Assay Diluent Research Articles

Development of enzyme-linked immunosorbent assay for detection of alkylphenol polyethoxylates and their biodegradation products

An enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative analysis of alkylphenol polyethoxylates (APnEOs) and their biodegradation products. To generate a specific monoclonal antibody (mAb) for the ELISA, hybridoma cells were produced by the fusion of mouse myeloma cells and spleen cells from mice immunized with nonylphenol polyethoxylate (NPnEO) derivatives coupled to bovine serum albumin. The developed ELISA showed the detection limits of 16 and 30 microg/L NP10EO when 10% and 60% (v/v) methanol solutions were used as assay diluent. The mAb was shown to be specific to APnEOs and their metabolites, such as short-ethoxy-chain APnEOs and alkylphenoxy carboxylic acids, except for nonylphenol. Moreover, no response was observed with non-APnEO surfactants as well as other compounds structurally similar to APnEOs. The percentage river water recoveries of 85-118% were obtained for 10 microg/L NP10EO fortification after preconcentration by C18 solid-phase extraction. The ELISA was also validated by comparing it with high-performance liquid chromatography for the analysis of APnEOs and their metabolites in river samples; the correlation coefficient between the values obtained by these assays was 0.96.

Analytical and Clinical Performance of the Immulite Cardiac Troponin I Assay

Cardiac troponin I (cTnI) is now regarded as one of the most specific markers for cardiac injury, and an increasing number of commercial methods are available. We report here a study of the Immulite cTnI assay manufactured by DPC. The Immulite cTnI assay is a sandwich immunoassay that uses a monoclonal antibody immobilized on beads and a goat polyclonal antibody labeled with alkaline phosphatase as a tracer (1); both antibodies recognize epitopes localized in the N-terminal part (residues 33–110) of the protein. The chemiluminescent substrate used for the enzymatic reaction is an ester of adamantyl dioxetane phosphate. We used as calibrators purified human cTnI in horse serum. The within- and between-run imprecisions (CV) were <3.9% and 3.6%, respectively, at a mean cTnI of 1.5 μg/L (n = 20). The minimal cTnI value quantified was 0.2 μg/L, and no detectable cTnI was observed in 20 runs of assay diluent. The lowest cTnI concentration …

Susceptibility of Vibrio cholerae O139 to antibody-dependent, complement-mediated bacteriolysis.

Volunteer studies with Vibrio cholerae O1 have shown that the best correlate of a vaccine's protective efficacy is its propensity to elicit serum bactericidal responses in its recipients. Attempts to detect such responses following infection with V. cholerae O139, however, have met with varying success. Using a tube-based assay which involves viable counting, we now report that strains of serogroup O139 can appear to be sensitive or resistant to a fixed concentration of complement in the presence of antibody, depending on assay conditions. Susceptibility to lysis is critically dependent on the availability of complement, but with O139 indicator strains this is not simply determined by the concentration of serum added to the reaction mix. The nature of the assay diluent and the concentration of indicator bacteria can also dramatically affect bactericidal end points, whereas such variables have minimal significance with O1 indicator bacteria. Although some laboratories use unencapsulated mutant strains to seek evidence of seroconversion following exposure to V. cholerae O139, this is not necessary, and our findings question the significance of capsule expression as a determinant of complement sensitivity when antibody is present. The medium used for growth of the indicator strain and the particular strain used appeared to be unimportant. Each of seven O139 isolates tested was found to be lysed by antibody and complement in our standard assay system, which allowed the detection of significant serum bactericidal responses in 9 of 11 cases of O139 disease.

Radioimmunoassay for the determination of alosetron in human urine and saliva.

The development of a radioimmunoassay (RIA) for the sub-ng ml-1 determination of alosetron, a potent and selective 5HT3 receptor antagonist, in human urine and saliva is described. The antiserum was raised in Soay sheep following primary and booster immunizations with an immunogen prepared by conjugating alosetron-p-azobenzoic acid to bovine serum albumin (BSA). The radioligand consisted of alosetron specifically 125-iodinated on the 2-position of the imidazole group. The mean (+/- standard deviation) theoretical sensitivity (minimum detectable dose corresponding to the imprecision of the zero standard) of the RIA is 3.2 +/- 2.6 pg ml-1 (n = 12) of alosetron in assay diluent (0.1% m/v gelatine-0.05% m/v sodium azide in 0.1 mol l-1 phosphate buffer solution, pH 7.4). The working calibration range using 0.1 ml samples of saliva and 20-fold diluted urine is 0.10-6.40 ng ml-1 of alosetron. Urine samples were diluted prior to assay to overcome adverse matrix effects; consequently, the lower limit of quantification for undiluted urine is 2.0 ng ml-1 of alosetron. Inter- and intra-assay bias and imprecision over the working calibration range were generally < +/- 12% and < 13%, respectively, except at the 0.10 ng ml-1 alosetron level, where the corresponding values were < +/- 17.3% and < 20.2%. The antiserum was free from adverse cross-reactivity with either a synthetic precursor of alosetron or with four major metabolites of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Reactivation of creatine kinase activity by preincubation with buffered NAC diluent.

