4. A Sensitive ELISA-Based Assay for Glycosyltransferases


Bruce A. Macher~Professor of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132


This procedure allows the detection and quantification of glycosyltransferase activity using an ELISA-based procedure and carbohydrate-specific monoclonal antibodies (1-3). The procedure avoids the use of radiolabeled substrates, and provides a method for the detection of a specific product. All of the steps of the assay are done in a single microtiter plate and it is possible to recover the product for further structural analyses (3). Our procedure was done with glycolipid substrate acceptors. A similar procedure was described by Palcic and coworkers (4) using neoglycoprotein acceptors substrate. A strategy for further adaptation of the assay to a highly sensitive chemiluminescence assay has recently been reported (5).

 

Materials:

Glycoconjugate acceptor

Nucleotide sugar donor

Enzyme source

Microtiter plate

Carbohydrate-specific monoclonal antibody

Secondary antibody

ELISA reagents

Phosphate buffered saline (PBS, 20mM sodium phosphate, 150mM NaCl, pH7.4)

Bovine serum albumin (BSA)

 

Protocol:

1. Glycoconjugate substrate is allowed to absorb to the surface of the microtiter plate wells.

2. Prepare reaction mixture including appropriate nucleotide sugar, buffer, and cofactors (e.g. metal ions) required for the glycosyltransferase of interest.

3. Add reaction mixture to microtiter well and initiate assay by adding enzyme source.

4. Incubation proceeds at room temperature or higher for appropriate time.

5. At the end of the incubation period, the reaction mixture is removed and the well containing unreacted substrate acceptor and product is washed several times with phosphate buffered saline (PBS).

6. The wells are treated with a 5% solution of BSA in PBS at room temperature to block nonspecific binding of antibodies. (Note: This step can be eliminated if the enzyme preparation is a crude homogenate).

7. A carbohydrate-specific monoclonal antibody (primary antibody) is added to each well and incubated at room temperature for 1 hr. or overnight at 4C. The well is washed with PBS.

8. A secondary antibody (e.g. biotinylated goat anti-mouse) is added to the wells and incubated for 30-60 min. at room temperature, followed by a PBS wash.

9. Detection of antibody binding to the glcosyltransferase product is done by the addition of avidin and a biotinylated enzyme (e.g. alkaline phosphatase) to the wells and incubation at room temperature.

10. Excess biotinylated enzyme is removed by washing the wells with PBS and the amount of bound biotinylated enzyme is measured by adding appropriate substrate (e.g. p-nitrophenyl phosphate) and monitoring product formed at 405 nm using an ELISA plate reader.

11. Absorbance is converted to units of product formation using a standard curve for the primary antibody. The standard curve is obtained by carrying out the ELISA procedure with known quantities of a glycoconjugate recognized by the carbohydrate-specific monoclonal antibody.

Note: This assay procedure can also be used to monitor the activity of glycosidases (1).

References:

(1) Stults, C.L.M., Wilbur, B.J., and Macher, B.A. (1988) Enzyme-linked immunosorbent assay (ELISA)-based quantification and identification of in vitro enzyme-catalyzed glycosphingolipid synthesis and degradation products with carbohydrate sequence-specific monoclonal antibodies. Analytical Biochemistry 174: 151-156.

(2) Stults, C.L.M., and Macher, B.A. (1990) Measurement of -galactosyltransferase activity in cell extracts with an ELISA-based assay. Archives of Biochemistry and Biophysics 280:20-26.

(3) Stults, C.L.M., and Macher, B.A. (1993) 1-3N-acetylglucosaminyltransferase in human leukocytes: Properties and role in regulating neolacto glycosphingolipid synthesis. Archives of Biochemistry and Biophysics 303: 125-133.

4) Crawley, S. C., Hindsgaul, O., Alton, G., Pierce, M., and Palcic, M. M. (1990) An enzyme-linked immunosorbent assay for N- acetylglucoaminyltransferase-V. Analytical Biochemistry 185:112-117.

5) Yan, L.Y., Smith, D.F., and Cummings, R.D. (1994) Determination of GDP-Fuc:Gal 1-4GlcNAc-R (Fuc to GlcNAc)1, 3fucosyltransferase activity by a solid-phase method Analytical Biochemistry 223: 111-118.

 


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