their primary task is to activate B cells and

killer T cells. However, the helper T cells themselves must be activated. This happens when a

macrophage or dendritic cell, which has eaten an invader, travels to the nearest lymph node

to present information about the captured pathogen. The phagocyte displays an antigen

fragment from the invader on its own surface, a process called antigen presentation. When

the receptor of a helper T cell recognizes the antigen, the T cell is activated. Once activated,

helper T cells start to divide and to produce proteins that activ ivate B and T cells as well as

other immune cells.

5.2.3 B - CELLS

- The B lymphocyte cell searches for antigen matching its receptors. If it finds such antigen it

connects to it, and inside the B cell a triggering signal is set off. The B cell now needs

proteins produced by helper T cells to become fully activated. When this happens, the B cell

starts to divide to produce clones of itself. During this process, two new cell types are

3

created, plasma cells and B memory cells. The plasma cell is specialized in producing a

specific protein, called an antibody, that will respond to the same antigen that matched the B

cell receptor. Antibodies are released from the plasma cell so that they can seek out intruders

and help destroy them. Plasma cells produce antibodies at an amazing rate and can release

tens of thousands of antibodies per second. When the Y-shaped antibody finds a matching

antigen, it attaches to it. The attached antibodies serve as an appetizing coating for eater

cells such as the macrophage. Antibodies also neutralize toxins and incapacitate viruses,

preventing them from infecting new cells. Each branch of the Y-shaped antibody can bind to

a different antigen, so while one branch binds to an antigen on one cell, the other branch

could bind to another cell - in this way pathogens are gathered into larger groups that are

easier for phagocyte cell to devour. Bacteria and other pathogens covered with antibodies

are also more likely to be attacked by the proteins from the complement system. The Memory

Cells are the second cell type produced by the division of B cells. These cells have a

prolonged life span and can thereby "remember" specific intruders. T cells can also produce

memory cells with an even longer life span than B memory cells. The second time an intruder

tries to invade the body, B and T memory cells help the immune system to activate much

faster. The invaders are wiped out before the infected human feels any symptoms. The body

has achieved immunity against the invader.

VERY SHORT ANSWER TYPE QUESTIONS

Q1 Define immunity.

Q2. What are the parts of immune system?

Q3.Name the blood cell which provides immunity.

Q4.Define innate immunity.

Q5.Define Acquired immunity.

SHORT ANSWER TYPE AND LONG ANSWER TYPE QUESTIONS

Q1.Write the difference between innate and acquired immunity.

Q2.Write the mechanism of innate immunity.

Q3. Write the mechanism of passive immunity.

Q4. Write the mechanism of acquired immunity.

Q5. Discuss the parts of immune system.

4

1

UNIT 6

ANTIGENS

LEARNING OBJECTIVE

To learn the concept of antigen

To learn the properties of antigen

To learn the types of antigen

6.1 INTRODUCTION

An antigen is a particle that stimulates the production of antibody and specifically

reacts with it. Antigens have the capability to bind with with the product of the

immune system. Antigens may be foreign such as cells, bacteria, viruses, fungi and

large proteins or polysaccharides molecules but I some conditions small molecules

and even self particles can become antigenic. The antigens are mostly the conjugated

proteins like lipoproteins , glycoproteins and nucleoproteins in nature. The whole

antigen does not activate the immune response and only a small part of it induces B

immune response. The small area of chemical grouping on the antigen molecule that

induces the specific immune response and reacts specifically with antibody is called

an antigenic determinant or combining site or epitope.

6.2 PROPERTIES

Foreignness: An antigen must be a foreign substance to the animal to activate

an immune response. The degree of foreignness influences the antigenicity.

Antigens from other individuals of the same species are less antigenic and less

antigenic than those from other species. Sometimes the mechanism of

recognising self antigens is impaired so that immune system produces

antibodies against its self antigen.

Molecular size and weight: Antigens with high molecular weight are generally

more antigenic than those with alow molecular weight. The most active

immunogens have a molecular mass of 14,000 to 6,00,000 daltons. Example:

tetanus toxoid, egg albumin, thyroglobulin are highly antigenic. Insulin have a

low molecular weight of 5700 and may be weakly antigenic or not.

Chemical nature and composition: The more chemically complex substance is

more immunogenic or antigenic. Antigens are mainly composed of proteins,

lipids and some polysaccharides.

Physical form: In general particulate antigens and denatured antigens are

more immunogenic than native form and soluble form.

Antigen specifity : Antigen specifity depends on the specific combining site

on the antigenic molecule. Antigenic determinants are the regions of

antigenwhich specifically binds with the antibody molecule. Each Y-shaped

2

anti body moleculehas atleast two binding sites that can attach to a specific

epitope or antigen.

Species specifity: Tissues of all individuals in a particular species having

species specific antigen. Individuals of related species having species specific

antigen. Individuals of related species may have some similar antigens which

may lead to cross reaction.

Organ Specifity: Heterogenic antigens that are found in the same organ of

different species. These are also known as tissue specific antigens.

Age: It may also affect antigenicity of an antigen. Generally very young and

the very old have a different ability to activate the immune response to against

an antigen.

Degradability: Antigens that are easily phagocytosed are generally more

antigenic.

Dose of the antigen: The dose of the administration of an antigen can

influence its antigenicity. There is a dose of antigen above or below which the

immune response will not be optimal.

Route of administration: Generally the subcutaneous route is to be used for

administration of an antigen than the intravenous or intragastric routes.

