Immunoassay procedures

5.4 Immunoassay procedures

To illustrate these different separation procedures a range of techniques is included below. The use of ammonium sulphate to separate bound from free antigen has already been discussed in Chapter 3.

5.4.1 Competition radioimmunoassay

The basic common feature of these assays is the separation of bound from free reactants. There are

a number of systems available, such as: • molecular sieving; • solvent and salt precipitation;

5.4IMMUNOASSAY PROCEDURES

• second antibodies; and • solid-phase systems.

In the radioimmunoassay for IgG, precipitation with polyethylene glycol (PEG) is used to separate bound from free antigen. One of the problems is that the antigen (human IgG) and the antibody (rabbit IgG) are of the same molecular weight and precipitability by PEG. This can be overcome by the use of human 125 I-Fab as the labelled antigen since it is soluble in PEG. Ideally, the labelled antigen and the ‘cold’ antigen to be assayed should be as alike as possible.

Radioimmunoassay of human IgG

Preparation of 125 I-Fab

MATERIALS AND EQUIPMENT Human IgG Sodium 125

I iodide, carrier free Bovine serum albumin (BSA), RIA grade

0.1 M tris(hydroxymethyl)-aminomethane (Tris)-buffered saline (TBS), pH 8.0 Chicken serum Whatman GF/B glass-fibre filter

METHOD

1 Obtain human IgG and prepare the Fab fragment.

2 Label 10 µg protein with 18.5 × 10 6 Bq 125

I and store in 5 ml TBS.

3 Immediately before use, dilute with TBS containing 10% chicken serum and filter under gentle pressure through a Whatman GF/B glass-fibre filter.

Determination of binding curve

MATERIALS AND EQUIPMENT Anti-human IgG 125 I-Fab (prepared as above) Bovine serum albumin (BSA), RIA grade

0.1 M Tris-buffered saline (TBS), pH 8.0 Polyethylene glycol (PEG) 6000 (15% w/v in TBS) Microtitre plates Cell-harvesting machine Whatman GF/B glass-fibre filter strips, for harvester Gamma spectrometer

METHOD

1 Dilute the anti-IgG serum 1 : 100 and then prepare a range of two- to three-fold dilutions in TBS containing 0.1% BSA.

2 Add 100 µl of 125 I-Fab (dilute to yield 10 5 c.p.m. in TCA precipitable material per well, as in step 3 above) to each well of the microtitre tray. (Use sufficient wells for triplicates of each antibody dilution.)

3 Add 50 µl aliquots of the diluted anti-IgG serum to appropriate wells, mix thoroughly and incubate at room temperature for 16 h.

Continued

C H A P T E R 5: Immunoassay

4 Add 200 µl of 23% w/v PEG solution to each well and incubate at room temperature for 2 h.

5 Collect the contents of each well onto Whatman GF/B glass-fibre filter strips using a cell- harvesting machine.

6 Wash each precipitate with 1.5 ml of PEG, 15% w/v in TBS.

7 Determine number of c.p.m. per sample using a g spectrometer and plot a curve of c.p.m. against antiserum dilution, i.e. an antibody-binding curve to determine the dilution of antibody that binds 50% of the antigen.

In all future assays with the same reagents use the dilution of antiserum binding 50% of the added radioactivity.

Construction of inhibition curve

MATERIALS AND EQUIPMENT Human IgG

Anti-human IgG serum (standardized as above) 125 I-Fab (diluted in chicken serum to half dilution used above)

0.1 M Tris-buffered saline (TBS) pH 8.0 Bovine serum albumin (BSA), RIA grade Polyethylene glycol (PEG) 6000, 23% w/v in TBS Cell-harvesting machine Whatman GF/B glass-fibre filter strips, for harvester

METHOD

1 Prepare a stock solution of IgG, final concentration 0.3 mg/ml in TBS containing 1% w/v BSA (aliquot and store at –20°C for use).

2 To perform the assay, prepare standard IgG solutions in the range 2 ng to 2 µg/ml in TBS containing 0.1% w/v BSA.

3 Add 50 µl aliquots of standard IgG to 50 µl of an appropriate dilution (determined as in section above) of anti-IgG serum (in triplicate).

4 Mix samples and incubate for 16 h at room temperature.

5 Add 50 µl of 125 I-Fab (diluted in 20% chicken serum to half dilution used above) and incubate for 4 h at room temperature.

6 Precipitate complex by the addition of 200 µl of PEG solution (23% w/v in TBS). Incubate and harvest precipitates as in steps 4–7 of the section above.

