Download - Bio 151 lectures for examination 2
VACCINESREFERENCES: a) Abbas; b) Kuby; and c) Lectures of Dr Nina Gloriani, MD, PhD
Monday, September 10, 2012
EDWARD JENNER
• Father of Vaccinology & Founder of Immunology
• Major contribution: first reliable method of conferring lasting immunity to a major contagious disease (smallpox)
• Initiated the technique of vaccination
Monday, September 10, 2012
LOOKING BACK...• 18th century: Small pox
• 19th century: Rabies; typhoid; cholera; plague
• Early 20th century: BCG; Yellow fever; pertussis: influenza; diphtheria; tetanus
• After world war II:
• OPV; MMR; Adenovirus; Salmonella Ty21a; varicella; Injected Polio; Rabies; Japanese encephalitis; Hepatitis A; Pneumococcus; meningococcus, Hemophilus influenza PRP, Hepatitis B; Acellular pertussis
Monday, September 10, 2012
RECALL: IMMUNITY
Monday, September 10, 2012
ACQUIRING PASSIVE AND ACTIVE IMMUNITY
Monday, September 10, 2012
ACTIVE & PASSIVE IMMUNITY
Monday, September 10, 2012
PASSIVE IMMUNIZATION
• Administration of preformed antibodies to treat infection
• Can be life-saving where toxins are already circulating : tetanus, diphtheria, snake bite
• Important where high titer specific antibody is required
Monday, September 10, 2012
PASSIVE IMMUNIZATION
• Preformed antibodies generally made in horses, but occasionally obtained from recovered patients
• Over 1000 donors used for each pool of ISG, and sera are screened for HIV and hepatitis B and C
Monday, September 10, 2012
Monday, September 10, 2012
COMMONLY USED AGENTSDISEASE AGENT
Black widow spider bite Horse antitoxin
Botulism Horse antitoxin
Diphtheria Horse antitoxin
Hepatitis A & B Pooled human IgG
Measles Pooled human IgG
Rabies Pooled human IgG
Snake bite Horse antivenin
Tetanus Pooled human IgG or horse antitoxin
Monday, September 10, 2012
PASSIVE IMMUNIZATION
Monday, September 10, 2012
NON-SPECIFIC IMMUNOTHERAPY• Same compounds that act as adjuvants for vaccines also
used on their own to boost the general level of immune activity
Monday, September 10, 2012
IMMUNOTHERAPY OF TUMORS
Monday, September 10, 2012
Monday, September 10, 2012
Monday, September 10, 2012
ACTIVE IMMUNITY
• The process of administering antigen to a live host to induce an immune response for either academic or public health reasons
• Vaccines have been developed as a prophylactic measure to prevent disease caused by infectious agents, provided their use caused only low levels of morbidity
• WORLD HEALTH ORGANIZATION: Expanded Program on Immunization (EPI) and Vaccine development programs/initiatives
Monday, September 10, 2012
GENERAL TYPE OF VACCINES
Monday, September 10, 2012
ANTIGENIC PREPARATIONS USED AS VACCINES
Monday, September 10, 2012
COMPARING NOTES
Monday, September 10, 2012
LIVE ATTENUATED VACCINES
Monday, September 10, 2012
EFFECT OF VACCINES ON VIRAL DISEASES
Monday, September 10, 2012
KILLED-WHOLE ORGANISM VACCINE
Monday, September 10, 2012
POLIO: LIVE VERSUS KILLED VACCINE
Monday, September 10, 2012
Monday, September 10, 2012
SUBCELLULAR FRAGMENTS VACCINES
Monday, September 10, 2012
Monday, September 10, 2012
TOXIN-BASED VACCINES
Monday, September 10, 2012
Monday, September 10, 2012
DNA VACCINES
Monday, September 10, 2012
THE MALARIA VACCINE STRATEGY
Monday, September 10, 2012
VACCINES IN GENERAL USE
Monday, September 10, 2012
VACCINES RESTRICTED TO CERTAIN GROUPS
Monday, September 10, 2012
SAFETY CONCERNS WITH VACCINES
Monday, September 10, 2012
WANTED VACCINES...
Monday, September 10, 2012
ADJUVANTS
Monday, September 10, 2012
EFFECTS OF ADJUVANTS
• Concentration of antigen in a site where lymphocytes are exposed to it
• the “DEPOT” effect
• Induction of cytokines which regulate lymphocyte function
• cytokines themselves shown to be effective adjuvants, particularly when coupled directly to antigen
Monday, September 10, 2012
START HERE
Monday, September 10, 2012
VACCINE CONSIDERATIONS #1
• Type
• Indications
• Target group
• Dose
• Route of Administration
• Monitoring Side Effects
Monday, September 10, 2012
TARGET GROUPSMonday, September 10, 2012
Monday, September 10, 2012
MONITORING
• Side effects: Fever
• LOCAL SYMPTOMS AT INJECTION SITE: Soreness, redness, swelling size
• GENERAL symptoms– headache, malaise, loss of appetite, nausea, vomiting
• Adverse Reactions
• ContraindicationsMonday, September 10, 2012
• SAFETY: Toxicity, teratogenicity, pyrogenicity, adverse reactions
• IMMUNOGENICITY: Seroconversion: levels of antibody before and after immunization; Geometric mean titer (GMT): antibody titer following vaccination
• EFFICACY: correlation between seroconversion and PROTECTION (Antibodies; CMI or both)
VACCINE CONSIDERATIONS #2
Monday, September 10, 2012
SAFETY: ADVERSE REACTIONS
• MILD: adverse reaction does not interfere with normal activities; may be bothersome
• MODERATE: adverse reaction produces some impairment of functioning but NOT hazardous to health; uncomfortable or disturbing
• SEVERE: adverse reaction produces significant impairment of functioning or incapacitation; a definite hazard to health
Monday, September 10, 2012
EFFICACY• Streptococcus pneumoniae; Neisseria meningitidis;
Hemophilus influenzae capsular polysaccharide (85%)
• Diphtheria and tetanus Toxoids (>90%)
• Bordetella pertussis (whooping cough) (>90%)
• Salmonella typhi (killed or live attenuated) (80%)
• Mycobacterium tuberculosis (BCG) (0-70%)Monday, September 10, 2012
VACCINE-PREVENTABLE
DISEASESMonday, September 10, 2012
VACCINES FOR YOU
Following primary series ofimmunizations, young adults should have a further dose of TD at age 15-16 years
Health care workers need evidence of two measles vaccinations or blood test conforming immunity
Meningitis vaccination recommended for students & staff in residential colleges of the university or in any group accommodation
Monday, September 10, 2012
VACCINES FOR YOU
Following primary series ofimmunizations, young adults should have a further dose of TD at age 15-16 years
Health care workers need evidence of two measles vaccinations or blood test conforming immunity
Meningitis vaccination recommended for students & staff in residential colleges of the university or in any group accommodation
Monday, September 10, 2012
VACCINES FOR YOU
Basic Vaccinations:
TetanusDiphtheriaPolioMeaslesMumpsRubellaHepatitis BMeningitis
Following primary series ofimmunizations, young adults should have a further dose of TD at age 15-16 years
Health care workers need evidence of two measles vaccinations or blood test conforming immunity