6.3 CLASSIFICATION OF ANTIGENS

Exogenous antigens

These antigens enters the body or system and start circulating in the body

fluids and trapped by the APCs (Antigen processing cells such as

macrophages, dendritic cells, etc.)The uptakes of these exogenous antigens by

APCs are mainly mediated by the phagocytosis Examples: bacteria, viruses,

fungi etc. Some antigens start out as exogenontigens, and later become

endogenous (for example, intracellular viruses)

Endogenous antigens

These are body’s own cells or sub fragments or compounds or the antigenic

products that are produced.The endogenous antigens are processed by the

macrophages which are later accepted by the cytotoxic T – cells.Endogenous

antigens include xenogenic (heterologous), autologous and idiotypic or

allogenic (homologous) antigens.

Examples: Blood group antigens, HLA (Histocompatibility Leukocyte

antigens), etc.

Autoantigens

An autoantigen is usually a normal protein or complex of proteins (and

sometimes DNA or RNA) that is recognized by the immune system of patients

suffering from a specific autoimmune diseaseThese antigens should not be,

3

under normal conditions, the target of the immune system, but, due mainly to

genetic and environmental factors, the normal immunological tolerance for

such an antigen has been lost in these patients. Examples: Nucleoproteins,

Nucleic acids, etc.

Complete Antigen or Immunogen

Posses antigenic properties denovo, i.e. ther are able to generate an immune

response by themselves. High molecular weight (more than 10,000). May be

proteins or polysaccharides

Incomplete Antigen or Hapten

These are the foreign substance, usually non-protein substances. Unable to induce an

immune response by itself, they require carrier molecule to act as a complete antigen

.The carrier molecule is a non-antigenic component and helps in provoking the

immune response. Example: Serum Protein such as Albumin or Globulin. Low

Molecular Weight (Less than 10,000). Haptens can react specifically with its

corresponding antibody. Examples: Capsular polysaccharide of pneumococcus,

polysaccharide “C” of beta haemolytic streptococci, cardiolipin antigens, etc.

VERY SHORT ANSWER TYPE QUESTIONS

Q1. Define antigen

Q2. Define hapten.

Q3 Define immunogen.

Q4 .Define molecular weight of antigen.

Q5. Define antigenicity.

SHORT ANSWER TYPE AND LONG ANSWER TYPE QUESTIONS

Q1.Discuss the concept of antigen.

Q2. Discuss the classification of antigen.

Q3.Discuss the properties of antigens.

Q4. Discuss the significance of antigens.

Q5. Discuss the epitope part of antigen.

Q6. Discuss haptens.

1

UNIT 7

ANTIBODIES

LEARNING OBJECTIVE

To learn the concept of structure of antibody

To learn the concept of classification of antibody

To learn the function of antibody

7.1 INTRODUCTION

Antibody (Ab) also know as Immunoglobulin (Ig) is the large Y shaped protein produced by

the body’s immune system when it detects harmful substances, called antigens like bacteria

and viruses. The production of antibodies is a major function of the immune system and is

carried out by a type of white blood cell called a B cell (B lymphocyte), differentiated B cells

called plasma cells. The produced antibodies bind to specific antigens express in external

factors and cancer cells.

7.2 STRUCTURE OF ANTIBODY

Antibodies are heavy (~150 kDa) globular plasma proteins. The basic structure of all

antibodies are same.

There are four polypeptide chains: two identical heavy chains and two identical light

chains connected by disulfide bonds. Light Chain (L) consists polypeptides of about 22,000

Da and Heavy Chain (H) consists larger polypeptides of around 50,000 Da or more. There

are five types of Ig heavy chain (in mammal) denoted by the Greek letters: α, δ, ε, γ, and

μ. There are two types of Ig light chain (in mammal), which are called lambda (λ) and kappa

(κ).

An antibody is made up of a variable region and a constant region, and the region that

changes to various structures depending on differences in antigens is called the variable

region, and the region that has a constant structure is called the constant region.

Each heavy and light chain in an immunoglobulin molecule contains an amino-terminal

variable (V) region that consists of 100 to 110 amino acids and differ from one antibody to

another. The remainder of each chain in the molecule – the constant (C) region exhibits

limited variation that defines the two light chain subtypes and the five heavy chains

2

subclasses. Some heavy chains (α, δ, γ) also contain a proline-rich hinge region. The amino

terminal portions, corresponding to the V regions, bind to antigen; effector functions are

mediated by the carboxy-terminal domains. The ε and μ heavy chains, which lack a hinge

region, contain an additional domain in the middle of the molecule. CHO denotes a

carbohydrate group linked to the heavy chain.

7.3 CLASSIFICATION OF ANTIBODY

Serum containing antigen-specific antibodies is called antiserum. The 5 types – IgG, IgM,

IgA, IgD, IgE – (isotypes) are classified according to the type of heavy chain constant region,

and are distributed and function differently in the body.

7.4 FUNCTIONS OF ANTIBODY

1. IgG provides long term protection because it persists for months and years after the

prescence of the antigen that has triggered their production.

2. IgG protect against bacteris, viruses, neutralise bacterial toxins, trigger compliment

protein systems and bind antigens to enhance the effectiveness of phagocytosis.

3. Main function of IgA is to bind antigens on microbes before they invade tissues. It

aggregates the antigens and keeps them in the secretions so when the secretion is

expelled, so is the antigen.

4. IgA are also first defense for mucosal surfaces such as the intestines, nose, and lungs.

5. IgM is involved in the ABO blood group antigens on the surface of RBCs.

6. IgM enhance ingestions of cells by phagocytosis.

7. IgE bind to mast cells and basophils wich participate in the immune response.

8. Some scientists think that IgE’s purpose is to stop parasites.

9. IgD is present on the surface of B cells and plays a role in the induction of antibody

production.

