Estradiol assay by microtitre plate enzyme immunoassay

Development of a simple enzyme linked immunosorbent assay (ELISA) for estradiol in serum extracts is described. The assay involves use of a 96-well microtitre plate, designed for immunoassay, as the support for a purified, high-titre antiserum, raised against estradiol-6(O)-carboxymethyloxime linked to bovine serum albumin, and using horseradish peroxidase-labelled estradiol-6-(O)-carboxymethyloxime as the labelled species, with 2,2'-azino-bis-(3-ethylbenzthiazoline sulfonic acid) diammonium salt (ABTS) as the chromogenic substrate. The assay characteristics rival those of radio- or chemiluminescence immunoassays for estradiol.     

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.     

Evaluation of a competitive antibody enzyme immunoassay for specific diagnosis of Chagas' disease

A competitive enzyme immunoassay based on the use of a monoclonal antibody (MAb) specific for "component 5" of Trypanosoma cruzi was evaluated. The antigenicity and immunogenicity of this component has been observed in natural and experimental infections. The studies were conducted in an area of Bolivia where mixed infections with Leishmania braziliensis are frequent and present a problem in the accurate diagnosis of T. cruzi infections. The specificity and sensitivity of this assay as compared to the indirect immunofluorescence and ELISA tests were demonstrated. The present test has proved to be more specific than the immunofluorescence and ELISA tests.     

Nonisotopic detection of RNA in an enzyme immunoassay using a monoclonal antibody against DNA-RNA hybrids

A sensitive nonisotopic solution hybridization assay for detection of RNA is described and characterized using a pSP65 plasmid model system. The assay procedure is based on a hybridization reaction in solution between a biotinylated DNA probe and a target RNA. The biotin-labeled hybrids are captured on a microtiter plate coated with an antibody to biotin. Bound DNA-RNA hybrids are detected by an immunoreaction with an enzyme-labeled monoclonal antibody specifically directed against DNA-RNA heteropolymers and the hybrids are quantitatively measured with the addition of a fluorogenic substrate. Optimal conditions under which to perform the assay were hybridization time, 1000 min; temperature, 75 degrees C; probe concentration, 0.2 microgram/ml; extent of probe biotinylation, 6.7%; buffer stringency, 2x SSC. A bisulfite-modified DNA probe was compared to nick-translated probes synthesized with reporter groups of different lengths (bio-11-dUTP or bio-19-dUTP). All probes could detect 10 pg/ml of target RNA. The presence of nonhomologous DNA or RNA sequences reduced the sensitivity of RNA detection by one half-log to 32 pg/ml (1.6 pg/assay).     

Quantitation of plasma membrane fatty acid-binding protein by enzyme dilution and monoclonal antibody based immunoassay

Summary A plasma membrane fatty acid-binding protein (h-FABPPm) has been isolated from rat hepatocytes. Analogous proteins have also been identified in adipocytes, jejunal enterocytes and cardiac myocytes, all cells with high transmembrane fluxes of fatty acids. These 43 kDa, highly basic (pl = 9.1) FABP_pm 's appear unrelated to the smaller, cytosolic FABP's (designated FABP's) identified previously in the same tissues. h-FABP_pm appears closely related to the mitochondrial isoform of glutamic-oxaloacetic transaminase (mGOT), and both the purified protein and liver cell plasma membranes (LPM) possess GOT enzymatic activity. From their relative GOT specific activities it is estimated that h-FABP_pm constitutes approximately 2% of LPM protein, or about 0.7 × 10⁷ sites per cell. A monoclonal antibody-based competitive inhibition enzyme immunoassay (CIEIA) for h-FABP_pm is described; it yields an estimate of 3.4 x 10⁷ h-FABP_pm sites per hepatocyte. Quantitated by either method, h-FABPPm appears to be a highly abundant protein constituent of LPM.     

Use of a monoclonal antibody for quantitation of rabies vaccine glycoprotein by enzyme immunoassay

The use of a monoclonal antibody specific for the envelope glycoprotein of rabies virus has been used in a direct enzyme immunoassay to quantify the glycoprotein content of rabies vaccines produced in two kinds of cell culture. The results of this direct enzyme immunoassay are well correlated with the in vivo NIH potency test. This test may be a useful tool to rapidly and accurately control the antigenic value of a vaccine. It could also be used for the "in process' control of vaccine production before deciding whether a vaccine batch may reasonably be subjected to the NIH potency test.     

Solid-phase enzyme immunoassay of urokinase using monoclonal antibodies

Using two monoclonal antibodies directed against urokinase, we have developed a micro enzyme-linked immunosorbant assay (ELISA) to detect and measure urokinase in biological fluids. The system presents the following characteristics: simple and rapid procedure, reproducibility, sensitivity (urokinase levels down to 1 ng/ml) and evaluation of the enzyme in biological fluids such as urine, pleural effusions, and ascitic fluids without preliminary purification.     

