Transcript Enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent
assay (ELISA)
ELISA
Immunoassay
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The technique of immunoassay using
labelled reagents for detecting antigens and
antibodies are exquisitely sensitive and
extremely economical in the
reagents(immunoassay for antibody).
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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.
Enzyme-linked immunosorbent
assay(ELISA)
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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.
A typical titration curve
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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.
Procedure of ELISA
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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.
Kids of Testing of SARS virus
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The recombinational protein of SARS virus
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Human serum(containing the antibody of SARS
virus?) (different dilution of serum)
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Polyclonal antibody against HumanIgG-HRP
(horseradish peroxidase)
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Substrate solution, OD490nm.
Kids of Testing of HBsAg
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Monoclonal antibody against HBsAg(antiHBsAg antibody)
Human serum(containing HBsAg?)
Polyclonal antibody against HBsAgHRP(horseradish peroxidase)(antiHBsAg-HRP)
Substrate solution, OD450nm.
Our Test of ELISA
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Antigen: Human IgG (5mg/well).
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First antibody: Rabbit anti-human IgG
antiserum (serum dilution).
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Secondary antibody:Goat anti-rabbit IgGHRP (1:20,000).
IMMUNOLOGICAL TECHNIQUES
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1. For recognizing and quantifying antigens in
tissues or fluids many immunological
techniques utilize the exquisite specificity of the
antigen-antibody bond.
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2. Cell populations can be identified and
characterized by their surface markers, using
the techniques of immunofluorescence or
immunohistochemistry
IMMUNOLOGICAL TECHNIQUES
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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.
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4. The principle assays for lymphocyte function
are by antibody or cytokine production, by
proliferation in response to antigen, or by
cytotoxicity.
ANTIGEN-ANTIBODY INTERACTION
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Precipitation reactions
Haemagglutination and complement fixation
Direct and indirect immunofluorescence
Immunoassay
Immunoblotting and immunoprecipitation
Immunochemical techniques
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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.
Antibodies
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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.
Antibodies
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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.
Complement
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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.
Complement
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Further reading:
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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: 177180.
Campbell RD, Law SKA, Reid KBM, Sim RB. Structure,
organisation, and regulation of the complement genes.
Annu Rev Immunol 1988; 6: 161-195.
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Polyclonal and monoclonal antibodies
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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 antigenbinding 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).
Production of antibodies
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Production of polyclonal antibodies(antisera)
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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.
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Production of antibodies
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Production of polyclonal antibodies(antisera):
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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.
Antibodies and sera
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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.
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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 PBS0.5mM EDTA.
From Bing Bin’s thesis(1999
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Production of monoclonal antibodies
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Monoclonal antibodies can be especially useful for
immunochemical methods. Such antibodies are secreted
by cloned, i.e. monoclonal cells, mature, antibodysecreting 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.
Production of monoclonal antibodies
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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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