3

VERY SHORT ANSWER TYPE QUESTIONS

Q1. Define antibodies.

Q2. Define immunoglobulins.

Q3. Name the types of antibodies.

Q4. Draw the structure of antibody.

SHORT ANSWER TYPE AND LONG ANSWER TYPE QUESTIONS

Q1.Discuss the classification of antibodies.

Q2. Discuss the function of antibodies.

Q3. Discuss the structure of antibody.

Q4. Discuss the importance of antibody.

UNIT 8

ANTIGEN-ANTIBODY REACTIONS

LEARNING OBJECTIVE

To learn the concept of antigen-antibody reactions.

To learn the classification of antigen-antibody reactions.

To learn the principle of agglutination

To learn the principle of precipitation

To learn the principle of flocculation

8.1 INTRODUCTION

Antigen-antibody interaction, or antigen-antibody reaction, is a specific chemical

interaction between antibodies of the white blood cells and antigens during immune

reaction. It is the fundamental reaction in the body by which the body is protected from

complex foreign molecules, such as pathogens and their chemical toxins. In the blood, the

antigens are specifically and with high affinity bound by antibodies to form an antigen-

antibody complex. The immune complex is then transported to cellular systems where it can

be destroyed or deactivated. There are several types of antibodies and antigens, and each

antibody is capable of binding only to a specific antigen. The specificity of the binding is due

to specific chemical constitution of each antibody. The antigenic determinant or epitope is

recognized by the paratope of the antibody, situated at the variable region of the polypeptide

chain. The variable region in turn has hyper-variable regions which are unique amino acid

sequences in each antibody. Antigens are bound to antibodies through weak and noncovalent

interactions such as electrostatic interactions, hydrogen bonds, Van der Waals forces,

and hydrophobic interactions. The principles of specificity and cross-reactivity of the

antigen-antibody interaction are useful in clinical laboratory for diagnostic purposes. One

basic application is determination of ABO blood group. It is also used as a molecular

technique for infection with different pathogens, such as HIV, microbes, and helminth

parasites.

8.2 CHARACTERISTICS OF ANTIGEN-ANTIBODY REACTIONS

Lock and Key Concept

The combining site of an antibody is located in the Fab portion of the molecule and is

constructed from the hypervariable regions of the heavy and light chains. X-Ray

crystallography studies of antigen-antibody interactions show that the antigenic

determinant nestles in a cleft formed by the combining site of the antibody. Thus, our

concept of antigen-antibody reactions is one of a key (i.e. the antigen) which fits into a

lock (i.e.the antibody).

Non-covalent Bonds

The bonds that hold the antigen to the antibody combining site are all non-covalent in

nature. These includehydrogen bonds, electrostatic bonds, Van der Waals

forces and hydrophobic bonds. Multiple bonding between the antigen and the antibody

ensures that the antigen will be bound tightly to the antibody.

Reversibility

Since antigen-antibody reactions occur via non-covalent bonds, they are by their

nature reversible.

Affinity

Antibody affinity is the strength of the reaction between a single antigenic

determinant and a single combining site on the antibody. It is the sum of the attractive

and repulsive forces operating between the antigenic determinant and the combining

site of the antibody .Affinity is the equilibrium constant that describes the antigen-

antibody reaction. Most antibodies have a high affinity for their antigens.

Avidity

Avidity is a measure of the overall strength of binding of an antigen with many

antigenic determinants and multivalent antibodies. Avidity is influenced by both the

valence of the antibody and the valence of the antigen. Avidity is more than the sum of

the individual affinities.To repeat, affinity refers to the strength of binding between a

single antigenic determinant and an individual antibody combining site whereas

avidity refers to the overall strength of binding between multivalent antigens and

antibodies.

Specificity

Specificity refers to the ability of an individual antibody combining site to react with only

one antigenic determinant or the ability of a population of antibody molecules to react

with only one antigen. In general, there is a high degree of specificity in antigen-antibody

reactions. Antibodies can distinguish differences in the primary structure of an antigen,

isomeric forms of an antigen and secondary and tertiary structure of an antigen

Cross reactivity

Cross reactivity refers to the ability of an individual antibody combining site to react with

more than one antigenic determinant or the ability of a population of antibody molecules

to react with more than one antigen. Cross reactions can arise. Cross reactions arise

because the cross reacting antigen shares anepitope in common with the immunizing

antigen or because it has an epitope which is structurally similar to one on the

immunizing antigen (multispecificity).

8.3Agglutination Reactions

The reaction between a particulate antigen and an antibody results in visible clumping called

agglutination. Antibodies that produce such reactions are known as agglutinins. The

principle of Agglutination reactions are similar to precipitation reactions; they depend on

the cross linking of polyvalent antigens. When the antigen is an erythrocyte it is called

hemagglutination. Theoretically all antibodies can agglutinate particulate antigens bu IgM,

due to its high specificity is a particularly good agglutinin.

There is no agglutination can be observed when the concentration of antibody is high, (lower

dilutions), and then the sample is diluted, agglutination occurs. Prozone effect is defined as

the invisibility of agglutination at high concentrations of antibodies. It is due to the reason

that excess antibody forms very minute complexes that do not clump to form visible

agglutination.

8.3.1Qualitative agglutination test

Agglutination tests can be used in a qualitative manner to assay for the presence of an

antigen or an antibody. The antibody is mixed with the particulate antigen and a positive test

is indicated by the agglutination of the particulate antigen.

