An enzyme-linked immunosorbent assay for measuring anti-sheep erythrocyte antibodies

A simple ELISA assay for detecting murine anti-SRBC antibodies of IgG class was developed and the variation of the results according to different experimental conditions was investigated. Erythrocytes were left to settle in flexible plastic microtiter plates, after which they were fixed with glutaraldehyde and the remaining binding sites in the plates saturated with ovalbumin. Serum or monoclonal IgG antibodies were then allowed to react with the erythrocytes. Protein A coupled to alkaline phosphatase caused a color change in the subsequently added enzyme substrate. The results proved to be of good reproducibility, specificity and sensitivity. The assay can be used for measuring IgG concentration, estimating antibody avidity and number of antigenic determinants on the SRBC, as well as screening IgG anti-SRBC hybridomas. The precision of concentration estimates was very good when standard curves were used.     

Quantitative enzyme-linked immunosorbent assay (ELISA) for non-enzymatically glycated serum protein

A competitive ELISA for quantitative determination of glucitollysine, the reduced hexose alcohol form of glucose conjugated to the epsilon amino group of lysine was developed. We applied it to measure non-enzymatically glycated serum proteins. The antiserum obtained by immunizing guinea pigs with reductively glycated human albumin was capable of identifying and quantitating glucitollysine residues of serum proteins in normal and diabetic subjects after reduction of the proteins with sodium borohydride. The ELISA assay developed here had satisfactory reproducibility as judged by the intra-assay precision of 2.3-7.6% and the interassay precision of 6.7-9.8%. Results from this assay procedure correlated well with those from the radioimmunoassay and the boronate affinity chromatography procedure. The data suggested that diabetic serum proteins contained at least three times as much immunochemically detectable glucitollysine residues as normal serum proteins after reduction of the proteins with sodium borohydride. This method allows to quantitate glucitollysine residues on any of the proteins that have been implicated in the pathological sequelae of diabetes.     

Quantitative enzyme-linked immunosorbent assay (ELISA) for hirudin

A competitive ELISA assay has been developed that permits reproducible quantitation of the anticoagulant hirudin in buffer and urine. Coupling of peroxidase and hirudin was performed with the heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio)-propionate. In both solvents the lower limit of sensitivity was 8 ng hirudin/ml (0.08 AT-U) while the upper limit was 7.7 micrograms/ml (78.45 AT-U).     

Direct binding enzyme-linked immunosorbent assay (ELISA) for serum amyloid A (SAA)

A solid-phase, direct binding ELISA for serum amyloid A (SAA) proteins is described, in which noncovalent interactions of SAA with other plasma constituents are disrupted to permit direct coating of the wells of flexible polyvinyl chloride microtitration plates with an amount of SAA antigen proportional to its concentration in plasma. The wells are coated overnight at 60 degrees C with plasma diluted in 3 M potassium bromide and 0.1 M sodium bicarbonate. pH 9.6. The next day, any remaining sites on the wells are blocked by incubation for 1 h at ambient temperature with a 5% solution of dry milk solids and 0.05% Tween 20 in 0.02 M phosphate buffer, pH 7.4. The wells are rinsed and incubated for 90 min at 37 degrees C with polyclonal rabbit or rat anti-human SAA antiserum. Then, the wells are rinsed and incubated with goat anti-rabbit or rat IgG antiserum to which has been conjugated horseradish peroxidase. o-phenylenediamine and hydrogen peroxide substrates are added to the wells, color is allowed to develop, and sulfuric acid is added to stop the enzyme-catalyzed reaction. The amount of SAA coated to wells is quantified by absorbance at 490 nm. Four or more serial three-fold dilutions of plasma samples are assayed simultaneously on separate plates. Each plate contains a set of wells with identical concentrations of SAA standard protein diluted in decreasing concentrations of plasma proteins corresponding to the dilution of sample. The method can detect SAA concentrations in plasma samples ranging from 1 microgram/ml to greater than 1000 micrograms/ml. The method is suited to serial monitoring of SAA concentration in patients undergoing treatment for inflammatory conditions and to the quantitative analysis of large numbers of samples.     

Estimation of serum IgE by an enzyme-linked immunosorbent assay (ELISA)

A reliable and relatively simple method for the estimation of serum IgE is presented. It is a modification of radioimmune inhibition methods in which the marker is an enzyme, alkaline phosphatase, not radioactivity. The method does not require radioactivity or expensive equipment, and the reagents are stable for long periods of time. As presented it has a minimum sensitivity of about 10 ng. per milliliter. The reproducibility of the method is ±3 per cent and for correlation with radioimmunoassay ? = 0.97.     