We have shown that several commercial control sera containing reversibly inactivated creatine kinase are reactivated by pre-incubation in N-acetyl cysteine assay diluent. The increase in activity can proceed for up to 4 h. This reactivation is not seen in patients' sera even after storage for 14 days at -20 degrees C, 7 days at 4 degrees C, or 2 days at 20 degrees C, during which time no loss of activity was evident. This study brings into question the validity of using consensus values to standardise enzyme assays.

Evaluation and Clinical Application of a Two-Site Immunoradiometric Assay for Alpha-1-Foetoprotein Using Readily Available Reagents

The development and evaluation in routine clinical practice of a simple, rapid, high-throughput, immunoradiometric assay (IRMA) for alpha-foetoprotein (AFP) in serum and amniotic fluid is described. The assay uses readily available reagents; a radioiodinated mouse monoclonal antibody produced by the authors and the immunoglobulin G (IgG) fraction of a polyclonal sheep anti-human-AFP serum covalently linked to Sepharose CL-4B. Both antibodies are distributed by the Scottish Antibody Production Unit (Law Hospital, Carluke). The results of an extensive comparative trial between the IRMA and a radioimmunoassay (RIA) are presented. A high-dose 'hook' effect is not a problem in this system. Quantitative recovery was obtained, independent of the assay diluent. The IRMA has high sensitivity and a wide working range. Improved assay precision allowed a clinically significant change in the intervention limits.

Specific High-Performance Liquid Chromatographic Determinatioin of Ampicillin in Bulks, Injectables, Capsules, and Oral Suspensions by Reverse-Phase Ion-Pair Chromatography

Abstract A rapid, specific, stability-indicating high-performance liquid chromatographic (HPLC) method has been developed for the assay of Ampicillin in Ampicillin Trihydrate bulk, capsules and oral suspensions and Sodium Ampicillin bulk and injectables. The assay is specific for Ampicillin in the presence of possible contaminants; Penicillin V, Phenylglycine, and 6-Aminopenicillanic Acid (6-APA); the degradation product, Penicilloic Acid of Ampicillin; and all excipients present in the formulations assayed. Ampicillin, Ampicillin formulations, and formulation excipient blends were force-degraded to further demonstrate specificity. The assay is precise, accurate, linear over the range 50% to 125% of expected Ampicillin sample level, and stability-indicating toward the described thermal, acid, base, aqueous, and light degradations. The procedure employs an ion-pairing eluent with UV detection at 254 nm. Ampicillin Trihydrate and Sodium Ampicillin bulk are stable in assay diluent for six hours allowing the ...

The regulation of calcitonin in normal human plasma as assessed by immunoprecipitation and immunoextraction.

We have studied the secretion of calcitonin in normal adults with a new procedure of increased sensitivity for the measurement of the hormone in peripheral plasma. In this method, endogenous calcitonin is immunoprecipitated with specific antibodies from a 10-ml plasma sample. The calcitonin is then dissociated from the antibodies and extracted into alcohol. The alcohol is evaporated, and the calcitonin is recovered in assay diluent for subsequent radioimmunoassay. The procedure produces a fivefold increase in immunoassay sensitivity and eliminates plasma proteins which can produce spurious immunoassay effects. This procedure was used to measure calcitonin in normal adults. The mean (+/- SE) plasma calcitonin in 43 subjects was 10 (+/- 2) pg/ml. A+ the end of a 3-h calcium infusion (12 mg/kg), mean plasma calcitonin in 10 subjects had risen to 114 (+/- 21) pg/ml. In 11 subjects, a 10-min infusion of 150 mg of calcium caused calcitonin to rise to a mean concentration of 28 (+/- 7) pg/ml at 20 min. EDTA infusion (50 mg/kg 2 h) caused a slight decrease in plasma calcitonin. These results are consistent with our previous reports of the low concentrations of calcitonin in adult plasma. Our data may underestimate calcitonin levels since not all of the heterogenous species of hormone may be extracted by this method. In any case, this procedure has allowed us to determine that the low concentrations of plasma calcitonin in normal adults are responsive to perturbations of calcium homeostasis. The immunoextraction method may be applicable to other assays in which it is necessary to increase sensitivity or define specificity.