CHAPTER 2 Basic Principles of ELISA

CHAPTER2 Basic Principles of ELISA Reaction Schemes This chapter introduces the basic test formats for the performance of solid-phase heterogeneous ELISA This form of ELISA has been so useful because: Substances,e.g., antibodies or antigens, may be passively adsorbed to solid surfaces, such as plastics Microtiter plates in a 96-well format are commercially available for use in ELISA, along with suitable equipment for easy manipulation and dispensing of reagents This allows use of small volumes and gives the ELISA the potential of handling high numbers of samples rapidly Smce one of the reactants in the ELISA is attached to a solid-phase, the separation of bound and free reagents is easily made by simple washing procedures The result of an ELISA is a color reaction that can be observed by eye and read rapidly using specially designed multichannel spectrophotometers This allows data to be stored and analyzed statistically The use of passive adsorption also allows a great deal of flexibility in assay design This chapter describes some basic test schemes These assays are dealt with in later chapters at the practical level Diagrams of the schemes are also included to reinforce the principles ELISAs may be classified under four headings: direct, indirect, sandwich, and competition In the following, the various ELISAs are represented using letters, as well as diagrams Ag = Antigen Ab = Antibody directed against antigen AB = Antibody from another animal species as compared to Ab Anti-Ab = Species-specific antiserum, e.g., if Ab was raised in a mouse, antiAb is anttmouse serum *E = Enzyme attached to a particular antibody (Anti-Ab*E = antispecies antibody linked to enzyme, e.g., antimouse) 35 Basic Principles of ELISA I = Solid-phase to which reagent is attached passively S = Substrate addition and color development + = Addition of reagents and incubation Read = Measurement of color using spectrophotometer W = Separation of bound and free reagents by washing Direct ELISA 2.1 Direct-Labeled I-Ag + Ab*E W + Antibody S + Read W Stage (i): Passive adsorption of antigen to plate by incubation in defined buffer Stage (ii): Wash Stage (iii): Addition and incubation of enzyme-labeled antibody Stage (iv): Wash Stage (v): Addition and incubation of color development system Stage (vi): Read Antigen attached to the solid phase is reacted directly with an enzymelabeled antiserum This has the disadvantage that sera raised against different antigens all have to be labeled Thus, this is a poor assay if used to detect antigen from “crude” samples (containing a high concentration of contaminating substances), since low levels of antigen attach to wells owing to competition for plastic sites by such contaminants This is a typical assay for use in the estimation of the titer of enzyme-labeled antispecies conjugates This is illustrated in Fig Thus, the Ag in this case might be the IgG from an animal of the appropriate species against which the Ab*E is made The interaction in this assay is made use of mainly in assays involving monoclonal antibodies (MAbs), which are directly labeled with enzyme In this case, a very important epitope might be detected by the MAb, so that the labeling of a single antibody might be justified and form the basis of other assays as described below 2.2 Direct-Labeled I-Ab + W Ag*E + Antigen S + Read W Stage (i): Passive adsorption of antibody to plate Stage (ii): Wash Stage(iii): Addition and incubation of enzyme-labeledantigen Direct ELISA 37 Fig Direct ELISA Antigen is attached to the solid phase After washing, enzyme-labeled antibodies are added After an incubation period and washing, the substrate system is added and the color allowed to develop Stage (iv): Wash Stage (v): Addition and incubation of color development system Stage (vi): Read Antibodies are adsorbed to the solid-phase usually after crude fractionation to obtain the immunoglobulin fraction (IgG) Enzyme-labeled antigens are then added and react with antibodies of suitable specificity This has a poor applicability to diagnostic problems Antigens are rarely labeled However, a modification of this assay is used to measure progesterone, using competitive conditions The scheme is illustrated in Fig 38 Basic Principles of ELISA Fig Direct-labeled antigen Antigen is labeled with enzyme and can be captured with antibodies attached to the solid phase This system can form the basis of other assays,e.g., competitive techniques where either antibody or antigen can be incubated with the labeled antigen and the degree of inhibition of binding of the labeled antigen with the solid phase measured This form of assay has been used in the estimation of hormone concentrations and is analogous to many radioimmunoassay methods