PART 11 Practical Exercises

PART 11 Practical Exercises The aim of Chapters 5-8 will be to illustrate the principles of ELISA fully by: Showing worked examplesof eachassay,including diagramsof platesand representationaldata from assays; Analyzing such data in terms of important rules that are learnedat each stage;and Providing full working instructronsfor workers to be ableto perform each assayso that they obtain their own data to be analyzedas describedin (1) and (2) This includes full instructionson the preparationand standardization of reagents The chapters can therefore be used in several ways Workers without accessto reagentswill obtain a working knowledge of ELISA through the examples The chapterscan also be used in training courses where reagents may be provided (as indicated in the text) The information will also be useful to workers who have already had some experience of the technique and who may have had difficulties in obtaining and analyzing data Remember that it is the application of the ELISA to specific problems, and not the methodology for its own sake, that is the most important reason the techniques should be mastered Test Schemes You may be already familiar with the concepts in ELISA, whereby an antigen binds to an antibody that can be labeled with an enzyme, or be in turn detected with a species-specific antibody (enzyme labeled) All the ELISAs are variations on this theme Inherent in the methods of ELISA described in these chapters is the fact that one of the reagents is attached to a solid-phase, making the separation of bound (reacted) and unbound (nonreacted) reagents simple by washing Before performing ELISA on disease agents, it is useful to train using reagents of defined reactivity, which are easily available and which provide security problems An ideal 115 116 Practical Exercises system is to use an imrnunoglobulin (Ig) and more particularly an immunoglobulin G (IgG) as an antigen Do not get confused here, since you have learned that the antibody population contains high levels of IgG acting as antibody In the context of learning the principles, we are using IgG as an antigenic protein, since: IgG from one animal species can be injected into another animal species so that a specific antiserum to that IgG is prepared Such antibodies can be labeled with enzyme, or detected with a second species-specific antibody labeled with enzyme Such reagents are defined, easy to standardize, stable, and available commercially The particular IgG system chosen in most of the chapters involves guinea pig, but similar tests can be performed with other species IgG using the appropriate antispecies reagents The practical elements of all the assays are very similar, i.e., reagents and equipment needed The systems described are analogous to the ones most commonly used to examine problems associated with diagnosis The schemes will be described using symbols where: I- = solid phase microtiter plate well Ag = antigen Agl, Ag2, etc = particular antigens highlighted in assay I-Ag = antigen passively adsorbed onto wells I-Ab, I-AB = particular antibodies passively coated onto wells Ab = antibody AB = antibody from a different species to Ab Abx, Aby = different antibodies identified by subscript letters Anti-Ab = antispecies specific antibody (against species in which Ab was produced) Anti-Ab*E = antispecies specific antibody labeled with enzyme W = washing step, involving separation of bound and free reagents + = addition of reagents and incubation step S = substratekhromophore addition Read = read test in spectrophotometer at 492 nm Throughout Chapters 5-8, many of the practical stages are the same The conjugates described are all made with horseradish peroxidase and the substratekhromophore is hydrogen peroxide/orthophenylamine diamine (OPD) The preparation and use of this is described in detail below Substratekhromophore: This is easiest made up from commercial tablets of OPD that are preweighed Commercial sources also supply citrate/phos- Test Schemes 117 phate buffer tablets (pH 5.0) Thus, the volume of OPD can be made as required by following the recommendations by the supplier As an example, 30 mg tablets are available that make 75 mL of chromophore solution in buffer Unused OPD solution (without added hydrogen peroxide) can be frozen at -20°C This can then be thawed and used later Close inspection should be made to ensure that the OPD is not drscolored Use complete chromophore/substrate as soon as possible Larger volumes of OPD in citrate/phosphate buffer can be made and frozen in a tightly