High-throughput determination of free D-aspartic acid in mammals by enzyme immunoassay using specific monoclonal antibody

A method for rapid determination of free d-aspartic acid (d-Asp) in mammals has been established using a highly specific mouse monoclonal antibody against d-Asp for the first time. An anti-d-Asp monoclonal antibody was obtained by the immunization of bovine-serum-albumin-conjugated d-Asp to BALB/c mice. The obtained antibody has a high specificity toward d-Asp but shows a slight cross-reactivity to all other d- and l- amino acids including l-Asp. The calibration range of the competitive enzyme linked immunosorbent assay (ELISA) is 0.016-16 μmol/mL d-Asp in rat serum samples. The precisions of this method were evaluated by inter-plate and intraplate assays, and the relative standard deviation values were 4.8% and 4.5%, respectively. The values of d-Asp determined by the present ELISA have a good correlation to those determined by high-performance liquid chromatography with the correlation coefficient of 0.963. Using this ELISA, the time course of d-Asp in the rat serum after intravenous administration was successfully demonstrated. The present method provides a simple and high-throughput determination of d-Asp in mammals, and is a useful tool for clarifying the physiological roles and diagnostic values of this d-amino acid.     

Development of a competitive enzyme immunoassay for factor VIII-related antigen

An enzyme immunoassay system basing on a competitive method has been developed to measure factor VIII related antigen (F. VIII R:Ag). A sufficient discrimination at low F. VIII R:Ag concentrations was gained. This method appears to be sensitive to 7,8 X 10(-3) U/ml F. VIII R:Ag showing an intraassay coefficient of variation (CV) of 0,11. In comparison to the commonly used Laurell electroimmunodiffusion assay for factor VIII significant less antisera per sample for the enzyme immunoassay technique is necessary.     

Measurement of phagocyte chemiluminescence using a microtitre plate luminometer

The use of chemiluminescence techniques to study the interaction between bacteria and phagocytes has been useful for examining the extent to which serum factors, such as opsonins, are important in internalization of the organisms and the response of the cell to phagocytosed bacteria. However, such methods have been limited by the number of experiments which can be performed at one time using most commercial luminometers. However, the recent introduction of the Amerlite microtitre plate luminometer allows the measurement of chemiluminescence responses in 96-well microtitre plates. Using this instrument, lucigenin-enhanced chemiluminescence can be detected from as few as 5000 cells (polymorphonuclear leukocytes or monocytes) per well with a 1:10 ratio of cells to zymosan particles opsonized with 10% serum. The opsonic capacity of up to 100 sera can be measured in triplicate wells in a single experiment using four microtitre plates and polymorphonuclear leukocytes prepared from less than 40 ml freshly obtained venous blood. We are currently using this technique to investigate the effect of serum opsonins on the interaction between normal human polymorphonuclear leukocytes and monocytes with mycobacteria of three species (Mycobacterium leprae, M. tuberculosis, and M. aviumintracellulare). Other possible applications of this method are discussed.     

An enzyme immunoassay for plasma betamethasone

A sensitive enzyme immunoassay for plasma betamethasone was developed using betamethasone-3-(O-carboxymethyl)oxime-β-D-galactosidase conjugate as a labelled antigen and 4-methylumbelliferyl-β-D-galactoside as a fluorescence substrate. The performances of the enzyme immunoassay were compared with that of a radioimmunoassay using ³H-betamethasone and the same antiserum. The minimal detectable level for the enzyme immunoassay was 0.15 pg/tube or 0.15 ng/ml of plasma, which was remarkably more sensitive than the radioimmunoassay level of 10 pg/tube or 2 ng/ml of plasma. The specificity was sufficient, in particular, the cross reactivity of cortisol was 0.008%. However, the precision of the enzyme immunoassay was inferior to that of the radioimmunoassay.     

Screening adenoviruses in stool samples: evaluation of a genus-specific monoclonal antibody based enzyme immunoassay

To screen fecal samples for adenovirus antigens a genus-specific monoclonal antibody based enzyme immunoassay was developed. In a comparative analysis with commercial latex agglutination test, high sensitivity was demonstrated. The assay did not detect other viruses usually found in faeces suggesting its specificity. One hundred and eighty stool samples collected in Baranya County were tested and 13 (7.2%) of them showed reactivity. The application of our immunoassay combined with other, more sophisticated methods may help us to determine the serotype specificity of these adenovirus isolates and assess the importance of adenoviruses in viral gastroenteritis.     

