This document describes various serological tests used to detect antigens and antibodies. It discusses primary tests like ELISA, IFAT, and RIA. Secondary tests include agglutination, complement fixation, precipitation, and neutralization tests. Tertiary tests determine antibody protective value. Agglutination tests can qualitatively and quantitatively detect particulate antigens. Coombs tests detect non-agglutinating antibodies. ELISA is then explained in detail, including indirect, sandwich, and competitive formats. ELISA is widely used to detect antigens and antibodies in applications like HIV and food allergen testing.
Karl Landsteiner discovered the A, B, and O blood groups in 1901, establishing the field of serology. Serology studies antigen-antibody reactions in blood serum to detect infections and other conditions. Precipitation tests like immunodiffusion and immunoelectrophoresis detect antigen-antibody interactions by forming visible precipitates in gels like agar. These tests are used diagnostically in microbiology and clinical immunology.
The document discusses antigen-antibody reactions. It begins by introducing antigens and antibodies and how they specifically combine in antigen-antibody reactions. The reactions occur in three stages: formation of an antigen-antibody complex, leading to visible events like precipitation or agglutination, and destruction or neutralization of the antigen. Key features of antigen-antibody reactions are their specificity, the formation of immune complexes, antigen binding sites called epitopes, and the binding force between antigens and antibodies. Common types of antigen-antibody reactions include precipitation, agglutination, complement fixation, ELISA, and immunofluorescence.
Reverse passive agglutination test coats antibodies onto carrier molecules that detect antigens in a patient's serum. For example, this test can be used to detect cholera toxin by coating cholera toxin antibodies onto carrier molecules which will agglutinate or clump if the cholera toxin antigen is present in a serum sample. This provides a detection method for certain antigens by reversing the roles of the antibody and antigen compared to a standard agglutination test.
This document summarizes various serological tests used to detect antigens and antibodies, including:
- Primary tests like ELISA, IFAT, RIA that detect markers
- Secondary tests like agglutination, complement fixation, precipitation that detect interactions
- Tertiary tests that assess protective value of antiserum in animals
It then provides details on specific tests like agglutination, Coombs test, hemagglutination inhibition, precipitation, complement fixation, ELISA and their applications in medicine, food/plant pathology, and quality control.
1) Agglutination tests detect antigens or antibodies by exploiting the ability of antibodies to cross-link antigen-coated particles, forming visible clumps or lattices.
2) There are several types of agglutination tests including direct, passive, and reverse passive agglutination as well as hemagglutination and hemagglutination inhibition.
3) Agglutination tests are useful, rapid techniques for detecting various infectious diseases and other analytes but can be limited by prozone effects at high antibody concentrations.
Immunochromatographic assays, also known as lateral flow strip tests, allow for the rapid detection of antigens or antibodies in a sample within 15 minutes. The test works by utilizing two types of antibodies - one immobilized on the test strip and one labeled with a detectable marker like colloidal gold. When a sample is applied, it migrates up the strip via capillary action, allowing any antigens/antibodies in the sample to bind to the labeled antibodies and form complexes. These complexes are then captured by the immobilized antibodies, producing a visible test line that confirms the presence of the target antigen or antibody. Lateral flow tests are commercially available, easy to use, and provide results quickly with no specialized equipment,
This document discusses the prozone phenomenon, serial dilution, titer, and titration. The prozone phenomenon occurs when there is too much antibody present, blocking the reaction. Serial dilution is used to solve this by diluting the sample until the reaction occurs. Titer refers to the reciprocal of the last dilution tube that shows a positive reaction, and is a measure of antibody concentration or antigen expression level. Titration is a semi-quantitative method used to determine these values through serial dilution and observing the reaction.
Antigen-Antibody Interactions -
Antigen-antibody interactions depend on four types
of noncovalent interactions: hydrogen bonds, ionic
bonds, hydrophobic interactions, and van der Waals
interactions.
The affinity constant, which can be determined by
Scatchard analysis, provides a quantitative measure of the
strength of the interaction between an epitope of the antigen
and a single binding site of an antibody. The avidity reflects
the overall strength of the interactions between a
multivalent antibody molecule and a multivalent antigen
molecule at multiple sites.
The interaction of a soluble antigen and precipitating antibody
in a liquid or gel medium forms an Ag-Ab precipitate.
Electrophoresis can be combined with precipitation
in gels in a technique called immunoelectrophoresis.
The interaction between a particulate antigen and agglutinating
antibody (agglutinin) produces visible clumping, or
agglutination that forms the basis of simple, rapid, and
sensitive immunoassays.
Radioimmunoassay (RIA) is a highly sensitive and quantitative
procedure that utilizes radioactively labeled antigen
or antibody.
The enzyme-linked immunosorbent assay (ELISA) depends
on an enzyme-substrate reaction that generates a
colored reaction product. ELISA assays that employ
chemiluminescence instead of a chromogenic reaction are
the most sensitive immunoassays available.
In Western blotting, a protein mixture is separated by electrophoresis;
then the protein bands are electrophoretically
transferred onto nitrocellulose and identified with labeled
antibody or labeled antigen.
Fluorescence microscopy using antibodies labeled with
fluorescent molecules can be used to visualize antigen on
or within cells.
Flow cytometry provides an unusually powerful technology
for the quantitative analysis and sorting of cell populations
labeled with one or more fluorescent antibodies.
Karl Landsteiner discovered the A, B, and O blood groups in 1901, establishing the field of serology. Serology studies antigen-antibody reactions in blood serum to detect infections and other conditions. Precipitation tests like immunodiffusion and immunoelectrophoresis detect antigen-antibody interactions by forming visible precipitates in gels like agar. These tests are used diagnostically in microbiology and clinical immunology.
The document discusses antigen-antibody reactions. It begins by introducing antigens and antibodies and how they specifically combine in antigen-antibody reactions. The reactions occur in three stages: formation of an antigen-antibody complex, leading to visible events like precipitation or agglutination, and destruction or neutralization of the antigen. Key features of antigen-antibody reactions are their specificity, the formation of immune complexes, antigen binding sites called epitopes, and the binding force between antigens and antibodies. Common types of antigen-antibody reactions include precipitation, agglutination, complement fixation, ELISA, and immunofluorescence.
Reverse passive agglutination test coats antibodies onto carrier molecules that detect antigens in a patient's serum. For example, this test can be used to detect cholera toxin by coating cholera toxin antibodies onto carrier molecules which will agglutinate or clump if the cholera toxin antigen is present in a serum sample. This provides a detection method for certain antigens by reversing the roles of the antibody and antigen compared to a standard agglutination test.
This document summarizes various serological tests used to detect antigens and antibodies, including:
- Primary tests like ELISA, IFAT, RIA that detect markers
- Secondary tests like agglutination, complement fixation, precipitation that detect interactions
- Tertiary tests that assess protective value of antiserum in animals
It then provides details on specific tests like agglutination, Coombs test, hemagglutination inhibition, precipitation, complement fixation, ELISA and their applications in medicine, food/plant pathology, and quality control.
1) Agglutination tests detect antigens or antibodies by exploiting the ability of antibodies to cross-link antigen-coated particles, forming visible clumps or lattices.
2) There are several types of agglutination tests including direct, passive, and reverse passive agglutination as well as hemagglutination and hemagglutination inhibition.