Meningitis vaccination recommended for students & staff in residential colleges of the university or in any group accommodation
Monday, September 10, 2012
VACCINES FOR YOU
• RUBELLA
• Lyophilized preparation of highly attenuated Wistar RA 27/3 strain rubella virus propagated in human diploid cells
• 0.5ml single dose , SC
• Side effects: rashes, malaise, temperature elevation, cough, coryza, headache, transient arthralgia & arthritis with or w/o joint effusion
Monday, September 10, 2012
VACCINES FOR YOU
• RUBELLA
• Lyophilized preparation of highly attenuated Wistar RA 27/3 strain rubella virus propagated in human diploid cells
• 0.5ml single dose , SC
• Side effects: rashes, malaise, temperature elevation, cough, coryza, headache, transient arthralgia & arthritis with or w/o joint effusion
Monday, September 10, 2012
VACCINES FOR YOU
Basic Vaccinations:
TetanusDiphtheriaPolioMeaslesMumpsRubellaHepatitis BMeningitis
• RUBELLA
• Lyophilized preparation of highly attenuated Wistar RA 27/3 strain rubella virus propagated in human diploid cells
• 0.5ml single dose , SC
• Side effects: rashes, malaise, temperature elevation, cough, coryza, headache, transient arthralgia & arthritis with or w/o joint effusion
Monday, September 10, 2012
VACCINES FOR YOU
• MMR
• Live attenuated measles (Edmonston –Zagreb strain), mumps (Rubini strain), rubella (Wistar RA 27/3 strain) viruses.
• Strains propagated on human diploid cells, no antibiotics
• Vaccinate infants only after 15 months = Repeat immunization after 15 years for infants immunized before age 12 months
Monday, September 10, 2012
VACCINES FOR YOU
• MMR
• Live attenuated measles (Edmonston –Zagreb strain), mumps (Rubini strain), rubella (Wistar RA 27/3 strain) viruses.
• Strains propagated on human diploid cells, no antibiotics
• Vaccinate infants only after 15 months = Repeat immunization after 15 years for infants immunized before age 12 months
Monday, September 10, 2012
VACCINES FOR YOU
Basic Vaccinations:
TetanusDiphtheriaPolioMeaslesMumpsRubellaHepatitis BMeningitis
• MMR
• Live attenuated measles (Edmonston –Zagreb strain), mumps (Rubini strain), rubella (Wistar RA 27/3 strain) viruses.
• Strains propagated on human diploid cells, no antibiotics
• Vaccinate infants only after 15 months = Repeat immunization after 15 years for infants immunized before age 12 months
Monday, September 10, 2012
VACCINES FOR YOU
• CHICKEN POX (VARICELLA)
• Live attenuated varicella vaccine
• For healthy infants from age 9 months onward, healthy subjects , high risk patients & healthy close contacts, patients with acute leukemia, under immunosuppressive treatment, patients with planned organ transplantation, patients with chronic diseases & healthy close contacts
• Dose recommended: 2 doses 4-6 weeks apart for adults
Monday, September 10, 2012
VACCINES FOR YOU
• CHICKEN POX (VARICELLA)
• Live attenuated varicella vaccine
• For healthy infants from age 9 months onward, healthy subjects , high risk patients & healthy close contacts, patients with acute leukemia, under immunosuppressive treatment, patients with planned organ transplantation, patients with chronic diseases & healthy close contacts
• Dose recommended: 2 doses 4-6 weeks apart for adults
Monday, September 10, 2012
VACCINES FOR YOU
• CHICKEN POX (VARICELLA)
• Live attenuated varicella vaccine
• For healthy infants from age 9 months onward, healthy subjects , high risk patients & healthy close contacts, patients with acute leukemia, under immunosuppressive treatment, patients with planned organ transplantation, patients with chronic diseases & healthy close contacts
• Dose recommended: 2 doses 4-6 weeks apart for adults
Additional Vaccinations:
Chicken poxHepatitis A InfluenzaPertussis
Salmonella typhi
CholeraPneumonia
Monday, September 10, 2012
VACCINES FOR YOU
• HEPATITIS A
• Inactivated hepatitis A virus for individuals > 15 years of age in pre-filled syringe
• Virosomal hepatitis A virus antigen (RG-SB strain) for adults & children over 12 months
• Basic immunization : Intramuscular injection
• Booster immunization: 6, 12 or 18 months after first immunization
• Simultaneous active & passive immunization at another site
Monday, September 10, 2012
VACCINES FOR YOU
• HEPATITIS A
• Inactivated hepatitis A virus for individuals > 15 years of age in pre-filled syringe
• Virosomal hepatitis A virus antigen (RG-SB strain) for adults & children over 12 months
• Basic immunization : Intramuscular injection
• Booster immunization: 6, 12 or 18 months after first immunization
• Simultaneous active & passive immunization at another site
Monday, September 10, 2012
VACCINES FOR YOU
• HEPATITIS A
• Inactivated hepatitis A virus for individuals > 15 years of age in pre-filled syringe
• Virosomal hepatitis A virus antigen (RG-SB strain) for adults & children over 12 months
• Basic immunization : Intramuscular injection
• Booster immunization: 6, 12 or 18 months after first immunization
• Simultaneous active & passive immunization at another site
Additional Vaccinations:
Chicken poxHepatitis A InfluenzaPertussis
Salmonella typhi
CholeraPneumonia
Monday, September 10, 2012
VACCINES FOR YOU
• INFLUENZA
• Highly purified surface antigens of influenza viruses types A & B constituted strains annually recommended by WHO
• Polyvalent whole virus vaccine against strains of groups A & B
• Purified split inactivated influenza virus
Monday, September 10, 2012
VACCINES FOR YOU
• INFLUENZA
• Highly purified surface antigens of influenza viruses types A & B constituted strains annually recommended by WHO
• Polyvalent whole virus vaccine against strains of groups A & B
• Purified split inactivated influenza virus
Monday, September 10, 2012
VACCINES FOR YOU
• INFLUENZA
• Highly purified surface antigens of influenza viruses types A & B constituted strains annually recommended by WHO
• Polyvalent whole virus vaccine against strains of groups A & B
• Purified split inactivated influenza virus
Additional Vaccinations:
Chicken poxHepatitis A InfluenzaPertussis
Salmonella typhi
CholeraPneumonia
Monday, September 10, 2012
VACCINES FOR YOU • CHOLERA
• Orochol E Berna contains not < 2x109 viable organisms of the attenuated strain Vibrio CVD 103-HgR in a lyophilized form
• For ORAL active immunization of adults & children >2 years against cholera
• Dosage: Resuspend contents of both chambers at the same time in cold or lukewarm water, mix carefully for 5-10 seconds & drink immediately.
