The Elisa guidebook

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7¡ª

Monoclonal Antibodies

The ability to produce and exploit monoclonal antibodies mAbs has revolutionized many areas of biological sciences. The unique property of an mAb is that it is a single species of immunoglobulin (IG) molecule. This means that the specificity of the interaction of the paratopes on the IG, with the epitopes on an antigenic target is the same on every molecule. This property can be used to great benefit in immunoassays to provide tests of defined specificity and sensitivity, which improve the possibilities of standardization. The performance of assays can often be determined relating the actual weight of antibody (hence the number of molecules) to activity. Often the production of an mAb against a specific epitope is the only way that biological entities can be differentiated. This chapter outlines the areas involving development of assays based on mAbs. The problems involved address the physical aspects of mAbs and how they may affect assay design and also the implications of results based on monospecific reagents. Often these are not fully understood, leading to assays that are less than satisfactory, which does not justify the relatively high cost of preparing and screening of mAbs.

There are many textbooks and reviews dealing with the preparation of mAbs, the principles involved, and various purification and manipulative methods for the preparation of fragments and conjugation. There has been little general information attempting to summarize the best approaches to assay design using mAbs. Much time can be wasted through bad planning, and this is particularly relevant to mAbs. A proper understanding of some basic principles is essential. It is beyond the scope of this chapter to discuss all aspects, but major areas are highlighted.

1¡ª

Purpose of the Assay Involving the mAb

The fundamental question, ''What is the purpose of the assay involving the mAb?" may never be properly considered in sufficient detail. The first consid-

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erations presented in the following list address the "state of knowledge" about a problem (disease organism, serology, antigenic structure, other tests) and available reagent base. These are in common with other test developments but are particularly relevant to mAbs:

1.How much knowledge do we have about other current assays?

2.How much information have do we about the target antigen (organism)?

3.What information is there in the literature?

4.What time frame have we got for development of an assay?

5.What advantages and disadvantages will an mAb-based assay have over conventional techniques or polyclonal systems?

6.What polyclonal reagents do we have available from their use in other tests?

1.2¡ª Planning

Clear planning involves consideration of the immunization regime that might best give a range of mAbs most relevant to their intended task. Again, reference to developments in this field will give clues as to the best methods. Basically, mice (usually) have to be exposed to a specific antigen(s) and must stimulate antibodyproducing cells. The approach can be "shotgun" in which a relatively complex and "crude" mixture of antigens is used as the immunizing agent, or it can involve administration of a pure, defined antigen. The first approach has implications when the antibody secreted from hybridomas (fused cells secreting antibodies) need to be screened for activity. Screening and selection of appropriate clones is time-consuming, requires assay development, and should always have the aim of finding as quickly as possible the antibodies of direct relevance to the preconceived objectives of the required function. Table 1 presents a typical scenario for the screening and eventual isolation and use of mAbs.

1.3¡ª

Screening mAbs after Fusion

Screening should be selective as early as possible. Thus, the appropriate antigen has to be used in screening to test for antibody binding. The ELISA systems are ideal for this and, often at this stage, rely on data from other

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polyclonal assays. As an example, the concentration of an antigen that gives a good level of coating and signal in a polyclonal serum-based assay might be known. This concentration can be used to screen for mAbs using an indirect ELISA.

The essential work in the early stages is to have actively growing hybridomas secreting IG, and to ensure that these stem from single cells. Usually at the early stages of producing stable hybridomas, only a small amount of tissue culture fluid is available for testing, so that assays are made using a single dilution. This limits the possibilities of screening against a variety of antigens, which would allow some differential analysis of binding to be made at this stage. Therefore, it may be more useful to select an antigenic mixture at this stage. The specificity of the mAbs could then be determined when they are available in higher amounts.

The antimouse conjugate used must detect all isotypes of mouse IG equally well (these are commercially available). Mouse IGs have a number of specific isotypes and in order not to miss any possible binding, the conjugate must detect IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 isotypes.

There is a similarity in stages 2 and 3 in Table 1. This indicates that screening can be made quite early when it is reasonably certain that colonies of cells have been derived from single cells, depending on the methods used.

When single cells have been picked, there may be fewer clones to examine, but confidence in their clonality is high. Thus, screening here may be more relevant than for limit dilution methods. From a practical point of view, it might be useful to give a few figures for good and bad fusion results. An initial target of 200¨C500 clones (representing the handling of two-to four-microtiter plates) is reasonable. These reflect my experience and depend greatly on exactly which methods are used and the experience of the operators involved as well as the biological system being examined. They also depend on exactly how much work is put into the production. There must always be a realistic balance between achieving a target mAb and the maintenance of thousands of clones. Of these clones, many will turn out to be unproductive for various reasons, and hence useless. The maintenance and testing of 200¨C500 clones in the early stages is manageable by a single person. Increasing this number runs the risk of poor management. The essential target of this part of the operation is to produce colonies of cells that are secreting mAb. The testing of the mAb as early as possible will highlight candidate mAbs for further testing, stabilization, and use.

