enzyme linked immunosorbent assay. immunoassays: the purpose of an immunoassay is to measure (or, in...

ELISAEnzyme linked immunosorbent assay

Immunoassays:The purpose of an immunoassay is

to measure (or, in a qualitative assay, to detect) an analyte. (An analyte is anything measured by a laboratory test. In immunoassay testing, the analyte may be either an antibody, or an antigen).

Immunoassay is the method of choice for measuring analytes normally present at very low concentrations that cannot be determined accurately by other less expensive tests.

The analytes being measured may be those that are naturally present in the body (such as a thyroid hormone), those that the body produces but are not typically present(such as a cancer antigen), or those that do not naturally occur in the body (such as an abused drug).

All immunoassays require the use of labeled material in order to measure the amount of antigen or antibody present.

Common uses include measurement of drugs, hormones, specific proteins, tumor markers, and markers of cardiac injury.

Qualitative immunoassays are often used to detect antigens on infectious agents and antibodies that the body produces to fight them. For example, immunoassays are used to detect antigens on Hemophilus, Cryptococcus , and Streptococcus organisms in the cerebrospinal fluid (CSF) of meningitis patients.

They are also used to detect antigens associated with organisms that are difficult to culture, such as hepatitis B virus and Chlamydia trichomatis . Immunoassays for antibodies produced in viral hepatitis, HIV, and Lyme disease are commonly used to identify patients with these diseases.

General Components of immunoassays

Labeled analyte: Used to detect reaction which has occurred, labeles can be Radioactive(RIA), Enzymes(ELISA), Fluorescent or Chemiluminescent.

Standards or Calibrators: Substance of exact known concentration, used to construct a standard curve for quantitative measurements of the analyte.

A method for detection of the label depending on the nature of the label used.

Definition Enzyme-linked Immunosorbent

assay, commonly known as ELISA (or EIA), are quantitative immunological procedures in which the Ag- Ab reaction is monitored by enzyme measurements. Is similar in principle to RIA but depends on an enzyme rather than a radioactive label. An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product. Such a substrate is called a chromogenic substrate.

ELISA techniques used for qualitative detection or quantitative measurement of either antigen or antibody.

Each type of ELISA can be used qualitatively to detect the presence of antibody or antigen by using cutt off value. Alternatively, a standard curve based on known concentrations of antibody or antigen is prepared, from which the unknown concentration of a sample can be quantitatively determined.ELISA reader

ELISA Kit:1. Microplate: Is adsorbent and container,

made usually from polystyrene which considered a strong protein adsorbent.

2. Calibrators or standard, positive and negative control and cutt off.

3. Conjugate enzyme:

It is an antibody linked with enzyme, it is the key substance in ELISA.

The good conjugate posses not only catalytic activity of enzyme but also immunological competence of antibody. The conjugate must has favorable stability.

Enzymes used in ELISA should meet requirements such as high purity, high conversion rate, favorable specificity, stable properties, rich resources, cheap price and remaining active component and catalytic capacity after becoming conjugated.

There are many types of enzymes used:

Horseradish peroxidase(HPR) Alkaline phosphatase(AP). β-Galactosidase. Urease

High purified IgG is usually used in order to avoid the interference of other proteins when it is linked with enzyme.

Conjugate is either ready to use or concentrated “need to dillute”.

(most commonly used)

4. Substrate bottle:

Contain substrate specific to conjugate enzyme and chromogenic material.

Substrate is selected according to the conjugate enzyme used.

5. Washing solution:

PBS-Tween 20 the most used. Must be dilluted 1:10 before using if dilution is not mentioned in the pamphlet.

6. Stop solution:

Used to stop the reaction and to develop the color.

H2SO4 is widely used to stop HRP reaction.

HCL and NaOH may be used.7. Occasionally diluent solution: used to

dilute the sample if necessary. STOPPING SUBSTRATE ENZYME

1 M NaOH p-NPP Alkaline Phosphatase

1 M H2SO4 TMBHorseradish Peroxidase

Types of ELISA

Indirect ELISA Antibody can be detected or

quantitatively determined with an indirect ELISA(e.g: HCV&HIV). Serum containing primary antibody is added to an antigen-coated microtiter well and allowed to react with the antigen attached to the well.