• Do not resuspend in milk, juice or carbonated beverages.
Monday, September 10, 2012
VACCINES FOR YOU • CHOLERA
• Orochol E Berna contains not < 2x109 viable organisms of the attenuated strain Vibrio CVD 103-HgR in a lyophilized form
• For ORAL active immunization of adults & children >2 years against cholera
• Dosage: Resuspend contents of both chambers at the same time in cold or lukewarm water, mix carefully for 5-10 seconds & drink immediately.
• Do not resuspend in milk, juice or carbonated beverages.
Monday, September 10, 2012
VACCINES FOR YOU • CHOLERA
• Orochol E Berna contains not < 2x109 viable organisms of the attenuated strain Vibrio CVD 103-HgR in a lyophilized form
• For ORAL active immunization of adults & children >2 years against cholera
• Dosage: Resuspend contents of both chambers at the same time in cold or lukewarm water, mix carefully for 5-10 seconds & drink immediately.
• Do not resuspend in milk, juice or carbonated beverages.
Additional Vaccinations:
Chicken poxHepatitis A InfluenzaPertussis
Salmonella typhi
CholeraPneumonia
Monday, September 10, 2012
VACCINES FOR YOU
• TYPHOID
• Vi capsular polysaccharide typhoid vaccine: Single IM or SC injection
• Oral Vaccine preparation > 109 viable organisms of attenuated Salmonella typhi strain Ty21a Berna; has cross immunity against S. paratyphi B
Monday, September 10, 2012
VACCINES FOR YOU
• TYPHOID
• Vi capsular polysaccharide typhoid vaccine: Single IM or SC injection
• Oral Vaccine preparation > 109 viable organisms of attenuated Salmonella typhi strain Ty21a Berna; has cross immunity against S. paratyphi B
Monday, September 10, 2012
VACCINES FOR YOU
• TYPHOID
• Vi capsular polysaccharide typhoid vaccine: Single IM or SC injection
• Oral Vaccine preparation > 109 viable organisms of attenuated Salmonella typhi strain Ty21a Berna; has cross immunity against S. paratyphi B
Additional Vaccinations:
Chicken poxHepatitis A InfluenzaPertussis
Salmonella typhi
CholeraPneumonia
Monday, September 10, 2012
VACCINES FOR YOU
• PNEUMOCOCCUS
• Purified polysaccharide of Streptococcus pneumoniae
• 25ug each of 23 serotypes: 1,2,3,4,5,6B,7F,8,9V,10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F
• Indications: Prevention of pneumococcal infections, particularly those of respiratory origin for ages over 2 years who are at risk of serious pneumococcal infection
Monday, September 10, 2012
VACCINES FOR YOU
• PNEUMOCOCCUS
• Purified polysaccharide of Streptococcus pneumoniae
• 25ug each of 23 serotypes: 1,2,3,4,5,6B,7F,8,9V,10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F
• Indications: Prevention of pneumococcal infections, particularly those of respiratory origin for ages over 2 years who are at risk of serious pneumococcal infection
Monday, September 10, 2012
VACCINES FOR YOU
• PNEUMOCOCCUS
• Purified polysaccharide of Streptococcus pneumoniae
• 25ug each of 23 serotypes: 1,2,3,4,5,6B,7F,8,9V,10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F
• Indications: Prevention of pneumococcal infections, particularly those of respiratory origin for ages over 2 years who are at risk of serious pneumococcal infection
Additional Vaccinations:
Chicken poxHepatitis A InfluenzaPertussis
Salmonella typhi
CholeraPneumonia
Monday, September 10, 2012
VACCINES FOR YOU
• PNEUMOCOCCUS
• Dosage: Initial Injections: Single IM or SC injection confers protection against the 23 types of pneumococci in the vaccine
• Revaccination should not be carried out for at least 5 years as severe local reactions (Arthus phenomena type) were observed if time between 2 injections is too short
Monday, September 10, 2012
VACCINES FOR YOU
• PNEUMOCOCCUS
• Dosage: Initial Injections: Single IM or SC injection confers protection against the 23 types of pneumococci in the vaccine
• Revaccination should not be carried out for at least 5 years as severe local reactions (Arthus phenomena type) were observed if time between 2 injections is too short
Monday, September 10, 2012
VACCINES FOR YOU
• PNEUMOCOCCUS
• Dosage: Initial Injections: Single IM or SC injection confers protection against the 23 types of pneumococci in the vaccine
• Revaccination should not be carried out for at least 5 years as severe local reactions (Arthus phenomena type) were observed if time between 2 injections is too short
Additional Vaccinations:
Chicken poxHepatitis A InfluenzaPertussis
Salmonella typhi
CholeraPneumonia
Monday, September 10, 2012
ISSUES TO CONSIDER
• Which vaccine to administer to whom?
• When to vaccinate: months before exposure?
• How many doses? Cost?
• Interval between doses in case of multi-dose regimen for primary immunization
• When to expect protection? Immunologic response vs protection?