Indirect I-Ag ELISA + Ab + Anti-Ab*E + S + Read W W W Stage (i): Passive adsorption of antigen Stage (ii): Wash Stage (iii): Addition of antibody directed against Ag Stage (iv): Wash Stage (v): Addition of enzyme-labeled antispecies antibody Stage (vi): Wash Sandwich ELISA 39 Stage(vii): Addition of color developmentsystem Stage(viii): Read This is extensively used for the detection and/or titration of specific antibodies from serum samples The specificity of the assay is directed by the antigen on the solid-phase, which, may be highly purified and characterized or relatively crude and noncharacterized After addition and incubation of the antigen, the wells are washed to get rid of unbound antigen Serum containing antibodies against this antigen can then be added and diluted in a buffer that prevents the nonspecific adsorption of protein for any free sites on the solid-phase not occupied by the antigen (blocking buffer) Sera, may be added as a single dilution (common in epidemiological testing of large number of sera)or as a dilution range After incubation, the wells are washed to get rid of unbound antibodies Bound antibody is then detected after incubation, with a single dilution of antispecies antibody conjugated to an enzyme This is diluted in blocking buffer The amount of specific antibody binding to the antigen is quantified after addition of color development reagents (enzyme substrate or substrate/ dye combination) Such assaysoffer immediate advantagesover the direct tests since only a single antispecies enzyme conjugate is needed to titrate antisera from many animals of a single species The scheme is shown diagrammatically in Fig Sandwich ELISA 4.1 Direct Sandwich I-Ab + W Ag W + AB*E + S + Read W Stage (i): Passive adsorption of antibody Stage (ii): Wash Stage (iii): Addition of antigen Stage (iv): Wash Stage (v): Addition of enzyme-labeled antIbody* against antigen Stage (vi): Wash Stage (vii): Addition of color development system Stage (viii): Read Enzyme-labeled antibody can be produced in the same animal (can be the same serum) that produced the passively adsorbed antibody, or from a different species immunized with the same antigen that is captured 40 Basic Principles of ELISA Fig Indirect ELISA Antibodies from a particular speciesreact with antigen attached to the solid phase Any bound antibodies are detected by the addition of an antispecies antiserum labeled with enzyme This is a widely used system in diagnosis This is similar to Section 2.2., except that antibody is attached to the solid-phase, usually as an IgG fraction of the whole serum A constant dilution of antibody is attached to the solid phase, and after incubation, unadsorped antibody is washed away Antigen at a single dilution or as a dilution range is then added, in a buffer that prevents nonspecific bind- Sandwich ELBA ing of the serum proteins to any available plastic sites After incubation, unbound antigen is washed away Bound antigen is then detected by the addition of enzyme-labeled antibody specific for the “trapped” or “captured” antigen This antibody can be: The same as that used on the solid phase Produced in the same species as the trapping serum Produced in a different speciesto that in which the trapping antibody was made After incubation and washing away of unreacted conjugate, the color detection system is added and color is measured Note that certain anti- gen preparations cannot be directly attached to microplates, since they are at low concentration and/or they are contained in high concentrations of contaminating protein The antibody attached to the plastic should bind antigen specifically, so that selection and concentration of the antigen takes place in the sandwich conditions Such assayshave been described as capture or trapping assays,referring to the property of the bound antibody to bind the antigen to the plastic surface Note also that antigens must contain at least two antigenic sites, capable of binding to antibody, since at least two antibodies act in the sandwich This is important where lowerTmo1 wt antigens are being used of limited antigenic potential, or where antigenic sites are concentrated on one surface Some difficulties can be encountered using the same MAb in sandwich assays, since the capture step may bind to the only epitope expressed on a “small” antigen As in the direct ELISA, this test has the disadvantage that all the detecting antisera have to be conjugated Figure shows the scheme diagrammatically 4.2 Indirect I-Ab + w Ag + w AB Sandwich + w Anti- AB *E + W S + Read Stage (i): Passive adsorption of Ab Stage (ii): Wash Stage (iii): Addition of antigen Stage (iv): Wash Stage (v) Addition of antibody from different species vs antigen Stage (vi): Wash Stage (vii): Addition of