stoppered brown bottle in small volumes The OPD solution should be made and frozen as quickly as possible Do not use solutions that show discoloration after freezing Hydrogen peroxide (HzO,) is the substrate for horseradish peroxidase enzyme This is purchased usually as 30 or 6% w/v and should be stored as recommended by the supplier The hydrogen peroxide should be kept refrigerated and not subjected to heating The addition of the hydrogen peroxide should be made immediately before the use of the OPD in the test Add PL of hydrogen peroxide (30% w/v) to every 10 mL of OPD solution (pH 5.0), or 25 pL of 6% hydrogen peroxide to every 10 mL of OPD solution Use the substrate/chromophore immediately OPD is a mutagen, so care is needed in its handling and disposal Washing solution used in washing steps: This is PBS without the addition of Tween 20 Washing requires the flooding and emptying of wells times with PBS Blocking buffer: This is PBS containing a final concentration of 1% bovine serum albumin (BSA) and 0.05% Tween 20 This should be made in small volumes as required, but can be stored at 4°C Care should be taken to avoid contaminated buffer Stopping solution: This 1M sulfuric acid in water Care should be taken in its preparation and handling Read: This implies reading plates using a multichannel spectrophotometer at the appropriate wavelength for the color developing in the ELISA In all cases for Chapters 5-8, this is 492 nm for OPD Plates should also be assessedby eye to ascertain whether the test results are as expected CHAPTER7 Use of Antibodies on Solid-Phase in Capture Use of Capture ELISA to Detect ELISA and Titrate Antigen In this exercise, the capture antibody, the detecting antibody, and the conjugate are used at optimal dilutions 1.1 Learning Principles 1, To optimize amounts of capture antibody attached to wells; and To optimize amount of detecting antibody Reaction Scheme I-AB + Ag + Ab + Anti-Ab*E + S + Read W w w W I- = Microplate AB x = Capture antibody (species X) specific for Ag Ag = Antigen Ab v = Detecting antibody (species Y) specific for Ag Anti-Abv*E = Antispecies-Y antibody linked to enzyme S = Substrate/color detection system W = Wash + = Addition and incubation of reactants Read = Read QD at 492 nm using spectrophotometer Basis of Assay Antigens may: Attach poorly to plastics Be present in low quantity, e.g., in tissure-culture fluids Be present as a low percentage of total protein in a “dirty” sample, e.g., in feces or in epithelium samples Be unavailable for purification and concentration, since they are antigenitally unstable when separated from other serum components 161 162 Use ofAntibodies on Solid Phase in Capture ELBA In these cases, the indirect assayis unsuitable for handling the antigen, since it relies on the attachment of the antigen directly to the wells The capture assay overcomes many of these problems, since the antigen is attached to the wells via specific antibodies The test relies on the availability of two antisera from different species, so that the conjugate reacting with the second (detecting) antibody does not react with the capture antibody It is also essential that the antigen has at least two antigenic sites, so that antibody may bind to allow the sandwich (the antigen being the filling) Thus, where small antigens are being used (e.g., peptides), they may not react in such assaysowing to their limited antigenic targets The test offers an advantage over the indirect assay in the quantification of antigens, since direct attachment of proteins to wells is often nonlinear, i.e., is not proportional to the amount of protein in the sample This is exaggerated if contaminating proteins are present with the antigen (e.g., serum components), since thesecompete for plastic sites in a nonlinear way Since the capture antibody is specific, it binds antigen in a proportional way over a large range of protein concentrations Thus, such assaysgive reproducible results where quantification is required (explained later more fully) The assay is really identical to the indirect assay, except that an extra step (the capture antibody) is added.Thus, we havethree parametersto optimize The captureantibody The detecting antibody The conjugateagainstthe detecting antibody Materials Capture antibody (AB,) = sheepantiguinea pig Ig (an IgG preparation [AB] mg/rnL in PBS) Antigen (Ag) = guineapig Ig at mg/mL or as preparedby worker (Ag) Detection antibody (Ab,) = rabbit antiguineapig Ig serum (Ab) Anti-AbY*E sheepantirabbit conjugate(HRPO) Microplates Multichannel