3) Agglutination tests are useful, rapid techniques for detecting various infectious diseases and other analytes but can be limited by prozone effects at high antibody concentrations.
Immunochromatographic assays, also known as lateral flow strip tests, allow for the rapid detection of antigens or antibodies in a sample within 15 minutes. The test works by utilizing two types of antibodies - one immobilized on the test strip and one labeled with a detectable marker like colloidal gold. When a sample is applied, it migrates up the strip via capillary action, allowing any antigens/antibodies in the sample to bind to the labeled antibodies and form complexes. These complexes are then captured by the immobilized antibodies, producing a visible test line that confirms the presence of the target antigen or antibody. Lateral flow tests are commercially available, easy to use, and provide results quickly with no specialized equipment,
This document discusses the prozone phenomenon, serial dilution, titer, and titration. The prozone phenomenon occurs when there is too much antibody present, blocking the reaction. Serial dilution is used to solve this by diluting the sample until the reaction occurs. Titer refers to the reciprocal of the last dilution tube that shows a positive reaction, and is a measure of antibody concentration or antigen expression level. Titration is a semi-quantitative method used to determine these values through serial dilution and observing the reaction.
Antigen-Antibody Interactions -
Antigen-antibody interactions depend on four types
of noncovalent interactions: hydrogen bonds, ionic
bonds, hydrophobic interactions, and van der Waals
interactions.
The affinity constant, which can be determined by
Scatchard analysis, provides a quantitative measure of the
strength of the interaction between an epitope of the antigen
and a single binding site of an antibody. The avidity reflects
the overall strength of the interactions between a
multivalent antibody molecule and a multivalent antigen
molecule at multiple sites.
The interaction of a soluble antigen and precipitating antibody
in a liquid or gel medium forms an Ag-Ab precipitate.
Electrophoresis can be combined with precipitation
in gels in a technique called immunoelectrophoresis.
The interaction between a particulate antigen and agglutinating
antibody (agglutinin) produces visible clumping, or
agglutination that forms the basis of simple, rapid, and
sensitive immunoassays.
Radioimmunoassay (RIA) is a highly sensitive and quantitative
procedure that utilizes radioactively labeled antigen
or antibody.
The enzyme-linked immunosorbent assay (ELISA) depends
on an enzyme-substrate reaction that generates a
colored reaction product. ELISA assays that employ
chemiluminescence instead of a chromogenic reaction are
the most sensitive immunoassays available.
In Western blotting, a protein mixture is separated by electrophoresis;
then the protein bands are electrophoretically
transferred onto nitrocellulose and identified with labeled
antibody or labeled antigen.
Fluorescence microscopy using antibodies labeled with
fluorescent molecules can be used to visualize antigen on
or within cells.
Flow cytometry provides an unusually powerful technology
for the quantitative analysis and sorting of cell populations
labeled with one or more fluorescent antibodies.
ELISA is a biochemical assay technique used in immunology to detect antibodies, antigens, or other substances. It works by immobilizing an antigen or antibody on a plate and detecting its presence with an enzyme-linked antibody or antigen. There are four common ELISA tests based on the binding structure between the antibody and antigen: direct ELISA, indirect ELISA, sandwich ELISA, and competition ELISA. ELISA provides a sensitive, specific, and quantitative method to detect antibodies or antigens in a sample.
Complement fixation tests (CFT) detect antibodies that do not agglutinate or precipitate by measuring their ability to fix complement. CFT involves incubating patient serum with antigen and complement, then determining if complement is still available to lyse indicator cells. If complement is fixed in the antigen-antibody complex, it cannot lyse the indicator cells, indicating antibody presence. CFT can detect antibody levels below 1 microgram/mL, but it is time-consuming and not sensitive enough for immunity screening due to occasional nonspecific reactions. Interpretation involves whether indicator cell lysis occurs, indicating the absence or presence of antibodies in the patient serum.
The ELISA (enzyme-linked immunosorbent assay) is a test that uses antibodies and color change to identify a substance. It involves using an enzyme to detect antigen-antibody binding, which converts a colorless substrate into a colored product. There are several types of ELISA including indirect, direct, sandwich, and competitive ELISA. ELISA can provide quantitative or qualitative results and has applications like screening donated blood and measuring hormone levels.
This document discusses principles of precipitation reactions and their applications in molecular immunogenetics testing. It describes how precipitation occurs when an antibody reacts with a soluble antigen, forming insoluble complexes. This reaction is used in tests like immunofixation electrophoresis and nephelometry. Nephelometry detects antigen-antibody complexes in solution by measuring light scattering. Immunofixation electrophoresis combines protein separation by electrophoresis with precipitation to identify monoclonal proteins. Precipitation reactions are useful for detecting interactions between antibodies and antigens.
Immunogens or antigens are foreign substances that elicit an immune response when introduced to the body. They are recognized by antibodies or T-lymphocytes. Immunogens can induce antibody formation themselves, while haptens require a carrier molecule to produce an immune response. Antigens are presented on antigen-presenting cells and recognized by B and T cells, initiating humoral or cell-mediated immunity. Exogenous antigens from bacteria, viruses, and other external sources are phagocytosed and processed, while endogenous antigens from infection or autoimmunity are presented via MHC I molecules.
This document discusses the diagnosis of autoimmune diseases. It describes several markers that provide evidence of autoimmune diseases, such as a positive family history, presence of other autoimmune diseases, infiltrating cells in affected tissues, and improvement with immunosuppressive drugs. It also discusses several mechanisms that can lead to autoimmunity, including antigenic alteration, sequestered antigens, molecular mimicry, and polyclonal B cell activation. Common diagnostic methods are also summarized, including initial laboratory evaluation, immunological studies, and detection of autoantibodies through enzyme-linked immunosorbent assays (ELISAs).
The lecture was presented to the students of Saudi board of Community Medicine to help them know about the various serological methods applicable in the diagnosis of infectious diseases in general with attention upon the specificity and sensitivity of various diagnostic modalities. The lecture covers the basic principles of each test and the clinical applications with the advantages and disadvantages of each.
Serology is the study of blood serum and the detection of antibodies and antigens. Key events in the history of serology include Karl Landsteiner's 1901 discovery of the A, B, and O blood groups. Serological tests can be classified as primary, secondary, or tertiary based on their level of sensitivity and directness of measurement. Common serological techniques include ELISA, immunofluorescence, agglutination tests, precipitation reactions, and complement fixation tests. These methods are used to detect infections and other medical conditions.
The complement fixation test (CFT) involves mixing antigen, antibody, and complement. If the antibody is specific to the antigen, it will bind and "fix" the complement, preventing it from lysing red blood cells. In a positive test, lysis does not occur, while a negative test results in lysis. CFT is used to detect antibodies against pathogens like syphilis, and can detect antibody levels below 1 microgram/ml. It has limitations like being time-consuming and labor-intensive. Variations involve using different complement sources or detection methods depending on the pathogen.
Immune tolerance is induced through central and peripheral mechanisms that eliminate or suppress self-reactive immune cells. Central tolerance occurs in the thymus and bone marrow where high-affinity self-reactive T and B cells undergo apoptosis or receptor editing. Peripheral tolerance includes anergy induction, suppression by regulatory T cells (Tregs), and inhibition by receptors like CTLA-4 and PD-1. Tregs expressing the transcription factor FoxP3 are critical for maintaining tolerance and preventing autoimmunity. Failure of these tolerance mechanisms can lead to autoimmune disease.
