• Home
  • Search Results
  • The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum.

The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum.

The Biochemical journal (1987-06-15)
E M Bailyes, R N Seabrook, J Calvin, G A Maguire, C P Price, K Siddle, J P Luzio
ABSTRACT

1. Liver and bone alkaline phosphatase isoenzymes were solubilized with the zwitterionic detergent sulphobetaine 14, and purified to homogeneity by using a monoclonal antibody previously raised against a partially-purified preparation of the liver isoenzyme. Both purified isoenzymes had a specific activity in the range 1100-1400 mumol/min per mg of protein with a subunit Mr of 80,000 determined by SDS/polyacrylamide gel electrophoresis. Butanol extraction instead of detergent solubilization, before immunoaffinity purification of the liver enzyme, resulted in the same specific activity and subunit Mr. The native Mr of the sulphobetaine 14-solubilized enzyme was consistent with the enzyme being a dimer of two identical subunits and was higher than that of the butanol-extracted enzyme, presumably due to the binding of the detergent micelle. 2. Pure bone and liver alkaline phosphatase were used to raise further antibodies to the two isoenzymes. Altogether, 27 antibody-producing cell lines were cloned from 12 mice. Several of these antibodies showed a greater than 2-fold preference for bone alkaline phosphatase in the binding assay used for screening. No antibodies showing a preference for liver alkaline phosphatase were successfully cloned. None of the antibodies showed significant cross-reaction with placental or intestinal alkaline phosphatase. Epitope analysis of the 27 antibodies using liver alkaline phosphatase as antigen gave rise to six groupings, with four antibodies unclassified. The six major epitope groups were also observed using bone alkaline phosphatase as antigen. 3. Serum from patients with cholestasis contains soluble and particulate forms of alkaline phosphatase. The soluble serum enzyme had the same size and charge as butanol-extracted liver enzyme on native polyacrylamide-gel electrophoresis. Cellulose acetate electrophoresis separated the soluble and particulate serum alkaline phosphatases as slow- and fast-moving forms respectively. In the presence of sulphobetaine 14 all the serum enzyme migrated as the slow-moving form on cellulose acetate electrophoresis. Monoclonal anti-(alkaline phosphatase) immunoadsorbents did not bind the particulate form of alkaline phosphatase in cholestatic serum but bound the soluble form. In the presence of sulphobetaine 14 all the cholestatic serum alkaline phosphatase bound to the immunoadsorbents. 4. The electrophoretic and immunological data are consistent with both particulate and soluble forms of alkaline phosphatase in cholestatic serum being derived from the hepatocyte membrane.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
1-Butanol, anhydrous, 99.8%
Sigma-Aldrich
1-Butanol, 99.9%
Sigma-Aldrich
1-Butanol, ACS reagent, ≥99.4%
Sigma-Aldrich
1-Butanol, for molecular biology, ≥99%
Sigma-Aldrich
1-Butanol, suitable for HPLC, ≥99.7%
Supelco
1-Butanol, analytical standard
Sigma-Aldrich
Butyl alcohol, natural, ≥99.5%, FCC, FG
Sigma-Aldrich
1-Butanol, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99.5% (GC)
Sigma-Aldrich
1-Butanol, BioRenewable, ACS reagent, ≥99.4%
Supelco
1-Butanol, suitable for HPLC, 99.8%