Rapid detection of antigen-specific mouse IgE by enzyme-linked immunosorbent assay (ELISA)

A rapid, sensitive, antigen-specific mouse IgE capture ELISA is described. A monoclonal rat anti-mouse IgE was used as the capture antibody, and a DNP-coupled BSA-biotinylated conjugate along with a peroxidase-avidin-biotin complex was utilized as the detection system. The lower detection limit of this assay is 8.5 ng/ml of antigen-specific IgE. With some modifications, this assay can be employed to screen for antigen specific antibodies of other isotypes and subtypes.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of chlamydial antibody in duck sera

An enzyme-linked immunosorbent assay (ELISA) for the detection of chlamydial antibody in duck sera is described. The assay was used to test sera from a flock of domestic Pekin ducks infected with Chlamydia psittaci, and sera from wildfowl (Mallard). Sera from 63% of the domestic birds and 75% of the wildfowl exhibited chlamydial antibody titres of >/= 1/32, indicative of current or past chlamydial infection. There was a good correlation between the results obtained with the ELISA and those obtained by a micro-immunofluorescence test. The application of the ELISA to large scale screening of flocks is suggested.     

An enzyme-linked immunosorbent assay (ELISA) for quantification of mouse surfactant protein D (SP-D)

Surfactant protein D (SP-D) is a pattern recognition molecule of the collectin family of C-type lectins. It is found in the airways and at mucosal surfaces. SP-D is part of the innate immune system where it neutralizes and leads to elimination of microorganisms. It regulates the functions of other innate immune cells, such as macrophages and neutrophils. It also modulates the adaptive immune response by interacting with antigen-presenting cells and T cells. Monoclonal anti-mouse-SP-D antibodies were raised from SP-D deficient mice using recombinant SP-D as antigen. Ten monoclonal antibodies were characterized and validated for use in sandwich enzyme-linked immunosorbent assay (ELISA). Based on two of these, we established an ELISA that allows for measurements of mouse SP-D in various body fluids. The final ELISA was optimized and calibrated with a standard of purified recombinant mouse SP-D, which was analyzed by quantitative amino acid analysis. The ELISA was validated with respect to parallelism, recovery, intra- and inter-assay variation. The practical working range was estimated to be 1.9–200 ng/ml. We measured SP-D in lung lavage to an average concentration of 435 ng/ml (3-ml lavage), and in mouse vaginal fluid (1-ml lavage) to an average concentration of 94 ng/ml, but could not detect SP-D in the serum or plasma of healthy mice (< 3.8 ng/ml). With this ELISA, it will be possible to study the regulation of SP-D in various mouse models and upon various stimuli.     

A solid-phase enzyme-linked immunosorbent assay (ELISA) for detection of antibody to rubella virus.

An enzyme-linked immunosorbent assay was established for detection of antibodies to rubella virus. In this system commercially available rubella antigen was attached to the wells of polystyrene microtitre plates after which sera were added and incubated to allow the formation of antigen-antibody complexes. The presence of bound antibody was detected by adding anti-human globulin coupled to horseradish peroxidase and visually observing the colour change produced after addition of an appropriate substrate. The test was reproducible and simple to perform and had a similar sensitivity to the widely used haemagglutination inhibition system.     

Automation of enzyme-linked immunosorbent assay (ELISA).

A prototype of automatized enzyme-linked immunosorbent assay (ELISA) in tubes is described, using a commercially available basic material, easily modified. Nine hundred samples could be completely studied in a day by only one person. The different steps of the automatized ELISA were systematically studied to obtain the best performance. Its application is described in toxoplasmosis serodiagnosis.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of trypanosomal antigens in goat serum using a monoclonal antibody.

An IgM murine monoclonal antibody (MAb) TEA 1/23.3.4.6 raised against circulating trypanosome antigens was used in a sandwich ELISA to assay trypanosomal antigens in a trypanosome lysate preparation and in sera from goats infected with Trypanosoma brucei evansi. As little as 1.25 ug/ml of trypanosomal antigen could be detected by this assay. Following infection, trypanosomal antigens were first detected in goat serum 24 hours after the intravenous (i/v) or 6 days after the intramuscular (i/m) inoculation of trypanosome parasites. Antigen levels remained detectable during the course of infection. After treatment with diminazene aceturate, antigens dropped to undetectable levels between day 12 to 41, suggesting that this assay offers a promising approach to the diagnosis of African Trypanosomiasis.     

Rapid enzyme-linked immunosorbent assay (ELISA) for Aspergillus fumigatus antibodies.

A rapid enzyme-linked immunosorbent assay (ELISA) where component incubation periods were shortened to one hour, was compared with agar gel double diffusion (AGDD) and a standard ELISA procedure for detecting antibodies to Aspergillus fumigatus in 28 asthmatic patients with suspected allergic aspergillosis. Using two A fumigatus antigens the rapid ELISA compared well with AGDD and the standard ELISA method. Eleven sera that reacted with both antigens in AGDD were all positive against antigen 1 in both forms of ELISA, but two failed to react with antigen 2 in the standard ELISA and three failed to react with this antigen in the rapid method. Thirteen AGDD-negative sera were also negative in both forms of ELISA. The rapid ELISA provides a sensitive and reproducible test for routine serological investigation of allergic aspergillosis.     