Central tolerance refers to deletion of self-reactive T and B cells in the thymus and bone marrow during maturation. T cells that recognize self antigens undergo apoptosis in the thymus. Peripheral tolerance uses backup mechanisms like clonal deletion through activation-induced cell death, clonal anergy from lack of co-stimulation, and suppression by regulatory T cells. These mechanisms help prevent autoimmune disease by silencing self-reactive cells that escape central tolerance.
The document discusses the antigen-antibody system. It defines antigen and antibody, and describes how they interact specifically. It explains different types of antigen-antibody reactions like precipitation, agglutination, neutralization, immunofluorescence, ELISA, and immunoelectron microscopy. These reactions form the basis of immunity and are used for disease diagnosis, identification of bacteria and viruses, forensic applications, and more. The antigen-antibody system plays an important role in both preventing and diagnosing infectious diseases.
This presentation discusses immunofluorescence techniques. Immunofluorescence involves tagging antibodies with fluorescent dyes so that antigen-antibody complexes can be visualized under a fluorescent microscope. It describes the history and principles of direct and indirect immunofluorescence. Direct immunofluorescence detects in vivo antibodies bound to tissue antigens, while indirect immunofluorescence detects antibodies in patient serum. Both techniques have advantages like sensitivity but also disadvantages like potential cross-reactivity.
The document discusses various properties of antigens that determine their antigenicity including molecular size, foreignness, chemical complexity, stability, and more. It also describes different types of antigenic determinants recognized by B cells and T cells as well as factors like dosage, route of administration, and adjuvants that influence immunogenicity. Finally, it covers antigen specificity and different types of antigens such as haptens, superantigens, and isoantigens.
This document discusses various precipitation reactions and immunological techniques used to detect antigens and antibodies, including: Ouchterlony double immunodiffusion, single radial immunodiffusion, immunoelectrophoresis, and rocket electrophoresis. It explains that precipitation reactions involve two soluble reactants forming an insoluble precipitate. These techniques use diffusion and electrophoresis of antigens and antibodies in semi-solid media like agar to form visible precipitin lines, rings, or rockets, allowing detection and sometimes quantification of proteins. The techniques have various applications in medicine and clinical laboratories.
This document provides an overview of immunohistochemistry methods. It defines key terms like antigens, antibodies, affinity, and sensitivity. It discusses the history of immunohistochemistry from the 1930s to current techniques. The principles of immunohistochemistry are described, including production of primary reagents, tissue fixation, antigen retrieval, staining, and limitations. Various immunohistochemistry methods are summarized such as direct, indirect, polymer, peroxidase-antiperoxidase, and alkaline phosphatase techniques.
The document discusses immunological tolerance and its breakdown which can lead to autoimmunity and autoimmune diseases. It explains the mechanisms of central and peripheral tolerance that normally prevent immune responses against self-antigens. Failure of these tolerance mechanisms can occur through various causes like a breakdown of T cell anergy or loss of regulatory T cells, resulting in an immune response against self-tissues and the development of autoimmune conditions.
Introduction, the principle of immunofluorescence, Technique, Fluorescent microscope and its components, Application and types of immunofluorescence, Direct and indirect immunofluorescence, FACS (Fluorescence-activated cell sorting), Uses and limitations of Immunofluorescence
The document summarizes the development of the male and female reproductive systems from the early undifferentiated gonads through sex determination and the formation of internal and external genitalia. It describes how in males, the testes secrete testosterone and MIF to develop the testes, penis, and scrotum. In females, the absence of these factors leads to ovarian development and the formation of the clitoris, labia, and uterus/vagina. It also discusses several congenital abnormalities that can occur during this development.
This document provides an overview of the male reproductive system. It begins with objectives and then describes key parts of the male reproductive system including the testes, epididymis, vas deferens, seminal vesicles, prostate gland, urethra, penis, and scrotum. It explains the functions of these organs such as sperm production, storage, and ejaculation. Diagrams are included to illustrate the anatomical structures. The document is intended to teach students the gross anatomy and functions of the male reproductive system.
ELISA is a biochemical assay technique used in immunology to detect antibodies, antigens, or other substances. It works by immobilizing an antigen or antibody on a plate and detecting its presence with an enzyme-linked antibody or antigen. There are four common ELISA tests based on the binding structure between the antibody and antigen: direct ELISA, indirect ELISA, sandwich ELISA, and competition ELISA. ELISA provides a sensitive, specific, and quantitative method to detect antibodies or antigens in a sample.
Complement fixation tests (CFT) detect antibodies that do not agglutinate or precipitate by measuring their ability to fix complement. CFT involves incubating patient serum with antigen and complement, then determining if complement is still available to lyse indicator cells. If complement is fixed in the antigen-antibody complex, it cannot lyse the indicator cells, indicating antibody presence. CFT can detect antibody levels below 1 microgram/mL, but it is time-consuming and not sensitive enough for immunity screening due to occasional nonspecific reactions. Interpretation involves whether indicator cell lysis occurs, indicating the absence or presence of antibodies in the patient serum.
The ELISA (enzyme-linked immunosorbent assay) is a test that uses antibodies and color change to identify a substance. It involves using an enzyme to detect antigen-antibody binding, which converts a colorless substrate into a colored product. There are several types of ELISA including indirect, direct, sandwich, and competitive ELISA. ELISA can provide quantitative or qualitative results and has applications like screening donated blood and measuring hormone levels.
This document discusses principles of precipitation reactions and their applications in molecular immunogenetics testing. It describes how precipitation occurs when an antibody reacts with a soluble antigen, forming insoluble complexes. This reaction is used in tests like immunofixation electrophoresis and nephelometry. Nephelometry detects antigen-antibody complexes in solution by measuring light scattering. Immunofixation electrophoresis combines protein separation by electrophoresis with precipitation to identify monoclonal proteins. Precipitation reactions are useful for detecting interactions between antibodies and antigens.
Immunogens or antigens are foreign substances that elicit an immune response when introduced to the body. They are recognized by antibodies or T-lymphocytes. Immunogens can induce antibody formation themselves, while haptens require a carrier molecule to produce an immune response. Antigens are presented on antigen-presenting cells and recognized by B and T cells, initiating humoral or cell-mediated immunity. Exogenous antigens from bacteria, viruses, and other external sources are phagocytosed and processed, while endogenous antigens from infection or autoimmunity are presented via MHC I molecules.
This document discusses the diagnosis of autoimmune diseases. It describes several markers that provide evidence of autoimmune diseases, such as a positive family history, presence of other autoimmune diseases, infiltrating cells in affected tissues, and improvement with immunosuppressive drugs. It also discusses several mechanisms that can lead to autoimmunity, including antigenic alteration, sequestered antigens, molecular mimicry, and polyclonal B cell activation. Common diagnostic methods are also summarized, including initial laboratory evaluation, immunological studies, and detection of autoantibodies through enzyme-linked immunosorbent assays (ELISAs).