• Possibility of adverse/side effects
Monday, September 10, 2012
EXPANDED IMMUNIZATION
PROGRAMS
Monday, September 10, 2012
MeaslesPoliomyelitisPertussisTetanusTuberculosis
DiphtheriaHepatitis B
The 7 EPI Diseases
Monday, September 10, 2012
1976 Official launching of EPI in the Philippines
1977 BCG & DPT2 for infants in priority areas
1979BCG & DPT2 expanded nationwideOPV3 in selected areas reporting outbreaksTT2 for pregnant women at 5 months gestation
1980 OPV3 & TT2 given nationwide
1982 Measles vaccine for 35% of the population
1983 Measles vaccination given nationwide
1984 DPT3 added; TT given anytime during pregnancy
1985 Comprehensive Program ReviewMonday, September 10, 2012
1986 Universal Child Immunization Goal by 1990-UN
1987 Start of Rotary International mass campaigns –Polio Plus
1989 TT3, TT4, TT5 added; Wednesday adopted as Immunization Day
1991 National Plan of Action for Polio Eradication launched; Polio Eradication Unit
1992 Hepatitis B immunization -40% of infants
1993 1st National Immunization Days
Neonatal Tetanus Elimination-Action Plan
Measles Control – National Plan of ActionMonday, September 10, 2012
Bacteria Disease Antigen Efficacy
Streptococcus pneumoniae Pneumonia Capsular
polysaccharide 85%
Neisseria meningitidis meningitis Capsular
polysaccharide 85%
Hemophilus influenzae meningitis Capsular
polysaccharide 85%
Corynebacterium diphtheriae Diphtheriae Toxoid >90%
Clostridium tetani tetanus Toxoid >90%
Bordetella pertussis Whooping cough Killed organism >90%
Mycobacterium bovis (BCG) Tuberculosis Live attenuated 0 – 70 %
Salmonella typhi Typhoid fever Killed/Live attenuated 80%
Monday, September 10, 2012
Vaccine Reason for Immunization
BCGGiven at the earliest possible age protects against the possibility of infection from
family members
DPT Early start with DPT reduces chance of severe pertussis
OPV Protection against polio increased the earlier the OPV is given
Hepatitis BEarly start of HBV vaccination reduces
chance of being infected and becoming a carrier
Measles At least 80% of measles can be prevented by immunization at this age
Monday, September 10, 2012
Age Diseases to be immunized against
Birth Tuberculosis
6 weeks DPT; Polio; HBV
10 weeks DPT; Polio; HBV
14 weeks DPT; Polio; HBV
9 months MeaslesMonday, September 10, 2012
Vaccine Minimum age/interval Percent protected Duration of protection
TT1 As early as possible during pregnancy
TT2 At least 4 weeks later 80%* Infants born to the
mother protected from neonatal tetanus; 3 years
protection to mother
TT3 At least 6 months later 95%* Infants protected;
5 years protection to mother
TT4 At least one year later 99%* Infants protected;
10 years protection to mother
TT5 At least one year later 99%Lifetime protection for
mother; * all infants born to that mother will be
protected
Tetanus toxoid Immunization Schedule for Women
Monday, September 10, 2012
Poliomyelitis Eradication Measles Elimination Maternal and Neonatal Tetanus Elimination
Control of Hepatitis B
Philippine Immunization Flagship Programs
Monday, September 10, 2012
TAKE-HOME QUIZ
• Next to Smallpox, why do you think that polio and measles are the next diseases targeted for global elimination?
• Differentiate the 4 Immunization Flagship Programs of the country in terms of:
• target group, vaccine to use and route, criteria for success
Monday, September 10, 2012
IMMUNOLOGIC TOLERANCE AND AUTOIMMUNITY
Monday, September 10, 2012
RECALL...
• Immunologic tolerance: unresponsiveness to self-antigens
•WHAT WE WILL LEARN:
• How does the immune system maintain its unresponsiveness to self-antigens
• What are the factors that may contribute to the development of autoimmunity?
Monday, September 10, 2012
ON ENCOUNTER WITH LYMPHOCYTES...
Monday, September 10, 2012
ON ENCOUNTER WITH LYMPHOCYTES...
The choice among lymphocyte activation, tolerance, and ignorance is determined by the nature of the antigen-specific lymphocytes and by the nature of the antigen and how it is displayed to the immune system
Monday, September 10, 2012
IMPORTANCE OF IMMUNOLOGIC TOLERANCE
• the knowledge that self-antigens normally induce tolerance
• if we learn how to induce tolerance in lymphocytes specific for a particular antigen, we may be able to use this knowledge to prevent or control unwanted immune reactions
• APPLICATIONS:
• treat allergic and autoimmune diseases
• prevent the rejection of organ transplants
• gene therapy
• prevent immune responses against the products of newly expressed genes or vectors
Monday, September 10, 2012
CENTRAL & PERIPHERAL
• central tolerance: induced when developing lymphocytes encounter these antigens in the generative lymphoid organs (bone marrow and thymus)
• peripheral tolerance: induces when mature lymphocytes encounter self antigens in peripheral tissues
Monday, September 10, 2012
AUTOIMMUNITY• Principal factors in the
development of autoimmunity:
• inheritance of susceptibility genes
• may contribute to failure of self-tolerance
• environmental triggers
• e.g. infections = may activate self-reactive lymphocytes
Monday, September 10, 2012
CENTRAL T-LYMPHOCYTE TOLERANCE
• Negative selection: principal mechanism of central tolerance
• Strong recognition of self antigens immature T-cells in the thymus may lead to death of the cells (negative selection or deletion)
• Self-antigen recognition in the thymus may also lead to the development of regulatory T-cells that enter peripheral tissues
Monday, September 10, 2012
PERIPHERAL T-LYMPHOCYTES & ANERGY
• Peripheral tolerance:
• induced when mature T cells recognize self antigens in peripheral tissues, leading to functional inactivation (anergy) or death
• induced when the self-reactive lymphocytes are suppressed by regulatory T cells
Monday, September 10, 2012
ANERGY
• ANERGY: functional inactivation of T-lymphocytes that occurs when these ceIls recognize antigens without adequate levels of the costimulators (second signals) that are needed for full T-cell activation
Monday, September 10, 2012
ACTIVATION-INDUCED CELL DEATH : DELETION
Monday, September 10, 2012
IMMUNE SUPPRESSION
Monday, September 10, 2012
Characteristics of self and foreign (e.g. microbial) protein antigens that determine why the self
antigens induce tolerance and microbial antigens stimulate T-cell mediated immune responses
Monday, September 10, 2012
CENTRAL TOLERANCE IN B-CELLS
Monday, September 10, 2012
PERIPHERAL TOLERANCE IN B-CELLS