This scenario is further complicated if mAbs against several antigens are being produced by different operators. It is extremely difficult to control operations in which several runs are being made, each requiring operative procedures at different times.

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The examination of whether colonies are secreting mouse IG might sometimes be useful, especially when there is little activity against target antigens in screening. Nonsecretors can also be discarded. This can be accomplished easily through developing a competitive assay. The competitive assay for determining mouse IgG is described next, for reference and to introduce techniques and terms that will be generally used.

2¡ª

Competitive Assay for Determination of Mouse IgG

2.1¡ª Requirements

1.Mouse Igs isolated from whole mouse serum.

2.Antimouse enzyme-labeled conjugate (commercially available reacting with all isotypes of mouse Igs).

3.Microtiter plates (96-well ELISA plates).

4.Micropipets (5- to 50-µL variable volume), single and multichannel pipets (12 channel), and tips.

5.Reservoirs for reagents.

6.5- to 20-mL bottles.

7.Relevant substrate/chromogen solution for enzyme conjugate.

8.Phosphate-buffered saline (PBS) (0.1 M, pH 7.4).

9.Blocking buffer: 5% skimmed milk powder in PBS.

10.Relevant agent/conditions for stopping reaction after addition of chromogen/substrate.

11.Multichannel spectrophotometer.

12.Samples for testing.

13.Washing solution (PBS, pH 7.4, 0.02¨C0.1 M).

2.2¡ª

Preparation of Mouse Ig

Preparation of mouse Ig requires a minimum of 2 mL of mouse serum. A relatively simple technique for the preparation of total mouse Ig from serum, and one that gives adequately pure Ig for this test, is to use ammonium sulfate precipitation. Once the Ig is obtained and dialyzed against, e.g., PBS as used in ELISA, the concentration can be assessed either by protein estimation using chemical kits or, more easily, by reading the absorbance at 280 nm in an ultraviolet (UV) spectrophotometer. The concentration of Ig can be related to the absorbance using the following formula:

However, the formula is only true for relatively pure IgG. Once this has been accomplished, a solution containing a known concentration of mouse Ig is available. It should be stored in small aliquots at ¨C20¡ãC. For the sake of this example, we can make the assumption that a sample with 1 mg/mL (i.e., 1000 µg/mL) has been obtained directly or has been diluted to this concentration.

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This process should take a few h to precipitate Ig followed by overnight dialysis allowing for changes in dialyzing solution.

2.3¡ª Stage 1:

Optimization of the Mouse/Antimouse System

The objective of this stage is to titrate the mouse Ig, when attached to the solid phase, against the antimouse conjugate. Optimal conditions specifying the dilution of mouse Ig (antigen) and conjugate will be determined. The system will then be challenged by the addition of known Ig concentrations to the conjugate on addition to the mouse Ig¨Ccoated wells. A standard curve relating the concentration of mouse Ig added and the degree of inhibition of pretitrated reaction between the mouse Ig antigen on the plate and the conjugate can be established. Readings of inhibition by samples possibly containing mouse Ig can then be used to estimate the concentration of mouse Ig. Although this may seem complicated, the test can be set up in 2 d. Such a system is also relevant for determination of concentrations of Ig for any antiserum species. The prepared Ig will act as the standard solution for all competitive tests, so it needs to be protected from contamination by storing at low temperature.

2.3.1¡ª

Practical Details of Stage 1: Chessboard Titration

1.Dilute the mouse Ig to 5 µg/mL in PBS. Prepare a volume of 1 mL of this solution. For our example, we have 1000 µg/mL so we add 5 µL of this to 1 mL (1000 µL) of PBS. Mix well without causing frothing.

2.Add 50 µL of PBS to columns 2¨C12 of a microtiter ELISA plate.

3.Add 50 µL of Ig solution to columns A and B, using a 50-µL pipet.

4.Place eight tips on a multichannel pipet, and set the volume to 50 µL. Place the tips in column 2. Mix the contents (50 µL of PBS and 50 µL of Ig solution) by gentle pipeting action four times. Take up 50 µL of mixed solution in column 2 and transfer to column 3. Repeat the mixing exercise in column 3. Transfer to column 4, repeat, and so on until column 11. Discard the last 50 µL after final pipeting action in column 11. We now have the same dilution range of Ig in 50-µL vol, added to rows A¨CH, beginning at 5 µg/mL and diluted twofold to column 11.

5.Incubate the plates for 2 h at 37¡ãC.

6.Wash the plates by flooding and emptying wells 5 times in PBS. Then blot almost dry by tapping plates onto a sponge of paper towel.