enzyme-labeled antispecies (directed against AB) 42 Basic Principles of ELISA Fig Sandwich ELISA-direct This systemexploits the antibodies attachedto the solid phase to capture antigen This is then detectedusing an enzyme-labeled serum specific for the antigen The detecting antibody is labeled with enzyme The capture antibody and the detecting antibody can be the same serum or from different sources The antigen must have at least two different antigenic sites Stage (viii): Wash Stage (ix): Addition of color development system Stage (x): Read Figure shows the scheme diagrammatically This is the same as Section l., except that the second antibody is produced in a different species from the trapping antibody Thus, the second antibody can be detected using a species-specific antiserum con- Competition ELISA 43 Fig Sandwich ELBA-indirect The detecting-antibody is from adifferent species than the capture antibody The antispecies enzyme-labeled antibody binds to the detecting antibody specifically and not to the capture antibody jugate that does not react with the antibody on the plastic The advantage is that many second antibodies, may be titrated with a single conjugate Competition ELISA Competition assays imply that two reactants are trying to bind to a third Proper competition assays involve the simultaneous addition of the two competitors 44 Basic Principles of ELISA Competition A Incubate Inhibition/Blocking B Wash + Incubate Incubate C Incubate Incubate No wash Fig Comparison of competition and inhibition ELISA The test scheme involves the reaction of two antibodies with an antigen attached to the solid phase Where one of the antibodies is incubated first, the assaysare called blocking or inhibition assays.Competition implies simultaneous addition of reagents Inhibition or blocking assays are similar, except that one of the reagents being examined for “competing” ability of a system is added and incubated before the second “competitor” is added This can be illustrated simply as shown in Fig 5.1 Direct I-Ag + Ab*E + W W +AB Antibody S + Read Competition Basic Principles 48 5.3 Indirect I-Ag + Ab + Antibody Competition Anti-Ab*E w +AB w of ELISA + S + Read w Stage (i): Passive adsorption of antigen Stage (ii): Wash Stage (iii): Addition of test antibody AB at various dilutions Stage (iv)*: (Optional washing step after incubation with AB alone) Stage (v): Addition of antibody (pretitrated): standard serum Stage (vi): Wash Stage (vii): Addition of antispectesconjugate against standard antiserum (Ab) Stage (viii): Wash Stage (ix): Addition of color development system Stage (x): Read This is essentially the same as the indirect ELISA, except that a competing antibody is added to the solid-phase antigen either before or simultaneously with pretitrated specific antibody The level of antibody used is usually about 70% maximal reactivity (solid-phase antigen excess) The competing antibody must be from a different species from the pretitrated antibody, since the antispecies conjugate must not react with both If the competing antibody is able to bind to the antigen, then it prevents the pretitrated antibody reacting, and this is observed as a decrease in the expected color as compared to controls without competitor The scheme is illustrated in Fig 5.4 Indirect I-Ag + W Ab + W Antigen Competition Anti-Ab*E + S + Read W +Ai3 Stage (i): Passive adsorption of antigen Stage (ii): Wash Stage (iii): Simultaneous incubation of free antigen (test sample): with antibody directed against antigen on plastic at pretitrated dilution Stage (iv): Wash Stage (v): Addition of enzyme-labeled antibody against Ab Stage (vi): Wash Stage (vii): Addition of substrate Stage (viii): Read Competition ELBA 49 Pre-titration Competition-addition of indirect system of samples containing antibodies? Serum contains antibodies which bind to antigen &, Serum contains NO antibodies which bind to antigen These block pre-titrated antibodies binding On addition of anti-species enzyme labelled conjugate A Conjugate does not bind No color COMPETITION Conjugate binds Color NO COMPETITION Fig Competition ELISA-indirect antibody A pretitrated system for antibody binding to antigen is challenged by the addition of another serum (test) sample If antibodies bind to the sites in common with the pretitrated antibodies, they block (if added before pretitrated antibody) or compete with (if added simultaneously) this reaction Since an antispecies conjugate is used, the competing sample serum cannot be from the same species This is an indirect ELISA where antibody is pretitrated against the solidphase bound antigen by the use of antispecies conjugate, which is challenged by the addition of dilution ranges of antigen in the liquid phase Again, the amount Basic Principles 