pipets, single-channelpipets, 10 and 1-mL pipets 0.05M Carbonate/bicarbonatebuffer, pH 9.6 PBS containing 1%BSA, 0.05% Tween 20 Solution of OPD in citrate buffer 10 Hydrogen peroxide 30%, v/v 11 Washing solution 12 1M sulfuric acid in water 13 Papertowels Methods 14 15 16 17 163 Small-volume bottles Multichannel spectrophotometer Clock Graph paper Methods 5.1 General Notes Since you are now familiar with the indirect assay, the steps in the optimization of the capture ELISA should be straightforward The first essential is to determine the amount of capture antibody to be attached to the wells We have two situations in the laboratory depending on the availability of specific reagents We can use capture antibody as an IgG preparation or, if sufficiently high titer serum is available against the antigen, as whole serum The easiest way to avoid serum effects is to prepare the IgG Salt fractionation is usually adequateand does not affect antibody activity Care must be taken to assessthe effect of chemical preparation of IgG from monoclonal antibodies (MAbs) 5.2 Use of Ig Preparations The advantage here is that the weight of IgG can be calculated, so that a defined quantity of reagent may be added to the plate In general, a maximum amount of protein will attach to the wells, so that the IgG at “saturating” level may be added in the knowledge that a maximum possible binding of subsequently added antigen may be expected Thus, a good estimation of the activity of a capture antibody (the particular dilution/concentration to be used) can be assessed.As an example, if capture antibody is added at pg/mL in 5O-l.rLamounts, this represents the saturating amount of antibody protein that will attach to the wells The ultimate activity will depend on the concentration of the specific IgG (against the Ag) in the capture antibody and the spacing of the capture molecules Some assays perform better at lower than saturating levels of capture antibody, so that a titration is needed Generally, the amount of specific antibodies in a serum as a percentage of the total protein is around l-5% The preparation of IgG eliminates a large percentage of the serum proteins not involved in the assay (e.g., serum albumins) Therefore, the activity of the IgG protein (relative increase in the IgG fraction that will attach to each well) is increased effectively In other words, there is a greater proportion of IgG sticking to the wells to act as trapping antibody if IgG preparations are used 164 Use of Antibodies 0' I 1234 I I on Solid Phase in Capture ELISA I / 567 I I \ 10 I I 11 12 Serum or IgG dilution (2 fold) I IgG -+4- Serum Fig Comparison of capureof IgG using whole serum or IgG as capture antibody 5.2 Use of Whole Serum Dilutions of untreated serum can be used However, as indicated above, the proportion of specific IgG is low, and other serum proteins attach in a competitive manner One cannot assume that putting on a low dilution of serum will give a good level of capture antibody The most usual event is that a bell-shaped curve of capture ability is obtained, with little activity at high concentrations of serum and a rise in activity as the serum is diluted In general, serum has to be diluted to around l/5001/2000 Thus, we have to have fairly high titers to be able to use whole serum Figure demonstrates the activity of whole and IgG capture antibodies as they are diluted down to illustrate the bell-shaped curve Titration of Capture Antibody Using IgG Dilute the sheepantiguineapig IgG preparationto ug/mL in carbonate buffer Add 50 uL to eachwell on the plate except column Add adsorptionbuffer alone to row 12 Incubate at 37°C for h or overnight if more convenient(rememberto put lids on plates,and so forth) Titration of Capture Antibody Using IgG 165 Wash the plates From now on, we are performing a similar procedure to the indirect ELISA demonstrated earlier Take microtiter plate with well Al at the top left-hand corner Add 50 PL of blocking buffer to each well Make a dilution range of guinea pig IgG (the antigen of mterest) from pg/mL from column 1(8 wells) to column 11 in blocking buffer Thus, add 50 ltL of guinea pig IgG at 10 pg/mL to first row using a multichannel pipet Mix, and double dilute across the plate (you should be competent at this now) Remember to discard the last 50 pL in the tips, so that each well should only contain 50 l.tL of fluid (check!) Incubate the plates at room temperature or at 37°C for h Wash the plates Add 50 pL of blocking buffer to each well Take rabbit antiguinea pig serum, and dilute to l/100 in blocking buffer (make up 1.OmL, add 10 l,tL of undiluted serum to 1.OmL of buffer) Mix Add 50 pL of the dilution to row A using a single-channel pipet Dilute across rows A-H using a multichannel pipet We now have a twofold dilution range from l/200 (row A) to l/25,600 (row H) 10 Incubate the plate at room temperature or at 37OCfor h 11 Wash the plate 12 Make up optimum dilution of sheep antirabbit conjugate This can be pretitrated as in Chapter Add 50 l.tL to each well using multichannel pipets 13 Incubate for standard time as used in optimization of conjugate (1 h at 37OCor room temperature) 14 Wash the plate 15 Add substrate and stop color at 10 6.1 Data Essentially we have made a chessboard titration of the antigen against the detecting antibody (as in the indirect assay) Thus, we have assumed that the capture antibody, put on the plate as an IgG, is at maximal reactivity The results are therefore similar to those obtained in the indirect assay and can be treated in a similar way Each of rows A-H had an identical dilution series of the antigen (guinea pig IgG) being captured by the same amount of antibody Thus the same amount of guinea pig IgG should be present and attached via antibody to wells l-l The rabbit antibody against the antigen has been titrated at different dilutions, so we can examine which dilution shows the best detection of IgG in rows A-H The use of pg/mL, of capture IgG was taken as that which from experience saturates the plastic sites available on the plate wells Once the antigen 166 Use of Antibodies on Solid Phase in Capture ELISA Table Chessboard Titration of Guinea Pig IgG vs Rabbit Antiguinea Pig IgG-Constant Capture Antibody, Constant Conjugate A B C D E F G H 10 11 12 1.67 1.68 1.56 1.12 1.00 0.78 0.54 0.34 1.67 1.68 1.54 1.09 0.97 0.74 0.51 0.34 1.68 1.65 1.52 1.00 0.89 0.71 0.51 0.32 1.65 1.56 1.43 0.94 0.78 0.56 0.42 0.21 1.54 1.51 1.34 0.87 0.67 0.51 0.36 0.18 1.34 1.31 1.23 0.78 0.56 0.43 0.32 0.15 1.09 1.04 0.99 0.67 0.43 0.32 0.21 0.16 0.89 0.84 0.76 0.56 0.34 0.21 0.16 0.09 0.67 0.59 0.52 0.45 0.23 0.19 0.14 0.08 0.54 0.51 0.43 0.34 0.21 0.14 0.09 0.07 0.34 0.32 0.23 0.19 0.17 0.09 0.08 0.08 0.23 0.17 0.09 0.08 0.08 0.10 0.09 0.09 Fig Diagrammatic representation of plate, data from Table and detecting serum optima have beenestablishedusing this level of capture IgG (as shown below), this can be altered to examine the effect on the assay Table shows the spectrophotometric plate readings A representation of the plate is also shown in Fig 6.2 Plots of Data Figure shows the data plotting the OD results obtained at the different Ag dilutions for each dilution of rabbit antiguinea pig serum Titration of Capture Antibody I I Using IgG Y I 10 0123456789 Antigen Rows 167 Dilution 11 12 + -A+B*C+DxE*FfG*H Fig 3, Titration of guinea pig IgG using constant capture conditions Each line represents titration of the same dilution range of IgG using a different concentration of rabbit antiguinea pig IgG The conjugate dilution is constant From data in Table 1, of Data Column 12 contained no antigen (guinea pig IgG) Therefore, examination of the color here gives a measure of the binding of the detection system to the plate or capture antibody Thus, rows A and B show higher levels of color than the rest The value around 0.09 appears to be the plate background expected in the presence of the same dilution of conju6.3 Assessment gate Thus, the end point detection of IgG is affected in rows A and B Examination of the plateau heights indicates that the trapping system is saturated in columns l-4, since we obtain similar OD values Thus as an example, we have around 1.67 for the first four wells using the l/200 detecting antibody Although the actual plateau height value reduces on dilution of the detecting rabbit antiguinea pig serum, examination of Fig 3, which relates the curves obtained for the detection of trapped Ig for different dilutions of the rabbit antiguinea pig Ig, shows most easily that the last dilution giving an optimal titration is in row C, After this dilution, the effect is to reduce more markedly the OD in the plateau region 168 Use of Antibodies on Solid Phase in Capture ELISA (where the trapped Ig is in excess) and also affect the sensitivity of detection of the Ig at higher dilutions, as indicated by a reduction in the end points where the test background is the same as the plate background of Capture IgG The optimal dilutions chosen will depend on how the test is to be used If an antigen is to be detected, then we might require high detection limits in the system, so that we