Abstract
A classical radioimmunoassay for hypoxanthine was developed and validated. Hypoxanthine, being a low molecular weight compound (M.W. 136.11) is not immunogenic unless first attached to a macromolecule such as a protein. Derivatives of hypoxanthine were synthesised for this purpose. In addition to the standard preparation of 6-trichloromethyl purine, a derivative of this compound with a 4-carbon spacer arm was prepared, namely purine-6-carboxypropanamide. Hypoxanthine itself showed an unusual degree of stability, having no reactivity towards reagents when considered in either the keto or the enol form. The use of 6-chloropurine, a far more reactive analogue of hypoxanthine, resulted in the synthesis of a novel carboxymethoxylamine derivative, purine-6-carboxymethyl oxime. Four conjugates were prepared using ovalbumin as carrier protein. Hypoxanthine derivatives with a free carboxyl group were conjugated using the mixed anhydride method. 6-trichloromethyl purine was reactive enough to be coupled directly. Hypoxanthine-9-B-D-arabinofuranoside was coupled using the periodate oxidation method for sugar derivatives with vicinal hydroxyl groups. As the hapten was linked via the E-amino groups of lysine residues in each case, the molar derivatisation of each conjugate was calculated by measuring the number of free amino groups in the protein before and after conjugation using 2, 4, 6-trinitrobenzene sulphonic acid. With one of the conjugates ultraviolet spectrophotometric analysis, and calculation of the amount of purine removed during dialysis were also used for comparison and confirmation of the value obtained. The antisera were affinity purified and twelve reagents were compared for their ability to elute anti-hypoxanthine antibodies, whilst retaining immunoreactivity of the eluted fractions. For the determination of hypoxanthine by radioimmunoassay two phase separation systems were investigated, namely chemical precipitation of antigen-antibody complexes using ammonium sulphate, and adsorption of free hypoxanthine using activated charcoal. The antisera were shown to be highly specific for hypoxanthine, with cross-reactivities to six analagous compounds being < 0.1%, and crossreactivity to two further compounds, adenine and allopurinol being 1.9 and 3.2% respectively. Recovery of hypoxanthine added to samples was of the order of 97.3%, and the limit of detection was > 150 nmole/g. Inter-assay coefficient of variation for the data points for the hypoxanthine standard curve was < 10% for hypoxanthine concentrations below 125 nmole/ml. Inter-assay coefficient of variation for samples of fish extract containing hypoxanthine was approximately 12%. Hypoxanthine levels increased with time in samples of trout and whitebait, so that its concentration was indicative of quality, but hypoxanthine levels in liver decreased with length of storage time. Hypoxanthine concentrations in fish samples, as determined by radioimmunoassay, were compared with the values obtained using a well established spectrophotometric method. The correlation coefficient for the two methods was 0.84507 (n = 45) using hypoxanthine solutions extracted from whitebait and 0.93298 (n = 33) for samples of trout muscle, so establishing the radioimmunoassay as a technique for measuring the quality of such foods.