50 Petitration Addition of same or similar to that on solid phase of indirect antigen of ELISA system Addition of antigen on solid phase different to that Wash and add conjugate Add substrate No color-100% competition Fig 10 Competition ELBA-indirect antigen The pretitrated indirect ELISA is competed for by antigen If the antigen shares antigenic determinants with that of the solid-phase antigen, it binds to the pretitrated antibodies preventing them from reacting with the solid-phase antigen If there is no similarity, the antibodies are not bound and can react with the solid-phase antigen Addition of the antispecies enzyme conjugate quantifies the bound antibodies of pretitrated antibody should be about 70% of the maximal reaction (solidphase antigen excess) Competition is reflected by a decrease in the expected color obtained without competitor The scheme is illustrated in Fig 10 Choice of Assays The most difficult question to answer when initiating the use of ELISAs is which system is most appropriate This section will attempt to 51 Choice of Assays C D Fig 11 Basic ELISA methods (A) Direct, (B) Indirect, (C) Sandwich (direct), (D) Sandwich (indirect) investigate the relationships between the various systems to aid in assessing their suitability Questions that must be addressed are: What is the purpose of the assay? What reagents I have? What I know about the reagents? Is the test to be developed for a research purpose to be used by me only, or for applied use by other workers? Is the test to be used in other laboratories? Is a kit required? These questions have a direct affect on the three phases that might be put forward as a general rule for the development of any assay, i.e.: Feasibility-proof that a test system(s) can work Validation-showing that test(s) is “stable” and that it is evaluated over time and under different conditions Standardization-quality control, establishment that the test is precise and can be used by different workers in different laboratories Figures 11-15 summarize the exploitation of ELISA methods, highlighting the relationship of assays to the relative purity and concentration of reactants, and indicating the use of direct and indirect methods for competition assays These will be relevant when examining the possibilities outlined in Section 6.1 Basic Principles 52 of ELISA *ENZ t A +ENZ t s- B Fig 12 Competition for direct ELISA 6.1 Phase in Developing an ELISA Feasibility Phase involves the trial of various systems of ELISA with existing and newly prepared reagents to be able to obtain the desired aim This phase includes identification of needs based on preliminary experiments and a good knowledge of the biology of the system The latter point may become more important when attempts at using ELISA fail because of lack of knowledge Thus, as an example, we may wish to estimate the antibody titer in cattle sera against a particular antigen The possibility of performing all the ELBA systems and obtaining the most appropriate system will depend on the availability of various reagents and their specificities Choice of Assays 53 INDIRECT A Fig 13 Competition for indirect ELBA 6.1.1 Assessing What Is Available As examples: We may have only the relevant antigen Figure 16 shows different types of antigens of ,varying complexity Thus, we may know a great deal about the antigen or very little We may have a high concentration of a defined protein/polypeptide/peptide of known amino acid sequence or have a thick soup of mixed proteins containing the antigen at a low concentration contaminated with “host cell” proteins We may have an antiserum against the antigen This could be against purified antigen or against the crude soup The antibody may have been raised in a given species, e.g., rabbit We may have an IgG fraction of the antiserum (or 54 Basic Principles of ELISA AG COMPETITION AB COMPETITION + +# + YI I, d ALTRRNATNE Yf Y llbii?S +o FOR ADDI’KION Fig 14 Competition for sandwich ELISA-same detection OF COMPETITORS antibody for capture and could easily make one) We may have field sera against the antigen (bovine sera) We may have an MAb We may have antisera from different species, e.g., rabbit and guinea pig sera ELISAs for similar systems may have been developed and can be found in the literature We will require an enzymic reaction in the assay Thus we will need an antispecies conjugate (commercial, most probably) or will have to label an antigen-specific serum with enzyme (facilities to this?) We have to decide which commercial conjugate to buy This will depend on the desired specificity of the conjugate (antiwhole molecule IgG, anti-Hchain IgG, anti-H chain IgM, and so forth) The choice is somewhat determined by the aims of the assay and its design Thus, we may wish to determine