Immunoenzymoassay of the hyaluronic acid-hyaluronectin interaction: Application to the detection of hyaluronic acid in serum of normal subjects and cancer patients

Abstract

The binding of a hyaluronic acid-binding glycoprotein, hyaluronectin (HN), isolated from human brain, to hyaluronic acid (HA) was investigated with the enzyme-linked immunosorbent assay technique using plastic microtest plates coated with a 50 mg/liter solution of HA in 0.1 m bicarbonate. Optimum conditions for HN binding to HA were in 0.2 m NaCl buffered with 0.1 m sodium phosphate at pH 7. An assay for HA in solution was set up exploiting the fact that HN binding could be inhibited by soluble HA. HA was preincubated for 1 h in a test tube with a 30 ng/ml HN solution ( v v ) in the buffer containing 0.1% bovine serum albumin. Incubation on HA-coated microtest plate lasted 4 h and maximum sensitivity was achieved when incubation was carried out at 4°C. HN bound to the plate was revealed by means of alkaline phosphatase-conjugated anti-HN antibodies. The test was used to measure HA inhibitory activity after depolymerization by ferrous ions. No difference was found between inhibitory activity of smaller fragments and that of high-molecular-weight HA. The assay was applied to determination of HA in sera. Specificity was demonstrated by Streptomyces hyaluronidase digestion of reactive material in sera. Other glycosaminoglycans did not interfere with the assay. Recovery of HA was good and intra- and interassay variation cofficients were 6±2.2 and 12%. In 103 blood donor sera, HA was found at 22.4 ± 16.7 μg/liter. HA was elevated in most of the cancer patient sera tested.