can use a dilution of detecting antiserum to maximize this, We will see later that capture assayswill be used in competitive situations where the amount of antigen to be captured needs to be reduced, so that a variation in reagent concentrations may be necessary and can be read from an exercise as described here The established optima for the antigen and detecting serum can be reassessedusing lower concentratons of capture IgG Thus, a full chessboard titration as described above can be performed using 2.5, 1.25, and 0.625 pg/mL of the capture IgG However, a simpler procedure is to coat plates with a dilution range of the capture IgG, and use constant antigen, detecting antiserum, and conjugate dilutions as found above Results of a typical titration of this sort are demonstrated in Table Here plates have been coated with capture anti-IgG at pg/mL in a twofold range from rows A-H, columns l-4 only, thus quadruplicate samples are being examined After incubation and washing, antigen (guinea pig IgG) at 0.625 pg/mL has been added in blocking buffer Following incubation and washing, the detecting antibody (rabbit antiguinea pig IgG) has been added at l/400 diluted in blocking buffer After incubation and washing, the antirabbit conjugate has been added at the dilution used to optimize the reagents Table shows that the capture IgG produces similar results at and 2.5 pg/mL, thus the latter dilution can be used in an assay to capture antigen Lower concentrations produce lower OD values, indicating that not all the available antigen is being captured The reduction in ability to bind antigen (when in excess) is accompanied by a loss in ability to detect small amounts of antigen (minimum detection limit is reduced) Similar titrations of the other reagents can be made where only one is diluted and the others are kept constant Thus, in the case above, we know we have three conditions optimized under experimental conditions with control sera and antigen The capture IgG can be used at 2.5 pg/mL, the antigen can be used at 0.625 yg/mL, and the rabbit detector at l/400, 6.4 Retitration Titration of Capture Antibody 169 Table Titration of Capture IgG Against Optimized Antigen, Detecting Antibody and Conjugate Capture IgG concentration, WmL 5.0 2.5 1.25 0.63 0.32 0.16 0.08 0.08 A B C D E F G H Mean 1.50 1.49 1.25 0.95 0.67 0.36 0.14 0.05 1.48 1.47 1.21 0.94 0.69 0.37 0.12 0.04 1.49 1.51 1.24 0.96 0.69 0.40 0.15 0.04 1.51 1.46 1.27 0.93 0.66 0.37 0.12 0.03 1.50 1.48 1.24 0.95 0.68 0.38 0.13 0.04 with the antirabbit conjugate at a constant dilution as assessedoriginally against the relevant IgG attached to a microplate We may wish to reassess the conjugate dilution under standardized conditions Thus, using the capture IgG, antigen, and detecting antiserum optima found above, replicate wells can be used to titrate different dilutions of conjugate An example is shown in Table Here a dilution of 2X)0 of conjugate gives similar results Effectively, a dilution of l/800 gives “optimal” results(OD value around 1.45)for an assay Titration of Capture Antibody when Used as Whole Serum As already stated, whole serum can be used to coat plates and act as a capture reagent This is not recommended, since we cannot measure the protein Ig because is contaminated with “blocking” serum proteins that compete for plastic binding sites preferentially over the IgG The simplest method is to perform a chessboard assay relating dilutions of capture serum to dilutions of detecting antibody and keep the antigen constant The diagram below illustrates this: I-Ab + w Ag + w AB + Anti-AB*E w I-Ab = Dilution range of trapping antibody Ag = Constant dilution of antigen (high concentration) AB = Dilution range of detecting antibody + W S + Read 170 Use of Antibodies on Solid Phase in Capture ELISA Table Assessment of Constant Capture System with Different Dilutions of Conlugate Conjugate dilution 200 400 800 1600 3200 6400 12,800 none A B C D E F G H Mean 1.95 1.84 1.45 0.95 0.77 0.36 0.15 005 1.87 82 1.41 0.94 0.79 0.37 14 0.04 87 1.84 1.44 0.96 0.79 0.40 0.15 04 1.95 82 1.47 0.93 0.76 0.37 0.14 0.03 192 83 1.44 0.95 78 038 0.15 04 Anti-AB*E = Conjugated antispecies antibody S = Substrate/chromophore Read = Read plate in spectrophotometer This assay will not be described in detail However, a description of the test will be given with relevant points highlighted You should now have enough experience to be able to set up the exact practical details yourself with help from the exercise titrating IgG as capture antibody 7.1 Method The serum containing capture