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Enzyme-linked immunosorbent a Enzyme-linked immunosorbent a ssay (ELISA) ssay (ELISA) ELISA ELISA

Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

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Page 1: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

Enzyme-linked immunosorbent assaEnzyme-linked immunosorbent assay (ELISA)y (ELISA)

ELISAELISA

Page 2: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

ImmunoassayImmunoassay

The technique of immunoassay using labThe technique of immunoassay using labelled reagents for detecting antigens and antiboelled reagents for detecting antigens and antibodies are exquisitely sensitive and extremely ecodies are exquisitely sensitive and extremely economical in the reagents(immunoassay for antibnomical in the reagents(immunoassay for antibody).ody).

Solid-phase assays for antibodies employSolid-phase assays for antibodies employing ligands labelled with radioisotopes or enzying ligands labelled with radioisotopes or enzymes(enzyme-linked immunosorbent assay; ELISmes(enzyme-linked immunosorbent assay; ELISA) are most widely used of all immunological asA) are most widely used of all immunological assays.says.

Page 3: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

Enzyme-linked immunosorbent assayEnzyme-linked immunosorbent assay(ELISA)(ELISA)

In this system, ligand is a molecule which In this system, ligand is a molecule which can detect the antibody and is covalently can detect the antibody and is covalently coupled to an enzyme such as peroxidase.coupled to an enzyme such as peroxidase.The amount of test antibody is measured The amount of test antibody is measured by assessing the mount of coloured end-pby assessing the mount of coloured end-product by optical density scanning of the roduct by optical density scanning of the plate.plate.

Page 4: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

A typical titration curveA typical titration curve

Antibody titres can only be detected correAntibody titres can only be detected correctly within the linear range. Typically the pctly within the linear range. Typically the plateau binding is 20-100 times the backgrolateau binding is 20-100 times the background. The sensitivity of the technique is usund. The sensitivity of the technique is usually about 1-50ng/ml of specific antibody.ually about 1-50ng/ml of specific antibody.

Page 5: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

Procedure of ELISAProcedure of ELISA

Incubate microtitre plate well with antigen;Incubate microtitre plate well with antigen; Wash off unbound antigen;Wash off unbound antigen; Incubate with antibody;Incubate with antibody; Wash off unbound antibody;Wash off unbound antibody; Incubate with labelled anti-immunoglobulin;Incubate with labelled anti-immunoglobulin; Wash off unbound labelled antibody;Wash off unbound labelled antibody; Count/incubate with enzyme substrate solution.Count/incubate with enzyme substrate solution.

Page 6: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

Kids of Testing of SARS virusKids of Testing of SARS virus

The recombinational protein of SARS virusThe recombinational protein of SARS virus

Human serum(containing the antibody of SARS vHuman serum(containing the antibody of SARS virus?) (different dilution of serum)irus?) (different dilution of serum)

Polyclonal antibody against HumanIgG-HRP (horPolyclonal antibody against HumanIgG-HRP (horseradish peroxidase)seradish peroxidase)

Substrate solution, OD490nm.Substrate solution, OD490nm.

Page 7: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

Kids of Testing of HBsAgKids of Testing of HBsAg

Monoclonal antibody against HBsAg(anti-Monoclonal antibody against HBsAg(anti-HBsAg antibody)HBsAg antibody)

Human serum(containing HBsAg?)Human serum(containing HBsAg?) Polyclonal antibody against HBsAg-HRPPolyclonal antibody against HBsAg-HRP

(horseradish peroxidase)(anti-HBsAg-HR(horseradish peroxidase)(anti-HBsAg-HRP)P)

Substrate solution, OD450nm.Substrate solution, OD450nm.

Page 8: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

Our Test of ELISAOur Test of ELISA

Antigen: Human IgG (5Antigen: Human IgG (5g/well). g/well).

First antibody: Rabbit anti-human IgG antiFirst antibody: Rabbit anti-human IgG antiserum (serum dilution).serum (serum dilution).