The lecture was presented to the students of Saudi board of Community Medicine to help them know about the various serological methods applicable in the diagnosis of infectious diseases in general with attention upon the specificity and sensitivity of various diagnostic modalities. The lecture covers the basic principles of each test and the clinical applications with the advantages and disadvantages of each.
Serology is the study of blood serum and the detection of antibodies and antigens. Key events in the history of serology include Karl Landsteiner's 1901 discovery of the A, B, and O blood groups. Serological tests can be classified as primary, secondary, or tertiary based on their level of sensitivity and directness of measurement. Common serological techniques include ELISA, immunofluorescence, agglutination tests, precipitation reactions, and complement fixation tests. These methods are used to detect infections and other medical conditions.
The complement fixation test (CFT) involves mixing antigen, antibody, and complement. If the antibody is specific to the antigen, it will bind and "fix" the complement, preventing it from lysing red blood cells. In a positive test, lysis does not occur, while a negative test results in lysis. CFT is used to detect antibodies against pathogens like syphilis, and can detect antibody levels below 1 microgram/ml. It has limitations like being time-consuming and labor-intensive. Variations involve using different complement sources or detection methods depending on the pathogen.
Immune tolerance is induced through central and peripheral mechanisms that eliminate or suppress self-reactive immune cells. Central tolerance occurs in the thymus and bone marrow where high-affinity self-reactive T and B cells undergo apoptosis or receptor editing. Peripheral tolerance includes anergy induction, suppression by regulatory T cells (Tregs), and inhibition by receptors like CTLA-4 and PD-1. Tregs expressing the transcription factor FoxP3 are critical for maintaining tolerance and preventing autoimmunity. Failure of these tolerance mechanisms can lead to autoimmune disease.
Central tolerance refers to deletion of self-reactive T and B cells in the thymus and bone marrow during maturation. T cells that recognize self antigens undergo apoptosis in the thymus. Peripheral tolerance uses backup mechanisms like clonal deletion through activation-induced cell death, clonal anergy from lack of co-stimulation, and suppression by regulatory T cells. These mechanisms help prevent autoimmune disease by silencing self-reactive cells that escape central tolerance.
The document discusses the antigen-antibody system. It defines antigen and antibody, and describes how they interact specifically. It explains different types of antigen-antibody reactions like precipitation, agglutination, neutralization, immunofluorescence, ELISA, and immunoelectron microscopy. These reactions form the basis of immunity and are used for disease diagnosis, identification of bacteria and viruses, forensic applications, and more. The antigen-antibody system plays an important role in both preventing and diagnosing infectious diseases.
This presentation discusses immunofluorescence techniques. Immunofluorescence involves tagging antibodies with fluorescent dyes so that antigen-antibody complexes can be visualized under a fluorescent microscope. It describes the history and principles of direct and indirect immunofluorescence. Direct immunofluorescence detects in vivo antibodies bound to tissue antigens, while indirect immunofluorescence detects antibodies in patient serum. Both techniques have advantages like sensitivity but also disadvantages like potential cross-reactivity.
The document discusses various properties of antigens that determine their antigenicity including molecular size, foreignness, chemical complexity, stability, and more. It also describes different types of antigenic determinants recognized by B cells and T cells as well as factors like dosage, route of administration, and adjuvants that influence immunogenicity. Finally, it covers antigen specificity and different types of antigens such as haptens, superantigens, and isoantigens.
This document discusses various precipitation reactions and immunological techniques used to detect antigens and antibodies, including: Ouchterlony double immunodiffusion, single radial immunodiffusion, immunoelectrophoresis, and rocket electrophoresis. It explains that precipitation reactions involve two soluble reactants forming an insoluble precipitate. These techniques use diffusion and electrophoresis of antigens and antibodies in semi-solid media like agar to form visible precipitin lines, rings, or rockets, allowing detection and sometimes quantification of proteins. The techniques have various applications in medicine and clinical laboratories.
This document provides an overview of immunohistochemistry methods. It defines key terms like antigens, antibodies, affinity, and sensitivity. It discusses the history of immunohistochemistry from the 1930s to current techniques. The principles of immunohistochemistry are described, including production of primary reagents, tissue fixation, antigen retrieval, staining, and limitations. Various immunohistochemistry methods are summarized such as direct, indirect, polymer, peroxidase-antiperoxidase, and alkaline phosphatase techniques.
The document discusses immunological tolerance and its breakdown which can lead to autoimmunity and autoimmune diseases. It explains the mechanisms of central and peripheral tolerance that normally prevent immune responses against self-antigens. Failure of these tolerance mechanisms can occur through various causes like a breakdown of T cell anergy or loss of regulatory T cells, resulting in an immune response against self-tissues and the development of autoimmune conditions.
Introduction, the principle of immunofluorescence, Technique, Fluorescent microscope and its components, Application and types of immunofluorescence, Direct and indirect immunofluorescence, FACS (Fluorescence-activated cell sorting), Uses and limitations of Immunofluorescence
The document summarizes the development of the male and female reproductive systems from the early undifferentiated gonads through sex determination and the formation of internal and external genitalia. It describes how in males, the testes secrete testosterone and MIF to develop the testes, penis, and scrotum. In females, the absence of these factors leads to ovarian development and the formation of the clitoris, labia, and uterus/vagina. It also discusses several congenital abnormalities that can occur during this development.
This document provides an overview of the male reproductive system. It begins with objectives and then describes key parts of the male reproductive system including the testes, epididymis, vas deferens, seminal vesicles, prostate gland, urethra, penis, and scrotum. It explains the functions of these organs such as sperm production, storage, and ejaculation. Diagrams are included to illustrate the anatomical structures. The document is intended to teach students the gross anatomy and functions of the male reproductive system.
Anatomy & Physiology Of The Reproductive Systemgueste481464
The document summarizes the anatomy and physiology of the female and male reproductive systems. It discusses how the female system produces eggs and the organs involved, including the ovaries, fallopian tubes, uterus, vagina, and breasts. It also describes the three stages of labor: dilation, expulsion, and placental delivery. Finally, it notes that the male system produces sperm and sex hormones through organs like the testes, epididymis, penis, and associated glands.
This document discusses different types of immunoprophylaxis, or protection against infectious diseases through immunization. It describes passive immunization, which provides temporary protection through transfer of antibodies from mother to fetus or through injection of antibodies such as antitoxins. It also describes active immunization, which provides long-lasting protection as the individual produces their own antibodies in response to vaccination or natural exposure. The document outlines various types of vaccines including killed, live attenuated, toxoids, and those produced through recombinant DNA technology. It emphasizes that immunization is most effective when administered as a combination of different vaccines.
Pencegahan transmisi perinatal Hepatitis B adalah salah satu langkah pencegahan utama timbulnya kasus Hepatitis Kronis pada dewasa. Beberapa langkah PMTCT pada hepatitis B akan dijelaskan dalam presentasi ini.
Dipresentasikan pada CME: 1st Surabaya Fetomaternal Update, 14 Mei 2016.
This document outlines the objectives and content of a lecture on the anatomy of the male and female reproductive systems. The objectives include identifying the structures of the external genitalia, reproductive tracts, blood and nerve supply in both sexes, and their roles in reproduction. The content will cover the anatomy and relationships of structures like the testes, vas deferens, prostate and penis in males, and vagina, uterus, uterine tubes and ovaries in females. It will also discuss the embryological development and ligaments of the reproductive organs.