the IgM response of cattle to our antigen, which will require an anti-IgM (specific) somewhere in the ELISA protocol Obviously the basic needs for performing the ELISA must be addressed in terms of plates, pipets, buffers, reader, and so on 55 Choice of Assays I &3ENZ +s-+ *ENZ AG COMPETITION I,2 & ALTERNATIVE TIIUES FORADDITION OF COMPET1mR.S J AB COMPETITION Fig 15 Competition for sandwich ELBA-different and detection antibody for capture 6.1.2 Examination of Possible Assays with Available Materials Obviously, the reagents available must be examined first as previously stated This section will deal with some extremes in order to illustrate the relationship of the assays available and their particular advantages As for Section 6.1.1.) perhaps we have to examine the level of antibodies in bovine serum Some scenarios are described with different available reagents These will probably cover most of those that are met in practice This assumes that there are sera to test from infected and noninfected animals Further subtleties can be examined by defining the specificities of the conjugates (anti-IgG, IgM, or whether they are H-chain-specific) The increase in choice of reagents and the possibilities for performing different ELBA configurations follow: a Crude antigen (multiple antigenic sites) b Antibody raised against crude antigen in rabbits c Anticow conjugate Basic Principles 56 Large multivalent antigen, sequential and conformational Smaller multivalent of ELISA epitopes antigen Smaller antigen, univalent Polypeptide, linear sequential epitopes Polypeptide, sequential and conformational epitope Peptide Peptide, linked to carrier protein with conformational epitope Fig 16 Different forms of antigens for use in ELBA and for antiserum production These antigens could be contaminated with “host cell” proteins d Postinfected and d (uninfected) cow sera a Purified antigen (small amount, e.g., 100 pg) b Crude antigen (large amount) c Antibody raised in rabbits against pure antigen d Antirabbit conjugate e Anticow conjugate f Postinfected and d (uninfected) cow sera a Crude antigen (as in 1.) b Antibody against pure antigen (rabbit) c Antibody against pure antigen (guinea pig) d Antiguinea pig conjugate e Postinfected and d (uninfected) cow sera f Anticow conjugate g Antirabbit conjugate Choice of Assays 57 6.1.2.1 SITUATION Here the use of crude antigen directly on an ELISA may well be unsuccessful, since it may be at a low concentration compared to other proteins and thus only attach at a low concentration This does not allow the ELISA approaches as shown in Fig lA,B and, thus, competitive methods based on these, as in Figs 12B and 13B Since a rabbit serum against the antigen is available, this may be used as a capture serum (or a capture IgG preparation) coated on wells to capture the crude antigen to give a higher concentration of antigen to allow the binding of antibody as in Fig 1lC,D This also allows competitive techniques as shown in Fig 15B The bound antibody would be from cows and would be detected using the antibovine conjugate There may be problems, since the crude antigen was used to raise the rabbit serum Thus, antibodies against the contaminating proteins may be produced in the rabbit The cow sera being tested may react with such captured contaminants However, where the antigen is an infectious agent, antibodies against the contaminating proteins may not be produced, thus eliminating the problem Where the antigen is used as a vaccine, whereby relatively crude preparations similar to the crude antigen are used to formulate the vaccine, then this problem will be present Attempts can be made to make the rabbit serum specific for the desired antigenic target Solid-phase immunosorbents involving the contaminating crude elements (minus the desired antigen) can be used to remove the anticrude antibodies from the rabbit serum, which could then be titrated as a capture serum An example can be taken from the titration of foot-and-mouth disease virus (FMDV) antibodies The virus is grown in tissue culture containing bovine serum Even when virus is purified from such a preparation, minute amounts of bovine serum contaminate the virus When this “purified” virus is injected into laboratory animals as an inactivated preparation, there is a large amount of antibovine antibodies, as well as antivirus antibodiesproduced.This serum cannot be used in a capture system for specifically detecting virus grown as a tissue-culture sample (containing bovine serum), since it also capturesbovine serum The capture serum is also unsuitable for capturing relatively pure virus for the titration of bovine antibodies from bovine serum samples, since the capture antibodies react strongly with the detecting cow serum Thus, the capture