After that any free primary Ab is washed away, the presence of antibody bound to the antigen is detected by adding an enzyme-conjugated secondary antibody, which binds to the primary antibody. Any free 2ry Ab then is washed away, and a substrate for the enzyme is added.

The amount of colored reaction product that forms is measured by specialized spectrophotometric plate readers, which can measure the absorbance of all of the wells of a 96-well plate in seconds called ELISA reader.

Sandwich ELISA (Direct) Antigen can be detected or measured by a sandwich ELISA(eg: Tumor markers & hormons). In this technique, the antibody (rather than the antigen) is immobilized on a microtiter well.

A sample containing antigen is added and allowed to react with the immobilized antibody. After the well is washed, a second enzyme-linked antibody (conjugate) specific for a different epitope on the antigen is added and allowed to react with the bound antigen.

After any free conjugate is removed by washing, substrate is added, and the colored reaction product is measured.

Competitive ELISA Competitive ELISA is another variation for measuring amounts of antigen (e.g Free testosteron), in which there is a competition between labeled and unlabeled antigen for a limited number of binding sites on the antibody.

Wells are coated with antibodies.

1 .Anti-analyte

2 .Analyte-E + sample

1 .Anti-analyte

Low [analyte] High [analyte]

E E E E E E E

3 .Wash 2 .Analyte- E

+ sample 1 .Anti-analyte

Low [analyte] High [analyte]

4 .Substrate 3 .Wash

2 .Analyte-E + sample

1 .Analyte

Low [analyte] High [analyte]

Competitive ELISA

In the competitive ELISA, the higher concentration of antigen in the original sample, the lower the absorbance (inversely proportional relationship), i.e.: The more intense color, the less Ag.

The less intense color, the more Ag.

Competitive vs. non competitive

Non-competitive competitive

an excess amount of an antibody is used to capture the analyte from the sample

competition between labeled and unlabeled antigen for a limited number of binding sites on the antibody

Mechanism

The measurement of labeled analyte is directly proportional to concentration of Ag or Ab in the sample

The amount of antigen in the test sample is inversely related to the amount of label measured

Relationship b/w label and analyte

more sensitive and reproducible

Less sensitive Sensitivity

Plate Preparation (coating and blocking)

1. Dilute the capture antibody to the working concentration. Immediately coat a 96-well microplate with 100μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4℃.

2. wash with at least 300μl wash buffer three times. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.

3. Block plates by adding 300 μL of blocking buffer to each well. Incubate at room temperature for a minimum of 1 hour.

4. Repeat the wash as in step 2. The plates are now ready for sample addition.

ELISA Protocol

Assay Procedure

1. Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at room temperature.

2. wash 3 times as in step 2 of plate preparation.

3. Add 100 μL of the detection(conjugate) antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1 hour at room temperature.

4. wash 3 times as in step 2 of plate preparation.

5. Add 200 μL of substrate solution to each well. Incubate for 20 minutes at room temperature .Avoid placing the plate in direct light.

6. Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.

7. Determine the Absorbance of each well immediately, using a microplate reader set to 450 nm.

APPLICATIONS OF ELISA1 -Hormones 7 -Vaccine Quality Control

2 -Proteins 8 -FOR GMO (Genetically modified organism)

3 -Infectious Agent ( Viral, Bacterial, Parasitic, Fungal )

9 -For Rapid Test

4 -Drug Markers 10 -IgG, IgM, IgA

5 -Tumor Markers 11 -In New Born Screening

6 -Serum Proteins 12 -In Clinical Research

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Advantages of ELISA:

Reagents are relatively cheap & have a long shelf life

ELISA is highly specific and sensitive.

No radiation hazards occur during labeling or disposal of waste(safe).

Easy to perform and quick procedures.

Equipment can be inexpensive and widely available.

Disadvantages of ELISA:

Measurement of enzyme activity can be more complex than measurement of activity of some type of radioisotopes.

Enzyme activity may be affected by plasma constituents.

False positives/negatives possible, especially with mutated/altered antigen