For example, to determine patient’s blood type the red blood cells of the person can be

mixed with antibody to a blood group antigen. Another example is that to assay the presence

of antibodies in a patient sample, the serum of the patient is mixed with the red blood cell

(RBC) of a known blood type.

8.3.2.Quantitative agglutination test

To measure the level of antibodies to particulate antigens, agglutination test can be widely

used. For this test, serial dilutions of the sample can be made and it is tested for antibody.

Then a fixed amount of particulate antigen or bacteria or red blood cells can be added to it.

Determine the maximum dilution which forms agglutination and this maximum dilution which

gives observable agglutination is known as the titer. The results is shown as the reciprocal of

the maximum dilution that forms visible agglutination.

8.3.3Passive Hemagglutination

The sensitivity and simplicity of agglutination reactions can be extended to soluble antigens

by the technique of passive agglutination. In this technique, antigen coated red blood cells

are prepared by mixing a soluble antigen with a red blood cells that have been treated with

tannic acid or chromium chloride, both of which promote adsorption of antigen to the surface

of the cells. However, it is possible to coat erythrocytes with a soluble antigen (e.g.. viral

antigen, a polysaccharide or a hapten) and use the coated red blood cells in an agglutination

test for antibody to the soluble antigen.

Serially diluted serum which contain antibody is loaded to each well of the microtiter plate,

after that antigen coated red blood cells is applied to each well. The characteristic pattern of

agglutinated red blood cells on the wells is used as a tool for assaying the agglutination

reactions. If the antigen is particulate, then the antigen can react with the antibody in the

serum and results in the clumping of antigen which shows a positive result.

Over the past several years, there has been a shift away from red blood cells to synthetic

particles, such as latex beads. The preparation can either be used immediately or stored for

later use. The use of synthetic beads offers the advantages of consistency, uniformity, and

stability. Furthermore, agglutination reactions employing synthetic beads can be read

rapidly, often within 3 to 5 minutes of mixing the beads with the test sample. Whether based

on red blood cells or the more convenient and versatile synthetic beads, agglutination

reactions. .The initial step in the test is the linking together of the latex particle by the

antibody molecules that specifically attach to the antigenic determinants on the surface of the

particles. There is a formation of large lattices through these cross links and these large

lattices sediment readily due to the large size of clumps and are visible to the unaided eye

within minutes. The degree of agglutination can be determined by plotting the aggutinant

concentration which gives a bell shaped curve. The antigen-antibody complexes can be

magnified using the latex particles. Many of the latex agglutination tests are performed

manually and detected by visual observation. To determine agglutination there must contain

about 100 clumps, and these clumps must be of about 50 micrometer in size to be seen by

eye.

8.3.4Agglutination Inhibition Reactions

If the antibody is incubated with antigen prior to mixing with latex, agglutination is inhibited;

this is because free antibodies are not available for agglutination. In agglutination inhibition,

the absence of agglutination is diagnostic of antigen, provides a high sensitive assay for

small quantities of antigen. For example home pregnancy kits contain human chorionic

gonadotropin (HCG hormone) coated latex particle and antibody to HCG. A pregnant

woman urine contain HCG which is secreted by the developing placenta after fertilization.

The addition of urine containing HCG, inhibits agglutination of latex particles when the anti-

HCG antibody is added; and thus the pregnancy is indicated by the absence of agglutination.

8.4PRINCIPLE AND APPLICATION OF PRECIPITATION

Precipitation Reaction is a type of antigen–antibody reaction, in which the antigen occurs in

a soluble form. When a soluble antigen reacts with its specific antibody, at an optimum

temperature and PH in the presence of electrolyte antigen antibody complex forms insoluble

precipitate. This reaction is called precipitation reaction. A lattice is formed between the

antigens and antibodies; in certain cases, it is visible as insoluble precipitate. Antibodies that

aggregate soluble antigens are called precipitins. They are based on the interaction of

antibodies and antigens i.e. two soluble reactants that come together to make one insoluble

product, the precipitate. These reactions depend on the formation of lattices (cross-links)

when antigen and antibody exist in optimal proportions. Excess of either component reduces

lattice formation and subsequent precipitation.

Antigen and antibody must be in appropriate concentration relative to each other.

Antigen access: Too much antigen prevents efficient crosslinking/lattice formation.

Antibody access: Too much antibody prevents efficient crosslinking/lattice formation.

Equivalent Antigen and Antibody: Maximum amount of lattice (Precipitate) is formed

The interaction of antibody with soluble antigen may cause formation of insoluble lattice

that will precipitate out of solution. Formation of an antigen–antibody lattice depends on the

valency of both the antibody and antigen. The antibody must be bivalent; a precipitate will

not form with monovalent Fab fragments. Antigen must be bivalent or polyvalent; that is it

must have at least two copies of same epitope or different epitopes that react with different

antibodies present in polyclonal sera.

8.4.1 Prozone and postzone phenomenon

Antigen and antibody reaction occurs optimally only when the proportion of the antigen and

antibody in the reaction mixture is equivalent. On either side of the equivalence zone,

precipitation is actually prevented because of an excess of either antigen or antibody. The

zone of antibody excess is known as the prozone phenomenon and the zone of antigen excess

is known as postzone phenomenon. Prozone: This phenomenon is a false negative response

resulting from high antibody titer which interferes with formation of antigen- antibody

lattice, necessary to visualize a positive precipitation test (i.e. there is too much antibody for

efficient lattice formation). This is because antigen combines with only few antibodies and no

cross-linkage is formed.

In postzone phenomenon, small aggregates are surrounded by excess antigen and again no

lattice network is formed. Thus, for precipitation reactions to be detectable, they must be run

in the zone of equivalence.

