The Elisa guidebook

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isotype-specific antibodies are applied for isotype determination in the hybridoma supernatants. Signal is detected by incubation with HRP labeled rabbit antigoat IgG. This step is followed by incubation with ready-to-use 3,3',5,5' tetramethylbenzidine chromogen.

Pierce offers ELISA-based mAb isotyping kits using HRP/2,2'-azino diethylbenzothiazoline sulfonic acid and Alkaline Phosphatase/p-nitro-phenylphosphate with two basic types of screening procedures. Antigen-dependent screening is as previously described above and involves coating the antigen on a microtiter plate. The hybridoma supernatant is then added, and the mAb is detected using an enzyme-conjugated antimouse antibody.

An antigen-independent screening method is also supplied when soluble antigens are difficult to obtain. ELISA plates are coated with an antibody to mouse immunoglobulin. This antibody then serves to capture the mAbs from the hybridoma supernatant. The presence of positive clones is proven with enzyme-conjugated antimouse immunoglobulin.

8¡ª

Processing of mAbs

Applications involving the use of Ig fractions may offer advantages. The proteolytic cleavage and purification of products from mAbs (particularly of mouse origin) can be accomplished through the use of kits designed to give good yields. Figure 7 gives the details of proteolytic cleavage of human IgG molecules. This illustrates the three major components of use and relevance in ELISA; the Fab2 and Fab and Fc fractions. Care must be taken concerning to the digestion of mouse IgG molecules since each isotype has a different degree of resistance to proteolytic cleavage. A good kit can be obtained from Pierce. These kits selectively cleave Ig molecules into Fab and F(ab')2 fragments using papain, pepsin, ficin, and trypsin immobilized on a crosslinked agarose support. Attaching the enzymes to a solid phase eliminates the problems often encountered with soluble enzymes to allow easy separation of enzyme from antibody fragments, no contamination with autodigestion products from the enzyme, and reproducible results.

9¡ª

Assay Formats Using mAbs

mAbs may be used in any assay format and they offer certain advantages over polyclonal reagents when used similarly. Care must be taken to assess the use in terms of the exact preparation (purity, isotype, fraction) of mAbs with due attention to the specificity of the interaction of the mAb and the epitope to which it binds and the nature of the antigen(s) that are involved in any assay. Certain formats used with mAbs can be used uniquely as compared to polyclonal sera. The valency of the mAb or mAb preparation can affect assays; Table 16 outlines parameters.

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Fig. 7.

Enzymatic cleavage of IgG. Pepsin cleaves the heavy chain to give F(Ab') and pFc' fragments. Further action results in greater fragmentation of the central protein to peptides. Papain splits the molecule in the hinge region to give two Fab fragments and the Fc fragment. Further action on the Fc can produce Fc'.

Table 17 shows the direct, indirect, and sandwich systems using symbols for the reactants. Each system can be challenged with antigen or antibody and thus, used to perform competitive or inhibition assays. Certain possible advantages and disadvantages of assays using mAb-based reagents as compared to polyclonal antibody reagents are shown.

The key features that can make mAbs potentially absolute reagents stem from their specificity. Therefore mAbs can be used effectively to measure only the epitopes against which they are directed and indirectly assess any other antibodies from other sources that bind with that epitope. The nature of the epitope (e.g., whether or not it is conformation dependent) and its overall ex-

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Table 16

Factors Involved

in Assay Design Using mAbs

Valency	Example
Monovalent	Fab fragments
Bivalent	Whole IgG1¨C3, Fab2
Multivalent	IgM/IgA
Mixture	Not successfully cloned

Table 17

Use of mAb Reagents in ELISA Systemsa

Direct ELISA

I-Ag + mAb*Enz

I-Ag + Fab2*Enz

I-Ag + Fab*Enz

Indirect ELISA

I-Ag + mAb + Antimouse*Enz

I-Ag + Fab2 + Antimouse*Enz or Anti-Fab*Enz

I-Ag + Fab + Antimouse*Enz or Anti-Fab*Enz

Direct sandwich ELISA

I-mAb1 + Ag + mAbl*Enz (same mAb detecting)

I-Fab2 + Ag + mAb*Enz (same mAb detecting)

I-mAb1 + Ag + mAb2*Enz (different mAb detecting)