Secondary antibody:Goat anti-rabbit IgG-Secondary antibody:Goat anti-rabbit IgG-HRP (1:20,000).HRP (1:20,000).

Page 9: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

IMMUNOLOGICAL TECHNIQUESIMMUNOLOGICAL TECHNIQUES

1. For recognizing and quantifying antigens in ti1. For recognizing and quantifying antigens in tissues or fluids many immunological techniques ssues or fluids many immunological techniques utilize the exquisite specificity of the antigen-antutilize the exquisite specificity of the antigen-antibody bond.ibody bond.

2. Cell populations can be identified and charact2. Cell populations can be identified and characterized by their surface markers, using the technierized by their surface markers, using the techniques of immunofluorescence or immunohistochques of immunofluorescence or immunohistochemistryemistry

Page 10: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

IMMUNOLOGICAL TECHNIQUESIMMUNOLOGICAL TECHNIQUES

3. Cell populations can be isolated according to 3. Cell populations can be isolated according to their surface markers, by techniques which inclutheir surface markers, by techniques which include fluorescence-activated cell sorting (FACS), pade fluorescence-activated cell sorting (FACS), panning and density-dependent centrifugations.nning and density-dependent centrifugations.

4. The principle assays for lymphocyte function 4. The principle assays for lymphocyte function are by antibody or cytokine production, by prolifare by antibody or cytokine production, by proliferation in response to antigen, or by cytotoxicity.eration in response to antigen, or by cytotoxicity.

Page 11: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

ANTIGEN-ANTIBODY INTERACTIONANTIGEN-ANTIBODY INTERACTION

1. Precipitation reactions1. Precipitation reactions 2.  Haemagglutination and complement fixation2.  Haemagglutination and complement fixation 3.  Direct and indirect 3.  Direct and indirect immunofluorescenceimmunofluorescence 4.  Immunoassay4.  Immunoassay 5.  Immunoblotting5.  Immunoblotting and immunoprecipitation and immunoprecipitation

Page 12: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

Immunochemical techniquesImmunochemical techniques

The study of antibodies(and some otThe study of antibodies(and some other immunologically important molecher immunologically important molecules such as complement componentules such as complement components) is known as immunochemistry.Sucs) is known as immunochemistry.Such methods are known as immunocheh methods are known as immunochemical techniques.mical techniques.

Page 13: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

AntibodiesAntibodies

Antibodies are a group of globular proteins kAntibodies are a group of globular proteins known as immunoglobulins.nown as immunoglobulins.

The basic four-chain model for immunogloThe basic four-chain model for immunoglobulin molecules is based on two distinct types of bulin molecules is based on two distinct types of polypeptide chain. The smaller(light) chain has a polypeptide chain. The smaller(light) chain has a molecular weight of 25,000 and is common to all molecular weight of 25,000 and is common to all classes, whereas the larger(heavy) chain has a mclasses, whereas the larger(heavy) chain has a molecular weight of 50,000-77,000 and is structuralolecular weight of 50,000-77,000 and is structurally distinct for each class or subclass. The polypely distinct for each class or subclass. The polypeptide chains are linked together by covalent and ptide chains are linked together by covalent and non-covalent forcesnon-covalent forces.

Page 14: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

AntibodiesAntibodies

Fab.(Fragment-antigen binding): Fab.(Fragment-antigen binding): The part of an antibody molecule which contaiThe part of an antibody molecule which contai

ns the antigen-binding site, consisting of a light chns the antigen-binding site, consisting of a light chain and part of the heavy chain; it is produced by eain and part of the heavy chain; it is produced by enzymatic digestion. (papain, pepsin)nzymatic digestion. (papain, pepsin)

Fc.(Fragment crystallisable): Fc.(Fragment crystallisable): The portion of an antibody that is responsiblThe portion of an antibody that is responsibl

e for binding to antibody receptors on cells and the for binding to antibody receptors on cells and the C1q component of complement.e C1q component of complement.

