Enzyme-linked immunosorbent assay (ELISA)

1. Enzyme-linked immunosorbent assay (ELISA) ELISA

2. Immunoassay • The technique of immunoassay using labelled reagents for detecting antigens and antibodies are exquisitely sensitive and extremely economical in the reagents(immunoassay for antibody). • Solid-phase assays for antibodies employing ligands labelled with radioisotopes or enzymes(enzyme-linked immunosorbent assay; ELISA) are most widely used of all immunological assays.

3. Enzyme-linked immunosorbent assay(ELISA) • In this system, ligand is a molecule which can detect the antibody and is covalently coupled to an enzyme such as peroxidase.The amount of test antibody is measured by assessing the mount of coloured end-product by optical density scanning of the plate.

4. A typical titration curve • Antibody titres can only be detected correctly within the linear range. Typically the plateau binding is 20-100 times the background. The sensitivity of the technique is usually about 1-50ng/ml of specific antibody.

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

6. Kids of Testing of SARS virus • The recombinational protein of SARS virus • Human serum(containing the antibody of SARS virus?) (different dilution of serum) • Polyclonal antibody against HumanIgG-HRP (horseradish peroxidase) • Substrate solution, OD490nm.

7. Kids of Testing of HBsAg • Monoclonal antibody against HBsAg(anti-HBsAg antibody) • Human serum(containing HBsAg?) • Polyclonal antibody against HBsAg-HRP(horseradish peroxidase)(anti-HBsAg-HRP) • Substrate solution, OD450nm.

8. Our Test of ELISA • Antigen: Human IgG (5mg/well). • First antibody: Rabbit anti-human IgG antiserum (serum dilution). • Secondary antibody:Goat anti-rabbit IgG-HRP (1:20,000).

9. IMMUNOLOGICAL TECHNIQUES • 1. For recognizing and quantifying antigens in tissues or fluids many immunological techniques utilize the exquisite specificity of the antigen-antibody bond. • 2. Cell populations can be identified and characterized by their surface markers, using the techniques of immunofluorescence or immunohistochemistry

10. IMMUNOLOGICAL TECHNIQUES • 3. Cell populations can be isolated according to their surface markers, by techniques which include fluorescence-activated cell sorting (FACS), panning and density-dependent centrifugations. • 4. The principle assays for lymphocyte function are by antibody or cytokine production, by proliferation in response to antigen, or by cytotoxicity.

11. ANTIGEN-ANTIBODY INTERACTION • 1. Precipitation reactions • 2.  Haemagglutination and complement fixation • 3.  Direct and indirect immunofluorescence • 4.  Immunoassay • 5.  Immunoblotting and immunoprecipitation

12. Immunochemical techniques • The study of antibodies(and some other immunologically important molecules such as complement components) is known as immunochemistry.Such methods are known as immunochemical techniques.

13. Antibodies • Antibodies are a group of globular proteins known as immunoglobulins. • The basic four-chain model for immunoglobulin molecules is based on two distinct types of polypeptide chain. The smaller(light) chain has a molecular weight of 25,000 and is common to all classes, whereas the larger(heavy) chain has a molecular weight of 50,000-77,000 and is structurally distinct for each class or subclass. The polypeptide chains are linked together by covalent and non-covalent forces.

14. Antibodies • Fab.(Fragment-antigen binding): • The part of an antibody molecule which contains the antigen-binding site, consisting of a light chain and part of the heavy chain; it is produced by enzymatic digestion. (papain, pepsin) • Fc.(Fragment crystallisable): • The portion of an antibody that is responsible for binding to antibody receptors on cells and the C1q component of complement.

15. Complement • The complement system is part of the innate immune system. There are two main pathways for complement activation, the classical and alternative pathway. The classical pathway links the adaptive immune system, antibody, to the innate immune system, complement, by the binding to immune complexes of C1q. Alternative pathway activation is initiated when C3, activated by the ‘tick-over’pathway, deposits on foreign surfaces, lacking regulatory molecules.

16. Complement • Further reading: • Porter RR, Reid, KBM. The biochemistry of complement. Nature 1978; 275: 699-704. • Reid KBM, Porter RR. The proteolytic activation systems of complement. Annu Rev Biochem 1981; 50: 433-464. • Reid KBM, Day AJ. Structure-function relationships of the complement components. Immunol Today, 1989; 10: 177-180. • Campbell RD, Law SKA, Reid KBM, Sim RB. Structure, organisation, and regulation of the complement genes. Annu Rev Immunol 1988; 6: 161-195.

17. Polyclonal and monoclonal antibodies • Usually, many different antibodies, recognising several different epitopes on each antigen are present. Such a response is described as polyclonal, as antibody is derived from more than one clone of B lymphocytes and shows heterogeneity in the amino acid sequences of the antigen-binding immunoglobulins present. • However, more recently, methods have been developed for deriving monoclonal antibodies, which are derived from a single B cell clone and show identical amino acid sequence. Monoclonal antibody preparations show homogeneous characteristics(including specificity and avidity for antigen, i.e. they recognise a single epitope).

18. Production of antibodies • Production of polyclonal antibodies(antisera) • In general, most immunochemical methods are devised for use with antibodies that recognise proteins and peptides. • In some cases, particular parts of the antigen produce very potent immune responses and such epitopes are known as immunodominant. Immunogenicity tends to increase with size; proteins with a molecular weight > 10,000 are usually immunogenic as long as they are recognised as foreign in responding animals.

19. Production of antibodies • Production of polyclonal antibodies(antisera): • For production of potent antibodies that perform well in immunochemical techniques, it is usually necessary to use an adjuvant as part of the immunogen. Such substances potentiate the immune response by forming a slow-release depot of antigen, by stimulating T cell help or by aiding antigen presentation.

20. Antibodies and sera • Rabbit polyclonals: rabbit antisera to human Factor H, bovine Factor H and human b21,and to the fusion protein GST-5th domain of b21 were available in our laboratory. • Affinity purfied rabbit IgG anti-human b21 was made by passing 2ml of rabbit antiserum on a column(2ml volume) of b21-Sepharose(1mg b21 covalently attached per ml of Sepharose). Bound antibodies were eluted with 3M MgCl2, pH 6.8 and dialysed into water, then into PBS-0.5mM EDTA. • From Bing Bin’s thesis(1999

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

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

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