antibody is diluted on plates in carbonate/ bicarbonate buffer (begin at l/100, twofold dilutions) Incubate and then wash plates Constant (excess) antigen is then added diluted in blocking buffer (difficult to specify here what excess might be for specific systems (e.g., an undiluted tissue culture sample containing vuus might be expected to have a high concentration of antigen) Incubate for 1h using standard conditions Wash and add dilutions of detecting antibodies in blocking buffer to obtam chessboard titration (dilute in the opposite direction to the capture serum) Incubate and then wash plates Add optimal conjugate, incubate, and then wash Add substrate and then stop at 10 mm 7.2 Data Typical results are shown in Table Rows A-H contain dilution ranges of the capture serum l/100-l/51,200, i.e., column = l/100, column 12 = l/51,200 Row A received the detecting antiserum at l/200, Use of Capture ELBA to Detect Antigens 171 Table Dilutions of Capture SerumMOO-l/51,200 A B C D E F G H 10 11 12 0.67 0.68 0.65 0.56 0.45 0.23 0.15 0.15 0.96 0.99 0.98 0.88 0.67 0.43 0.23 0.19 1.34 1.42 1.36 1.23 1.00 0.78 0.34 0.18 1.35 1.37 1.34 1.19 1.09 0.76 0.35 17 32 1.29 1.15 1.01 0.98 0.56 0.21 0.16 1.11 1.09 0.99 0.88 78 0.45 0.15 0.10 0.98 0.89 0.87 0.74 0.56 0.40 0.16 0.09 0.76 0.75 0.72 0.65 0.45 33 0.09 0.08 0.56 0.54 0.52 0.43 0.34 23 0.08 0.09 0.45 0.36 0.31 0.26 0.21 16 0.07 0.07 0.23 0.22 0.17 0.14 0.12 0.12 0.09 0.07 0.12 0.11 0.09 0.09 0.07 0.08 0.09 0.09 row B l/400, and so on, to row H at l/12,800 See Fig for further clarification 7.3 Conclusions Optimal dilution of capture serum is around column (last column show- ing maximum OD) Optimal dilution of detecting second antibody is around row C/D (last showing maximal titration curve of antigen) Bell-shapedcurvesareobtainedwherelow dilutions of captureserumgive low OD values (columns and 2) Use of Capture ELBA to Detect Antigens Once the optimal conditions have been established, the capture assay can be used in several ways as indicated below 8.1 Diagnosis I-Ab + w Ag + w of Specific AB + W Disease Agents Anti-Ab*E + S + Read W Here a sample possibly containing antigen is added to a capture system (microtiter plate wells coated with an antiserum against a specific disease) Any bound antigen is then detected by another antibody from a different species Such assays are important in serotyping where the second antibody may further “divide” the disease agent into a serological grouping, e.g., as is used routinely to serotype foot-and-mouth disease viruses (FMDV) into one of seven distinct serotypes The use of capture antibody means that relatively crude or contaminated samples can be used Use of Antibodies on Solid Phase in Capture ELBA 16 08 06 Dilution of capture ambody (l/100,2 fold) Rows -+A Dhtlon l/100 +B l/200 *C l/400 *D 11800 *E *F l/1600 l/3200 *G l/6400 *H l/12800 Fig Graph of data in Table Columns 1-12 contain dilutions of capture antibody on wells Rows A-H have different dilutions of detecting antibody Quantification of antigens may be made with reference to a standard antigen titration on the same plate Single dilutions of material containing the Ag can then he titrated in the same system and the developing OD read against the standard titration Again, the use of the capture antibody ensures an efficient and proportional uptake of the antigen onto the plate, which is unaffected by contaminating proteins Use of Capture to Detect and Titrate ELISA Antibodies 9.1 Learning Principles Optimization of capture antibodies; and Optimization of detecting antibody 9.2 Reaction Scheme + Abv + Anti-Abv*E + I-ABx + Ag w w w w I- = Microplate AB, = Trapping antibody (species X) Ag = Antigen Abv = Test or control sera (species Y) S + Read Capture ELBA to Detect and Titrate Antibodies 173 Anti-Abv*E = Antispecies Y antibody conjugated with enzyme S = Substrate/color detection system W = Wash + = Addition and incubation of reagents Read = Measure OD in spectrophotometer of Method Essentially the same parameters have to be standardized as for capture ELISA for antigen detection, However, the test is used to measure antibodies against a fixed amount of antigen captured on the plate Thus, we have to optimize the system to have the correct amount of capture anti9.3 Principle body and antigen necessary to bind any test or control antisera The test offers the ability to capture antigen specifically using a solid-phase antibody Thus, relatively crude preparations can be used where the required antigen concentration may be low Care has to be taken to avoid reactions