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.     

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 for N-acetylglucosaminyltransferase-V

The development of an enzyme-linked immunosorbent assay (ELISA) for uridine 5'-diphospho-N-acetyl-glucosamine: alpha mannoside beta 1----6 N-acetylglucosaminyltransferase (GnT-V) is reported. The assay quantitates the enzymatic conversion of the specific synthetic GnT-V acceptor GlcNAc beta 1----2Man alpha 1----6Man beta-R (5) to the product GlcNAc beta 1----2[GlcNAc-beta 1----6]Man alpha 1----6Man beta-R (6) when these oligosaccharide structures were covalently attached to bovine serum albumin which was then coated on microtiter wells. Conversion of 5 to 6 was detected using a polyclonal antiserum raised against the product 6 and from which antibodies cross-reacting with acceptor 5 had been removed by affinity adsorption. GnT-V activity detected by ELISA was linearly proportional to both enzyme concentration and time under appropriate experimental conditions where 50-300 fmol of product was formed per microtiter well. GnT-V activity could be measured by ELISA in Triton X-100 extracts of hamster kidney acetone powder and in human serum. The twofold increase in GnT-V activity which is known to accompany Rous sarcoma virus transformation of baby hamster kidney cells could also be quantitated using the ELISA.     

An enzyme-linked immunosorbent assay for detection of pyrene and related polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) can form DNA-binding compounds that show genotoxicity and carcinogenicity. Pyrene, as a PAH, was covalently linked to carrier protein bovine serum albumin and ovalbumin. A monoclonal antibody (McAb) was produced that showed high cross-reactivity values with chrysene (169.73%), benzo[a]pyrene (693.34%), benzo[a]anthracene (16.36%), and indeno[1,2,3-cd]pyrene (40.96%) and showed no significant cross-reactivity values with other homologues (<0.1%). A competitive enzyme-linked immunosorbent assay (ELISA) was developed for detection of pyrene and some homologues in water samples. The detection limit of the assay was 65.08 pg ml⁻¹. The average recoveries of PAHs from tap water, lake water, and mineral water were 99.13, 99.74, and 99.19%, respectively, indicating that matrices of water samples do not interfere with the assay. The results demonstrated that the developed ELISA seems to be a potential method for monitoring of pyrene and some homologous PAHs in water samples.     

An enzyme-linked immunosorbent assay for zein and other proteins using unconventional solvents for antigen adsorption

An enzyme-linked immunosorbent assay (ELISA) performed in polystyrene microtiter plates that can detect and quantitate the maize prolamin zein is described. The assay yields positive reactions with as little as 1 ng of antigen and uses solvents not ordinarily employed in ELISA methods. A systematic investigation of zein adsorption to polystyrene in various solvents supports the hypothesis that antigen binding occurs through nonpolar interactions. The method was also used to determine structural relationships among three zein polypeptides differing in size and charge. Additional experiments indicate that a number of soluble proteins are absorbed to polystyrene in the denaturing agent urea and retain immunological reactivity. The retention of antigen reactivity after solubilization in 6-8 M urea suggests that ELISA methods may be applicable to other proteins which are insoluble, or rendered insoluble, in aqueous buffers.     

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.     

An enzyme-linked immunosorbent poly(ADP-ribose) polymerase biomarker assay for clinical trials of PARP inhibitors

Many established cancer therapies involve DNA-damaging chemotherapy or radiotherapy. The DNA repair capacity of the tumor represents a common mechanism used by cancer cells to survive DNA-damaging therapy. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that is activated by DNA damage and has critical roles in DNA repair. Inhibition of PARP potentiates the activity of DNA-damaging agents such as temozolomide, topoisomerase inhibitors and radiation in both in vitro and in vivo preclinical models. Recently, several PARP inhibitors have entered clinical trials either as single agents or in combination with DNA-damaging chemotherapy. Because PARP inhibitors are not cytotoxic, a biomarker assay is useful to guide the selection of an optimal biological dose. We set out to develop an assay that enables us to detect 50% PAR reduction in human tumors with 80% power in a single-plate assay while assuring no more than a 10% false-positive rate. We have developed and optimized an enzyme-linked immunosorbent assay (ELISA) to measure PARP activity that meets the above-mentioned criterion. This robust assay is able to detect PAR levels of 30-2000 pg/ml in both tumor and peripheral blood monocyte samples. In a B16F10 mouse syngeneic tumor model, PARP inhibitor ABT-888 potentiates the effect of temozolomide in suppressing tumor growth, and PARP activity is greatly reduced by ABT-888 at efficacious doses. In summary, the ELISA assay described here is suitable for biomarker studies in clinical trials of PARP inhibitors.     