When precipitate remains suspended as floccules, instead of sedimentation, reaction is

known as flocculation. Precipitation reactions differ from agglutination reactions in the size

and solubility of the antigen. Antigens are soluble molecules and larger in size in

precipitation reactions. There are several precipitation methods applied in clinical

laboratory for the diagnosis of disease. These can be performed in semi-solid media such as

agar or agarose, or non-gel support media such as cellulose acetate.

8.4.2 APPLICATIONS

1. Detection of unknown antibody to diagnose infection e.g. VDRL test for syphilis.

2. Identification of bacterial component e.g Ascoli’s thermoprecipitin test for Bacillus

anthracis

3. Identification of Bacteria e.g. Lancified grouping of streptococci.

4. Standardisation of toxins and antitoxins.

VERY SHORT ANSWER TYPE QUESTIONS

Q1.Define agglutination.

Q2. What is precipitation?

Q3 Define flocculation.

Q4. Define lattice.

Q5. Define antitoxins.

SHORT ANSWER TYPE AND LONG ANSWER TYPE QUESTIONS

Q1. Discuss the lattice theory.

Q2.Discuss the mechanism of precipitation.

Q3.Discuss the mechanism of agglutination.

Q4.Discuss the mechanism of flocculation.

Q5. What are the characteristics of antigen-antibody reactions.

UNIT 9

SEROLOGICAL TESTS

LEARNING OBJECTIVE:

To learn the concept of widal test.

To learn the concept of Antistreptolysin O test.

To learn the concept of C- Reactive protein test.

To learn the concept of Rheumatoid factor.

To learn the concept of VDRL

To learn the concept of ELISA

9.1 WIDAL TEST

9.1.1 INTRODUCTION

Widal Test is an agglutination test which detects the presence of serum agglutinins (H

and O) in patients serum with typhoid and paratyphoid fever.

When facilities for culturing are not available, the Widal test is the reliable and can

be of value in the diagnosis of typhoid fevers in endemic areas.

It was developed by Georges Ferdinand Widal in 1896.

The patient’s serum is tested for O and H antibodies (agglutinins) against the

following antigen suspensions (usually stained suspensions):

S. Typhi 0 antigen suspension, 9, 12

S. Typhi H antigen suspension, d

S. Paratyphi A 0 antigen suspension, 1, 2, 12

S. Paratyphi A H antigen suspension, a

S. Paratyphi B 0 antigen suspension, 1, 4, 5, 12

S. Paratyphi B H antigen suspension, b, phase 1

S. Paratyphi C 0 antigen suspension, 6, 7

S. Paratyphi C H antigen suspension, c, phase 1

Salmonella antibody starts appearing in serum at the end of first week and rise

sharply during the 3rd week of endemic fever. In acute typhoid fever, O agglutinins

can usually be detected 6–8 days after the onset of fever and H agglutinins after 10–

12 days.

It is preferable to test two specimens of sera at an interval of 7 to 10 days to

demonstrate a rising antibody titre.

Salmonella antigen suspensions can be used as slide and tube techniques.

9.1.2 PRINCIPLE OF WIDAL TEST

Bacterial suspension which carry antigen will agglutinate on exposure to antibodies

to Salmonellaorganisms. Patients’ suffering from enteric fever would possess antibodies in

their sera which can react and agglutinate serial doubling dilutions of killed,

coloured Salmonella antigens in a agglutination test.

The main principle of widal test is that if homologous antibody is present in patients serum, it

will react with respective antigen in the reagent and gives visible clumping on the test card

and agglutination in the tube. The antigens used in the test are “H” and “O” antigens

of Salmonella Typhi and “H” antigen of S.Paratyphi. The paratyphoid “O” antigen are not

employed as they cross react with typhoid “O” antigen due to the sharing of factor 12. “O”

antigen is a somatic antigen and “H” antigen is flagellar antigen.

9.1.3 PREPARATION

H suspension of bacteria is prepared by adding 0.1 per cent formalin to a 24 hours

broth culture or saline suspension of an agar culture.

For preparation of O suspensions of bacteria, the organisms is cultured on phenol

agar (1:800) to inhibit flagella.

Standard smooth strains of the organism are used; S Typhi 901, O and H strains are

employed for this purpose.

The growth is then emulsified in small volume of saline, mixed with 20 times its

volume of alcohol, heated at 40° C to 50° C for 30 minutes and centrifuged.

The antigens are treated with chloroform (preservative) and appropriate dyes are

added for easy identification of antigens.

9.1.4. PROCEDURE

o Place one drop of positive control on one reaction circles of the slide.

o Pipette one drop of Isotonic saline on the next reaction cirlcle. (-ve Control).

o Pipette one drop of the patient serum tobe tested onto the remaining four

reaction circles.

o Add one drop of Widal TEST antigen suspension ‘H’ to the first two reaction

circles. (PC & NC).

o Add one drop each of ‘O’, ‘H’, ‘AH’ and ‘BH’ antigens to the remaining four

reaction circles.

o Mix contents of each circle uniformly over the entire circle with separate

mixing sticks.

o Rock the slide, gently back and forth and observe for agglutination

macroscopically within one minute.

9.1.5 INTERPRETATION OF TEST RESULTS

Agglutination is a positive test result and if the positive reaction is observed with 20 ul of test

sample, it indicates presence of clinically significant levels of the corresponding antibody in

the patient serum. No agglutination is a negative test result and indicates absence of

clinically significant levels of the corresponding antibody in the patient serum.