Page 15: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

ComplementComplement

The complement system is part of the innate imThe complement system is part of the innate immune system. There are two main pathways for cmune system. There are two main pathways for complement activation, the classical and alternatiomplement activation, the classical and alternative pathway. The classical pathway links the adapve pathway. The classical pathway links the adaptive immune system, antibody, to the innate immtive immune system, antibody, to the innate immune system, complement, by the binding to immuune system, complement, by the binding to immune complexes of C1q. Alternative pathway activatne complexes of C1q. Alternative pathway activation is initiated when C3, activated by the ‘tick-ovion is initiated when C3, activated by the ‘tick-over’pathway, deposits on foreign surfaces, lacking er’pathway, deposits on foreign surfaces, lacking regulatory molecules.regulatory molecules.

Page 16: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

ComplementComplement

Further reading:Further reading:

Porter RR, Reid, KBM. The biochemistry of complement. Porter RR, Reid, KBM. The biochemistry of complement. NNature ature 1978; 275: 699-704.1978; 275: 699-704.

Reid KBM, Porter RR. The proteolytic activation systems of Reid KBM, Porter RR. The proteolytic activation systems of complement. complement. Annu Rev BiochemAnnu Rev Biochem 1981; 50: 433-464. 1981; 50: 433-464.

Reid KBM, Day AJ. Structure-function relationships of the Reid KBM, Day AJ. Structure-function relationships of the complement components. complement components. Immunol TodayImmunol Today, 1989; 10: 177-1, 1989; 10: 177-180.80.

Campbell RD, Law SKA, Reid KBM, Sim RB. Structure, orgCampbell RD, Law SKA, Reid KBM, Sim RB. Structure, organisation, and regulation of the complement genes. anisation, and regulation of the complement genes. Annu Annu Rev ImmunolRev Immunol 1988; 6: 161-195 1988; 6: 161-195.

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Polyclonal and monoclonal antibodiesPolyclonal and monoclonal antibodies

Usually, many different antibodies, recognising several diffUsually, many different antibodies, recognising several different epitopes on each antigen are present. Such a responerent epitopes on each antigen are present. Such a response is described as polyclonal, as antibody is derived from se is described as polyclonal, as antibody is derived from more than one clone of B lymphocytes and shows heterogmore than one clone of B lymphocytes and shows heterogeneity in the amino acid sequences of the antigen-binding ieneity in the amino acid sequences of the antigen-binding immunoglobulins present.mmunoglobulins present.

However, more recently, methods have been develHowever, more recently, methods have been developed for deriving monoclonal antibodies, which are deriveoped for deriving monoclonal antibodies, which are derived from a single B cell clone and show identical amino acid d from a single B cell clone and show identical amino acid sequence. Monoclonal antibody preparations show homogsequence. Monoclonal antibody preparations show homogeneous characteristics(including specificity and avidity for eneous characteristics(including specificity and avidity for antigen, i.e. they recognise a single epitope).antigen, i.e. they recognise a single epitope).

Page 18: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

Production of antibodiesProduction of antibodies

Production of polyclonal antibodies(antisera)Production of polyclonal antibodies(antisera)

In general, most immunochemical methods In general, most immunochemical methods are devised for use with antibodies that recognise proteins are devised for use with antibodies that recognise proteins and peptides.and peptides.

In some cases, particular parts of the antigeIn some cases, particular parts of the antigen produce very potent immune responses and such epitopn produce very potent immune responses and such epitopes are known as immunodominant. Immunogenicity tends tes are known as immunodominant. Immunogenicity tends to increase with size; proteins with a molecular weight > 10,o increase with size; proteins with a molecular weight > 10,000 are usually immunogenic as long as they are recognis000 are usually immunogenic as long as they are recognised as foreign in responding animals.ed as foreign in responding animals.

