Enzyme immunoassay for oxytocin

A competitive, double antibody enzyme immunoassay for oxytocin in a heterologous system was developed. Horseradish peroxidase was conjugated with oxytocin using N-succinimidyl 3-(2-pyridyldithio) propionate, and rabbit anti-oxytocin serum was produced by immunization of oxytocin-bovine serum albumin complex which was prepared by the carbodiimide method. The sensitivity of the assay was 4 microIU/tube, which corresponded to 10 microIU per ml using 400 microliters of the sample which was extracted from the same volume of plasma by means of SEP-PAK C18 cartridges. The coefficients of variation for different levels of oxytocin ranged from 6.8-15.9% and 8.5-16.7%, for intra- and inter-assay. Recovery of oxytocin added to plasma after extraction was 99-117%. No or little cross-reaction with arginine- and lysine-vasopressin was found. Plasma oxytocin concentrations determined by the proposed enzyme immunoassay were well correlated with those determined by radioimmunoassay (r = 0.90).     

Enzyme channeling immunoassay: A new homogeneous enzyme immunoassay technique

A homogeneous enzyme immunoassay has been developed in which an antigen and glucose-6-phosphate dehydrogenase are coimmobilized on agarose beads. Binding of hexokinase-labeled antibody to the bead-bound antigen results in an accelerated conversion of glucose, ATP, and NAD⁺ to 6-phosphogluconolactone, ADP, and NADH. Critical parameters affecting assay response are discussed.     

Enzyme immunoassay of serum testosterone.

An enzyme immunoassay of serum testosterone using the testosterone-glucoamylase complex is described. Testosterone was estimated by the enzyme immunoassay after extraction with hexan: ether (4:1) for serum from men and additional thin layer chromatographic step for serum from women. The within and between assay errors, measured as the coefficient of variation were 11.1 percent (n=8) and 12.0 percent (n=12). The sensitivity of this assay was 0.25 ng. The mean testosterone concentration (+/- SD) in 19 normal men and 4 normal cycling women were 5.3 +/- 1.8 and 0.52 +/- 0.12 ng/ml, respectively. The level of testosterone found by the present assay compared favorably with those obtained by other methods.     

Enzyme immunoassay of HBeAg employing beta-D-galactosidase

An enzyme-linked immunosorbent assay (ELISA) system for hepatitis B e antigen (HBeAg) was developed employing beta-D-galactosidase conjugated with antibody to HBeAg (anti-HBe) and using m-maleimidobenzoyl-N-hydroxysuccinimide ester as the coupling reagent. The experimental conditions for quantitative assay of HBeAg were determined. The presence of rheumatoid factor in test sera did not affect the results. This assay system is more sensitive than the micro-Ouchterlony method and as sensitive as radioimmunoassay. The use of beta-D-galactosidase for ELISA in the field of virology is recommended.     

Enzyme immunoassay of thromboxane B2

An immunoassay for thromboxane B2 was developed in which the hapten molecule was labeled with beta-galactosidase. The immunoprecipitate formed after competition between enzyme-labeled and unlabeled thromboxane B2 was subjected to a fluorometric assay of beta-galactosidase. Thromboxane B2 was detectable in the range of 0.1-30 pmol. Both enzyme immunoassay and radioimmunoassay showed essentially the same cross-reactivities with other prostaglandins and their metabolites when the same antibody was used. Known amounts of thromboxane B2 were added to human plasma, and the sample was applied to an octadecyl silica column. The extract was analyzed by enzyme immunoassay to examine the correlation between the added (x) and measured (y) thromboxane B2 (y = 1.09x + 11.07 pmol/ml, r = 0.99). A satisfactory correlation was observed between radioimmunoassay (x) and enzyme immunoassay (y) (y = 0.92x + 4.64 pmol/ml, r = 0.96). The validity of enzyme immunoassay was also confirmed by gas chromatography-mass spectrometry of a dimethylisopropylsilyl ether derivative of thromboxane B2 methyl ester. The method was applicable to the assay of thromboxane B2 produced from endogenous precursor during thrombin-induced aggregation of human platelets.     

Enzyme immunoassay of ampicillin in milk

An indirect immunoassay for quantitative determination of ampicillin (range, 10-1000 ng/ml) in buffer or milk has been developed. Polyclonal antibodies were obtained against ampicillin conjugated with bovine serum albumin; the conjugate was synthesized by direct condensation using carbodiimide. The antibodies were specific for ampicillin and exhibited low cross-reactivity to other penicillins (azlocillin, 17%; penicillin G, 10%; piperacillin, 5%; and carbenicillin, 4%). Matrix effects were minimized by combining the use of a casein-supplemented buffer (content of casein, 1%) with sample dilution. The threshold of ampicillin detection in milk (diluted tenfold) was equal to 5.0 ng/ml (which corresponded to 50 ng/ml of the original sample).     