Monday, September 10, 2012
AUTOIMMUNE DISEASES & MHC MOLECULES
Monday, September 10, 2012
Monday, September 10, 2012
MOLECULAR MIMICRY & AUTOIMMUNITY
Monday, September 10, 2012
• Immunologic tolerance: specific unresponsiveness to an antigen induced by exposure of lymphocytes to that antigen
• All individuals are tolerant of (unresponsive to) their own (self)antigens
• Tolerance against antigens may be induced by administering that antigen in particular ways = strategy may be useful for treating immunologic disease and for preventing the rejection of transplants
SUMMARY
Monday, September 10, 2012
• Autoimmune diseases: result from a failure of self-tolerance
• Multiple factors contribute to autoimmunity:
• immunologic abnormalities
• susceptibility genes
• infections
SUMMARY
Monday, September 10, 2012
• Central tolerance: induced by the death of immature lymphocytes that encounter antigens in the generative lymphoid organs while Peripheral tolerance: results from the recognition of antigens by mature Iymphocytes in peripheral tissues
• T-CELLS:
• Central tolerance (negative selection): result of high-affinity recognition of antigens in the thymus, which tend to be widely disseminated self-antigens AND may eliminate the potentially most dangerous T-cells, which express high-affinity receptors for disseminated self-antigens
SUMMARY
Monday, September 10, 2012
• T-CELLS:
• Peripheral tolerance: induced by multiple mechanisms
• Anergy (functional inactivation) results from the recognition of antigens without costimulators (second signals) or when T cells use inhibitory receptors to recognize costimulators
• Deletion (death by apoptosis) occurs when T cells repeatedly encounter self antigens
• Some self-reactive T cells suppress potentially pathogenic T-cells
SUMMARY
Monday, September 10, 2012
•B-CELLS
• central tolerance: induced when immature cells recognize self antigens in the bone marrow
• peripheral tolerance: (by anergy) induced when mature B cells recognize self antigens without T cell help
SUMMARY
Monday, September 10, 2012
• AUTOIMMUNITY
• Many genes contribute to the development of autoimmunity
• The strongest associations are between HLA genes and various T cell-mediated autoimmune diseases
• Infections predispose to autoimmunity, by causing inflammation and inducing the aberrant expression of costimulators or because of cross-reactions between microbial and self antigens
SUMMARY
Monday, September 10, 2012
IMMUNE RESPONSE AGAINST TUMORS/TRANSPLANTS
vanderbilt.edu
Reference; Immunology by Abbas
Monday, September 10, 2012
CALENDAR FOR BIO 151DATE ACTIVITY
September 11 TUMORS, TRANSPLANTS & SERODIAGNOSIS
September 18 EXAMINATION 2 (VACCINES TO SEROLOGICAL DIAGNOSTICS)
September 25- October 2 IMMUNITY TO MICROBIAL INFECTIONS: Pneumoniaa.Fungib.Virusc.Bacteria
IMMUNITY TO MICROBIAL INFECTIONS: Schistosomiasis
October 9 EXAMINATION 3: Take-Home (24 hours)
Questions available October 9, 2012 at 12 noon...Submission should be October 10, 2012 at 12 noon (at DB, received and
stamped by DB staff)
*** Printed and well-cited
Monday, September 10, 2012
WHAT ARE TUMORS?
• Simply means a mass of cells
• Can be either benign or malignant
• Benign tumors: not a threat to life or long-term health; made up of related but different cells
• Malignant tumors: threat to life or long-term health; clonal or all identical to the cell of origin
webmd.com
Monday, September 10, 2012
IMMUNE RESPONSE TO TUMORS
• T cells generally mount effective surveillance against tumors associated with oncogenic viruses or UV induction (these are strongly immunogenic)
• More weakly immunogenic tumors are not controlled by T cell surveillance, although sometimes low grade responses are evoked
joseph-birch.livejournal.com
Monday, September 10, 2012
IMMUNE RESPONSE TO TUMORS
• NK cells play a role in containing tumor growth and metastases;
• The importance of the immune system in preventing tumor growth can be seen in immunocompromised patients - AIDS patients have a much higher chance of developing many types of cancer
Monday, September 10, 2012
WHAT IS CANCER?• diseases in which there is loss
of regulation of the proliferative process
• characterized by excessive, uncontrolled growth of abnormal cells, which invade and destroy other tissues
• hyperproliferation of cells that have violated the basic rules of social cell behavior (i.e, lost the ability to be controlled by normal cell signals)
nursingcrib.com
Monday, September 10, 2012
CANCER• Control is important to
ensure that cells only divide when needed (organs and tissues should maintain their correct shapes and sizes)
• Should this system fail, several backup safety mechanisms prevent the cell from dividing uncontrollably
topnews.in
Monday, September 10, 2012
DEFENSE AGAINST UNCONTROLLED PROLIFERATION
• A cell can initiate cell death (suicide) when a defect is detected through a process called apoptosis
• A cell can repair the error and become a normal cell
• Immune system (e.g., natural killer cells can detect abnormal cells and kill them)
genengnews.com
Monday, September 10, 2012
WHAT ARE ORGANS TRANSPLANTS?
• surgical operation in which a failing or damaged organ in the human body is removed and replaced with a functioning one
• donated organ may be from a deceased donor, a living donor, or an animal
• NOTE: In some cases an artificial organ is used
Monday, September 10, 2012
WHAT ARE ORGANS TRANSPLANTS?
• Cadaveric organ donation involves removing organs from a recently deceased donor
• Living organ donation involves the donation of one of a paired organ (such as kidneys) or a portion of an organ (such as a lobe of the liver or lung)
• The donor's organ system is still able to function after the donation
• Living donors are often related to the patient, but that is not always the case
Monday, September 10, 2012
SO WHAT’S IT FOR IMMUNOLOGY?
• CANCER: enhancing immunity against the tumors holds much promise for treatment
•TRANSPLANTS: immune responses against transplants are a barrier for a successful transplantation
Monday, September 10, 2012
WHAT WE WILL LEARN...
• What are the antigens in tumors and tissue transplants that are recognized as foreign by the immune system?
• How does the immune system recognize and react to tumors and transplants?
• How can the immune responses to tumors and grafts be manipulated to enhance tumor rejection and inhibit graft rejection?