I-mAb + Ag + PC*Enz (polyclonal antibody detector)

I-PC + Ag + mAb*Enz (polyclonal capture antibody)

Indirect sandwich ELISA

I-mAbisotype1 + Ag + mAb isotype2 + Antiiso2*Enz

I-Fab2 + Ag + mAb + AntiFc*Enz

I-mAb + Ag + PC + AntiPC*Enz

I-Fab2 + Ag + PC + AntiPC*Enz

I-PC + Ag + mAb (+Fab2 or Fab) + Anti mAb*Enz (or Anti-Fab*Enz)

aI- = solid phase with attached reagent; mAb = whole mAb molecules; Fab2 =

bivalent mAb minus Fc; Fab = monovalent fraction; + = addition of reagent in sequence; Ag = antigen; PC = polyclonal antibodies; *Enz = reagent conjugated to enzyme; Fc = Fc fraction of mAb Ig.

pression are also important, and the antigen used in any assay has to be considered carefully. Factors affecting affinity are changes in antigen (alterations of epitopes by physical factors or differences among samples and their density of expression) and the valency of the mAb preparation. The epitope density also affects the binding according to the exact systems used. Such factors

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can be examined with reference to some of the assay systems described in Table 17.

9.1¡ª

Direct ELISA

The following list is a reminder of the systems as given in Table 17:

1.I = Ag + mAb*Enz.

2.I = Ag + Fab2*Enz.

3.= Ag + Fab*Enz.

Direct ELISA depends on being able to successfully bind antigen to a solid phase while maintaining a reaction with mAb. Binding of antigen can affect the availability and orientation of epitopes (presentation to antibody) and also alter the antigenicity, particularly in which mAbs are directed against conformation-dependent epitopes. The mAb or fractionated mAb also has to be conjugated to enzyme. Conjugation may affect the binding of mAbs. The specific activity of the mAb detector is related to the amount of enzyme. This type of assay is best suited for a competitive/inhibition format in which an mAb has been identified that provides useful information about a single property. Since the initial screening of mAbs probably involves the indirect ELISA, it is likely that mAbs identified will bind to the coated-well antigen (if the same antigen preparation is used). The key is that several mAbs may have to be labeled until the best is identified. This is laborious and the use of the indirect ELISA, utilizing an antispecies conjugate, is recommended when many mAbs can be assessed in competition format.

9.2¡ª

Indirect ELISA

The following list is a reminder of the systems as given in Table 17.

1.I-Ag + mAb + Anti-mouse*Enz.

2.I-Ag + Fab2 + Anti-mouse*Enz or anti-Fab*Enz.

3.I-Ag + Fab + Anti-mouse*Enz or anti-Fab*Enz.

mAbs screened by indirect ELISA already should be suitable for this test. The main application is in competitive tests in which both antigen and antibodies can be assessed in samples. This involves pretitration of the system (Ag and mAb). Samples can then be added and mixed with the pretitrated concentration of mAb and incubated, or added simultaneously. The binding of Fab fractions may well show different characteristics to whole mAb since bivalency of antibody molecules is lost. This affects the relative affinity of binding to the antigen. It may allow a greater density of binding of Fab molecules as compared to whole molecules. The use of Fab molecules in competition assays may reduce or enhance sensitivities depending on the exact distribution and effective relative affinities of binding to antigen.

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9.3¡ª

Direct Sandwich ELISA

The following list is a reminder of the systems as shown in Table 17:

1.I-mAb1 + Ag + mAbl*Enz (same mAb detecting).

2.I-Fab2 + Ag + mAb*Enz (same mAb detecting).

3.I-mAb1 + Ag + mAb2*Enz (different mAb detecting).

4.I-mAb + Ag + PC*Enz (polyclonal antibody detector).

5.I-PC + Ag + mAb*Enz (polyclonal capture antibody).

The advantage of this system is linked with the specificity of the mAb in possibly capturing only the targeted antigen. Bivalent mAbs usually capture well, but have to be tested individually for required performance.

The use of the same Mab for capture and detection (as in examples 1 and 2) can lead to problems in which there is a limited number or single (e.g., peptide) epitope being expressed since the capture antibody may effectively preclude any further reaction. However, the use of the same Ab can be exploited to achieve highly specific assays for the detection of particular complexes bearing an antigen, as is illustrated for specific detection of whole particles of foot-and-mouth disease virus (FMDV) from subunits. In this example, the key is that the initial capture by the mAb orientates the antigen complexes. The consideration of the effects of orientation is necessary in all tests involving mAb capture.