7.Obtain conjugate and read the titer recommended by the commercial company. Make a dilution of approximately eight times that recommended; for example, if the recommended dilution is 1/400, make up 1/500. A final volume of 1 mL (1000 µL) is needed in this stage. Thus, add 2 µL of conjugate to 1000 µL of PBS. An examination of the appropriate pipet to use with small volumes is required. Where there is no pipet to deliver 2 µL accurately, then a 5-µL vol can be used into the appropriate volume; however, this does waste conjugate. Mix the conjugate solution without undue force.

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Table 2

Data from CBT of Ig and Conjugatea

aConjugate diluted from A to H (e.g., 1/500 in above example). Mouse Ig is diluted from 1 to 11 (5 µg/mL starting concentration in this example).

8.Add 50 µL of blocking buffer to all the wells of the Ig-coated microplates using a multichannel pipet.

9.Add 50 µL of the diluted conjugate to each well of row A.

10.Using a multichannel pipet with 12 tips, mix the contents of row A. Transfer 50 µL to row B, mix, transfer to row C, and so on until row H. Discard the last 50 µL in the tips. We now have a dilution of conjugate in blocking buffer (50 µL per well) from 1/1000 (since we added 50 µL at 1/500 to 50 µL of buffer) to 128,000.

11.Incubate the plates for 2 h at 37¡ãC.

12.Wash the plates, and blot.

13.Add the appropriate substrate/chromogen solution, leave for the prescribed time, and stop the reaction.

14.Read the OD values on a spectrophotometer.

2.3.2¡ª Results

Table 2 gives typical results obtained with a horseradish peroxidase (HRP) conjugate/H2O2-ortho- phenylenediamine (OPD) system.

Chessboard titrations (CBTs) have already been considered in some detail (see Chapter 4). In Table 2, row E in this plate indicates that there is a good level of color development with the conjugate at 1/8000. The plateau maximum value in the presence of excess antigen (mouse Ig) is acceptable, and the Ig is detected to column 11 (with respect to a plate background value of 0.1).

For the final competitive assay we require a single dilution of coating antigen. Ultimately, the sensitivity of the assay depends on the concentrations of conjugate and Ig, so that we are seeking a level of coating antigen Ig that does not saturate the wells, and a conjugate dilution that just reacts with all the coating Ig. The box in Table 2 reflects the area where this is relevant. Stage 2

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involves closer examination of the concentrations under competitive conditions to assess the best system in practice. This procedure takes only 1 d. The box in Table 2 covers three antigen dilutions and two conjugate dilutions, which can be examined in stage 2.

2.4¡ª

Practical Details Stage 2:

Competition of Standard IgG with Titrated Conjugate and Coating Ig

2.4.1¡ª Materials

The materials needed are the same as those used in stage 10. Two microtiter plates are needed: plate 1 for the competition conditions, and plate 2 to set up controls for 100% competition values.

2.4.2¡ª Method

The diagram in Fig. 1 shows a template indicating the addition of reagents to plate 1.

1. Coat the wells of the plate (50 µL per well in PBS) with IgG at the three dilutions identified in stage 1. The microplate can be rotated so that the long edge is vertical and to the left of the operator (row H). Add the first dilution of IgG to wells 1¨C4, A¨CH. Add the second dilution to wells 5¨C8, A¨CH. Add the third dilution to wells 9¨C12, A¨CH. We have 1.5, 0.75, and 0.375 µg/mL for the three dilutions of Ig to coat the wells. The same conditions for coating concerning incubation and washing are as already described.

2.Wash the wells with PBS.

3.Add competing Ig as a dilution range from 5 µg/mL across seven wells. The Ig is diluted in blocking buffer to prevent it from coating the wells.

4.Add the conjugate (50 µL per well) as shown in Fig. 1. The dilutions x and y represent the dilutions identified in stage 1.

5.Incubate the plates for 2 h at 37¡ãC as in stage 1.

6.Wash the wells.

7.Add the appropriate substrate/chromogen, and stop the reaction.

8.Read the results on a spectrophotometer.

Plate 2

1.In place of the coating step with Ig, add 50 µL of coating buffer to wells H¨CA, 1¨C4. Incubate as for coating.

2.After washing, add PBS diluent (50 µL) to each well as a substitute for the competing Ig.

3.Add the two dilutions of conjugate used in plate I in 50-µL vol to the wells: 1/x dilution in blocking buffer from H¨CA, 1 and 2; 1/y dilution to wells H to A, 3 and 4. Incubate as for the competition stage in plate 1.

4.Wash and add substrate/chromogen, incubate, and stop the reaction as for plate 1. Read the results.

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Fig. 1.

Scheme for addition of reagents to assess optimal concentrations for competition assay.

2.4.3¡ª Results

Typical results are given in Table 3. The mean values for the replicates are shown in bold. In plate 1, the data show that the addition of Ig as a competitor for the labeled antimouse conjugate had the effect of reducing color when it was added at high concentrations, as indicated in the wells H, G, and F. As the

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Table 3

Plate 1 and 2 Data

1/y mean of 16 wells = 0.05