of the conjugate with components of the assay 9.4 Materials And Methods Capture antibody (ABx) = sheep antiguinea pig Ig at mg/mL in PBS Antigen (Ag) = guinea pig Ig at mg/mL Test antisera (Ab,) = antirabbit antiguinea pig Ig (Ab) Also sero-negative rabbit sera (as used in Chapter 6, Section 5.5.) Anti-Abv*E = sheep antirabbit IgG conjugated to horseradish peroxidase Microplates Multichannel, single-channel, lo- and 1-mL pipets 0.05M Carbonate/bicarbonate buffer, pH 9.6 PBS containing 1% BSA, 0.05% Tween 20 10 11 12 13 14 15 16 17 Solution of OPD in citrate buffer Hydrogen peroxide Washing solution Paper towels 1M sulfuric acid in water Small-volume bottles Multichannel spectrophotometer Clock Graph paper 9.5 Optimization We need to know: What dilution of captureantibody to use What dilution of antigen to use of Test 174 Use of Antibodies on Solid Phase in Capture ELBA What dilution of conjugate to use The aim is to have a constant system involving capture antibody (AB), antigen (Ag), and conjugate (Anti-Ab*E), which can then be usedto titrate test sera (Ab) The use of capture antibody as whole serum or as IgG has been dealt with This exercise will deal with the use of sheep antiguinea pig IgG (or the equivalent in an individual’s system) Thus, examination of the data in Table allows an estimation of the optimum capture IgG and antigen levels required to allow detection of antibodies 9.5.1 Data Using the titrations established in Section 6.1.) we can obtain the optimal amount of antigen (guinea pig Ig in this case) that gives a high pla- teau OD where the detecting antiserum is in excess Turn to the data showing the plate readings for Section 6.1 (Table 1) We can see the plateau height is maintained to around column 4, showing that there is a maximum level of antigen to react with the antibodies in the positive serum This concentration (or dilution) can be used in the capture assay under the same conditions to titrate antibodies from any sera From the data obtained in Section 6.1.) we can use: Tbe capture antibody at 2.5 l.tg/rnL if used as an Ig preparation or at the titrated level as found in Table 2 The antigen at the concentration or dilution used in columns and (Table 1) The conjugate as titrated initially in Table 9.6 Methods for Titration of Antibodies As described in Chapter 6, we can examine serafor antibodies by using full dilution ranges or as single dilutions The methodology in the capture ELISA is the same, except that after initial optimization of capture and antigen-coating conditions, capture plates are set up, coated with antigen, and then used to assess antibodies Thus, optimization of the capture antibody and antigen concentration (as shown above), allows the possibility of determining specific antibodies from test and control sera Perform the capture assay based on the addition of the same rabbit antiguinea pig sera, as from stage 4, in the direct assay described in Chap- ter 6, Section 5.5 Add the various dilutions of positive and negative rabbit antiguinea pig sera to plates containing an optimal concentration of captured guinea pig IgG After incubation and washing of plates, add the antirabbit conjugate (optimal dilution as measured in Table 3), incubate, Capture ELLSA to Detect and Titrate Antibodies 175 wash, and develop color Plot the data from the spectrophotometer and compare these to the data obtained in Chapter 6, Section 5.5 (Table 2) Repeat the exercise in Chapter 6, Section 6.6., using the capture ELISA, capturing IgG under optimal conditions to determine the positivity of the samerabbit seraat a single dilution Read and plot the data Compare these to the indirect assay results and assessthe sera as positive or negative using the statistical criteria outlined before Note any differences 9.6 Problems Using Capture Assays Care must be taken to examine whether any of the reagents interact Unexpected crossreactions can be found with immunological reagents, e.g., the conjugated antibodies might react with species other than those for which they were prepared There are crossreactionsbetween certain species,so that conjugates against cow proteins will react with sheepand goat proteins Thus, a system using sheep or goat Ig as a capture antibody will preclude the use of antibovine conjugates to detect the reaction of bovine antibodies with a particular antigen Where relatively crude antigens are captured, contaminating proteins may also be trapped that interfere with the assay.As an example of difficulties, when purified FMDV is injected into an animal, there is a specific