Page 19: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

Production of antibodiesProduction of antibodies

Production of polyclonal antibodies(antisera):Production of polyclonal antibodies(antisera):

For production of potent antibodies that perform For production of potent antibodies that perform well in immunochemical techniques, it is usually well in immunochemical techniques, it is usually necessary to use an adjuvant as part of the immunecessary to use an adjuvant as part of the immunogen. Such substances potentiate the immune rnogen. Such substances potentiate the immune response by forming a slow-release depot of antigesponse by forming a slow-release depot of antigen, by stimulating T cell help or by aiding antigen en, by stimulating T cell help or by aiding antigen presentation.presentation.

Page 20: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

Antibodies and seraAntibodies and sera

Rabbit polyclonals: rabbit antisera to human Factor H, boviRabbit polyclonals: rabbit antisera to human Factor H, bovine Factor H and human b21,and to the fusion protein GST-ne Factor H and human b21,and to the fusion protein GST-5th domain of b21 were available in our laboratory.5th domain of b21 were available in our laboratory.

Affinity purfied rabbit IgG anti-human b21 was madAffinity purfied rabbit IgG anti-human b21 was made by passing 2ml of rabbit antiserum on a column(2ml volue by passing 2ml of rabbit antiserum on a column(2ml volume) of b21-Sepharose(1mg b21 covalently attached per ml me) of b21-Sepharose(1mg b21 covalently attached per ml of Sepharose). Bound antibodies were eluted with 3M MgClof Sepharose). Bound antibodies were eluted with 3M MgCl2, pH 6.8 and dialysed into water, then into PBS-0.5mM ED2, pH 6.8 and dialysed into water, then into PBS-0.5mM EDTA.TA.

From Bing Bin’s thesis(1999From Bing Bin’s thesis(1999

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Production of monoclonal antibodiesProduction of monoclonal antibodies

Monoclonal antibodies can be especially useful for immunMonoclonal antibodies can be especially useful for immunochemical methods. Such antibodies are secreted by cloneochemical methods. Such antibodies are secreted by cloned, i.e. monoclonal cells, mature, antibody-secreting lymphod, i.e. monoclonal cells, mature, antibody-secreting lymphocytes from immunised animals can be cloned, but these sucytes from immunised animals can be cloned, but these survive for only a very short period in culture, and therefore drvive for only a very short period in culture, and therefore do not provide useful amounts of antibody. However, proceo not provide useful amounts of antibody. However, procedures have been developed to allow production of large qudures have been developed to allow production of large quantities of monoclonal antibodies by producing continouslantities of monoclonal antibodies by producing continously growing(immortal) cell lines that secrete antibody efficiey growing(immortal) cell lines that secrete antibody efficiently. These involve generation of hybrid cells, transformatintly. These involve generation of hybrid cells, transformation of lymphocytes with a virus, or recombinant DNA proceon of lymphocytes with a virus, or recombinant DNA procedures.dures.

Page 22: Enzyme-linked immunosorbent assay (ELISA) ELISA ELISA

Production of monoclonal antibodiesProduction of monoclonal antibodies

Animals(usually mice or rats) are immunized with antigen. Animals(usually mice or rats) are immunized with antigen. Once the animals are making a good antibody response thOnce the animals are making a good antibody response their spleens are removed and a cell suspension is prepared. eir spleens are removed and a cell suspension is prepared. These cells are fused with a myeloma cell line by the additiThese cells are fused with a myeloma cell line by the addition of polyethylene glycol(PEG) which promotes membrane on of polyethylene glycol(PEG) which promotes membrane fusion. Only a small proportion of the cell fuse successfullfusion. Only a small proportion of the cell fuse successfully. The fusion mixture is then set up in culture with medium y. The fusion mixture is then set up in culture with medium containing “HAT”. HAT is a mixture of hypoxanthine, amincontaining “HAT”. HAT is a mixture of hypoxanthine, aminopterin and thymidine. Aminopterin is powerful toxin whicopterin and thymidine. Aminopterin is powerful toxin which blocks a metabolic pathway. This pathway can be bypassh blocks a metabolic pathway. This pathway can be bypassed if the cell is provided with the intermediate metabolites ed if the cell is provided with the intermediate metabolites hypoxanthine and thymidine.hypoxanthine and thymidine.

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THANKS