The development of an enzyme-linked immunosorbent assay (ELISA) for cephalexin.

Cephalexin was structurally modified by the attachment of a spacer at the carboxylic acid through which it was subsequently covalently attached to BSA. This method permitted the molecule to be attached without cleavage of the beta-lactam ring giving a conjugate distinct from previously described immunogenic preparations of penicillins and cephalosporins. This approach required the development of a novel spacer molecule, and its synthesis and characterisation are reported. Rabbits were used to raise antisera and the antibodies produced were characterised with respect to their reactivity with cephalexin and various analogues, other cephalosporins, and a number of penicillins.     

Statistical considerations in the quantitation of serum immunoglobulin levels using the enzyme-linked immunosorbent assay (ELISA)

Several methods for analyzing ELISA data have been evaluated using optical density values derived by reacting serial two-fold dilutions of a rhesus monkey (Macaca mulatta) reference serum with dilutions of peroxidase-conjugated monospecific antisera to human IgG and IgM in the micro-ELISA 'sandwich' technique using microplates coated with appropriately diluted antisera to human IgG and IgM. Representation of optical density as a linear function of log serum dilution was shown to be inappropriate and potentially misleading. Weighted non-linear least squares analysis of a 4-parameter logit was demonstrated to be inappropriate because it required the specification of a somewhat arbitrary variance function and weight estimates varied substantially depending on the function used. Representation of optical density as a 4-parameter logistic function with estimation carried out on the log scale was shown to be the most appropriate procedure for determining the concentration of antigens or antibodies by the ELISA method.     

Assay of factor VII antigen by enzyme-linked immunosorbent assay (ELISA)

This paper introduces a newly developed enzyme-linked immunosorbent assay (ELISA) method, using monospecific anti-human factor VII rabbit IgG, to quantify antigen levels of factor VII. The procedure is relatively simple, its method is remarkably specific and reproducible. Its sensitivity to detect factor VII antigen is as low as 0.015 U/ml, when factor VII antigen in 1 ml normal plasma is arbitrarily defined as 1 unit. The level of factor VII antigen in 4 patients with congenital factor VII deficiency and their family members was investigated through this ELISA and the results correlated well with those obtained by electroimmunoassay previously described. The significant correlation between the antigen levels and activity of factor VII was demonstrated in 4 patients with congenital factor VII deficiency and their family members, normal individuals and patients with liver disease. However, the level of factor VII antigen in patients treated with warfarin was higher than their coagulation activity.     

Comparison of enzyme-linked immunosorbent assay (ELISA) technique and complement-fixation test for estimation of cytomegalovirus IgG antibody.

The ELISA technique has been found to be reliable for the detection and titration of cytomegalovirus-specific IgG antibody in serum. It is about six times more sensitive than the CF test although some discrepancies were found between the antibody titres determined by the two methods.     

Development of a sensitive monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for mitozantrone

A mouse monoclonal antibody (NO-1) with specificity for the anti-cancer drug mitozantrone (MZ) (Novantrone) was produced by immunization of a BALB/c mouse with mitozantrone-keyhole limpet haemocyanin (MZ-KLH) conjugate. When used in an indirect competitive enzyme-linked immunosorbent assay (ELISA), NO-1 permitted the accurate and reproducible detection of between 0.25-50 ng/ml of MZ in pooled human serum, the standard curve obtained within this range being virtually linear. The assay demonstrated good reproducibility with intra-assay coefficients of variation (CV) of between 1.41% and 7.02% and an inter-assay CV of 3.45%. Regression analysis of levels of MZ detected by ELISA vs. the actual amounts added to pooled human serum gave a very good correlation coefficient of r = 0.995. NO-1 showed no cross-reactivity with either bisantrene or daunorubicin. A simple pharmacokinetic study was undertaken in rabbits given MZ intravenously at a dose of 0.5 mg/kg of body weight. Levels of MZ in rabbit serum measured with the assay ranged between 82 and 170 ng/ml for rabbits 1 and 2, respectively at 15 min falling to 1.25 ng/ml by 48 h for rabbit 1 and falling to undetectable levels by 120 h for rabbit 2.     

A novel enzyme-linked immunosorbent assay (ELISA) for the detection of beryllium antibodies

A novel immunological method has been developed for detecting antibodies (IgG molecules) specific to beryllium, a light metal used in industry and capable of causing chronic beryllium disease. Beryllium metal was vacuum deposited onto commercially available immunological microsticks, which were then exposed to test plasma containing the putative antibodies. Antigen-antibody complexes were located using a biotin-avidin amplification method. One employee diagnosed with chronic beryllium disease and one diagnosed as "sensitized" (lymphocyte transformation positive) exhibited antibody titers graphically and statistically different and higher than a pooled baseline control population. Plasma from these two employees (former beryllium workers) was used in four different approaches to validate the presence of beryllium antibodies. The assay proved to be reproducible.