Monday, September 10, 2012
IMMUNE SURVEILLANCE
• 1950s: a physiologic function of the adaptive immune system is to prevent the outgrowth of transformed cells or to destroy these cells before they become harmful tumors
london-research-institute.org.uk aihealthsolutions.ca
Monday, September 10, 2012
EVIDENCES SUPPORTING THE CONCEPT OF
IMMUNO SURVEILLANCE
Monday, September 10, 2012
TUMOR ANTIGENS
• Malignant tumors express various types of molecules that may be recognized by the immune system as foreign antigens
Monday, September 10, 2012
IMMUNE MECHANISM FOR TUMOR REJECTION
• The principal immune mechanism of tumor eradication is killing of tumor cells by cytolytic T-lymphocytes (CTLs) specific for tumor antigens
• endogenously synthesized cytosolic proteins displayed as Class I MHC-associated peptides
• SO: recognized by class I MHC-restricted CD8+ CTLs (kills the cell producing the antigens)
Monday, September 10, 2012
IMMUNE MECHANISM FOR TUMOR REJECTION
• CTLs responses against tumor cells are often induced by recognition of tumor antigens on host-antigen presenting cells (APCs) which ingest tumor cells or their antigens and present the antigens to T-cells
Monday, September 10, 2012
IMMUNE MECHANISM FOR TUMOR REJECTION
• anti-tumor CD4+ T-cell responses and antibodies have been detected in patients (but may not be protective)
• activated macrophages and NK cells are capable of killing tumor cells in vitro (unclear protection)
Monday, September 10, 2012
EVASION OF IMMUNE RESPONSE
BY TUMORS
• Immune response often fail to check tumor growth because these responses are ineffective or because tumors evolve to evade immune attack
• Immune responses against tumors may be weak because many tumor antigens are weakly immunogenic, perhaps because they differ only slightly from self antigens
Monday, September 10, 2012
EVASION OF IMMUNE RESPONSE BY TUMORS
• Growing tumors also develop mechanisms for evading immune responses
• stop expressing the antigens that are targets of immune attack (antigen loss variants) = continue to grow and spread
• stop expressing class I MHC molecules = cannot display antigens to CD8+ T-cells (cross-priming)
• may produce molecules, e.g. transforming growth factor-beta, that suppress immune responses
Monday, September 10, 2012
IMMUNOTHERAPY FOR TUMORS
• MAIN GOAL:
• provide antitumor effectors (antibodies and T-cells) to patients
• actively immunize patients against their tumors
• stimulate the patients’ own antitumor immune response
Monday, September 10, 2012
IMMUNOTHERAPY VS CHEMOTHERAPY
• Chemotherapy/Irradiation: may have devastating effects on normal non-tumor tissues
• Immunotherapy: highly-specific for the tumor health.umn.edu
Monday, September 10, 2012
STRATEGY 1: PASSIVE IMMUNITY
• immune effectors are injected into cancer patients
• e.g. monoclonal antibodies to tumor antigens + potent toxins
• HOW: antibodies bind to tumor antigens and activate host effector mechanisms (e.g. phagocytes or complement system)
• HOW: toxins delivered to tumor cells
Monday, September 10, 2012
EXAMPLE: BREAST CANCER IMMUNOTHERAPY
• ALL information from: Critical Reviews in Oncology and Hematology, October 2010 (Emde et al. Therapeutic strategies and mechanisms of tumorigenesis of HER2-overexpressing breast cancer)
• HER2 (tyrosine kinase): overexpressed in approximately 25% of breast cancers; acts as a signal amplifier for its siblings = transmembrane receptors that collectively bind with 11 distinct growth factors of the EGF family
• THUS: overexpression of HER2 confers aggressive invasive growth in preclinical models and in patients
• “Specific therapies targeting HER2 include monoclonal antibodies, antibody–drug conjugates, small molecule tyrosine kinase inhibitors, as well as heat shock protein and sheddase inhibitors. Two of these drugs have shown impressive – yet mostly transient – efficacy in patients with HER2 overexpressing breast cancer.”
Monday, September 10, 2012
Signal transduction pathways instigated by HER2, co-receptors and EGF-like growth factors (epidermal) and NRGs (neuregulins)
Monday, September 10, 2012
Clinically approved and experimental therapeutic strategies targeting ErbB-2/HER2 in carcinomas:
a. Trastuzumab: humanized monoclonal antibody directed against the extracellular domain of HER2; recruits immune effector mechanisms and can induce apoptosis, block angiogenesis and inhibit tumor cell proliferation.
b. Pertuzumab: prevent heterodimerization of HER2 with other family members
RECENTS:
a. Lapatinib: tyrosine kinase inhibitors; reversible inhibitor
b. Neratinib: irreversible inhibitor; variably inhibit a broad range of tyrosine kinases
c. 17-AAG: block the ATP/ADP binding pocket of HSP90 and target HER2 for proteasomal degradation (Note; HSP90: molecular chaperone required for proper folding of protein kinases like HER2
Monday, September 10, 2012
STRATEGIES FOR ENHANCING ANTITUMOR
IMMUNE RESPONSE
• 1. Tumor-antigen pulsed DC: mimic normal pathway of cross-presentation to induce specific T-cell response
Monday, September 10, 2012
STRATEGIES FOR ENHANCING ANTITUMOR
IMMUNE RESPONSE
• 2. DNA or transfected DC: the host produce the tumor antigen thus inducing specific T-cell response
Monday, September 10, 2012
STRATEGIES FOR ENHANCING ANTITUMOR
IMMUNE RESPONSE
• 3. tumor cell expressing costimulators (e.g. B7) or cytokines (e.g. IL-2): cytokines or costimulators to stimulate the generation of own tumor-specific immune response
Monday, September 10, 2012
IMMUNE RESPONSE AGAINST TRANSPLANTS
Monday, September 10, 2012
• transplants exchanged between animals of the same and other inbred are accepted
• grafts among different strains are rejected
IMMUNE RESPONSE AGAINST TRANSPLANTS
wenliang.myweb.uga.edu
• GENES for graft rejection: products are expressed in ALL tissues
Monday, September 10, 2012
• Allogeneic (allografts) and Xenogeneic (xenografts) grafts are always rejected
• Alloantigens (alloreactive antibodies and T-cells)
• Xenoantigens (xenoreactive antobodies and T-cells)
•CLINICAL Scenario: mostly allogeneic
IMMUNE RESPONSE AGAINST TRANSPLANTS
Monday, September 10, 2012
Transplantation Antigens
• The antigens of allografts that serve as the principal targets of rejection are proteins encoded in the MHC
• Human MHC = human leukocyte antigen or HLA
Monday, September 10, 2012
INDUCTION OF IMMUNE RESPONSES AGAINST
TRANSPLANTS
• T-cells may recognize allogeneic MHC molecules in the graft by professional APCs, or graft alloantigens may be processed and presented by hosts’ professional APCs
Monday, September 10, 2012
INDUCTION OF IMMUNE RESPONSES AGAINST
TRANSPLANTS
• T-cells may recognize allogeneic MHC molecules in the graft by professional APCs, or graft alloantigens may be processed and presented by hosts’ professional APCs
if the graft does not contain professional APCs, how does it stimulate T cells?