This document discusses immunoprophylaxis and vaccination. It defines prophylaxis and vaccine preventable diseases. There are two types of immunization: active and passive. Active immunization involves inducing immunity through vaccination and works through humoral and cellular immunity. Vaccines can be live attenuated, killed/inactivated, toxoids, cellular fractions, subunit vaccines, or recombinant vaccines. Passive immunization provides immediate short-term protection through administration of antibodies or immunoglobulins. The document also discusses vaccination schedules and recent vaccination missions.
HIV structure,pathogenesis, classification and transmiss216191912
The document discusses HIV classification, structure, pathogenesis, and modes of transmission. It describes the CDC classification system for HIV based on CD4 cell counts and conditions, with categories A, B, and C. It explains HIV's structure including its envelope, matrix proteins, core, and RNA. HIV pathogenesis involves binding to and fusing with host cells, reverse transcribing its RNA into DNA, and using the host cell to replicate. HIV is typically transmitted via unprotected sex, contaminated needles, or from mother to child during birth or breastfeeding.
The document discusses the life cycle of HIV and the stages of HIV disease progression if left untreated. It describes the five stages of the HIV life cycle: (1) entry, (2) reverse transcription, (3) integration, (4) transcription, and (5) maturation. It also includes timelines of drug development and the typical course of untreated HIV disease progression from initial infection to AIDS.
The human immunodeficiency virus (HIV) is a lentivirus (a subgroup of retrovirus) that causes HIV infection and over time acquired immunodeficiency syndrome (AIDS).[1][2] AIDS is a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive. Without treatment, average survival time after infection with HIV is estimated to be 9 to 11 years, depending on the HIV subtype.[3] Infection with HIV occurs by the transfer of blood, semen, vaginal fluid, pre-ejaculate, or breast milk. Within these bodily fluids, HIV is present as both free virus particles and virus within infected immune cells.
HIV infects vital cells in the human immune system such as helper T cells (specifically CD4+ T cells), macrophages, and dendritic cells.[4] HIV infection leads to low levels of CD4+ T cells through a number of mechanisms, including pyroptosis of abortively infected T cells,[5] apoptosis of uninfected bystander cells,[6] direct viral killing of infected cells, and killing of infected CD4+ T cells by CD8 cytotoxic lymphocytes that recognize infected cells.[7] When CD4+ T cell numbers decline below a critical level, cell-mediated immunity is lost, and the body becomes progressively more susceptible to opportunistic infections.
This document provides an overview of AIDS/HIV including:
- HIV is a retrovirus that causes AIDS by destroying immune cells. It has a long asymptomatic period before symptoms appear.
- It is transmitted through blood, semen, vaginal fluids. High risk groups include men who have sex with men, IV drug users, and those with other STDs.
- Symptoms progress from acute infection, to asymptomatic carrier state, to AIDS-related complex with opportunistic infections, and finally AIDS with life-threatening infections like PCP.
- Diagnosis involves antibody tests, viral tests, and CD4 counts. Treatment involves antiretroviral drugs to suppress viral load. Prevention focuses on safe sex
This document discusses the epidemiology and pathogenesis of HIV. It begins with global epidemiology statistics, including that there are 35.3 million people living with HIV worldwide. It then provides more specific statistics on new infections, deaths, at-risk groups, and prevalence by region. Regarding pathogenesis, it explains that HIV primarily attaches to CD4 receptors on cells and integrates its genetic material, leading to infection. It also discusses the body's immune response and how HIV evades detection through high mutation rates. Prevention methods discussed include behavior change, condoms, testing, and antiretroviral treatment and prophylaxis.
The document summarizes the male and female reproductive systems. The male reproductive system includes the testes, which produce sperm, and the duct system including the vas deferens and epididymis, which transports and matures sperm. The female reproductive system includes the ovaries, which produce eggs, the fallopian tubes where fertilization occurs, and the uterus which nourishes a developing fetus. Fertilization happens when a sperm enters an egg in the fallopian tubes, forming a zygote that implants in the uterus.
The document provides information on HIV/AIDS, including:
1. HIV was first identified in 1981 and there have been two major strains identified, HIV-1 and HIV-2.
2. HIV is transmitted through bodily fluids and can be transmitted sexually or through contact with infected blood.
3. There are three phases of HIV infection eventually resulting in AIDS if not treated. Antiretroviral treatment can suppress the virus and prevent AIDS.
The skin has three main layers - the epidermis, dermis, and subcutaneous layer. The epidermis is a thin outer layer made of stratified squamous epithelium with several layers including the basal and stratum corneum layers. Below this is the thicker dermis layer made of dense connective tissue containing blood vessels, nerves, hair follicles, and sweat and sebaceous glands. The deepest layer is the subcutaneous tissue containing fat and connective tissue. The skin provides protection from damage and infection, contains sensory receptors, regulates body temperature, and plays a role in fluid balance and vitamin D synthesis through sweat and sebaceous glands.
This document summarizes various serological tests used to detect antigens and antibodies. It describes primary, secondary and tertiary serological tests including ELISA, immunofluorescence, radioimmunoassay and more. It also details different types of agglutination tests like qualitative and quantitative tests. Additional tests covered are precipitation tests, complement fixation tests, passive hemagglutination and sandwich ELISA. The document provides information on applications and procedures for many antibody and antigen detection techniques.
Anatomy of the female reproductive systemraj kumar
The document provides an overview of the anatomy of the female reproductive system, including:
1) The pelvis contains bones that form the base of the pelvis and support the organs. Within the pelvis are the internal genitalia.
2) The internal genitalia include the uterus, fallopian tubes, ovaries, and cervix. The vagina connects the internal genitalia to the external genitalia.
3) The external genitalia include the labia, clitoris, and openings of the urethra and vagina. Surrounding structures like blood vessels, lymph nodes, and adjacent organs like the bladder and rectum are also discussed.
A and B antigens help determine blood types and are present in various body fluids and tissues. Antigens and antibodies can form lattices through bivalent and polyvalent interactions, resulting in precipitation. Precipitation reactions can be measured through techniques like immunodiffusion assays and radial immunodiffusion to analyze antigen-antibody interactions and concentrations. Agglutination, another antigen-antibody reaction, involves antibody-mediated clumping of particulate antigens and is used for applications like blood typing and bacterial infection diagnosis.
The document discusses antigen-antibody techniques used for disease diagnosis. It defines antigens and antibodies, and describes various agglutination tests like bacterial agglutination and hemagglutination which detect antibodies through visible clumping. Precipitation tests detect insoluble antigen-antibody complexes. ELISA and immunoelectrophoresis are also summarized, with ELISA being a sensitive and specific immunoassay and immunoelectrophoresis separating antigens based on charge and mobility. Monoclonal versus polyclonal antibodies are compared.
Some Clinical Laboratory Measurement of Immune FunctionsAmany Elsayed
This document describes several clinical laboratory techniques for measuring immune functions, including antibody-based assays that detect antigens or quantify them. It discusses methods like agglutination assays, precipitation assays, immunoassays using radioisotopes, enzymes or fluorescence to label antibodies or antigens. It also summarizes techniques like immunofluorescence and flow cytometry to detect epitopes on or within cells, assays to assess immune functions like phagocytosis and proliferation, and methods for evaluating hypersensitivity reactions.
