Immunodiagnosis of bovine fascioliasis by enzyme-linked immunosorbent assay and immunoprecipitation methods

Calves were infected with 1,000 Fasciola hepatica metacercariae and the antibodies which developed were monitored by gel diffusion, counterelectrophoresis (CEP) and the enzyme-linked immunosorbent assay (ELISA) using crude or tegument antigen preparations of the adult fluke. Serologic diagnosis was positive as early as 2-4 wk of infection by gel diffusion, by 4 wk by CEP, and by 6-8 wk by ELISA, whereas parasitologic diagnosis via the detection of fluke eggs in the feces was possible only after 8-10 wk of infection. In general, both gel diffusion and ELISA were superior to CEP in detecting active infection.     

An enzyme-linked immunosorbent assay for phytochrome

A double-antibody sandwich, enzyme-linked immunosorbent assay has been developed for phytochrome in Avena sativa L. cv. Saladin. An immunoglobulin fraction of rabbit antiserum raised to 118 kdalton phytochrome was used with alkaline phosphatase as the enzyme label. The assay detected as little as 0.2 ng phytochrome in extracts of dark-grown plant material. No evidence for specific or non-specific measurement of proteins other than phytochrome was found. The assay detected phytochrome in extracts of Avena grown in the light. Dilution curves for light-grown phytochrome extracts had a reduced slope and saturated at a lower level of enzyme activity than those for dark extracts. These differences were not caused by an inhibitor in extracts from light-grown plants. Phytochromes from dark- and light-grown plants may be immunologically different.     

An enzyme-linked immunosorbent assay for rat prolactin

A sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) for rat prolactin was developed using reagents from the National Institute of Arthritis, Diabetes, Digestive Diseases and Kidney. In this assay soluble prolactin and prolactin adsorbed to a solid-phase support compete for rabbit anti-prolactin antibody binding sites. Therefore, a high concentration of soluble prolactin in the sample will result in a low concentration of antibody immobilized to the adsorbed prolactin. The immobilized antibody-prolactin complex is detected and quantified using goat anti-rabbit immunoglobulin G covalently conjugated to the enzyme horseradish peroxidase. Assay parameters were optimized by investigating the concentration of reagents and the reaction kinetics in each of the assay steps. The assay can be performed in 24 h. A sensitivity range of 0.06 to 6 ng in the region of 90 to 10% binding was obtained. Near 50% binding (0.6 ng), the intraassay coefficient of variation (CV) was 4.2% and the interassay CV was 7.6%. The correlation between radioimmunoassay and the ELISA was 0.868. Selected applications of the assay are described. The assay should prove a useful alternative to the radioimmunoassay in those instances where steps involving the use of 125I become limiting, for example, iodination facility and gamma counter availability or prolonged reagent storage.     

A quantitative enzyme-linked immunosorbent assay for bovine herpesvirus type 1 (BHV-1) antibody

A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure antibody to bovine herpesvirus type 1 (BHV-1) in cattle sera. The optical density produced from a single dilution of test serum was compared with a standard curve and the results were read and printed out from a computer interfaced to a multichannel ELISA reader. The printed results were expressed in ELISA units. The ELISA results obtained on 370 cattle sera were compared with those of the serum neutralisation test (SNT). An agreement of 90.5% was obtained when reciprocal SNT titres equal to or greater than 4 and IgG ELISA units equal to or greater than 50 were taken as indicative of a specific reaction. Of the 370 sera, 35 gave discrepant results of which 21 were SNT positive/IgG ELISA negative and 14 were SNT negative/IgG ELISA positive. When the SNT positive sera negative in the IgG ELISA were tested in an IgM ELISA, 19 were found to be positive. Thus, when the IgG and IgM ELISA results were combined the overall agreement between the ELISA and SNT increased to 95.7%. The IgG ELISA had a sensitivity of 82.4% and specificity of 94.4% relative to the SNT, whereas the combined IgG and IgM ELISA results gave a sensitivity and specificity of 98.3% and 94.4% respectively. There was a good positive correlation between the two tests (r = 0.86).     

A quantitative enzyme-linked immunosorbent assay for Dermatophagoides pteronyssinus-specific immunoglobulin G

A monoclonal mouse anti-human IgG was used to develop an enzyme-linked immunosorbent assay (ELISA) for the measurement of Dermatophagoides pteronyssinus (DP)-specific IgG in human sera. This monoclonal antibody (HG2-25) binds to all subclasses of IgG but not to IgA, IgM, or IgE. For the assay, the DP antigen is coated firmly on polystyrene beads through physical adsorption and any leakable antigen is washed off. The assay gives satisfactory reproducibility and parallelism of the dilution curves. Using 0.1% human serum albumin as a substitute for the DP-specific IgG preabsorbed diluent gave extremely low backgrounds and high sensitivity. Horseradish peroxidase-labeled HG2-25 prepared with the optimum degree of conjugation and free of polymerized conjugates gave responses fairly proportional to the doses. This ELISA gives a satisfactory recovery and is not affected by nonspecific IgG levels in human sera.     