When more than one mAb is available, sandwich assays can be made by labeling mAbs and using them as detectors and can alleviate problems of orientation and limited epitopes. This can also lead to very specific assays.

Examples 4 and 5 show the use of a polyclonal serum to either capture or detect. As a capture serum, mAbs can be used to detect specific epitopes and increase the specificity of assays. Again, with antigens showing limited epitopes, the polyclonal capture may result in prevention of any more binding saturation of epitopes. When polyclonal antibodies are used as a general detector, they allow a number of mAbs to be screened for effective capture of antigens. The specificity of the initial capture depends on the mAb. Here, orientation effects are more limited (as shown in data, e.g., with foot-and-mouth disease virus (FMDV) in Subheading 10.1).

The assays developed (pretitrated) can all be used with competitive/inhibition systems for the detection of antibodies. Examination of antigens is more difficult since it must be ensured that the capture antibody is saturated with antigen because addition of competing antigen increases the effective concentration and free capture antibodies will bind to this. The same is applicable in indirect sandwich systems.

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9.4¡ª

Sandwich ELISA¡ªIndirect

The following list is a reminder of the systems given in Table 17:

1.I-mAbisotype1 + Ag + mAb isotype2 + Anti-iso2*Enz.

2.I-Fab2 + Ag + mAb + Anti-Fc*Enz.

3.I-mAb + Ag + PC + Anti-PC*Enz.

4.I-Fab2 + Ag + PC + Anti-PC*Enz.

5.I-PC + Ag + mAb(+Fab2 or Fab) + Anti mAb*Enz (or Anti-Fab*Enz).

In cases in which mAbs are available in pairs and their isotype is known, mAbs with different isotypes can be used to capture and detect antigens, as shown in example 1. This is made possible by the use of an antimouse isotype-specific conjugate, which allows a higher level of screening of mAbs provided that the enzyme conjugates are affordable. When the bivalent Fab2 is prepared, the whole molecule mAb or different mAbs can be used. These are detected using an anti-Fc specific conjugate (example 2). Thus, a large number of mAbs can be screened using a single successful capture reagent.

The use of polyclonal antibodies and mAbs is shown in examples 1¨C3. Examples in 3 and 4 show the benefit of screening mAbs for capture activity using a general detecting reagent and antispecies conjugate, and example 3 is probably the most widely used application.

When polyclonal antibodies are used to capture antigens, the screening of mAbs is relatively easy, and whole mAb or fractions can be used with appropriate antispecies conjugates (example 5).

The preparation of polyclonal reagents in experimental animals or characterization from field sera is important in the development of assays. Such sera can be used directly as components of assays and also as reagents defining mAb reactivity. This is particularly important in research areas. Often the best assays involve the use of polyclonal and mAb reagents, one allowing generalized reactivity and the other high specificity.

The systems can all be used in competition/inhibition ELISA for examination of antibodies. The target antigen can be captured first and then competition performed for the detecting antibody.

In summary, the use of mAbs and mAb-polyclonal systems offers a large number of possibilities. The particular advantage of any one has to be determined in the feasibility stages of the development of assays. The production of polyclonal reagents is recommended to allow greater flexibility and possibly avoid too great a specificity of reaction at the various phases of the ELISAs; for example, polyclonal reagents may serve as a general capture reagent for a polyvalent antigen and the specificity of the mAb detector for a particular epitope exploited. Assays can be evolved with limited reagents, e.g., the use of

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mAb combinations using isotype-specific conjugates. Care must be taken to examine the use of mAbs in combination with respect to the orientation of antigens and subsequent elimination of binding of the detector. Some knowledge of the antigen(s) should be sought (molecular weight, density) to aid the designing of the assays.

10¡ª

Examples of the Use of mAbs

Examples of the use of mAbs are now given in detail and involve studies on FMDV:

1.The quantification of whole virus particles in the presence of subunits bearing the same epitope using the same mAb as capture and detector (direct sandwich ELISA).

2.The use of panels of mAbs at a single dilution to differentiate antigenic differences among many virus isolates, involving polyclonal capture sera, mAb detectors and antimouse conjugate (indirect sandwich).