Monday, September 10, 2012
INDUCTION OF IMMUNE RESPONSES AGAINST
TRANSPLANTS
• T-cells may recognize allogeneic MHC molecules in the graft by professional APCs, or graft alloantigens may be processed and presented by hosts’ professional APCs
if the graft does not contain professional APCs, how does it stimulate T cells?
graft cells are ingested by professional APCs in the recipient and the donor alloantigens are processed and presented by the self MHC molecules on recipient APCs
Monday, September 10, 2012
MIXED LYMPHOCYTE REACTION (MLR)
• an in vitro model of T-cell recognition in alloantigens
wenliang.myweb.uga.edu
Monday, September 10, 2012
MIXED LYMPHOCYTE REACTION (MLR)
• T-cells from one individual are cultured with leukocytes of another individual and the responses of the T-cells are assayed
• magnitude of response is proportional to the extent of MHC differences between these individuals
• rough predictor of the outcomes of grafts exchanged between these individuals
Monday, September 10, 2012
IMMUNE MECHANISM OF GRAFT REJECTION
• occurs within minutes of transplantation; characterized by thrombosis of graft vessels and ischemic necrosis of the graft
• mediated by circulating antibodies specific for antigens on graft endothelial cells
Monday, September 10, 2012
IMMUNE MECHANISM OF GRAFT REJECTION
• occurs within minutes of transplantation; characterized by thrombosis of graft vessels and ischemic necrosis of the graft
• mediated by circulating antibodies specific for antigens on graft endothelial cells
not common BUT major barrier to xenotransplantation
Monday, September 10, 2012
IMMUNE MECHANISM OF GRAFT REJECTION
• occurs within days or weeks after transplantation
• principal cause of early graft failure
• mediated mainly y T-cells which react against alloantigens in the graft or may react agains cells in graft vessels leading to vascular damage
Monday, September 10, 2012
IMMUNE MECHANISM OF GRAFT REJECTION
• occurs within days or weeks after transplantation
• principal cause of early graft failure
• mediated mainly y T-cells which react against alloantigens in the graft or may react agains cells in graft vessels leading to vascular damage
antibodies may also play a role
Monday, September 10, 2012
IMMUNE MECHANISM OF GRAFT REJECTION
• occurs over months or years that leads to progressive loss of graft function
• manifested as fibrosis or narrowing of vessels (arteriosclerosis)
• T-cells secrete cytokines which stimulate proliferation of fibroblasts and vascular smooth muscle cells in the graft
Monday, September 10, 2012
PREVENTION & TREATMENT OF GRAFT REJECTION
e-steroid.com
Monday, September 10, 2012
BONE MARROW TRANSPLANTS
• elicit strong rejection reactions: must perform cross-matches
• carry the risk of graft-versus-host disease: If mature allogeneic T cells are transplanted with the marrow cells, these mature T cells can attack the recipient's tissues
• lead to temporary immunodeficiency in recipients
Monday, September 10, 2012
QUESTIONS?Monday, September 10, 2012
SERODIAGNOSIS
elitechgroup.com
Reference; Jawetz Medical Microbiology (http://accessmedicine.com/resourceTOC.aspx?resourceID=6)
Monday, September 10, 2012
BIOSAFETY
2008.igem.org
ehrs.upenn.edu
busytrade.com
Monday, September 10, 2012
OVERVIEW OF DIAGNOSTICS
• 3 CATEGORIES:
• Direct: clinical specimen is examined directly for the presence of virus particles, virus antigen or viral nucleic acids
• Indirect: the specimen into cell culture, eggs or animals in an attempt to grow the virus
• Serology: detection of rising titres of antibody between acute and convalescent stages of infection, or the detection of Ig
Monday, September 10, 2012
ANTIGEN DETECTION: DIRECT
• Applications:
• immunofluorescence testing of nasopharyngeal aspirates for respiratory viruses e.g.. RSV, flu A, flu B, and adenoviruses
• detection of rotavirus antigen in feces
• the pp65 CMV antigenaemia test
• the detection of HSV and VZV in skin scraping
rotavirus, adenovirus, astroviruses, Norwalk-like viruses
Monday, September 10, 2012
ANTIGEN DETECTION: DIRECT
wclassproducts.com
norgenbiotek.com
geneaid.com
Monday, September 10, 2012
ANTIGEN DETECTION: INDIRECT
Monday, September 10, 2012
ANTIGEN DETECTION: INDIRECT (enhanced)
Monday, September 10, 2012
ANTIBODY DETECTION: SEROLOGY
usada.org
http://virology-online.com
Monday, September 10, 2012
COMMON TESTS• Classical Techniques:
• Complement fixation tests (CFT)
• Hemagglutination inhibition tests (HAI)
• Immunofluorescence techniques (IF)
• Neutralization tests
• Single Radial Hemolysis
• Newer Techniques:
• Radioimmunoassay (RIA)
• Enzyme linked immunosorbent assay (EIA)
• Particle agglutination
• Western Blot (WB)
• Recombinant immunoblot assay (RIBA)
Monday, September 10, 2012
DIAGNOSING A PRIMARY (ACUTE) INFECTION
• A significant rise in titre of IgG/total antibody between acute and convalescent sera
• CFT and HAI, it is normally taken as a four-fold or greater increase in titre
• diagnosis is usually retrospective because by the time the convalescent serum is taken, the patient had probably recovered
Monday, September 10, 2012
DIAGNOSING A PRIMARY (ACUTE) INFECTION
• Presence of IgM
• EIA, RIA, and IF may be used (rapid)
• BUT:
• interference by rheumatoid factor,
• re-infection by the virus
• unexplained persistence of IgM years after the primary infection
Monday, September 10, 2012
DIAGNOSING A PRIMARY (ACUTE) INFECTION
• Seroconversion
• changing from a previously antibody negative state to a positive state
• e.g. seroconversion against HIV following a needle-stick injury, or against rubella following contact with a known case
Monday, September 10, 2012
DIAGNOSING A PRIMARY (ACUTE) INFECTION
• A single high titre of IgG (or total antibody)
• this is a very unreliable means of serological diagnosis since the cut-off is very difficult to define
Monday, September 10, 2012
DIAGNOSING A RE-INFECTION / RE-ACTIVATION
• It is often very difficult to differentiate re-infection/re-activation from a primary infection
• When important: rubella infection in the first trimester of pregnancy: primary infection is associated with a high risk of fetal damage whereas re-infection is not
• RE-INFECTION: sharp large rise in antibody titres
• RE-INFECTION/ RE-ACTIVATION: IgM is usually low or absent
