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b-Galactosidase Labeling of IgG Antibody

G. Brian Wisdom

1. Introduction

The Eschericia coli β-galactosidase (EC 3.2.1.23) is a large enzyme (465 kDa) with a high turnover rate and wide specificity which allows it to be measured conveniently in several different ways. Unlike several other enzyme labels β-galactosidase is not found in mammalian tissues or fluids hence background contributions are negligible when this label is measured at a neutral pH. This can be advantageous as peroxidase activities (due to various heme proteins) and alkaline phosphatase are frequently present in these samples.

The heterobifunctional reagent, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), is of value when one of the components of a conjugate has no free thiol groups eg. IgG. In this method (1) the IgG antibody is first modified by allowing the N-hydroxysuccinimide ester group of the MBS to react with amino groups in the IgG; the β-galactosidase is then added and the maleimide groups on the modified IgG react with thiol groups in the enzyme to form thioether links. This procedure produces conjugates with molecular weights in the range 0.6-1 ×106.

F(ab′)2 fragments of IgG have been labeled via the β-galactosidase’s

amino groups with sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohex-ane-1-carboxylate (2) and there are various other heterobifunctional reagents that may be used for β-galactosidase labelling (3).

2. Materials 1. β -Galactosidase from E. coli, 600 U/mg or greater (with 2-nitrophenyl-β -galacto-

pyranoside as substrate) (see Note 1).

From: The Protein Protocols Handbook, Third EditionEdited by: J.M. Walker © Humana Press, a Part of Springer Science + Business Media, LLC 2009

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678 Wisdom

2. IgG antibody. This should be the pure IgG fraction or, better, affinity-purified antibody from an antiserum or pure monoclonal antibody.

3. 0.1 M Sodium phosphate buffer, pH 7.0, containing 50 mM NaCl 4. MBS. 5. Dioxan. 6. Sephadex G25 (GE Healthcare Bio-sciences, Uppsala, Sweden and Piscataway,

NJ) or an equivalent gel. 7. 10 mM Sodium phosphate buffer, pH 7.0, containing 50 mM NaCl and 10 mM

MgCl2.

8. 2-Mercaptoethanol. 9. DEAE-Sepharose (GE Healthcare Bio-sciences) or an equivalent ion-exchange

medium.10. 10 mM Tris-HCl buffer, pH 7.0, containing 10 mM MgCl

2 and 10 mM 2-mercap-

toethanol.11. Item 10 containing 0.5 M NaCl.12. Item 10 containing 3% bovine serum albumin (BSA) and 0.6% NaN

3.

3. Methods 1. Dissolve 1.5 mg of IgG in 1.5 mL of 0.1 M sodium phosphate buffer, pH 7.0,

containing 50 mM NaCl. 2. Add 0.32 mg of MBS in 15 μL of dioxan, mix, and incubate for 1 h at 30°C. 3. Fractionate the mixture on a column of Sephadex G25 (approx. 0.9 × 20 cm) equil-

ibrated with 10 mM sodium phosphate buffer, pH 7.0, containing 50 mM NaCl and 10 mM MgCl

2, and elute with the same buffer. Collect 0.5 mL fractions, measure

the A280

and pool the fractions in the first peak (about 3 mL in volume). 4. Add 1.5 mg of enzyme, mix, and incubate for 1 h at 30°C. 5. Stop the reaction by adding 1 M 2-mercaptoethanol to give a final concentration of

10 mM (about 30 μL). 6. Fractionate the mixture on a column of DEAE-Sepharose (approx. 0.9 × 15 cm)

equilibrated with 10 mM Tris-HCl buffer, pH 7.0, containing 10 mM MgCl2 and

10 mM 2-mercaptoethanol; wash the column with this buffer (50 mL) followed by the buffer containing 0.5 M NaCl. (50 mL). Collect 3 mL fractions in tubes with 0.1 mL of Tris buffer containing 3% BSA and 0.6% NaN

3. Pool the major peak

(this is eluted with 0.5 M NaCl), and store at 4°C (see Notes 2 and 3).

4. Notes

1. The thiol groups of β-galactosidase may become oxidized during storage thus diminishing the efficacy of the labeling. It is relatively easy to meas-ure these groups using 5,5 -dithiobis(2-nitrobenzoic acid) (see Chapter 93); about 10 thiol groups per enzyme molecule allow the preparation of satisfactory conjugates.

b-Galactosidase Labeling of IgG Antibody 679

2. The conjugates are stable for a year at 4°C as the NaN3 inhibits microbial

growth and the BSA minimizes denaturation and adsorption losses. 3. The activity of the conjugate can be checked by the method described in

Note 4 of Chapter 64.

References 1. O’Sullivan, M. J., Gnemmi, E., Morris, D., Chieregatti, G., Simmonds, A. D.,

Simmons, M., Bridges, J. W., and Marks, V. (1979) Comparison of two methods of preparing enzyme-antibody conjugates: Application of these conjugates to enzyme immunoassay. Anal. Biochem. 100, 100–108.

2. Luz, Z., Gurlo, T., and von Grafenstein, H. (2000) Cell-ELISA using β-galactosidase conjugated antibodies. J. Immunol. Methods 234, 153–167.

3. Hermanson, G. T. (1996) Bioconjugate Techniques (Academic Press, San Diego), pp 229–248.