An enzyme-linked immunosorbent assay for 6-keto PGF

An enzyme-linked immunosorbent assay for 6-keto prostaglandin F_1α, a stable metabolite of prostacyclin, has been developed. The assay allows quantitation of 6-keto PGF_1α 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 PGF_1α 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 for plant cadmium-binding peptide

Polyclonal antibodies raised against Cd-binding peptide from roots of Agrostis gigantea Roth were used with an enzyme-linked immunosorbent assay (ELISA). The antigen was best adsorbed to Immulon 2 “U” microtitre plates in 50 mM acetic acid. The antibodies to the antigen from Agrostis cross-reacted with Cd-binding peptides from the roots of maize and tomato, but not with glutathione nor metallothioneins I and II from rabbit liver. The antibodies reacted specifically with peptides rich in cysteine and glutamate, and having glycine or serine in the least amount. Reaction of antibodies was limited to ELISA; the antiserum did not form antigen-antibody precipitates when tested by standard diffusion and immunoelectrophoretic methods.     

Creating highly amplified enzyme-linked immunosorbent assay signals from genetically engineered bacteriophage

For early detection of many diseases, it is critical to be able to diagnose small amounts of biomarkers in blood or serum. One of the most widely used sensing assays is the enzyme-linked immunosorbent assay (ELISA), which typically uses detection monoclonal antibodies conjugated to enzymes to produce colorimetric signals. To increase the overall sensitivities of these sensors, we demonstrate the use of a dually modified version of filamentous bacteriophage Fd that produces significantly higher colorimetric signals in ELISAs than what can be achieved using antibodies alone. Because only a few proteins at the tip of the micron-long bacteriophage are involved in antigen binding, the approximately 4000 other coat proteins can be augmented—by either chemical functionalization or genetic engineering—with hundreds to thousands of functional groups. In this article, we demonstrate the use of bacteriophage that bear a large genomic fusion that allows them to bind specific antibodies on coat protein 3 (p3) and multiple biotin groups on coat protein 8 (p8) to bind to avidin-conjugated enzymes. In direct ELISAs, the anti-rTNFα (recombinant human tumor necrosis factor alpha)-conjugated bacteriophage show approximately 3- to 4-fold gains in signal over that of anti-rTNFα, demonstrating their use as a platform for highly sensitive protein detection.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antisperm antibodies in horse serum

An enzyme-linked immunosorbent assay (ELISA) was developed and evaluated to detect equine antisperm antibodies (ASA) in horse serum. Six maiden mares between 12 and 18 mo of age were immunized with stallion sperm cells (SC group, N=2), seminal plasma (SP group, N=2), or phosphate-buffered saline (PBS) as a control (C group, N=2). Horses received a second injection of the same antigen 2 wk after the first. Blood was collected weekly for 10 wk after initial immunization and again at Week 15. Serum ASA levels (IgG and IgA) were measured by ELISA using two assay systems, one containing stallion SC as the plate antigen and another containing SP.

In horses immunized with SC, peak IgG levels were detected by ELISA during Wk 2 and 3 after first injection using either plate antigen. The antibody levels persisted through Week 5 and then slowly declined until Week 15. Horses immunized with SP had IgG levels that did not differ from control horses using either ELISA plate antigen. The only significant elevation in serum IgA ASA occured during Week 5 after initial immunization and only in mares immunized with SC as detected by ELISA using SC as the plate antigen. Attachment of ASA to stallion spermatozoa was confirmed by an indirect immunofluorescence assay.     

An enzyme-linked immunoadsorbent assay for rat ceruloplasmin

A noncompetitive, colorimetric enzyme-linked immunoadsorbent assay (ELISA)† was developed for measuring rat ceruloplasmin in serum and in medium from culture hepatocytes. The assay utilized polystyrene immobilized antibody which bound ceruloplasmin which then bound biotinylated antibody. The biotinylated antibody-antigen complex was detected with strepavidin-alkaline phosphatase conjugate. Standard curves for rat ceruloplasmin were constructed in the range between 10 and 50 ng/mL. Increases of 10 ng produced an increase inA ₄₀₃ of more than 0.2. With this immunoassay, serum ceruloplasmin levels were found to average 35 mg Cp/dL in control rats and 87 mg/dL in rats with experimental inflammation. Liver parenchymal cells secreted 1.6μg Cp/5×10⁵ cells/24h. This ELISA assay for ceruloplasmin will facilitate studies on the regulation of ceruloplasmin synthesis and secretion in both intact rats and isolated hepatocytes.     