serum has to be adsorbed with solid-phase immunosorbents, e.g., those produced through the attachment of bovine serum to agarosebeads, Basic Principles 58 of ELISA Solid Phase Coating with Guinea pig IgG Wash Addition of anti-guinea pig IgG conjugate Wash Addition of substrate/chromogen Scheme Once the specificity of the capture serum is established, the optimization of the crude antigen concentration can be made using a known or several known positive cow sera using full dilution ranges Inclusion of dilution ranges of negative sera allows an assessment of the difference between negative and positive sera at different dilutions of serum The diagram below illustrates the use of the reagents to set up a sandwich ELISA The assay is made possible through the specific capture of enough antigen by the solid-phase rabbit serum as in Fig 11D I-Ab + Ag + AB + Anti-AB*Enz Rabbit Crude Cow Anticow + Substrate + Read 6.1.2.2 SITUATION This situation is not very different from the first However, we have more reagents! We have the antigen purified and used to raise antibodies in rabbits (see Scheme 1) Thus, with due reference to the reservations Choice of Assays 59 already described in Section 6.1.2 l., we have the basis of setting up a capture ELISA, since the rabbit antibodies may capture the antigen at a high concentration from the crude antigen, which we have in a large amount The development of the capture ELISA as shown in Fig lC,D is as described above The availability of the antirabbit conjugate may allow development of competitive assays if enough specific antigen binds to plates, although this is unlikely, as indicated above The antigen and rabbit serum could be titrated in an indirect ELISA (Fig 11B) in a chessboard fashion, enabling the optimization of the antigen and serum These optimal dilutions could be used to set up competitive ELISAs, whereby cow sera would be competed for the pretitrated antigen/rabbit/antirabbit conjugate system, as in Fig 13B Again it must be emphasized that this is unlikely since the antigen is crude and some form of capture system will be needed to allow enough antigen to be presented on the wells Since this situation has some purified antigen, this could be used in the development of a similar competitive assay This will depend on the availability of this antigen, which can be determined after the initial chessboard titrations where the optimal dilution of antigen is calculated, The chief benefit of obtaining purified antigen was to obtain a specific serum in rabbits, allowing specific capture of antigen from the crude sample In many cases, there is enough antigen of sufficient purity to be used in such assays Another alternative, as shown in Fig 14B, is available if the rabbit serum can be conjugated with enzyme 6.1.2.3 SITUATION Here we have all the possibilities of the first two situations plus the production of a second species (guinea pig) serum against the purified antigen (seeScheme2) This allows the development of competitive assays as in Fig 15B using either the rabbit or guinea pig as capture serum or detector with the relevant antispecies conjugate Different species may have better properties in acting as capture reagents and also show varying specificities This can be assessed in chessboard titrations This is relevant since we require results on the detection and titration of cattle sera so that the competitive phase relies on the interruption of a pretitrated antibody as close to the reaction of cattle serum with antigen as possible The rabbit or guinea pig serum may differ in their specificities as compared to cattle sera 60 Basic Principles of ELISA Solid Phase Coating with dilution of sheep anti-Guinea pig IgG Wash Addition of guinea pig IgG Wash Addition of rabbit anti-guinea pig serum Wash Addition of sheep anti-rabbit enzyme conjugate Wash Addition of substrate/chromogen Scheme 6.1.2.4 FURTHER COMMENTS Assays shown in Fig 12B (competition for direct ELISA) are probably inappropriate owing to the possession of crude antigen (ior reasons described above) However, if it can be shown that enough antigen can attach and that cattle sera react specifically (and not through excess antibodies directed against contaminants in the crude antigen), then we can Choice of Assays 61 set up assays based on this system This requires identification of a positive cow serum and labeling of this serum with an enzyme Of more practical value could be the use of positive cow serato develop a system as shown in Fig 14B Here a positive cow serum, as identified from other tests (or samples of different positive cow sera), can be selected and labeled with enzyme The serum can then be used for both capture (particularly as an IgG fraction) and detection In this way, the competitive assay shown in Fig 14B is feasible and may have an advantage in that the reaction being competed against is homologous (cow antibody against antigen), thus avoiding any complications of the use of second species antisera produced by vaccination The system is suitable to measure the competition by other cow sera, since the detecting antibody is labeled Thus, a worker with relatively few reagents and the ability to label antibodies with an enzyme may have enough materials to develop assays In this brief description of system possibilities, we have concentrated on antibody detection It should be noted that most of these comments are relevant to antigen detection [...]