Solid-phase radioimmunoassay for cell-surface antigens

5.5 Solid-phase radioimmunoassay for cell-surface antigens

Cells may be used in solid-phase assays; centrifugation and fixation are necessary to firmly attach the cells to the plastic surface. Enzyme detection systems can be used but care must be taken that cellular enzymes do not interfere with the assay. To avoid these problems, complete radiolabelled antibodies may be used as shown below.

5.5.1 Preparation of cell-coated assay plates

MATERIALS AND EQUIPMENT Cells carrying antigen of interest Glutaraldehyde Phosphate-buffered saline (PBS) PBS containing 5% w/v bovine haemoglobin and 0.2% w/v sodium azide Microtitre plate with U-shaped wells, flexible polyvinyl chloride

Note: Azide is a dangerous chemical—do not discard down the sink.

METHOD

1 Harvest the cells by centrifugation and wash three times in PBS by centrifugation (150 g for 10 min at 4°C).

2 Count and adjust the cell numbers to 2 × 10 7 /ml.

3 Dispense 50 µl aliquots of fresh 0.25% glutaraldehyde in PBS into each well of the microtitre plate.

4 Add 50 µl of cell suspension to each of 95 wells of the plate and centrifuge at 100 g for 5–10 min at 4°C. The 96th well is used as a control for non-specific binding in the final assay.

5 Remove the glutaraldehyde solution by tapping the inverted plate over a sink.

6 Flood the plate with PBS and roll a glass rod over the surface to remove air bubbles. Washing may also be performed by immersing the plate in a beaker of PBS.

7 Flood the plate with PBS containing bovine haemoglobin (5% w/v) and sodium azide (0.2% w/v). Again, roll a glass rod over the surface to remove air bubbles.

Continued on p. 174

5.5SOLID-PHASE RADIOIMMUNOASSAY FOR CELL-SURFACE ANTIGENS

8 Incubate the plate for 1 h at room temperature. This will saturate the protein-binding sites on the plastic.

9 The plates may be used immediately or stored for up to 10 weeks without removing the haemoglobin buffer.

TECHNICAL NOTES • Soluble proteins will adsorb directly to these polyvinyl plates. Add 50 µl of protein solution (at

50–200 fmol/ml) in PBS to each well and incubate for at least 1 h at room temperature. Remove the supernatant (keep for re-use) and wash the plate three times with PBS containing bovine haemoglobin (5% w/v) and sodium azide (0.2% w/v). The protein solution must be free of detergent as this will inhibit binding.

• Antibody may also be linked to the plate and used to adsorb viable cells which are then fixed with glutaraldehyde. • The relatively low concentration of glutaraldehyde used to fix the cells does not seem to alter surface antigens.

5.5.2 Radioiodinated anti-mouse immunoglobulin antibody

(see Section 4.9)

Either: Prepare antibody to mouse immunoglobulin by affinity chromatography and then label with 125

I using Iodogen. Or alternatively: Label anti-mouse immunoglobulin antibody while it is still attached to the affinity column using the chloramine T technique. Elute the 125 I-labelled antibodies with

0.2 m glycine–HCl buffer, pH 2.5, containing carrier protein.

5.5.3 Binding assay

MATERIALS AND EQUIPMENT Cultures of fused cells

Assay plates coated with cells Phosphate-buffered saline (PBS) PBS containing 5% w/v bovine haemoglobin, and 0.2% w/v sodium azide 125 I-labelled anti-mouse immunoglobulin antibody Plate sealers Vacuum trap for radioactive washings Nichrome wire, electrically heated, for cutting up plates Gamma spectrometer

METHOD

1 Remove the haemoglobin buffer by tapping the inverted test plate.

2 Remove 50 µl of supernatant from each hybrid well to be tested and transfer to the assay plate according to the following.

Continued

C H A P T E R 5: Immunoassay

Protocol. Well number

Test antigen

Antibody

125 I-anti-mouse immunoglobulin

Hybrid supernatant

Hybrid supernatant

Positive control*

Negative control*

Positive control*

* See Technical notes, item 3.

3 Incubate for 1 h at room temperature.

4 Wash the plate three times by immersing it in PBS and emptying it into a sink.

I labelled anti-mouse immunoglobulin antibody to each well and incubate for 1 h at room temperature.

5 Add 25 µl of haemoglobin buffer containing 5 × 10 4 c.p.m. 125

6 Remove the unbound radioactive antibody using a Pasteur pipette attached to a suction trap.

7 Wash five times by adding 3 drops of PBS to each well and then suck the solution into a vacuum trap.

8 Leave the plates to dry in a fume cupboard.

9 Cut up the tray with an electrically heated Nichrome wire to release the wells. For convenience, a plate sealer can be stuck to the bottom of the tray during cutting.