3.The use of mAb-based competition assay for detection of antibodies against rinderpest virus and the development of kits.

The methods illustrate the interface of different technologies and disciplines needed to produce a successful ELISA for a specific purpose. This typifies the interaction of research facilities necessary to develop assays and a thorough understanding of the biological entities being examined.

10.1¡ª

Quantification of Whole Virus Particles of FMDV in the Presence of Virus Subunits, Using mAbs in a Sandwich ELISA

10.1.1¡ª Background

The immunogenicity (ability to elicit protective antibodies in animals) of FMDV vaccines depends, to a large extent, on the production of whole virions (146S particles, so named because of their sedimentation characteristics in sucrose density gradients) in tissue culture and the stability of these particles after virus inactivation procedures and formulation into vaccines. The immunogenicity of subunits (12S particles) is very poor, weight for weight, compared to the 146S particles. Both whole and subunit particles are produced in the infectious process during the manufacture of vaccines. The specific quantification of 146S particles is made using physical methods using either CsCl2 or linear sucrose density centrifugation methods. Both these methods are laborious, take a relatively long time, are subject to standardization problems, require expensive equipment, and do not assess whether virus has been affected by proteolytic enzymes.

Serological methods for estimating the specific weight of 146S are complicated since whole particles and subunits share most of the same epitopes. Thus,

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polyclonal sera produced against purified 146S particles cross react with subunits and, therefore, cannot be used directly to assess the weight of whole particles specifically.

Virus-neutralizing mAbs offer serological systems that can overcome the problems of crossreactivity. Neutralizing mAbs against most serotypes of FMDVs have been prepared and characterized in many laboratories worldwide. Such reagents have been used to compare virus isolates antigenically, to prepare and characterize mAb escape mutants allowing the identification of epitopes at the amino acid level, and as reagents in assays to measure antibodies. From such studies, the antigenic makeup of the surface of FMDVs is becoming more clear, particularly when studied in conjunction with X-ray crystallographic data.

One strategy for the specific detection of 146S would be to select an mAb that bound only to the whole virion and not to the subunit particle. This has been proved possible, but such mAbs are not commonly isolated. Another strategy is to use the same mAb as capture and detector. This strategy has another advantage when commonly produced mAbs of a particular specificity have been defined. The use of centrifugation methods involves the sedimentation of virus and its assessment by reading the absorbance of fractions at 259 nm in a spectrophotometer. This measures the RNA content of the fraction, which is then used to calculate the protein weight using a formula. The association of protein to RNA of the correct sedimentation value indicates that virions are being quantified, however, it does not indicate whether the proteins in the virion are cleaved. Cleavage of protein VP1 in the virion capsid can dramatically alter the immunogenicity of the vaccine. If cleavage is to be estimated, then the peak fractions measured from the gradient have to be analyzed by polyacrylamide gel electrophoresis (PAGE), which is a laborious and limiting procedure. The complete procedure of centrifugation and analysis on PAGE, which gives full confidence in the vaccine, does not allow the prospect of on-line testing for virus as it is being produced during the vaccine run. The use of mAbs similar to those identified in this chapter will not only quantify 146S specifically but will identify whether the VP1 protein has been cleaved. The system could also be adapted to the on-line continuous testing for 146S virus, which would allow a greater control of the manufacturing process so that virus could be harvested at the time of maximum production.

10.1.2¡ª

Materials and Methods

10.1.2.1¡ª Viruses

Type O1 Kaufbeuren FMDV was grown in BHK-21 cells in the absence of bovine serum. Infected tissue culture fluid was clarified by low-speed centrifu-

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gation and the protein precipitated by the addition of an equal volume of saturated ammonium sulfate (pH 7.4, controlled by the addition of NaOH). After 1 h at room temperature, the precipitated protein was collected by centrifugation (approx 6000g; Mistral 6L centrifuge). The sediment was resuspended in a minimum volume of PBS and clarified by centrifugation at 10,000g. The supernatant was then centrifuged at 100,000g for 2.5 h to pellet the virus. One milliliter of PBS was added to cover the pellet, which was then left overnight at 4¡ãC to allow the pellet to rehydrate. The pellets were then resuspended by agitation with a pipet and brief sonication in a water bath sonicator. Purification was made on linear 15¨C45% sucrose density gradients after the addition of sodium dodecyl sulfate (SDS) to a final concentration of 0.1%. The concentration of purified virus (146S) was established by examination of the RNA adsorption at 259 nm. Peak samples were stored at ¨C70¡ãC without further additions.