Dot-enzyme-linked immunosorbent assay (dot-ELISA) for the rapid diagnosis of human fascioliasis

A dot-enzyme-linked immunosorbent assay (dot-ELISA) was developed as a fast and field applicable antibody detection tool for the diagnosis of human fascioliasis. The assay is performed using partially purified antigens from a species of Fasciola at 180 ng protein/dot (2 microliters) and serum samples at 1:20 dilution (1 microliter). Dot-ELISA results completely agreed with those of micro-ELISA. Antigen-coated nitrocellulose sheets stored for 3 mo at -20 C showed results identical to fresh sheets. Sera from patients with fascioliasis (n = 30) and other parasitic or viral infections (n = 120) were compared with sera from healthy controls (n = 14). Ninety samples can be tested within 90 min. The sensitivity, specificity, and speed of the assay may justify its use in laboratory and field studies.     

Clickable enzyme-linked immunosorbent assay

Click chemistry is explored as a potential cost-effective and selective immobilization method for the production of an enzyme-linked immunosorbent assay (ELISA). Coatings were formulated containing either a terminal alkyne or a bicyclo[6.1.0]non-4-yne (BCN) chemical handle, and a diagnostic peptide was subsequently immobilized onto these coatings by the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) or copper-free strain-promoted azide-alkyne 1,3-dipolar cycloaddition (SPAAC), respectively. The terminal alkyne-containing coating showed high background levels in subsequent ELISA's due to the copper catalyst used in the immobilization step. The BCN-containing coating, however, was successfully employed and presents a cost-effective alternative to existing (strept)avidin-biotin immobilization methods. This technology was illustrated with an ELISA used for the diagnosis of rheumatoid arthritis (RA) but could be easily applied to a wide range of diagnostic tests.     

An enzyme-linked immunosorbent assay (ELISA) for plasma testosterone

A rapid, single extraction ELISA for testosterone in plasma is described, using a standard 96 well microtitre plate. Testosterone is covalently bonded to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to the ELISA plate, giving an immobilised antigen approach which simplifies subsequent assay standardisation for steroid hormone assays. The addition of standard, sample and first antibody (rabbit anti-testosterone), which is unique for each different assay, is followed by a general procedure which includes washing, addition of peroxidase labelled goat antirabbit IgG, further washing and finally, addition of o-phenylenediamine substrate with colour development and reading of the plate at 492 nm on an automatic ELISA processor. The ELISA assay is compared to a testosterone RIA with 125I-label and has similar specificity and precision to the latter with a quicker processing time, and is more cost effective. The added advantages that ELISA assays confer over RIAs in terms of isotope purchase and disposal make this an ideal procedure for use in a routine steroid laboratory.     

An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures

We report a quantitative method by which a single microculture can be examined for (i) cell morphology; (ii) cell number; (iii) DNA synthesis; and (iv) expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens.     

An enzyme-linked immunosorbent assay for quassin and closely related metabolites

A microtitration plate enzyme-linked immunosorbent assay has been developed for quassin and closely related seco-triterpenes. The method is much more sensitive than conventional chromatographic techniques and enables the routine analysis of large numbers of samples with a detection limit of 5 ng quassin. Antibody production was elicited by injecting a conjugate of iso-quassinic acid linked to bovine serum albumin into rabbits. Important features of the technique are (a) the use of a double-antibody, noncompetitive enzyme-linked immunosorbent assay for a low-molecular-weight, nonantigenic compound using microtitration plates; and (b) that the initial incubation of samples with primary antiserum can be carried out in buffer containing up to 20% ($\text{v}\text{v}$) methanol with minimal loss of sensitivity. The structural requirements for recognition of the hapten by the antiserum are considered based on the levels of cross-reaction found with a wide range of other quassinoids.     

An enzyme-linked immunosorbent assay (ELISA) for plasma cortisol

A direct ELISA for plasma cortisol is described which is carried out in a standard 96 well microtitre plate. In this ELISA cortisol-thyroglobulin conjugate is immobilised to the microtitre plate and competes with cortisol in the standard or plasma sample for antibody binding sites. Following washing the rabbit cortisol antibody bound to immobilised cortisol is incubated with peroxidase labelled goat antirabbit IgG. Following further washing o-phenylenediamine is added, colour developed, and the plate read at 492 nm on a standard ELISA plate reader. This ELISA shows good agreement with RIA and its sensitivity, specificity and precision allow its use in the routine steroid laboratory.     

An enzyme-linked immunosorbent assay for 6-keto PGF1 alpha

An enzyme-linked immunosorbent assay for 6-keto prostaglandin F1 alpha, a stable metabolite of prostacyclin, has been developed. The assay allows quantitation of 6-keto PGF1 alpha in the range 1-200 pg/0.1 ml and shows very low cross reactivity to nine other prostaglandins. Dose dependent stimulation by thrombin of 6-keto PGF1 alpha formation in human endothelial cells in culture has been used to verify the assay. Quantitation by the enzyme linked immunosorbent assay agrees closely with determination by radioimmunoassay.     