Characterization by enzyme-linked immunosorbent assay of monoclonal antibodies to pisum and Avena phytochrome

Nine monoclonal antibodies to pea (Pisum sativum L.) and 16 to oat (Avena sativa L.) phytochrome are characterized by enzyme-linked immunosorbent assay against phytochrome from six different sources: pea, zucchini (Cucurbita pepo L.), lettuce (Lactuca sativa L.), oat, rye (Secale cereale L.), and barley (Hordeum vulgare L.). All antibodies were raised against phytochrome with a monomer size near 120,000 daltons. Nevertheless, none of them discriminated qualitatively between 118/114-kilodalton oat phytochrome and a photoreversible, 60-kilodalton proteolytic degradation product derived from it. In addition, none of the 23 antibodies tested discriminated substantially between phytochrome—red-absorbing form and phytochrome—far red-absorbing form. Two antibodies to pea and six to oat phytochrome also bound strongly to phytochrome from the other species, even though these two plants are evolutionarily widely divergent. Of these eight antibodies, two bound significantly to all of the six phytochrome preparations tested, indicating that these two may recognize highly conserved regions of the chromoprotein. Since the molecular function of phytochrome is unknown, these two antibodies may serve as unique probes for regions of this pigment that are important to its mode of action.     

Interference from xylem sap in an enzyme-linked immunosorbent assay for zeatin riboside

The extent of interference from xylem sap in an enzyme-linked immunosorbent assay was determined for a woody perennial [ Populus trichocarpa Torr. & Gray x P deltoides Bart, ex Marsh (Hybrid 1l–ll)] and a herbaceous annual ( Phasesolus vulgaris L. cv. Contender). Crude xylem sap collected from excised roots from both species interfered with the assay for zeatin riboside. Assays for zeatin riboside in xylem sap collected from Popidus overestimated endogenous levels, and added standards could not be accurately measured from a range of sap dilutions. When Phaseolus plants were grown under various nutrient regimens, interference in the assay was dependent on nutrient availability. Of xylem sap components (inorganic minerals, amino acids and sucrose) which may vary with environmental conditions or among species, only sucrose interfered at the concentrations tested. Since the pH of xylem sap varies it was necessary to buffer samples prior to analysis. Partial purification using anion exchange columns and Sep-Paks cffectively eliminated interference. These results demonstrate that estimates of plant growth regulators in xylem sap by the ELISA (enzyme-linked immunosorbant assay) method can be influenced by species and environmental conditions such as plant nutritional status.     

Development of a fibrinogen-specific sandwich enzyme-linked immunosorbent assay microarray assay for distinguishing between blood plasma and serum samples

We have developed a fibrinogen-specific sandwich enzyme-linked immunosorbent assay (ELISA) microarray assay for use in qualitatively distinguishing between blood plasma and serum samples. Three capture antibodies (49D2, HPA001900, and F8512) were evaluated in conjunction with 1D6 as the detection antibody. The data show that 49D2 and (to a lesser extent) F8512 successfully identify previously unknown plasma and serum samples based on approximately a 28-fold difference in signal intensity between the sample types. This assay has utility in rapidly identifying previously archived clinical samples with incomplete annotation in a high-throughput manner prior to proteomic analyses.     

An enzyme linked immunosorbent assay for detecting palytoxin-producing bacteria

A competitive ELISA using the intact toxin as a coating antigen for detecting palytoxin was developed. This immunoassay allows palytoxin (PTX) to be determined in the range of 6–250 ng/ml. In sensitivity, this determination is comparable with RIA but is three times inferior to ELISA using monoclonal antibodies. Inhibition experiments using some toxins of marine invertebrates proved the serological specificity of the palytoxin binding to antibodies. Both the indirect and competitive ELISA were used to find PTX-producing bacteria among 420 isolates of sea bacteria. It was found that gram-negative bacteriaAeromonas sp. andVibrio sp. associated with toxic samples of the soft coralPalythoa sp. produced compounds antigenically related to PTX.     

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 (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.