Antigen-antibody interactions can be quantified using various serological tests. Common types include precipitation tests like immunodiffusion that form visible precipitate lines, agglutination tests where antigens clump together, neutralization tests using viruses and complement fixation assays. Enzyme-linked immunosorbent assays (ELISAs) are now widely used as they are sensitive, specific and can be quantitative or qualitative. Fluorescent antibody techniques use fluorescent dyes to label antibodies or cells for detection under a microscope.
This document provides an overview of antigen-antibody interactions and serological testing. It discusses the specific binding between antigens and antibodies, the formation of immune complexes, and the properties of antigen-antibody reactions including affinity, avidity, cross-reactivity, and specificity. It also describes several types of antigen-antibody reactions like precipitation, agglutination, complement fixation, ELISA, and immunofluorescence. The document is intended to guide students in understanding the basics of antigen-antibody interactions and their applications.
This document provides an overview of antigen-antibody reactions, including definitions, general features, measurement techniques, types of reactions such as precipitation, agglutination, neutralization, immunofluorescence, radioimmunoassay, and enzyme-linked immunosorbent assay (ELISA). It describes techniques like precipitation reactions in liquids and gels, single and double diffusion, electrophoresis, latex agglutination, complement fixation, and microtitration agglutination tests. The document outlines the applications and uses of these various antigen-antibody reaction techniques.
elisaLecture_based on new way to medical lab.pptmainakg09
The document describes the enzyme-linked immunosorbent assay (ELISA) technique. ELISA uses antibodies and antigen-enzyme conjugates to detect the presence and quantify antigens or antibodies in a sample. It is a sensitive, specific immunoassay that is usually performed in microwell plates, where antibodies or antigens bind to the wells and are detected using enzyme-linked antibodies and color-changing substrates. There are different types of ELISA including sandwich, competitive, and indirect formats.
This document discusses antigen-antibody reactions including factors that affect their measurement and techniques used to measure them in the lab. Key points covered include: affinity and avidity being measures of strength of antigen-antibody binding; specificity and cross-reactivity relating to reaction with single or multiple antigens; and techniques including precipitation tests, agglutination, ELISA, radioimmunoassay, immunofluorescence, and complement fixation. Both qualitative and quantitative applications are discussed.
Antigen and antibody interaction is the basis of serological testing. There are several types of serological tests that detect this interaction, including precipitation, agglutination, hemagglutination, enzyme-linked immunosorbent assay (ELISA), Western blot, hemagglutination inhibition, and immunofluorescence. These tests exploit the formation of antigen-antibody complexes to diagnose diseases, identify pathogens, and detect proteins.
This document discusses antigen-antibody reactions. It begins by defining antigen-antibody reactions as the specific interaction between antigens and antibodies. It then describes the three stages of antigen-antibody reactions: initial formation of antigen-antibody complexes, followed by visible events like precipitation or agglutination, and finally neutralization or destruction of antigens. The document also discusses various types of antigen-antibody reactions like precipitation, agglutination, and immunoassays. It provides examples of applications of antigen-antibody reactions like disease detection, identification of bacteria, and standardization of toxins.
This document discusses immunity and antigen-antibody reactions. It begins with definitions of immunity, antigens, and antibodies. It describes the components of the immune system including antigen specificity and types of antigens. It then explains antigen-antibody reactions and how they are used for diagnostic tests. Different diagnostic tests are also summarized, including precipitation reactions, agglutination, immunofluorescence, radioimmunoassay, and ELISA. Potential sources of markers for periodontal disease activity are also listed.
The document discusses antigen-antibody reactions. It describes how antigens interact specifically with antibodies produced against them, forming antigen-antibody complexes. The complexes can then undergo precipitation, agglutination, complement fixation, or cytolysis depending on whether the antigen is soluble or insoluble. It also discusses the properties, types, techniques and applications of common antigen-antibody reactions like precipitation, agglutination, complement fixation tests and opsonization.
Immunological tests use antigen-antibody reactions to determine the presence of antigens or antibodies. Some common tests include agglutination tests which cause antigens to clump in the presence of antibodies, hemagglutination tests using red blood cells, precipitation tests forming aggregates, ELISA measuring antibodies or antigens bound to an enzyme, immunofluorescence using fluorescent dyes to identify antigens, and complement fixation testing antigen-antibody activation of complement. These various serological tests are important in the diagnosis of diseases.
This document summarizes antigen-antibody interactions. It describes how antibodies specifically bind to antigens via epitopes and paratopes. The binding is due to non-covalent interactions like hydrogen bonds and van der Waals forces. This interaction forms the basis of serological tests like precipitation, agglutination, complement fixation and ELISA, which are used to detect infectious diseases. The document also discusses properties of antigen-antibody reactions like affinity, avidity and specificity.
The document provides an overview of the enzyme-linked immunosorbent assay (ELISA) technique. It describes three main types of ELISA - the sandwich ELISA, indirect ELISA, and competitive ELISA. For each type, it provides a brief example of clinical applications, such as detecting neural proteins in urine to diagnose Alzheimer's disease (sandwich ELISA) or detecting HIV antibodies in serum (indirect ELISA). It emphasizes that ELISA is a sensitive, cost-effective, and widely used assay for diagnosing infections and diseases.
ELISA, principle and method by kk sahuKAUSHAL SAHU
What is ELISA.
Principle.
History.
Types of ELISA method.
1.Direct ELISA.
2.Indirect ELISA.
3.Sandwhich ELISA.
Conclusion.
References.
Antibodies (also known as immunoglobulins abbreviated Ig) are gamma globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.
Antigen ,Antibody and Ag-Ab reactions ppt by DR.C.P.PRINCEDR.PRINCE C P
An immunogen refers to a molecule that is capable of eliciting an immune response, whereas an antigen refers to a molecule that is capable of binding to the product of that immune response (Ab).
So, an immunogen is necessarily an antigen, but an antigen may not necessarily be an immunogen
The terms immunogen and antigen are often used interchangeably but the later is more common.
Antibodies are Globulin Protein (Immunoglobulin) that are synthesized in the Serum and Tissue fluids.
It reacts specifically with the antigen that stimulated their production.
There are two types serum proteins: albumin and globulin
There are Three types of globulins .
1. Alpha globulin
2. Beta globulin
3. Gamma globulin (Antibodies)
Gamma globulins are responsible for immunity. So they are called as Immunoglobulin (Ig)
The binding of an antibody with an antigen of the type that stimulated the formation of antibody that results in the following reaction
Agglutination
Precipitation
Complement fixation
Phagocytosis
Neutralization of an exotoxin
Opsonization
Tissue fixation
Chemotaxis
Activation of mast cells and basophils
PPT prepared by:
DR.PRINCE C P
Associate Professor , Department of Microbiology,
Mother Theresa Post Graduate & Research Institute of Health Sciences (Government of Puducherry Institution)
Antigen ,Antibody and Ag-Ab reactions ppt by DR.C.P.PRINCE
Serological tests
1. Serological tests
(Antigen antibody interactions)
Classification of antigen-antibody interactions:
1. Primary serological tests: (Marker techniques) e.g.
a. Enzyme linked immuono sorben assay (ELISA)
b. Immuno flurescent antibody technique (IFAT)
c. Radio immuno assay (RIA)
2. Secondary serological tests: e.g.
a. Agglutination tests
b. Complement fixation tests (CFT)
c. Precipitation tests
d. Serum neutralization tests (SNT)
e. Toxin-antitoxin test
3. Tertiary serological test: e.g.
a. Determination of the protective value of an anti serum in an
animal.