... purity and concentration of reactants, and indicating the use of direct and indirect methods for competition assays These will be relevant when examining the possibilities outlined in Section 6.1 Basic Principles 52 of ELISA *ENZ t A +ENZ t s- B Fig 12 Competition for direct ELISA 6.1 Phase 1 in Developing an ELISA Feasibility Phase 1 involves the trial of various systems of ELISA with existing and... same species This is an indirect ELISA where antibody is pretitrated against the solidphase bound antigen by the use of antispecies conjugate, which is challenged by the addition of dilution ranges of antigen in the liquid phase Again, the amount Basic Principles 50 Petitration Addition of same or similar to that on solid phase of indirect antigen of ELISA system Addition of antigen on solid phase different... titration of cattle sera so that the competitive phase relies on the interruption of a pretitrated antibody as close to the reaction of cattle serum with antigen as possible The rabbit or guinea pig serum may differ in their specificities as compared to cattle sera 60 Basic Principles of ELISA Solid Phase Coating with dilution of sheep anti-Guinea pig IgG Wash Addition of guinea pig IgG Wash Addition of. .. are of increasing importance particularly where MAbs are used Note that any serum from any species can be used as competitor The scheme is illustrated in Fig 7 5 .2 Direct I-Ag Antigen + Ab*E W + S + Read W + Ag as dilution range Stage (i): Passive adsorption of antigen, Stage (ii): Wash Competition 46 Basic Principles Pre-titration I of antigen and labelled of ELISA antibodies Competition-addition of. .. reservations Choice of Assays 59 already described in Section 6.1 .2 l., we have the basis of setting up a capture ELISA, since the rabbit antibodies may capture the antigen at a high concentration from the crude antigen, which we have in a large amount The development of the capture ELISA as shown in Fig 1 lC,D is as described above The availability of the antirabbit conjugate may allow development of competitive... specificity of the conjugate (antiwhole molecule IgG, anti-Hchain IgG, anti-H chain IgM, and so forth) The choice is somewhat determined by the aims of the assay and its design Thus, we may wish to determine the IgM response of cattle to our antigen, which will require an anti-IgM (specific) somewhere in the ELISA protocol Obviously the basic needs for performing the ELISA must be addressed in terms of plates,... virus for the titration of bovine antibodies from bovine serum samples, since the capture antibodies react strongly with the detecting cow serum Thus, the capture serum has to be adsorbed with solid-phase immunosorbents, e.g., those produced through the attachment of bovine serum to agarosebeads, Basic Principles 58 of ELISA Solid Phase Coating with Guinea pig IgG 1 Wash Addition 1 of anti-guinea pig IgG... Wash Addition 1 of substrate/chromogen Scheme 1 Once the specificity of the capture serum is established, the optimization of the crude antigen concentration can be made using a known or several known positive cow sera using full dilution ranges Inclusion of dilution ranges of negative sera allows an assessment of the difference between negative and positive sera at different dilutions of serum The diagram... should be about 70% of the maximal reaction (solidphase antigen excess) Competition is reflected by a decrease in the expected color obtained without competitor The scheme is illustrated in Fig 10 6 Choice of Assays The most difficult question to answer when initiating the use of ELISAs is which system is most appropriate This section will attempt to 51 Choice of Assays C D Fig 11 Basic ELISA methods (A)... aim This phase includes identification of needs based on preliminary experiments and a good knowledge of the biology of the system The latter point may become more important when attempts at using ELISA fail because of lack of knowledge Thus, as an example, we may wish to estimate the antibody titer in cattle sera against a particular antigen The possibility of performing all the ELBA systems and obtaining