An alternative method for Staphylococcus aureus DNA isolation

This study describes a rapid procedure for the isolation of genomic DNA from Staphylococcus aureus that yielded a good amount of high quality DNA for the amplification of staphylococcal enterotoxins genes (A, B, C, D, and E) and the TSST-1 gene as well as enzymatic restriction (HaeIII) from environmental isolates. With this method, it was possible to detect these genes in a sample containing as little as 10(5) cells with positive PCR reactions obtained from approximately 10pg of DNA in a final reaction volume of 25µl.

A double-monoclonal immunoenzymometric assay for alpha 1-fetoprotein modified for increased analytical precision.

We modified a one-step, two-site, double monoclonal immunoenzymometric assay (Abbott Laboratories) for serum alpha1-fetoprotein (AFP) to increase its sensitivity and improve test precision at the low end. We increased sample size, incubation interval, and reaction time and temperature, and decreased the final reaction volume. Interassay CVs for the modified method ranged from 6.2 to 8.0% for mean concentrations of AFP in serum of 5.2 to 34.2 micrograms/L--substantially better than those for the unmodified monoclonal method--and agreed well with those of the comparison method (modified Abbott polyclonal immunoenzymometric assay). AFP values by the modified monoclonal procedure (y) correlated well with results by the polyclonal method (x): y = 0.983x + 1.84 micrograms/L (r = 0.927, n = 59). The detection limit of the modified monoclonal test was 0.2 microgram/L, as compared with 1.0 and 1.4 micrograms/L, respectively, for the modified polyclonal and the unmodified monoclonal procedures. We recommend using the modified monoclonal method for monitoring cancer patients with low tumor burden.

A tableted enzymic reagent for salicylate, for use in a discrete multiwavelength analytical system (Paramax).

A fully enzymic reagent for determination of salicylate in serum has been developed for use in the Paramax analytical system. The assay, run as an equilibrium determination, is based on the reaction of salicylate with NADPH and oxygen in the presence of salicylate hydroxylase (EC 1.14.13.1) to form catechol, NADP+, carbon dioxide, and water. The reaction is complete within 7 min, after which time the resulting absorbance change at 340/405 nm is measured. The sample:reagent ratio is 1:60 (5 microL of sample in a 300 microL final reaction volume). A single 30-mg tablet contains all of the reactants with tableting excipients. The use of NADPH eliminates interferences from reactions involving NADH. The large sample:reagent ratio, high sensitivity, and choice of bichromatic wavelengths minimize sample error. Results are linearly related to salicylate concentration to 1500 mg/L. Precision (CV) is 1.7% at 540 mg/L and 2.4% at 280 mg/L.

Measurement of serum magnesium with a centrifugal analyzer.

We show how serum magnesium can be determined with a centrifugal analyzer. The method is based upon a manual procedure involving magnesium/calmagite complex formation in an alkaline reagent mixture. The assay, performed at 30 degrees C, requires 5 mul of serum in a final reaction volume of 405 mul, including 350 mul of reagent mixture. The stable color produced at 1 min is measured at 520 nm. Linearity studies showed that Beer's law was followed to 50 mg/liter. Average analytical recovery was 97.3%. Within-day studies showed CV's of 3.36% and 1.09%, compared to day-to-day variations of 5.32% and 3.04% at 18.8 and 46.6 mg of magnesium per liter, respectively. Correlation studies with the manual method, the Du Pont aca (methylthymol blue compleximetric) procedure, and atomic absorption spectroscopy showed correlation coefficients of 0.996, 0.976, and 0.950, respectively. Results compare excellently with those by common present methods. The method is fast, economical, reliable, and applicable to small specimen volumes.

Measurement of Erythrocyte Glucose-6-Phosphate Dehydrogenase Activity with a Centrifugal Analyzer

We describe two improved methods for determination of erythrocyte glucose-6-phosphate dehydrogenase activity. One method is an"enzyme-linked" procedure in which an excess of 6-phosphogluconate dehydrogenase is used to produce two moles of NADPH for each mole of glucose-6-phosphate oxidized. In the other method 2,3-diphosphoglycerate is used to inhibit the variable contribution of endogenous 6-phosphogluconate dehydrogenase to glucose-6-phosphate dehydrogenase activity in erythrocyte lysates. These assays require 100 µl of blood and are performed on a centrifugal analyzer in a final reaction volume of 410 µl at 37 °C. NADPH formation is monitored at 340 nm. Hemoglobin is measured as oxyhemoglobin in the same reaction mixture used to determine enzyme activity by changing the wavelength to 550 nm. Results are expressed as international units of glucose-6-phosphate dehydrogenase activity per gram of hemoglobin. The coefficient of correlation between "enzyme-linked" assay and "standard" assay was .992, the slope of the regression line was 1.07, and the intercept was at -0.76. When results of the "enzyme-linked" assay were compared to those of the "nonlinked" assay with added 2,3-diphosphoglycerate, the slope of the regression line was .994, the intercept 0.109, and the correlation coefficient .994.