Enzyme modulator mediated immunoassay (EMMIA)

1. Enzyme modulator mediated immunoassay (EMMIA) is a separation-free (homogeneous) enzyme amplified immunoassay. 2. The assay is based on the ability of an analyte labeled enzyme modulator to modify the activity of an indicator enzyme and on ability of an anti-analyte antibody to abrogate the modifying action of the analyte-labeled enzyme modulator upon its binding to anti-analyte antibody. 3. The principle of EMMIA is elaborated in detail. The stages in the development of an EMMIA are described. 4. Criteria in selecting enzymes and modulators suitable for EMMIA are discussed. 5. Examples of EMMIA's using different kinds of modulators for different classes of analytes are presented.     

Enzyme immunoassay for determination of sulfamethoxypyridazine in honey

An enzyme immunoassay technique for the detection of sulfamethoxypyridazine in honey, developed using rabbit polyclonal antibodies raised against N-sulfonyl-4-aminobutyric acid, which contains a structural group characteristic of sulfonamides, is proposed. Under the optimized conditions, the sulfamethoxypyridazine detection limit was 0.05 ng/ml, with the entire analysis procedure taking 2 h. In total, 24 honey samples were tested using the protocol based on tenfold dilutions of samples without their preliminary treatment.     

Enzyme immunoassay of (24R)-brassinosteroids

Brassinosteroids are a new group of phytohormones that are widely distributed in plants and play an important role in the processes of plant growth and development. Physiological concentrations of brassinosteroids in plants are extremely low, and their analysis in organs and tissues is very difficult. This study is devoted to the chemical aspects of elaboration and to bioanalytical parameters of an immunoenzymatic system for quantitative determination of the phytohormones 24-epicastasterone and 24-epibrassinolide.     

Enzyme immunoassay of (24R)-brassinosteroids

Brassinosteroids are a new group of phytohormones that are widely distributed in plants and play an important role in the processes of plant growth and development. Physiological concentrations of brassinosteroids in plants are extremely low, and their analysis in organs and tissues is very difficult. This study is devoted to the chemical aspects of elaboration and to bioanalytical parameters of an immunoenzymatic system for quantitative determination of the phytohormones 24-epicastasterone and 24-epibrassinolide.     

Enzyme immunoassay for the determination of hexestrol in meat

An indirect competitive enzyme-linked immunosorbent assay (ELISA) of hexestrol (HES), an anabolic hormone forbidden for use in livestock farming, has been developed. Conditions of ELISA have been optimized by varying the concentrations of the coating conjugate (HES-ovalbumin), anti-HES antiserum, casein, and Tween 20. In the absence of Tween 20 in the reaction mixture, the detection limit (IC₁₀) equaled 0.01 ng/ml, IC₅₀ equaled 0.17 ng/ml, and the working range (IC₂₀–IC₈₀) equaled 0.03–0.86 ng/ml, while, in the presence of 0.05% Tween 20, these values equaled 0.05, 2.9, and 0.26–32.0 ng/ml, respectively. Standard deviation of the analysis results did not exceed 5.4%. If ELISA was performed in the absence of detergents, the recovery value upon HES determination in spiked beef samples ranged from 74 to 147%.     

Enzyme immunoassay for detection of Salmonellae in foods.

An enzyme immunoassay was developed to detect Salmonella in foods. Indirect test protocols were developed for use with microtitration plates or Gilford microcuvettes. Samples from enrichment cultures were mixed with H-specific immunoglobulin G and allowed to react; unbound antibody was removed by three 5-min centrifugation washes; goat anti-rabbit antibody conjugated to alkaline phosphatase was added and allowed to react; and unbound conjugate was removed by centrifugation washing as before. Salmonella-positive samples were indicated by the production of a chromogenic reaction product after the addition of alkaline phosphatase substrate. The color could be read visually or quantified by absorbance. Ninety-eight food samples were examined to compare the enzyme immunoassay with enrichment serology, immunofluorescence, and the Food and Drug Administration pure culture technique. The enzyme immunoassay was sensitive and specific, and it possessed advantages over methods currently in use. Furthermore, when the enzyme immunoassay was used to screen preenrichment media, the results indicated that it might be decidedly more sensitive than the conventional pure culture technique.     