7 Plot the inhibition of binding of 125 I-labelled Fab on a linear scale (i.e. c.p.m. in precipitate) against the concentration of unlabelled IgG added on a log scale.

Use this standard curve to determine the concentration of IgG in unknown solutions. TECHNICAL NOTE

If an anti-immunoglobulin antibody is used to enhance complex formation, only a 2% solution of PEG is required in this method. This offers the significant advantage that whole IgG may then be used as a labelled antigen.

5.4IMMUNOASSAY PROCEDURES

5.4.2 Immunometric assays

Immunometric assays almost invariably require solid-phase systems. They can be performed in many different ways, for example: (a) with antibody on the solid phase to capture antigen, which is then detected by a second labelled antibody directed against another epitope on the antigen; or (b) for the detection of antibody by adsorption of antigen on the solid phase, followed by binding of the antibody to be determined, which, in turn, is detected by the addition of a labelled second antibody directed against the Fc region. We describe a method based on the latter type of assay.

5.4.3 Enzyme-linked immunosorbent assay (ELISA) General method for ELISA

MATERIALS AND EQUIPMENT Antigen; for example, human serum albumin (HSA)

0.05 M carbonate–bicarbonate buffer, pH 9.6 Phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBS–Tween) Hydrogen peroxide (30%)

0.18 M citrate–phosphate buffer, pH 4.0 2,2′-azinobis (3′ ethylbenzthiazoline sulphonic acid, ABTS) Casein Bovine serum albumin (BSA) Normal sheep serum Sodium fluoride (80 mg in 25 ml distilled water) Horseradish peroxidase–anti-immunoglobulin conjugate; for example, sheep anti-mouse Ig conjugate Test sera; for example, sera from mice immunized with HSA Enzyme immunoassay microtitre plates ELISA reader

Preparation in advance

PREPARATION OF ENZYME SUBSTRATE Prepare this just before adding to the plates.

1 Add 50 mg ABTS to 100 ml 0.18 M citrate–phosphate buffer, pH 4.0.

2 Add 30 µl of 30% hydrogen peroxide. Hydrogen peroxide is gradually lost from the stock solution with storage. Therefore it is advisable to calculate the exact amount needed to be added each week.

3 Make a 1 : 1000 dilution of hydrogen peroxide by adding 50 µl H 2 O 2 to 50 ml distilled water.

4 Determine the absorbance at 240 nm in a 1-cm cell against a distilled water blank.

5 The percentage concentration of original H 2 O 2 = absorbance 240 × 77.98.

6 Volume of original H 2 O 2 solution needed per 100 ml substrate solution: =

1 ml

% concentration

0.37, therefore percentage concentration =

For example, absorbance of 1 : 1000 dilution of H 2 O 2 =

0.37 × 77.98 = 28.9% therefore volume of H 2 O 2 needed per 100 ml = 1/28.9 = 0.035 ml.

C H A P T E R 5: Immunoassay

METHOD

1 Dissolve the antigen in carbonate–bicarbonate buffer. The optimum concentration should

be determined for each antigen but a concentration of 1–10 µg/ml should give acceptable results for most antigens.

2 Add 200 µl to each well of a micro-ELISA plate cover and incubate overnight at 4°C in a humid chamber.

3 Wash to remove unbound antigen and fill the wells with 250 µl 1% w/v casein to block any remaining protein-binding sites (gelatin, BSA or skimmed milk powder are often used instead of casein).

4 Incubate at room temperature for 1 h.

5 Wash the plates twice with PBS–Tween by filling, then inverting and shaking the plates.

6 Dilute the test sera in PBS–Tween containing 1% BSA. (The optimum dilution must be determined in advance; it will generally be about 1 : 1000.)

7 Add 200 µl diluted test serum and incubate for 2 h at room temperature in a humid chamber.

8 Wash the plates three times with PBS–Tween.

9 Prepare the peroxidase–antibody conjugate by mixing 100 mg casein, 1 ml sheep serum, 100 µl Tween 20 with 50 µl peroxidase–antibody and adjust to a final volume of 100 ml with PBS. Allow to dissolve with gentle stirring. (The exact dilution of conjugate will vary and must be determined by experiment. As a guide, this will generally be between 1 : 1000 and 1 : 10 000 for good antibody preparations.)

10 Add 200 µl diluted conjugate to each well.

11 Incubate at room temperature for 1 h.

12 Wash three times with PBS–Tween.

13 Prepare the substrate solution and add 200 µl substrate to each well. Leave in the dark at room temperature for the colour to develop, usually 10–30 min.