9.2 ANTISTREPTOLYSIN O TEST

9.2.1 INTRODUCTION

It is a rapid latex agglutination test for the qualitative and semi-quantitative determination of

anti-streptolysin-O antibodies (ASO) in serum. In infections caused by β-haemolytic

streptococci, streptolysin-O is one of the two hemolytic exotoxins liberated from the

bacteria that stimulates production of ASO antibodies in the human serum. The presence

and the level of these antibodies in a serum may reflect the nature and severity of infection.

ASO latex reagent is a stabilized buffered suspension of polystyrene latex particles that have

been coated with Streptolysin O.

9.2.2 MATERIALS USED

ASO Antigen: A stabilized buffered suspension of polystyrene latex particles coated with Streptolysin O and 0.1% sodium azide as preservative. Shake well prior to use.

ASO Positive Control: Human serum containing more than 200 IU/ml ASO and 0.1%

sodium azide as preservative.

ASO Negative Control: Human serum containing 0.1% sodium azide as preservative.

Sufficient disposable pipettes.

Glass test slide.

9.2.3 PROCEDURE

Bring all test reagents and samples to room temperature.

Use a disposable pipette to draw up and place one free-falling drop of each undiluted

sample into its identified circle of the slide. Retain each pipette for mixing in step 5.

Deliver one free-falling drop of positive and negative control into its identified circle.

Mix the ASO latex reagent by gently shaking. Add one free-falling drop of reagent to

each control and sample.

Using the flattened end of the appropriate plastic pipette as a stirrer (step 2),

thoroughly mix each sample with reagent within the full area of the circle.

Discard the disposable pipette.

Slowly rock the slide for exactly two (2) minutes and observe for agglutination under a high intensity light.

Record results.

Re-wash glass slide for future use

9.2.4 TEST RESULT

A test sample is considered to contain ASO antibodies in excess of 200 IU/ml when

agglutination (clumping) is observed when compared to the result of the negative control.

9.3 C-REACTIVE PROTEIN TEST

9.3.1 INTRODUCTION

Rheumatoid Factors are autoantibodies that react with individuals own immunoglobulin.

These antibodies are usually directed against the Fc fragment of the human IgG. RF have

been associated with three major immunoglobulin classes: IgM, IgG, and IgA. Of these IgM

and IgG are the most common. The formation of immune complex in the joint space leads to

the activation of complement and destructive inflammation, causing rheumatoid arthritis

(RA).

As indicated by its name, RF has particular application to diagnosis and monitoring of

rheumatoid arthritis. Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting

primarily the joints and periarticular tissues. Rheumatoid factor is detected in 60-80% of

cases of diagnosed rheumatoid arthritis. However, it is also detectable sometimes in the

serum of patients with Systemic Lupus Erythematosus (SLE) and in certain non-rheumatic

conditions. Elevated values may also be observed in normal elderly population.

9.3.2 PRINCIPLE

A number of methods are available for testing of RF. The most commonly used serological

method is based on latex agglutination test. As RF is an IgM class of antibody directed

against the Fc portion of the IgG molecule, it is detected by it’s ability to agglutinate

the latex. Reagent used is a suspension of polystyrene latex particles in glycine-saline buffer.

9.3.3 PROCEDURE

Bring all reagents and specimens to room temperature.

Place one drop of the positive control and 40ul of the patient serum into separate circles on the slide.

Gently and add one drop of RF latex reagent on each circle of sample to be tested and

control.

Use separate Applicator sticks/stir sticks to spread reaction mixture over entire area of the particular field.

Tilt the slide back and forth for 2 minutes in a rotary shaker so that the mixture rotates slowly.

Observe for agglutination after 2 minutes under bright artificial light.

9.3.4 INTERPRETATION

Agglutination of latex particles is considered a positive reaction, indicating the presence of

rheumatoid factor at a significant and detectable level.

Positive result: An agglutination of the latex particles suspension will occur within two

minutes, indicating a RF level of more than 18 IU/ml.

Negative result: No agglutination of the latex particles suspension within two minutes.

9.5 VENERAL DISEASE RESEARCH LABORATORY

9.5.1 INTRODUCTION

Venereal Disease Research Laboratory (VDRL) Test is a slide flocculation test

employed in the diagnosis of syphilis. Since the antigen used in this test is cardiolipin,

which is a lipoidal extracted from beef heart, it is not a specific test. This test is also

classified as non-specific or non-treponemal or standard test. The antibodies reacting

with cardiolipin antibodies have been traditionally (but incorrectly) termed “regain”.

Principle: Patients suffering from syphilis produce antibodies that react with cardiolipin

antigen in a slide flocculation test, which are read using a microscope. It is not known if

the antibodies that react with cardiolipin are produced against some lipid component of

Treponema pallidum or as a result of tissue injury following infection. Requirements:

Patient’s serum, water bath, freshly prepared cardiolipin antigen, VDRL slide,

mechanical rotator, pipettes, hypodermic syringe with unbeveled needle and

microscope. Known reactive and non-reactive serum controls are also required.

VDRL antigen: The cardiolipin antigen is an alcoholic solution composed of 0.03%

cardiolipin, 0.21% lecithin and 0.9% cholesterol. The cardiolipin antigen must be

freshly constituted each day of test. The working antigen is a buffered saline suspension

of cardiolipin.

VDRL slide: This is a glass slide measuring 2 X 3 inch with 12 concave depressions,

each measuring 16 mm in diameter and 1.78 mm deep.

9.5.2 PROCEDURE

Patients’ serum is inactivated by heating at 56o C for 30 minutes in a water bath to

remove non-specific inhibitors (such as complement)

The test can be performed both qualitatively and quantitatively.

Those tests that are reactive by qualitative test are subjected to quantitative test to

determine the antibody titres.