A. Agglutination tests:
1. Agglutination/Hemagglutination
When the antigen is particulate, the reaction of an antibody with the antigen can be
detected by agglutination (clumping) of the antigen. The general term agglutinin is
used to describe antibodies that agglutinate particulate antigens. When the antigen is
an erythrocyte the term heamagglutination is used. All antibodies can theoretically
agglutinate particulate antigens but IgM, due to its high valence, is particularly good
agglutinin and one sometimes infers that an antibody may be of the IgM class if it is a
good agglutinating antibody.
a. Qualitative 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, a patient's red blood cells can be mixed with antibody to a blood group
antigen to determine a person's blood type. In a second example, a patient's serum is
mixed with red blood cells of a known blood type to assay for the presence of
antibodies to that blood type in the patient's serum
.
2. b. Quantitative agglutination test
Agglutination tests can also be used to measure the level of antibodies to
particulate antigens. In this test, serial dilutions are made of a sample to be
tested for antibody and then a fixed number of red blood cells or bacteria or
other such particulate antigen is added. Then the maximum dilution that gives
agglutination is determined. The maximum dilution that gives visible
agglutination is called the titer. The results are reported as the reciprocal of the
maximal dilution that gives visible agglutination.
Prozone effect - Occasionally, it is observed that when the concentration of antibody
is high (i.e. lower dilutions), there is no agglutination and then, as the sample is
diluted, agglutination occurs.
The lack of agglutination at high concentrations of antibodies is called the prozone
effect. Lack of agglutination in the prozone is due to antibody excess resulting in very
small complexes that do not clump to form visible agglutination.
c. Applications of agglutination tests
1. Determination of blood types or antibodies to blood
group antigens.
2. To assess bacterial infections
e.g. A rise in titer of an antibody to a particular bacterium indicates an infection
with that bacterial type. N.B. a fourfold rise in titer is generally taken as a
significant rise in antibody titer.
2-Passive hemagglutination:
The agglutination test only works with particulate antigens. 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. This is called passive hemagglutination.
The test is performed just like the agglutination test. Applications include detection of
antibodies to soluble antigens and detection of antibodies to viral antigens.
3-Coomb's Test (Antiglobulin Test):
a. Direct Coomb's Test
When antibodies bind to erythrocytes, they do not always result in
agglutination. This can result from the antigen/antibody ratio being in antigen
excess or antibody excess or in some cases electrical charges on the red blood
cells preventing the effective cross linking of the cells. These antibodies that
bind to but do not cause agglutination of red blood cells are sometimes
referred to as incomplete antibodies. In no way is this meant to indicate that
the antibodies are different in their structure, although this was once thought to
be the case. Rather, it is a functional definition only. In order to detect the
presence of non-agglutinating antibodies on red blood cells, one simply adds a
second antibody directed against the immunoglobulin (antibody) coating the
red cells. This anti-immunoglobulin can now cross link the red blood cells and
result in agglutination.
b. Indirect Coomb's Test
If it is necessary to know whether a serum sample has antibodies directed
3. against a particular red blood cell and you want to be sure that you also detect
potential non- agglutinating antibodies in the sample, an Indirect Coomb's test
is performed.
This test is done by incubating the red blood cells with the serum sample, washing
out any unbound antibodies and then adding a second anti-immunoglobulin
reagent to cross link the cells.
c. Applications
These include detection of anti-rhesus factor (Rh) antibodies. Antibodies to
the Rh factor generally do not agglutinate red blood cells. Thus, red cells from
Rh+ children born to Rh- mothers, who have anti-Rh antibodies, may be
coated with these antibodies. To check for this, a direct Coombs test is
performed. To see if the mother has anti-Rh antibodies in her serum an
Indirect Coombs test is performed.
4-HemagglutinationInhibition
The agglutination test can be modified to be used for the measurement of soluble
antigens. This test is called hemagglutination inhibition. It is called hemagglutination
inhibition because one measures the ability of soluble antigen to inhibit the
agglutination of antigen-coated red blood cells by antibodies. In this test, a fixed
amount of antibodies to the antigen in question is mixed with a fixed amount of red
blood cells coated with the antigen. Also included in the mixture are different
amounts of the sample to be analyzed for the presence of the antigen. If the sample
contains the antigen, the soluble antigen will compete with the antigen coated on the
red blood cells for binding to the antibodies, thereby inhibiting the agglutination of
the red blood cells.
B. Precipitation tests:
1-RadialImmunodiffusion(Mancini)
In radial immunodiffusion antibody is incorporated into the agar gel as it is poured
and different dilutions of the antigen are placed in holes punched into the agar. As the
antigen diffuses into the gel, it reacts with the antibody and when the equivalence
point is reached a ring of precipitation is formed.
2-Immunoelectrophoresis:
In immunoelectrophoresis, a complex mixture of antigens is placed in a well punched
out of an agar gel and the antigens are electrophoresed so that the antigen are
separated according to their charge. After electrophoresis, a trough is cut in the gel
and antibodies are added. As the antibodies diffuse into the agar, precipitin lines are
produced in the equivalence zone when an antigen/antibody reaction occurs.
This tests is used for the qualitative analysis of complex mixtures of antigens,
although a crude measure of quantity (thickness of the line) can be obtained. This test
is commonly used for the analysis of components in a patient' serum. Serum is placed
in the well and antibody to whole serum in the trough. By comparisons to normal
serum, one can determine whether there are deficiencies on one or more serum
components or whether there is an overabundance of some serum component
(thickness of the line). This test can also be used to evaluate purity of isolated serum
proteins.
4. 3- Countercurrent electrophoresis:
In this test the antigen and antibody are placed in wells punched out of an agar gel and
the antigen and antibody are electrophoresed into each other where they form a
precipitation line.
This test only works if conditions can be found where the antigen and antibody have
opposite charges. This test is primarily qualitative, although from the thickness of the
band you can get some measure of quantity. Its major advantage is its speed.
C. Complement fixation test:
• The complement fixation test is an immunological medical test looking for
evidence of infection. It tests for the presence of either specific antibody or
specific antigen in a patient's serum. It uses sheep red blood cells (sRBC),
anti-sRBC antibody and complement, plus specific antigen (if looking for
antibody in serum) or specific antibody (if looking for antigen in serum).
• If either the antibody or antigen is present in the patient's serum, then the
complement is completely utilized, so the sRBCs are not lysed. But if the
antibody (or antigen) is not present, then the complement is not used up, so it
binds anti-sRBC antibody, and the sRBCs are lysed.
• The Wassermann test is one form of complement fixation test.
