Practical automation and interpretation of quantitative assays of antibodies to therapeutic proteins, illustrated with human growth hormone.

To quantify concentrations of anti-growth hormone antibody in less than 600 serum samples by radioimmunoassay, we devised a system ("BOLAC") to process data and to execute the curve-fitting program LIGAND-PC automatically with an IBM PC-compatible computer. We fit data from each sample to four binding and one or two-antibody binding sites. Total antibody concentration is then calculated from the model that is statistically "best". This process occasionally selects a two-binding-site model that severely overestimates the antibody concentration. Errors of this kind are discarded by constraining the product of the second-site antibody's affinity and its concentration to exceed a minimum value (0.05). We evaluated the performance of the BOLAC system by assaying controls and by using computer simulations to demonstrate the high confidence levels attainable in estimation of antibody concentrations. Between-assay variability (CV) was less than 25%, and analytical recovery exceeded 90%. These figures are acceptable for an assay based on curve-fitting of competitive radioimmunoassay data, allowing clinically relevant assessments of antibody responses in patient's samples. The advantages of the BOLAC system include high throughput and the reporting of results in absolute units of affinities and concentrations.

Stability of frozen solutions of cefamandole nafate.

The chemical, microbiological and visual stability of frozen solutions of cefamandole nafate was studied. Solutions of cefamandole nafate were prepared by diluting 1 g of drug with 3 ml of Water for Injection, USP, or 0.9% Sodium Chloride Injection, USP, or 5% Dextrose Injection, USP (i.m. dilutions); or with 50 or 100 ml of the latter two diluents (i.v. dilutions). Stability of samples stored in glass and polyvinyl chloride plastic containers for up to 52 weeks at -10 and -20 C was measured by microbiologic, polarographic, iodometric, nephelometric and chromatographic assay and pH was measured. In mice, LD50 tests were performed using the i.m. dilutions. I.M. dilutions of cefamandole nafate were stable for 52 weeks when stored at -20 C; at -10 C, however, some samples did not freeze completely and were turbid when thawed. I.V. dilutions were stable for 26 weeks when stored at -20 C. I.V. dilutions with D5W stored at -10 C developed a transient haze. A gradual decrease in pH, which was a function of storage time, was noted for the frozen solutions. Six months of freezing did not alter the LD50 in mice. Solutions of cefamandole nafate are stable for at least 26 weeks when stored at -20 C in glass or PVC containers.

Comparison of cefamandole nafate to cefamandole by microbiological assay.

Before microbiological assay, cefamandole nafate should be hydrolyzed for 1 h at 37 degrees C in pH 8 buffer (0.1 M) or for 30 min at room temperature in aqueous solutions containing 1.25 molar equivalents of sodium carbonate.

Substitued 5-nitro-1, 3-dioxanes: correlation of chemical structure and antimicrobial activity.

Various derivatives of 5-nitro-1, 3-dioxane were synthesized to determine the relative effect of chemical substitution in the 2-and 5-positions on broad spectrum antimicrobial activity. Each compound was evaluated quantitatively by calculation of a microbiocidal index, which measured the time to kill several different microorganisms. This test system indicated that 5-bromo-5-nitro substitution was essential for significant activity. Optimal activity was effected by 2-methyl substitution in the alkyl series and 2-hydroxy-phenyl substitution in the aryl series. The antimicrobial activity of the substituted dioxanes was not related directly to water solubility or hydrolysis to microbiocidal diols or aldehydes.

Effects of pesticides on nitrite oxidation by Nitrobacter agilis.

The influence of pesticides on the growth of Nitrobacter agilis in aerated cultures and on the respiration of N. agilis cell suspensions and cell-free extracts was studied. Two pesticides, aldrin and simazine, were not inhibitory to growth of Nitrobacter, but five compounds [isopropyl N-(3-chlorophenyl) carbamate (CIPC), chlordane, 1,1-dichloro-2,2-bis (p-chlorophenyl) ethane (DDD), heptachlor, and lindane] prevented growth when added to the medium at a concentration of 10 mug/ml. Whereas CIPC and eptam prevented nitrite oxidation by cell suspensions, the addition of DDD and lindane resulted in only partial inhibition of the oxidation. Heptachlor and chlordane also caused only partial inhibition of oxidation, but were more toxic with cell-free extract nitrite oxidase. None of the pesticides inhibited the nitrate reductase activity of cell-free extracts, but most caused some repression of cytochrome c oxidase activity. Heptachlor was the most deleterious compound.