The production of antibodies to pentosanpolysulfate (ELMIRON, SP-54).

Pentosanpolysulfate (PPS) represents the product obtained after sulfation of xylan and is composed of beta 1----4-D-xylopyranose residues sulfated at C2 and C3. Studies have shown that this compound can often be effective in relieving the symptoms of interstitial cystitis (IC). In order to elucidate the mode of action of PPS in IC, a sensitive and reliable assay was needed. To this end we prepared an immunogenic form of PPS by coupling it to methylated bovine serum albumin (MBSA). This complex was used to immunize NZW rabbits (1 mg, IM). Four of five animals responded with anti-PPS antibodies, three of which had high titer (greater than 1/2000) as measured by an enzyme-linked immunosorbent assay (ELISA). All sera were routinely absorbed with an MBSA-Sepharose immunoadsorbent to remove anti-MBSA antibodies. ELISA inhibition tests were used to determine the sensitivity and specificity of the sera. At least 50 ng/ml of PPS could be routinely detected by this assay. A number of naturally occurring proteoglycans, polysaccharides, monosaccharides and disaccharides were examined for reactivity with the antibodies but only heparin was an effective inhibitor. Absorption with heparin immunoadsorbents reduced, but did not eliminate, the ability of heparin to inhibit anti-PPS binding. This activity could be destroyed by treatment with heparinase without affecting PPS inhibition. Normal urine did not affect the ELISA or ELISA inhibition tests and thus allowed the determination of PPS levels in IC patient urines. Initial analysis of seven IC patients receiving oral PPS revealed urine concentration of 0.8-16.0 micrograms/ml. No inhibition could be detected in pre-treatment urine samples.

A monoclonal antibody that recognizes 2,3-, 2,6-, and 4,6-disulphate ester ring substitution in pyranose-containing polysaccharides. Its production, characterization and application for the quantitation of pentosan polysulphate, dextran sulphate, glycosaminoglycan polysulphate and chondroitin sulphate E.

A method is described for the preparation of a monoclonal antibody (MAb) which binds specifically to polysaccharides which contain 2,3-, 2,6- and 4,6-disulphate ester pyranose ring substitution. Such molecules include the semisynthetic heparinoids, pentosan polysulphate (PPS), dextran sulphate (DS) and glycosaminoglycan polysulphates (GAGPS), as well as the naturally occurring polysulphated polysaccharides, chondroitin sulphate E, and the 2,6-disulphated galactoses of carrageenans. The antibody (MAb 5-B-10) did not significantly cross-react with other sulphated polysaccharides such as heparin, heparan sulphate, the chondroitin sulphates, A, B, C or D, or keratan sulphate. No cross-reactivity was found with non-sulphated polysaccharides or polyanions including hyaluronic acid, xylan, or DNA. MAb 5-B-10 was characterized as IgM and kappa light chains, and was to develop an amplified enzyme-linked immunosorbent inhibition assay (ELISIA) to detect and quantitate some of the polysulphated polysaccharides in biological fluids. Using this assay, the lower limits of detection of these compounds in serum were in the order of 50 ng/ml; however 50% inhibition was obtained between 200-500 ng/ml. The intra- and inter-assay coefficients of variation for PPS were 4.2 +/- 2.8 and 16.7 +/- 13.8% respectively. The MAb 5-B-10 and the ELISIA were used to determine the levels of PPS in plasma of three healthy non-fasted volunteers for up to 120 min post-intravenous infusion (1.0 mg/kg). The results obtained compared favourably with kinetic data reported by others using a competitive binding assay method for this drug.