D. Enzyme-Linked ImmunoSorbent Assay
(ELISA):
• Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical
technique used mainly in immunology to detect the presence of an antibody or
an antigen in a sample. The ELISA has been used as a diagnostic tool in
medicine and plant pathology, as well as a quality control check in various
industries. In simple terms, in ELISA an unknown amount of antigen is
affixed to a surface, and then a specific antibody is washed over the surface so
that it can bind the antigen. This antibody is linked to an enzyme, and in the
final step a substance is added that the enzyme can convert to some detectable
signal. Thus in the case of fluorescence ELISA, when light is shone upon the
sample, any antigen/antibody complexes will fluoresce so that the amount of
antigen in the sample can be measured.
1.Indirect ELISA:
• The steps of the general, "indirect," ELISA for determining serum antibody
concentrations are:
1. Apply a sample of known antigen of known concentration to a surface,
often the well of a microtiter plate. The antigen is fixed to the surface
to render it immobile. Simple adsorption of the protein to the plastic
surface is usually sufficient. These samples of known antigen
concentrations will constitute a standard curve used to calculate
antigen concentrations of unknown samples. Note that the antigen
itself may be an antibody.
5. 2. The plate wells or other surface are then coated with serum samples of
unknown antigen concentration, diluted into the same buffer used for
the antigen standards. Since antigen immobilization in this step is due
to non-specific adsorption, it is important for the total protein
concentration to be similar to that of the antigen standards.
3. A concentrated solution of non-interacting protein, such as Bovine
Serum Albumin (BSA) or casein, is added to all plate wells. This step
is known as blocking, because the serum proteins block non-specific
adsorption of other proteins to the plate.
4. The plate is washed, and a detection antibody specific to the antigen of
interest is applied to all plate wells. This antibody will only bind to
immobilized antigen on the well surface, not to other serum proteins or
the blocking proteins.
5. The plate is washed to remove any unbound detection antibody. After
this wash, only the antibody-antigen complexes remain attached to the
well.
6. Secondary antibodies, which will bind to any remaining detection
antibodies, are added to the wells. These secondary antibodies are
conjugated to the substrate-specific enzyme. This step may be skipped
if the detection antibody is conjugated to an enzyme.
7. Wash the plate, so that excess unbound enzyme-antibody conjugates
are removed.
8. Apply a substrate which is converted by the enzyme to elicit a
chromogenic or fluorogenic or electrochemical signal.
9. View/quantify the result using a spectrophotometer,
spectrofluorometer, or other optical/electrochemical device.
2.Sandwich ELISA :
• A sandwich ELISA:
Plate is coated with a capture antibody
sample is added, and any antigen present binds to capture antibody
detecting antibody is added, and binds to antigen
enzyme-linked secondary antibody is added, and binds to detecting
antibody
substrate is added, and is converted by enzyme to detectable form.
A less-common variant of this technique, called "sandwich" ELISA, is used to detect
sample antigen. The steps are as follows:
1. Prepare a surface to which a known quantity of capture antibody is
bound.
2. Block any non specific binding sites on the surface.
3. Apply the antigen-containing sample to the plate.
4. Wash the plate, so that unbound antigen is removed.
5. Apply primary antibodies that bind specfically to the antigen.
6. Apply enzyme-linked secondary antibodies which are specific to the
primary antibodies.
7. Wash the plate, so that the unbound antibody-enzyme conjugates are
removed.
8. Apply a chemical which is converted by the enzyme into a color or
fluorescent or electrochemical signal.
6. 9. Measure the absorbance or fluorescence or electrochemical signal
(e.g., current) of the plate wells to determine the presence and quantity
of antigen.
3.Competitive ELISA:
• A third use of ELISA is through competitive binding. The steps for this
ELISA are somewhat different than the first two examples:
1. Unlabeled antibody is incubated in the presence of its antigen.
2. These bound antibody/antigen complexes are then added to an antigen
coated well.
3. The plate is washed, so that unbound antibody is removed. (The more
antigen in the sample, the less antibody will be able to bind to the
antigen in the well, hence "competition.")
4. The secondary antibody, specific to the primary antibody is added.
This second antibody is coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a chromogenic or
fluorescent signal.
• For competitive ELISA, the higher the original antigen concentration, the
weaker the eventual signal.
4.Applications:
• Because the ELISA can be performed to evaluate either the presence of
antigen or the presence of antibody in a sample, it is a useful tool both for
determining serum antibody concentrations (such as with the HIV test[1] or
West Nile Virus) and also for detecting the presence of antigen. It has also
found applications in the food industry in detecting potential food allergens
such as milk ,peanuts ,walnuts ,almonds , and eggs [2]The ELISA test, or the
enzyme immunoassay (EIA), was the first screening test commonly employed
for HIV. It has a high sensitivity. In an ELISA test, a person's serum is diluted
400-fold and applied to a plate to which HIV antigens have been attached. If
antibodies to HIV are present in the serum, they may bind to these HIV
antigens. The plate is then washed to remove all other components of the
serum. A specially prepared "secondary antibody" — an antibody that binds to
human antibodies — is then applied to the plate, followed by another wash.
This secondary antibody is chemically linked in advance to an enzyme. Thus
the plate will contain enzyme in proportion to the amount of secondary
antibody bound to the plate. A substrate for the enzyme is applied, and
catalysis by the enzyme leads to a change in color or fluorescence. ELISA
results are reported as a number; the most controversial aspect of this test is
determining the "cut-off" point between a positive and negative result.
7. 9. Measure the absorbance or fluorescence or electrochemical signal
(e.g., current) of the plate wells to determine the presence and quantity
of antigen.
3.Competitive ELISA:
• A third use of ELISA is through competitive binding. The steps for this
ELISA are somewhat different than the first two examples:
1. Unlabeled antibody is incubated in the presence of its antigen.
2. These bound antibody/antigen complexes are then added to an antigen
coated well.
3. The plate is washed, so that unbound antibody is removed. (The more
antigen in the sample, the less antibody will be able to bind to the
antigen in the well, hence "competition.")
4. The secondary antibody, specific to the primary antibody is added.
This second antibody is coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a chromogenic or
fluorescent signal.
• For competitive ELISA, the higher the original antigen concentration, the
weaker the eventual signal.
4.Applications:
• Because the ELISA can be performed to evaluate either the presence of
antigen or the presence of antibody in a sample, it is a useful tool both for
determining serum antibody concentrations (such as with the HIV test[1] or
West Nile Virus) and also for detecting the presence of antigen. It has also
found applications in the food industry in detecting potential food allergens
such as milk ,peanuts ,walnuts ,almonds , and eggs [2]The ELISA test, or the
enzyme immunoassay (EIA), was the first screening test commonly employed
for HIV. It has a high sensitivity. In an ELISA test, a person's serum is diluted
400-fold and applied to a plate to which HIV antigens have been attached. If
antibodies to HIV are present in the serum, they may bind to these HIV
antigens. The plate is then washed to remove all other components of the
serum. A specially prepared "secondary antibody" — an antibody that binds to
human antibodies — is then applied to the plate, followed by another wash.
This secondary antibody is chemically linked in advance to an enzyme. Thus
the plate will contain enzyme in proportion to the amount of secondary
antibody bound to the plate. A substrate for the enzyme is applied, and
catalysis by the enzyme leads to a change in color or fluorescence. ELISA
results are reported as a number; the most controversial aspect of this test is
determining the "cut-off" point between a positive and negative result.