Protein Protocols Protein Protocols

344 Wisdom
4. Glutaraldehyde.
5. 50 mM Tris-HCl buffer, pH 7.5, containing 1 mM MgCl 2, 0.02% NaN 3, and 2% bovine
serum albumin (BSA).
3. Methods
1. Add 0.5 mg of IgG antibody in 100 µL of PBS to 1.5 mg of alkaline phosphatase.
2. Add 5% glutaraldehye (about 10 µL) to give a final concentration of 0.2% (v/v), and stir
the mixture for 2 h at room temperature.
3. Dilute the mixture to 1 mL with PBS and dialyze against PBS (2 L) at 4°C overnight (see Note 1).
4. Dilute the solution to 10 mL with 50 mM Tris-HCl buffer, pH 7.5, containing 1 mM MgCl2, 0.02% NaN3, and 2% BSA, and store at 4°C (see Notes 2,–4).
4. Notes
1. Dialysis of small volumes can be conveniently done in narrow dialysis tubing by placing
a short glass tube, sealed at both ends, in the tubing so that the space available to the
sample is reduced. Transfer losses are minimized by carrying out the subsequent steps
in the same dialysis bag. There are also various microdialysis systems available
commercially.
2. The conjugates are stable for several years at 4°C as the NaN3 inhibits microbial growth
and the BSA minimizes denaturation and adsorption losses. These conjugates should not
be frozen.
3. Purification of the conjugates is usually unnecessary; however, if there is evidence of the
presence of free antibody it can be removed by gel filtration in Sepharose CL-6B
(Amersham Pharmacia Biotech, Uppsala, Sweden) or a similar medium with PBS as
solvent.
4. The efficacy of the enzyme-labeled antibody may be tested by immobilizing the appropriate
antigen on the wells of a microtiter plate or strip, incubating various dilutions of the
conjugate for a few hours, washing the wells, adding substrate, and measuring the amount
of product formed. This approach may also be used for monitoring conjugate purification
in chromatography fractions.
References
1. Engvall, E. and Perlmann, P. (1972) Enzyme-linked immunosorbent assay, ELISA. III.
Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulin in antigencoated
tubes. J. Immunol. 109, 129–135.
2. Mahan, D. E., Morrison, L., Watson, L., and Haugneland, L. S. (1987) Phase change
enzyme immunoassay. Analyt. Biochem. 162, 163–170.