An enzyme-linked immunosorbent assay (ELISA) for bovine LH capable of monitoring fluctuations in baseline concentrations

Using a Fab'-horseradish peroxidase conjugate, an ELISA for bovine LH was developed and validated. It had good specificity (less than or equal to 0.2% cross-reactivity with FSH or TSH), was not susceptible to interference from plasma components and exhibited very low non-specific binding. It was carried out on microtitre plates and had incubation times totalling 4 or 18 h, not including the initial antibody coating step. The longer incubations gave a lower detection limit (10 pg, 300 amol) and were used when fluctuations in baseline concentrations were being monitored. The values determined were independent of the plasma volume employed, standard added to plasma samples was accurately determined and the results obtained from the analysis of plasma samples correlated closely with those obtained by radioimmunoassay. The assay was applied to the analysis of plasma samples taken from heifers with normal ovarian activity or treated in a number of ways, and in every case the results obtained were similar to those determined by radioimmunoassay and reported in the literature.     

An enzyme-linked immunosorbent assay for the detection of cytoplasmic polyhedrosis virus

An enzyme-linked immunosorbent assay (ELISA) for the detection of Euxoa scandens cytoplasmic polyhedrosis virus (EsCPV) is described. Antisera to EsCPV, produced in rabbits and guinea pigs, are specific to EsCPVs when used in an indirect assay. This indirect assay approach permits the detection of homologous antigens at a concentration of about 1 μg/ml; however, this procedure is not suitable to test large numbers of unpurified specimens. For this type of analysis we used a double antibody sandwich assay which can detect 10 ng/ml of homologous antigen in unpurified material without nonspecific reactions. This assay is used to diagnose EsCPV infections in field and laboratory studies.     

An enzyme-linked immunosorbent assay (ELISA) for human corticosteroid-binding globulin

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) of the "sandwich-type" for human corticosteroid-binding globulin was developed. A polyclonal rabbit anti-CBG antibody is immobilised to a microtitre plate. Following incubation of standards and samples a second monospecific rabbit anti-CBG antibody, labelled with alkaline phosphatase, is added. After colour development the microtitre plate is read at 405 nm wavelength. The assay shows good agreement to CBG binding capacity assay and commercially available RIA.     

Microwave-mediated enzyme-linked immunosorbent assay procedure

Here we demonstrate a novel microwave-mediated enzyme-linked immunosorbent assay (MELISA) method that has dramatically reduced the enzyme-linked immunosorbent assay (ELISA) timing to less than 5 min with a result comparable to that obtained by 18-h conventional ELISA. Efficacy of the MELISA procedure is demonstrated by detecting human immunoglobulin G (IgG), rabbit IgG, human immunoglobulin E (IgE), human interleuken 1β (IL-1β), Entamoeba histolytica antibody, and Aspergillus fumigatus antibody. MELISA could be an excellent substitute for time-consuming conventional ELISA for rapid diagnosis of diseases in cases of medical urgency, outbreak of infectious diseases, and screening of samples in blood banks or emigration counters.     

A simple enzyme-linked immunosorbent assay for testosterone

A new, enzyme-linked, immunosorbent assay for testosterone is described. The assay uses horseradish peroxidase coupled to testosterone as the tracer and offers the same sensitivity and reliability but greater convenience than radioimmunoassay. The assay is also simpler and more rapidly completed than previously described assays for testosterone.     

Bionanocapsule-based enzyme-antibody conjugates for enzyme-linked immunosorbent assay

Macromolecules that can assemble a large number of enzyme and antibody molecules have been used frequently for improvement of sensitivities in enzyme-linked immunosorbent assays (ELISAs). We generated bionanocapsules (BNCs) of approximately 30 nm displaying immunoglobulin G (IgG) Fc-binding ZZ domains derived from Staphylococcus aureus protein A (designated as ZZ-BNC). In the conventional ELISA using primary antibody and horseradish peroxidase-labeled secondary antibody for detecting antigen on the solid phase, ZZ-BNCs in the aqueous phase gave an approximately 10-fold higher signal. In Western blot analysis, the mixture of ZZ-BNCs with secondary antibody gave an approximately 50-fold higher signal than that without ZZ-BNCs. These results suggest that a large number of secondary antibody molecules are immobilized on the surface of ZZ-BNCs and attached to antigen, leading to the significant enhancement of sensitivity. In combination with the avidin-biotin complex system, biotinylated ZZ-BNCs showed more significant signal enhancement in ELISA and Western blot analysis. Thus, ZZ-BNC is expected to increase the performance of various conventional immunoassays.     

An enzyme-linked immunosorbent assay (ELISA) for measurement of heterophile antibody

An enzyme-linked immunosorbent assay (ELISA) test has been developed for measurement of heterophile antibody. The microtiter test utilizes a bovine erythrocyte monolayer as antigen and anti-human IgM antiserum conjugated with horseradish peroxidase to measure the degree of binding of the heterophile antibody in the test serum with the erythrocytes. A single serum dilution yields quantitative results when read in a spectrophotometer. The ELISA test showed a sensitivity comparable with the immune adherence hemagglutination assay (IAHA) and other heterophile tests, good reproducibility, and high specificity.