0.05 ml of inactivated serum is taken into one well.

1/60th ml (or 1 drop from 18 gauge needle) of the cardiolipin antigen is then added

with the help of a syringe (unbeveled) to the well and rotated at 180 rpm for 4

minutes.

Every test must be accompanied with known reactive and non-reactive controls.

The slide is then viewed under low power objective of a microscope for flocculation.

The reactive and non-reactive controls are looked first to verify the quality of the

antigen.

Depending on the size the results are graded as weakly reactive (W) or reactive (R).

Reactive samples are then subjected to quantitative test.

9.5.3 QUANTITATIVE TEST

This is performed to determine the antibody titres. The serum is doubly diluted in saline

from 1in 2 to 1:256 or more. 0.05 ml of each dilution is taken in the well and 1/60 ml of

antigen is added to each dilution and rotated in a rotator. The results are then checked

under the microscope. The highest dilution showing flocculation is considered as

reactive titre. Sometimes, due to very high level of antibodies in the serum (prozone

phenomenon) the qualitative test may be non-reactive. If the clinical findings are

strongly suggestive of syphilis, a quantitative test may be directly performed on the

serum specimen.

9.5.4 CSF VDRL

VDRL test may also be performed on CSF samples in the diagnosis of neurosyphilis.

Quantitative VDRL is the test of choice on CSF specimens. However, there are some

variations in this test.

The antigen is diluted in equal volumes with 10% saline, CSF must not be heated (or

inactivated), the volume of antigen solution taken is 0.01 ml (or 1 drop from 21 gauge

needle) and rotation time is 8 minutes.

Rest of the procedure remains same. Significance of VDRL test: VDRL test becomes

positive 1-2 weeks after appearance of (primary lesion) chancre. The test becomes

reactive (50-75%) in the late phase of primary syphilis, becomes highly reactive

(100%) in the secondary syphilis and reactivity decreases (75%) thereafter.

Treatment in the early stages of infection may completely suppress production of

antibodies and result in non-reactive tests. Effective treatment in the primary or

secondary stages results in rapid fall in titre and the test may turn non-reactive in few

months.

Treatment in latent or late syphilis has very little effect on the titre and the titres may

persist at low levels for long periods.

Since the titre falls with effective treatment, it can be used for assessment of prognosis.

VDRL test is more suitable as a screening agent than a diagnostic tool. VDRL test is

also helpful in the diagnosis of congenital syphilis. Since passively transferred

antibodies through placenta may give false reactive test in serum of the infant, a repeat

test after a month showing no increase in titre may help rule out congenital syphilis.

Since the test employs a non-treponemal antigen, there are many chances of false

positive results. False positivity (other than technical) may be due to physiological of

pathological conditions. These are called biological false positives (BFP). If the remain

positive for less than 6 months it is considered acute and they remain positive for longer

than 6 months it is called chronic BFP. The physiological reasons for BFP include

pregnancy, menstruation, repeated blood loss, vaccination, severe trauma etc while the

reasons for pathological BFP include malaria, infectious mononucleosis, hepatitis,

relapsing fever, tropical eosinophilia, lepromatous leprosy, SLE, rheumatoid arthritis

etc. A reactive VDRL test does not necessarily imply that the person is syphilitic. The

diagnosis must be made in conjunction with clinical findings. Any reactive VDRL test

must be confirmed with a specific or treponemal test such as TPHA, FTA-ABS test.

9.6 ELISA

9.6.1 INTRODUCTION

ELISA is an antigen antibody reaction. In 1971, ELISA was introduced by Peter Perlmann

and Eva Engvall at Stockholm University in Sweden. It is a common laboratory technique

which is usually used to measure the concentration of antibodies or antigens in blood.

ELISA is a plate based assay technique which is used for detecting and quantifying

substances such as peptides, proteins, antibodies and hormones. An enzyme conjugated with

an antibody reacts with colorless substrate to generate a colored product. Such substrate is

called chromogenic substrate. A number of enzymes have been used for ELISA such as

alkaline phosphatase, horse radish peroxidase and beta galactosidase. Specific substrate

such as ortho-phenyldiamine dihydrochloride (for peroxidase), paranitrophenyl phosphate

(for alkaline phosphatase) are used which are hydrolysed by above enzymes to give colored

end product.

9.6.2 PRINCIPLE

ELISAs are typically performed in 96-well polystyrene plates. The serum is incubated in a

well, and each well contains a different serum. A positive control serum and a negative

control serum would be included among the 96 samples being tested. Antibodies or antigens

present in serum are captured by corresponding antigen or antibody coated on to the solid

surface. After some time, the plate is washed to remove serum and unbound antibodies or

antigens with a series of wash buffer. To detect the bound antibodies or antigens, a

secondary antibodies that are attached to an enzyme such as peroxidase or alkaline

phosphatase are added to each well. After an incubation period, the unbound secondary

antibodies are washed off. When a suitable substrate is added, the enzyme reacts with it to

produce a color. This color produced is measurable as a function or quantity of antigens or

antibodies present in the given sample. The intensity of color/ optical density is measured at

450nm. The intensity of the color gives an indication of the amount of antigen or antibody.

9.6.3TYPES OF ELISA

Frequently there are 3 types of ELISA on the basis of binding structure between the Antibody

and Antigen.

Indirect ELISA

Sandwich ELISA

Competitive

9.6.4 INDIRECT ELISA

Antibody can be detected or quantitatively determined by indirect ELISA. In this technique,

antigen is coated on the microtiter well. Serum or some other sample containing primary

antibody is added to the microtiter well and allowed to react with the coated antigen. Any

free primary antibody is washed away and the bound antibody to the antigen is detected by

adding an enzyme conjugated secondary antibody that binds to the primary antibody.