Monday, September 10, 2012
USE DEPENDS ON PATHOGEN
• onset of clinical symptoms coincide with the development of antibodies: e.g. rubella and hepatitis A (IgM or rising titres of IgG in the serum of the patient would indicate active disease)
• pathogen produce clinical disease before the appearance of antibodies: e.g. respiratory and diarrheal viruses (serological diagnosis would be retrospective and therefore will not be that useful)
• pathogen produce clinical disease months or years after seroconversion: e.g. HIV and rabies (the mere presence of antibody is sufficient to make a definitive diagnosis)
Monday, September 10, 2012
READING ASSIGNMENT:
Q: WHAT DO YOU THINK ARE SOME PROBLEMS WITH SEROLOGY
Monday, September 10, 2012
THE SEROLOGICAL DIAGNOSTICS
medicineworld.org
immunology.utoronto.ca
Monday, September 10, 2012
COMPLEMENT FIXATION TEST
dshs.state.tx.us
path.cam.ac.uk
http://virology-online.com
Monday, September 10, 2012
HEMAGGLUTINATION/ HEMAGGLUTINATION INHIBITION
TESTS (HAI)
virology.ws
motifolio.com
Monday, September 10, 2012
HEMAGGLUTINATION/ HEMAGGLUTINATION INHIBITION
TESTS (HAI)
qmark2.leeds.ac.uk path.cam.ac.uk
Monday, September 10, 2012
IMMUNOFLUORESCENCE TECHNIQUE (IF)
• DIRECT: known labeled antibody interacts with unknown antigen
• INDIRECT: known antigen is attached to a slide, unknown serum is added and then washed = An/Ab fixed onto slide and can be detected by antibody-specific reagent
Monday, September 10, 2012
IMMUNOFLUORESCENCE TECHNIQUE (IF)
Respiratory Syncytial Virus (RSV)
(a) Indirect technique: polyclonal rabbit serum and a conjugated sheep anti-rabbit serum; both were extensively absorbed to remove unwanted antibodies
(b) Direct technique: mixture of three monoclonal conjugated antibodies and Evans blue counterstain (which, paradoxically, appears red) = staining only those antigenic concentrations for which there are antibodies in the mixture
CREDITS: (Both micrographs were taken by Joyce McQuillin personally, SCIENCE DIRECT)
Monday, September 10, 2012
NEUTRALIZATION TESTS
• potential of the serum of neutralizing the biological activity of an antigen can also be known
• assessment of the capability of a serum against bacterial toxins, bacteria or viruses
• laborious, need cell cultures, sterile conditions, and usually require more time
http://www.sanidadanimal.info
biobest.co.uk
Monday, September 10, 2012
NEUTRALIZATION TESTS
biobest.co.uk
culture negative to CA
Positive MDCC-MSB1 infected cells culture showing fluorescence at the point where CAV
antigen was detected
Monday, September 10, 2012
SINGLE RADIAL HEMOLYSIS
• E.g. Rubella antibody.
• LEFT PLATE: Control Plate
• RIGHT PLATE: well in the middle of the plate contains the 15 miu/ml control serum = clear zone of lysis surrounding the well on the test plate which is absent on the control plate
• NOTE: Specimens which gives a zone of lysis equal or greater than the 15 miu/ml control well are regarded as positive for rubella antibody. = Should a zone of similar size is present on the control plate, then the result is not valid
Monday, September 10, 2012
RADIOIMMUNOASSAY (RIA)
• used to quantitate antigens or haptens taht can be radioactively-labeled
• based on competition for specific antibody between the labeled (known) and the unlabeled (unknown) concentration of the material
• complex that form between An/Ab can then be separated and the amount of radioactivity measured
Monday, September 10, 2012
RADIOIMMUNOASSAY (RIA)
• concentration of the unlabeled (unknown) antigen or hapten is determined by comparing the results with those obtained using several concentrations of a predetermined standard antigen
• highly sensitive for:
• assay of hormones or drugs from serum
• measure amount of IgE with known allergenMonday, September 10, 2012
users.rcn.com
Monday, September 10, 2012
RADIOALLERGOSORBENT ASSAY (RAST)
lookfordiagnosis.com
pennstatehershey.adam.com
Monday, September 10, 2012
ENZYME IMMUNOASSAY (EIA)
• depends on the conjugation of an enzyme to either an antigen or an antibody
• enzyme is detected by assaying for enzyme activity with its substrate
• no need for radioactive labelspiercenet.com
Monday, September 10, 2012
ELISAtechnologyinscience.blogspot.com
• can detect the ANTIGEN-ANTIBODY reaction by the use of enzyme-linked antibodies
• based on the use of labeled antibodies (usually done with the enzyme peroxidase) so that the resulting conjugates have both immunological and enzymatic activity
Monday, September 10, 2012
ELISA• one of the components of the conjugate
(antibody or antigen) is attached to the plate, so the antigen-antibody reaction can be easily measured when adding the reaction substrate
• This substrate produces a colored reaction product when it comes into contact with the enzyme
• The color change can be seen or quantified with a colorimeter
Monday, September 10, 2012
ELISA: ANTIGEN DETECTION
• Sandwich ELISA
• plates are usually coated with an antibody (monoclonal or polyclonal antibody) against the unknown antigen
• sample that needs to be tested is added to the wells, and if the antigen is present, the antigen-antibody reaction will take place
• add another antibody linked to an enzyme.
• when the reaction substrate is added it turns a color
Monday, September 10, 2012
ELISA: ANTIBODY DETECTION
• INDIRECT ELISA: coating of the ELISA plate with the antigen against the specific antibodies that may be present in the serum
• NEXT: addition of serum, incubation and washing; addition of the conjugate, incubation and washing and finally, the addition of the substrate, stopping the reaction and reading the results
Monday, September 10, 2012
ELISA: ANTIBODY DETECTION
• COMPETITIVE ELISA: have an antibody (monoclonal of polyclonal) of a known antigen which has previously been bound to the plate
• competitive because the serum is incubated with the antigen previous to its incubation with the antiserum bound to the plate = both compete for the antigen
• NEXT: addition of the conjugate, incubation, washing, and finally, substrate addition and reading the results
Monday, September 10, 2012
ELISA & HIV
virology-online.com
hivinfosource.orgMonday, September 10, 2012
DENGUE SEROLOGY
Monday, September 10, 2012
QUESTIONS?Monday, September 10, 2012
END OF EXAM 2 COVERAGE
Monday, September 10, 2012