Enzyme immunoassay for plasma estradiol using a monoclonal antibody

A microtitre plate enzyme immunoassay (EIA) for plasma estradiol is described, involving competition between sample estradiol and an immobilized estradiol-bovine serum albumin complex for a monoclonal anti-estradiol antibody, followed by immobilized antibody quantitation using enzyme-labelled antiglobulins. The assay dose-response curve covered a range of 6-1500 fmol/well. The intra- and inter-assay coefficient of variation for the assay of three plasma pools ranged from 3.1 to 4.7% and from 4.7 to 10.6% respectively. The assay showed satisfactory correlation with a standard estradiol radioimmunoassay. Pre-coated microtitre plates were stable, dried, at 4 degrees C for up to 3 months and the anti-estradiol was stable to lyophilization and also was stable in solution at 4 degrees C for up to 1 month.     

The Determination of Phaseic Acid by Monoclonal Antibody-Based Enzyme immunoassay

A monoclonal antibody PA3-2-B3, IgG₁ (Λ) is described which specifically recognizes phaseic acid and shows very little cross-reactivity (0.14%) with abscisic acid or dihydrophaseic acid (0.88%). Based on this antibody, an enzyme immunoassay was developed which displays a linearity range from 15 pg to 3 ng of phaseic acid. Results obtained with this assay agree with those obtained by gas chromatography-electron capture detection. Using the novel enzyme immunoassay, as well as an established immunoassay for abscisic acid, levels of these two compounds in leaves of Phaseolus vulgaris were determined as a function of plant age, water stress, recovery from stress, and feeding of abscisic acid through the transpiration stream. The production of phaseic acid in a microsomal system from bean leaves was demonstrated. The results show a regulation of the plant's capacity to metabolize abscisic acid to phaseic acid as a function of water stress.     

Enzyme immunoassay for serum 18-hydroxycorticosterone and its clinical application

An enzyme immunoassay for serum 18-hydroxycorticosterone was established using alkaline phosphatase as a label. The antiserum for 18-hydroxycorticosterone was produced by immunization of rabbits with 18-hydroxycorticosterone 3-(O-carboxymethyl)oxime conjugated to bovine serum albumin. Sephadex LH-20 column chromatography was used to separate 18-hydroxycorticosterone from other steroids in serum samples. The minimal detectable amount of 18-hydroxycorticosterone was 50 pg/tube and the measurable range was from 5 to 1000 ng/dl when a 1.0 ml serum sample was used. Intra- and inter-assay coefficients of variance were 5.0% (n=6) and 5.8% (n=6), respectively. Four of 5 patients with aldosterone-producing adenoma had above-normal serum 18-hydroxycorticosterone levels.     

Enzyme immunoassay of the receptors for modified low density lipoprotein

Mouse macrophages (line J 774) were incubated with monospecific goat anti-low density lipoprotein antibodies, which were conjugated to horseradish peroxidase (AB-HRP). Addition of low density lipoprotein (LDL) modified by treatment with malondialdehyde to cultures of these cells resulted in a dose-dependent increase in the amount of cell-associated enzyme activity. The concentration curve was hyperbolic with half-saturation of modified LDL at a concentration of about 3 micrograms/ml. This effect was completely blocked by polyinosinic acid and was not observed in experiments with human fibroblasts, which do not exhibit high affinity binding sites that recognize chemically modified LDL. Our data indicate that receptor-mediated endocytosis of AB-HRP in the presence of native or modified LDL may be used as very simple, efficient, and sensitive assay for investigation of the scavenger receptors for modified LDL.     

Enzyme immunoassay for cortisol in serum using cortisol 21-amine

Cortisol 21-amine (21-amino-11β,17-dihydroxy-4-pregnene-3,20-dione) was prepared and an enzyme immunoassay for cortisol in serum was established using cortisol 21-amine conjugated with alkaline phosphatase. The minimal amount of cortisol detected was 1ng/tube and the measurable range was from 1 to 80 μg/d1, using 10 μ1 of serum sample. This enzyme immunoassay satisfied the standard criteria of dilution, accuracy and precision. The values correlated well with those obtained by radioimmunoassay. This enzyme Immunoassay is applicable to the routine determination of serum cortisol in any clinical laboratory. Cortisol 21-amine was found to be a useful derivative for preparing cortisol-enzyme conjugate in enzyme immunoassay.