A microtitre plate assay for measuring glycosidase activity

Glycosidases perform a wide range of functions in physiology and pathology, and are potential targets for the treatment of diseases such as influenza, cancer, AIDS and diabetes. This paper reports a convenient discontinuous colourimetric assay for the measurement of glycosidase activity. The assay utilises 4-nitrophenyl- substrates and quantities of product are determined by measuring absorbance at 405 nm. This assay is performed in a 96 well microtitre plate and has been used to characterise the properties of seven different glycosidases from bacteria, yeast and higher eukaryotes and their kinetic parameters determined. Assays in the presence of known inhibitors showed that inhibition modes can be determined, and IC(50) and K(i) values calculated. This assay appears to be of widely applicable and of general utility for the measurement of glycosidase activity and the evaluation of inhibitors.     

A microtitre plate assay for measuring glycosidase activity

Glycosidases perform a wide range of functions in physiology and pathology, and are potential targets for the treatment of diseases such as influenza, cancer, AIDS and diabetes. This paper reports a convenient discontinuous colourimetric assay for the measurement of glycosidase activity. The assay utilises 4-nitrophenyl- substrates and quantities of product are determined by measuring absorbance at 405 nm. This assay is performed in a 96 well microtitre plate and has been used to characterise the properties of seven different glycosidases from bacteria, yeast and higher eukaryotes and their kinetic parameters determined. Assays in the presence of known inhibitors showed that inhibition modes can be determined, and IC₅₀ and K_i values calculated. This assay appears to be of widely applicable and of general utility for the measurement of glycosidase activity and the evaluation of inhibitors.     

Simultaneous competitive enzyme immunoassay for human chorionic gonadotropin.

A simultaneous competitive enzyme immunoassay (SICEIA) for hCG was developed using beta-D-galactosidase (beta-Gal) as a labelled enzyme and anti-hCG antibody coated sheep red blood cells (SRBC) as a solid phase. In this report, a new coupling agent, MCAE, was used to couple beta-Gal with hCG. The sensitivity was improved to the degree of 2.5 mIU/ml, equal to that of RIA. The present procedure was safer and rapider than RIA. The value of hCG in urine by our procedure had good correlation with that by RIA.     

Development of an enzyme immunoassay for cryptococcal antibody

An enzyme immunoassay (ELISA) for measurement of cryptococcal Ig G antibody in human serum is described. Clinical studies indicate that the assay is a useful addition to the currently available techniques for measuring antibodies in cryptococcosis. IgG-specific antibody (titers 4 to 1,024) was detected in the serum of 78 % of the cryptococcosis patients tested and in 61 % of the serum from healthy individuals with positive delayed skin hypersensitivity to cryptococcin. The micro-ELISA for cryptococcal antibody is of potential value in patient management, and in epidemiological studies.     

A one-step enzyme immunoassay for human manganese superoxide dismutase with monoclonal antibodies

A one-step enzyme immunoassay for the determination of manganese superoxide dismutase in serum has been developed with two kinds of monoclonal antibodies. Proposed method had high sensitivity (assay range, 0.4-200 ng/ml), good recovery (recovery percentage, 102.9-106.2%) and reproducibility (intraassay, C.V. = 1.87-3.66%; interassay, C.V. = 3.03-10.4%). From these results, it is possible to apply this method to routine clinical analysis and biochemical research with various purposes.     

Production and characterization of anti-nisin Z monoclonal antibodies: suitability for distinguishing active from inactive forms through a competitive enzyme immunoassay

As a pre-requisite to monoclonal antibody development, an efficient purification strategy was devised that yielded 72 mg of nisin Z from 14.5 1 of Lactococcus lactis subsp. lactis biovar. diacetylactis UL 719 (L. diacetylactis UL719) culture in supplemented whey permeate. Specific monoclonal antibodies (mAbs) were produced in mice against the purified nisin Z using keyhole limpet hemocyanin as a carrier protein. These antibodies did not recognize nisin A, suggesting that the asparagine residue at position 27 is involved in antibody recognition to nisin Z. However, the high reactivity of mAbs against biologically inactive nisin Z degradation products, produced during storage of freeze-dried pure nisin Z at -70 degrees C, indicated that the dehydroalanine residue at position 5 (Dha5), required for biological activity, is not necessary in nisin Z recognition by the mAb. A competitive enzyme immunoassay (cEIA) using the specific anti-nisin Z mAb was developed and used for rapid and sensitive detection and quantification of nisin Z in fresh culture supernatant, milk and whey. Detection limits of 78 ng/ml in phosphate-buffered saline, 87 ng/ml in culture supernatant, 106 ng/ml in milk and 90.5 ng/ml in whey were obtained for this assay. The cEIA using specific mAbs can be used to quantify nisin Z in food products.