Enzyme labelling of immunoglobulins

4.6 Enzyme labelling of immunoglobulins

Improvements in the technology of derivatization and detection of enzymes are such that their sensitivity has come to match that of radioisotopes for many applications. Moreover, their safety has been greatly improved, beyond that of radioisotopes, by the replacement of the commonly used carcinogenic substrates. Technically, enzyme labels compare favourably with fluorescent probes in that they do not require special microscopes and darkened rooms for visualization, and offer both speed and simplicity in detection compared with radioisotopes.

4.6.1 Conjugation of horseradish peroxidase

Peroxidase is a glycoprotein with about 18% carbohydrate that is not necessary for its enzymic activity. This carbohydrate can be converted to aldehyde groups by oxidation with sodium peri- odate. The periodate aldehyde can then form Schiff bases with immunoglobulin.

MATERIALS Horseradish peroxidase IgG fraction of antiserum or monoclonal antibody (8 mg/ml in carbonate buffer)

0.1 M sodium periodate 0.001 M acetate acetic buffer, pH 4.4

0.1 M sodium carbonate buffer, pH 9.5 Sodium borohydride (4 mg/ml in distilled water)

0.1 M borate buffer, pH 7.4 Glycerol Bovine serum albumin (BSA)

METHOD

1 Dissolve 4 mg horseradish peroxidase in 1 ml distilled water.

2 Add 200 µl freshly prepared sodium periodate solution and stir gently for 20 min at room temperature. The mixture should turn greenish brown.

3 Dialyse overnight at 4°C against sodium acetate buffer.

Continued on p. 130

4.6ENZYME LABELLING OF IMMUNOGLOBULINS

4 Add 20 µl of sodium carbonate buffer to raise the pH to approximately 9–9.5 and immediately add 1 ml (8 mg) of the protein to be conjugated.

5 Leave at room temperature for 2 h with occasional stirring.

6 Add 100 µl of freshly prepared sodium borohydride solution (4 mg/ml in distilled water) and leave for 2 h at 4°C. This reduces any free enzyme.

7 Dialyse against borate buffer.

8 To store, add an equal volume of 60% glycerol in borate buffer and store at 4°C. (Carrier protein such as BSA may be added to 1% w/v if required.)

TECHNICAL NOTES • This conjugate should be stable for at least 1 year. • Although it is not usually necessary to separate conjugated immunoglobulin from uncon-

jugated immunoglobulin or enzyme, this may be done by gel filtration on Sephacryl S-200.

4.6.2 Conjugation of alkaline phosphatase

Alkaline phosphatase can generally be successfully conjugated to immunoglobulin with glutaraldehyde.

MATERIALS AND EQUIPMENT Alkaline phosphatase suspension in ammonium sulphate Phosphate-buffered saline (PBS) IgG fraction of antiserum (minimum initial concentration 2 mg/ml in PBS) Glutaraldehyde, 25% v/v in PBS (Caution: Wear protective glasses)

0.1 M tris(hydroxymethyl)-aminomethane (Tris)–HCl buffer, pH 7.4 Bovine serum albumin (BSA) Sodium azide Dialysis tubing

Note: Azide is a dangerous chemicalado not discard down the sink. Alkaline phosphatase is supplied in various formats. This method assumes that the enzyme was

purchased as an ammonium sulphate precipitate and the salt must be removed by dialysis. The method is easily adapted for use of dried or PBS solution of the enzyme by omitting the dialysis step.

METHOD

1 Place 5 mg alkaline phosphatase in a test tube, centrifuge and discard supernatant.

2 Add 2 mg of the IgG fraction of antiserum to the enzyme pellet.

3 Dialyse overnight at 4°C against PBS.

4 Adjust to 1.25 ml with PBS.

5 Add 10 µl of 25% v/v glutaraldehyde. Mix and allow to stand for 2 h at room temperature.

6 Dialyse exhaustively against PBS; five changes of 1 litre at 4°C.

7 Dialyse for 8 h against two changes of Tris–HCl buffer.

8 Adjust volume to 4 ml with Tris–HCl buffer containing 1% BSA and 0.02% sodium azide.

C H A P T E R 4: Antibodies as probes

4.6.3 SPDP conjugation of enzymes

Very effective conjugates between antibody and enzymes can be made using the heterobifunc- tional cross-linking reagent N-succinimidyl-3-(2-pyridylthio) propionate (SPDP). This reagent can be coupled separately to both antibody and enzyme through free amino groups on the proteins. The resultant antibody–pyridyldithio groups can then be reduced to thiol groups and disulphide linked to the enzyme–pyridyldithio groups with the release of 2-pyridinethione (Fig. 4.4).

–NH 2 (HRP) O

SPDP

HRP N C (CH 2 )

HRP–PDP

Dithiothreitol reduction O HRP N C CH 2 CH 2 + SH S

A HRP with free sulphydryl group

(b)

Immunoglobulin (Ig) + SPDP

Ig–PDP

Formation of disulphide linkage between HRP and Ig

Fig. 4.4 Disulphide coupling of enzyme–immunoglobulin conjugates. The amino groups of a protein may be reacted with N-succinimidyl-3-(2-pyridylthio) propionate (SPDP) to form a PDP derivative, which in turn may be reduced with dithiothreitol to form sulphydryl groups. This modified protein may be reacted with other PDP-bearing proteins and coupled via disulphide linkages.

4.6ENZYME LABELLING OF IMMUNOGLOBULINS

MATERIALS AND EQUIPMENT

0.2 M phosphate–saline coupling buffer, pH 7.5 IgG fraction of antiserum (2 mg/ml in coupling buffer) Horseradish peroxidase (1.0 mg/ml in coupling buffer) N-succinimidyl-3-(2-pyridylthio) propionate (SPDP) Methanol

1 M dithiothreitol

0.1 M iodoacetamide (stock) Sephadex G-25 column for rapid buffer exchange Rotator Bovine serum albumin (BSA)

METHOD

1 Dissolve 3 mg of SPDP in 0.3 ml methanol. This solution must be prepared freshly each time.

Carry out steps 2–4 A and B in parallel.

2 Add 50 µl SPDP solution to the horseradish peroxidase (0.5 mg in 0.5 ml coupling buffer).

3 Rotate gently for 1 h at room temperature.

4 Isolate the labelled enzyme on the Sephadex G-25 buffer-exchange column equilibrated with coupling buffer.

2 Add 8 µl of SPDP solution to the antibody (1 mg in coupling buffer).

3 Rotate for 1 h at room temperature.

4 Isolate the labelled antibody on the Sephadex G-25 buffer-exchange column equilibrated in coupling buffer (see Appendix B.1).

5 Measure the volume of antibody solution and add 1 M dithiothreitol to a final concentration of 50 m M .