Unbound secondary antibody is then washed away and a specific substrate for the enzyme is

added. Enzyme hydrolyzes the substrate to form colored products. The amount of colored end

product is measured by spectrophotometric plate readers that can measure the absorbance of

all the wells of 96-well plate.

9.6.5 PROCEDURE OF INDIRECT ELISA

Coat the micro titer plate wells with antigen.

Block all unbound sites to prevent false positive results.

Add sample containing antibody (e.g. rabbit monoclonal antibody) to the wells and incubate the plate at 37°c.

Wash the plate, so that unbound antibody is removed.

Add secondary antibody conjugated to an enzyme (e.g. anti- mouse IgG).

Wash the plate, so that unbound enzyme-linked antibodies are removed.

Add substrate which is converted by the enzyme to produce a colored product.

Reaction of a substrate with the enzyme to produce a colored product.

9.6.6 ADVANTAGES OF INDIRECT ELISA

Increased sensitivity, since more than one labeled antibody is bound per primary

antibody.

A wide variety of labeled secondary antibodies are available commercially.

Maximum immunoreactivity of the primary antibody is retained because it is not labeled.

Versatile because many primary antibodies can be made in one species and the same

labeled secondary antibody can be used for detection.

Flexibility, since different primary detection antibodies can be used with a single labeled

secondary antibody.

Cost savings, since fewer labeled antibodies are required.

Different visualization markers can be used with the same primary antibody.

9.6.7 DISADVANTAGES OF INDIRECT ELISA

Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal.

An extra incubation step is required in the procedure.

9.6.8 SANDWICH ELISA

Antigen can be detected by sandwich ELISA. In this technique, antibody is coated on the

microtiter well. A sample containing antigen is added to the well and allowed to react with

the antibody attached to the well, forming antigen-antibody complex. After the well is

washed, a second enzyme-linked antibody specific for a different epitope on the antigen is

added and allowed to react with the bound antigen. Then after unbound secondary antibody

is removed by washing. Finally substrate is added to the plate which is hydrolyzed by enzyme

to form colored products.

9.6.9 PROCEDURE OF SANDWICH ELISA

Prepare a surface to which a known quantity of antibody is bound.

Add the antigen-containing sample to the plate and incubate the plate at 37°c.

Wash the plate, so that unbound antigen is removed.

Add the enzyme-linked antibodies which are also specific to the antigen and then incubate at 37°c.

Wash the plate, so that unbound enzyme-linked antibodies are removed.

Add substrate which is converted by the enzyme to produce a colored product.

Reaction of a substrate with the enzyme to produce a colored product.

9.6.10 ADVANTAGES

High specificity, since two antibodies are used the antigen is specifically captured and

detected.

Suitable for complex samples, since the antigen does not require purification prior to

measurement.

Flexibility and sensitivity, since both direct and indirect detection methods .

9.6.11 COMPETITIVE ELISA

This test is used to measure the concentration of an antigen in a sample. In this test, antibody

is first incubated in solution with a sample containing antigen. The antigen-antibody mixture

is then added to the microtitre well which is coated with antigen. The more the antigen

present in the sample, the less free antibody will be available to bind to the antigen-coated

well. After the well is washed, enzyme conjugated secondary antibody specific for isotype of

the primary antibody is added to determine the amount of primary antibody bound to the

well. The higher the concentration of antigen in the sample, the lower the absorbance.

9.6.12 PROCEDURE

Antibody is incubated with sample containing antigen.

Antigen-antibody complex are added to the microtitre well which are pre-coated with

the antigen.

Wash the plate to remove unbound antibody.

Enzyme linked secondary antibody which is specific to the primary antibody is added.

Wash the plate, so that unbound enzyme-linked antibodies are removed.

Add substrate which is converted by the enzyme into a fluorescent signal.

9.6.13 ADVANTAGES

High specificity, since two antibodies are used.

High sensitivity, since both direct and indirect detection methods can be used.

Suitable for complex samples, since the antigen does not require purification prior to

measurement.

VERY SHORT ANSWER TYPE QUESTIONS

Q1. Define widal test.

Q2. Write the full form of ASO.

Q3. Write the full form of CRP.

Q4. Define RF FACTOR.

Q5. Write the full form of VDRL.

Q6. Write the full form of ELISA.

Q7. . Write the full form of RF factor.

SHORT ANSWER TYPE QUESTIONS AND LONG ANSWER TYPE QUESTIONS

Q1.Discuss the principle and procedure of widal test

.Q2. Discuss the procedure and interpretation of widal test.

Q3. Discuss the principle and procedure of ASO test.

Q4. Discuss the procedure and interpretation of ASO test.

Q5 Discuss the principle and procedure of CRP test

Q6 Discuss the procedure and interpretation of CRP test.

Q7 Discuss the principle and procedure of RF test

Q8 Discuss the procedure and interpretation of RF test.

Q9 Discuss the principle and procedure of VDRL test

Q10 Discuss the procedure and interpretation of VDRL test.

Q11 Discuss the principle and procedure of ELISA test

Q12 Discuss the procedure and interpretation of ELISA test.

APPLICATION OF ELISA

1. Presence of antigen or the presence of antibody in a sample can be evaluated.

2. Determination of serum antibody concentrations in a virus test. 3. Used in food industry when detecting potential food allergens. 4. Applied in disease outbreaks- tracking the spread of disease e.g. HIV, bird

flu, common, colds, cholera, STD etc.