10.1.2.2¡ª

Preparation of 12S Subunit Particles

One milliliter of purified virus containing 200 µg of virus was acidified by the addition of 2 mL of 0.1 M NaH2PO4. Phenol red indicator solution was added (0.05 mL), and the mixture was left at room temperature for 10 min, after which the pH was adjusted to 7.4 by the addition of 0.1 M NaOH.

10.1.2.3¡ª

Preparation of Trypsin-Treated Virus

To 200 µg of purified virus in 1.0 mL of sucrose was added to 50 µL of trypsin solution (2 mg/mL in 0.1 M phosphate buffer, pH 7.4). The mixture was incubated at 37¡ãC for 15 min. The virus was then diluted to the assay concentrations in the relevant buffer with no further treatment.

10.1.2.4¡ª

Preparation of Denatured Virus

To 1 mL of purified virus containing 200 µg was added 10 mg of SDS (giving a final concentration of 1%) and 20 µL of mercaptoethanol (to a final concentration of 2%). The mixture was heated for 3 min in a boiling water bath. The virus preparation was dialyzed against PBS, and the volume after dialysis was noted to allow an accurate determination of concentration of the protein relative to the starting material.

10.1.2.5¡ª Antisera

Guinea pig and rabbit polyclonal antisera against type O and SAT 2 FMDVs were prepared after multivaccination of animals with purified inactivated virus containing antibodies with a wide spectrum of activity against all FMDVs components.

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10.1.2.6¡ª

Monoclonal Antibodies

mAbs were prepared as described in ref. 2. Acites fluids were prepared by ip inoculation of the hybridoma cells into mice previously sensitized with Freunds' Complete Adjuvant (FCA). Ascites fluids were clarified by centrifugation and stored at ¨C20¡ãC. The anti¨Ctype O mAbs B2, C8, C9, and D9 have been extensively characterized using serological tests and mAb escape mutant studies (3¨C5).

10.1.2.7¡ª

Enzyme Conjugation of mAbs

Ascites fluids were labeled with HRP using the method described in ref. 6.

10.2¡ª ELISAs

10.2.1¡ª

Titration of mAbs as Capture Antibodies

The ascites and the polyclonal rabbit serum were diluted in 0.05 M carbonate /bicarbonate buffer, pH 9.5, in 50µL vol into wells of a microtiter ELISA plate (Nunc Maxisorb). Twofold dilution series from 1/20 were made across 11 wells, in quadruplicate. The plates were incubated at 4¡ãC overnight or at 37¡ãC for 2 h. Plates were then washed by flooding and emptying the wells four times with PBS. Plates were blotted almost dry and 50 µL of the respective purified FMDV, 12S, trypsin-treated virus (TTV), or denatured virus (DNV) were added to each well at 2 µg/mL, diluted in PBS containing 5% bovine skimmed milk powder (Marvel) and 0.1 % Tween-20 (blocking buffer to prevent nonspecific attachment of protein). The plates were incubated at 37¡ãC for 1 h while being rotated. Plates were then washed and 50 µL of the relevant type of specific polyclonal guinea pig antiFMDV serum was added at optimal dilution to each well, diluted in the blocking buffer just described. Plates were then incubated at 37¡ãC for 1 h while being rotated. Anti-guinea pig whole IgG HRP conjugate was then added, 50 µL per well diluted in blocking buffer, and the plates were incubated for 1 h at 37¡ãC while being rotated. Plates were then washed and 50 µL per well of OPD/H2O2 chromogen/substrate was added. Color was allowed to develop for 10 min ,and then the reaction was stopped by the addition of 50 µL of 1 M H2SO4. The results were quantified by reading the plates on a multichannel spectrophotometer. Data relating the activity of each mAb dilution to capture virus as detected by the polyclonal system were plotted. Optimal dilutions of each mAb were measured to allow a single dilution to be assessed in virus quantification studies. The effect of using different concentrations of mAbs as capture reagents was also examined when assessing the various antigen preparations.