14 Stop the reaction by adding 50 µl sodium fluoride solution to each well.

15 Quantify the colour reaction in an ELISA reader set at 650 nm.

TECHNICAL NOTES • Strictly, each assay should include dilutions of a standard reference serum for the calibration of

unknown samples. In practice, however, the test is reasonably reproducible and some workers record their results directly in absorbance units.

• The same assay could be performed with radiolabelled antibody. In this case flexible

polystyrene plates should be used so that each well may be punched out and the bound radioactivity measured in a g spectrometer after step 12, instead of processing for enzyme activity.

• An alternative substrate for the peroxidase enzyme is 34 mg O-phenylene diamine and 50 µl

hydrogen peroxide (20 volumes) to 100 ml 0.1 M citrate–phosphate buffer, pH 5.0. The reac- tion is stopped by the addition of 50 µl 12.5% sulphuric acid and the absorbance measured at 492 nm.

• If an alkaline phosphatase-labelled enzyme is used, the substrate should be made up as follows:

50 mg 4-nitrophenyl phosphate in 50 ml diethanolamine buffer, pH 9.8. The reaction is stopped by the addition of 50 µl 3 M NaOH and the absorbance is measured at 405 nm.

5.4IMMUNOASSAY PROCEDURES

• Material from detergent-solubilized cells binds very poorly to ELISA plates because of the sur- factant effect: for example, protein dissolved in < 0.1% Triton X-100 shows little and variable binding; > 0.1% detergent inhibits binding completely. The problem of poor adherence may

be overcome (for many antigens) by denaturation with Bouin’s fixative: add 50 µl antigen solu- tion to each well (approximately 40 µg/ml initial protein concentration) and 200 µl Bouin’s fluid. Centrifuge at 500 g for 10 min, remove the fixative, wash once with 50% v/v ethanol and twice with PBS. Block plates with PBS containing 3% w/v BSA and 0.01% w/v thiomersal for 1 h. Such plates can be stored at 4°C for 1 week. This does not work for all cell-derived antigens and needs to be determined empirically. Marked increases in sensitivity can be obtained by substituting a bioilluminescent detection

system for the enzyme in solid-phase assays. Jackson et al. (1996), using identical assay systems, have shown a > 10 4 -fold increase in sensitivity with luminometry, using a covalent conjugate of streptavidin and aequorin. Aequorin is a bioluminescent molecule that releases a blue light upon the addition of Ca 2+ .

Matrices for solid-phase assays

Plastic surfaces The majority of assays are now performed with the antigen or antibody passively adsorbed onto

a plastic solid phase. Improved manufacturing processes have resulted in plastic supports with reproducible binding characteristics, although there is batch-to-batch variation, so researchers must optimize each system. Various proteins adsorb to differing degrees to the same plastic so trials must be carried out to determine the best support for a particular protein. Plates may now be obtained which have been irradiated to allow covalent interactions following the generation of free radicals. Some proteins (especially smaller molecules), bind very differently to the various makes of plate, whereas others seem to be ‘plate-indifferent’. For radioassays, use flexible polystyrene plates which can be easily cut into separate wells for

counting. For enzyme assays, use flat-bottomed plates with good optical as well as binding properties.

Particles Cellulose, Sephadex, Sepharose, Sephacryl and many other particles are available as supports that

have the advantage of a large surface area for derivatization. For assay quantification, the particles must be recovered from suspension by centrifugation or, alternatively, the use of particles containing iron oxide, that can be rapidly sedimented with a magnet prior to decantation of the supernatant.

Papers and membranes Cellulose comes in a convenient form as everyday paper, small discs of which can be coupled

with antigen. Nitrocellulose membranes have a strong surface charge and bind proteins tightly, a property taken advantage of in Western blotting. They are suitable for the dot blotting of samples; the paper is blocked with non-specific protein such as albumin, then assayed with specific antibodies.

C H A P T E R 5: Immunoassay

For use in radioisotopes, the paper may be cut for counting. In enzyme assays, an insoluble coloured product is deposited on the membrane, which can be quantified in a densitometer.

ELISA optimization

ELISA systems are so robust that there is a strong temptation to select an ‘average’ set of conditions, find the assay that works, and stick with it. Marked increases in sensitivity and reproducibility can be obtained by optimizing each of the steps. Sittampalam et al. (1996) have applied experimental design techniques to optimize an ELISA to reproducibly measure in the 10–1000 pg/ml range.