response against the virus, but also a response against contammating bovine serum proteins that are present in extremely low amounts, coming from the tissue-culture medium Such sera used as capture reagent will capture not only virus, but also bovine proteins Thus, in typing exercises using tissueculture or bovine epithelial samples, a high quantity of bovine protein is captured The use of antibovine conjugates to detect bound bovine serum in a trapping assay,therefore, also binds to the trapped bovine protein giving high backgrounds In the typing assay proper, guinea pig sera are prepared as the second typing detecting sera.These also bind bovine proteins and therefore, detect bound bovine protein to the capture antiserum Again, specific typing is affected However, the second antibody can be treated to remove the crossreactivity either by adding a high concentration of the crossreactiveprotein to the reagent (in this case, mL of normal nonimmune bovine serum is added to mL of typing guinea pig serum), or by using affinity reagents where bovine serum is attached to a solid-phase, e.g., agarose beads, which can be incubated with the serum, so that the crossreactive antibodies are removed after incubation and separation of the beads by centrifugation, or as is most common, the test may be made using blocking buffers containing high levels (around 5%) of the crossreactive protein [...]... Antigens 171 Table 4 Dilutions of Capture SerumMOO-l/51,200 A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 0.67 0.68 0.65 0.56 0.45 0.23 0.15 0.15 0.96 0.99 0.98 0.88 0.67 0.43 0.23 0.19 1.34 1.42 1.36 1.23 1.00 0.78 0.34 0.18 1.35 1.37 1.34 1.19 1.09 0.76 0.35 0 17 1 32 1.29 1.15 1.01 0.98 0.56 0.21 0.16 1 .11 1.09 0.99 0.88 0 78 0.45 0.15 0.10 0.98 0.89 0.87 0.74 0.56 0.40 0.16 0.09 0.76 0.75 0.72 0.65... spectrophotometer This assay will not be described in detail However, a description of the test will be given with relevant points highlighted You should now have enough experience to be able to set up the exact practical details yourself with help from the exercise titrating IgG as capture antibody 1 2 3 4 5 7.1 Method The serum containing capture antibody is diluted on plates in carbonate/ bicarbonate buffer...Titration of Capture Antibody I I Using IgG Y I 10 0123456789 Antigen Rows 167 Dilution 11 12 + -A+B*C+DxE*FfG*H Fig 3, Titration of guinea pig IgG using constant capture conditions Each line represents titration of the same dilution range of IgG using a different concentration of rabbit... 0.89 0.87 0.74 0.56 0.40 0.16 0.09 0.76 0.75 0.72 0.65 0.45 0 33 0.09 0.08 0.56 0.54 0.52 0.43 0.34 0 23 0.08 0.09 0.45 0.36 0.31 0.26 0.21 0 16 0.07 0.07 0.23 0.22 0.17 0.14 0.12 0.12 0.09 0.07 0.12 0 .11 0.09 0.09 0.07 0.08 0.09 0.09 row B l/400, and so on, to row H at l/12,800 See Fig 4 for further clarification 7.3 Conclusions 1 Optimal dilution of capture serum is around column 5 (last column show-... that relatively crude or contaminated samples can be used Use of Antibodies on Solid Phase in Capture ELBA 16 08 06 Dilution of capture ambody (l/100,2 fold) Rows -+A Dhtlon l/100 +B l/200 *C l/400 *D 118 00 *E *F l/1600 l/3200 *G l/6400 *H l/12800 Fig 4 Graph of data in Table 4 Columns 1-12 contain dilutions of capture antibody on wells Rows A-H have different dilutions of detecting antibody Quantification... antirabbit IgG conjugated to horseradish peroxidase Microplates Multichannel, single-channel, lo- and 1-mL pipets 0.05M Carbonate/bicarbonate buffer, pH 9.6 8 PBS containing 1% BSA, 0.05% Tween 20 9 10 11 12 13 14 15 16 17 Solution of OPD in citrate buffer Hydrogen peroxide Washing solution Paper towels 1M sulfuric acid in water Small-volume bottles Multichannel spectrophotometer Clock Graph paper 9.5... proteins will react with sheepand goat proteins Thus, a system using sheep or goat Ig as a capture antibody will preclude the use of antibovine conjugates to detect the reaction of bovine antibodies with a particular antigen 2 Where relatively crude antigens are captured, contaminating proteins may also be trapped that interfere with the assay.As an example of difficulties, when purified FMDV is injected... serum proteins that are present in extremely low amounts, coming from the tissue-culture medium Such sera used as capture reagent will capture not only virus, but also bovine proteins Thus, in typing exercises using tissueculture or bovine epithelial samples, a high quantity of bovine protein is captured The use of antibovine conjugates to detect bound bovine serum in a trapping assay,therefore, also