Detection of serum antibodies to Bacillus piliformis in mice and rats using an enzyme-linked immunosorbent assay.

Rats and mice were infected with Bacillus piliformis organisms at a dosage which resulted in clinical signs of Tyzzer's disease in gerbils. Although rats and mice did not show clinical signs of disease, rising antibody titers to B. piliformis were detected by enzyme-linked immunosorbent assay (ELISA) 2 to 6 weeks post-inoculation and remained at positive levels 11 weeks post-inoculation. Western blot analyses of sera from experimentally infected animals revealed banding patterns nearly identical to those obtained using hyperimmune serum. Results indicated that elevated ELISA titers reflected production of specific antibodies directed against antigens of B. piliformis. ELISA and Western blot analyses of naturally infected animals yielded similar results. These findings suggest that immunoassays such as ELISA can be used to detect subclinically infected rats and mice in the absence of clinical signs or histopathologic evidence of Tyzzer's disease.     

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 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 (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 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 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.     

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 assay for fetal steroid binding protein of human serum

The binding characteristics and quantitation of the recently reported fetal steroid binding protein (FSBP) cannot be determined on unpurified samples; an immunoassay was therefore desirable. The protein was purified to homogeneity in order to raise a highly specific polyclonal antibody. An enzyme-linked immunosorbent assay applicable to unpurified samples was developed. Intra- and inter-assay coefficients of variation are 8.0% and 9.2% respectively; there is a sensitivity of 30 fmol FSBP per well, and there is no cross-reactivity with other binding proteins. Results obtained with the assay correlate with the more complex ligand binding assay (r = 0.85; p less than 0.02). Measurement of sera showed that FSBP levels are higher in women than in men (51.2 +/- 10.62 nM; 41.2 +/- 13.65 respectively; p less than 0.05) and are elevated in cirrhotic women (66.4 +/- 18.67; p less than 0.05) and in males with hepatocellular carcinoma (62.2 +/- 13.05; p less than 0.002). Use of the enzyme-linked immunosorbent assay confirmed the identity of FSBP separate from sex hormone binding globulin.     

Enzyme-linked immunosorbent assay for detection of serum antibody to CAR bacillus

An enzyme-linked immunosorbent assay (ELISA) for detection of CAR bacillus antibody in rat sera was developed by Ganaway et al., in 1985 although the ELISA method was not described in detail. We investigated antigen preparation and test procedures of the ELISA using two strains of CAR bacillus which we isolated from a mouse (CB-M) and a rat (CB-R). Allantoic fluids containing 2.4 X 10(8)/ml of CB-M and 2.0 X 10(8)/ml of CB-R were washed with sterile phosphate buffered saline (PBS), resuspended in a 1/5 volume of sterile carbonate buffer (pH 9.8) and sonicated. Then 1/40 and 1/80 dilutions of CB-M and CB-R lysates in PBS, respectively, were used for antigen solutions of ELISA. Briefly, antibodies in sera are reacted with antigens coated on the surface of microtiter plates. The amount of horse radish peroxidase labeled protein-A or anti-rat IgG bound to the antigen-antibody complexes is measured on the spectro photometer at wave length of 492 nm. A total of 180 mouse and 205 rat sera were tested against both antigens. The optical density (OD) values of 140 mouse and 161 rat sera obtained from SPF mice and rats free from CAR bacillus infection were on the average 0.005 and 0.019, respectively. On the other hand, OD values of the sera collected from CB-M or CB-R infected animals ranged from 0.20 to 1.52. According to these results, the cut-off OD value for positive reaction was set at 0.1.(ABSTRACT TRUNCATED AT 250 WORDS)     

An enzyme-linked immunosorbent assay to detect serum IgG to Pasteurella multocida in naturally and experimentally infected rabbits

An enzyme-linked immunosorbent assay (ELISA) was evaluated for efficacy in detecting serum IgG against Pasteurella multocida in both naturally and experimentally infected rabbits. Blood samples and nasal cultures were taken concurrently from 58 rabbits from four conventional rabbitries. Nine rabbits from a pasteurella-free colony served as negative controls. Fifty-six rabbits were ELISA positive. Of these, 46 were P. multocida culture positive, 10 were culture negative. Two rabbits were ELISA negative, culture negative. There were no ELISA negative, culture positive animals. Serotyping by the gel diffusion precipitin test demonstrated that of the 44 typed P. multocida isolates, 57% were serotype 4, 27% were serotype 12 and 16% were serotype 3. In rabbits experimentally infected intranasally with P. multocida, serum IgG against P. multocida began to rise 21 to 33 days after infection and remained elevated until the animals were euthanized 90 days post infection. Two enzyme-linked immunosorbent assays were compared which used potassium thiocyanate extracts of different serotypes of P. multocida as antigen. The results obtained were similar, suggesting the presence of antigens common to both serotypes.     

双抗体夹心酶联免疫吸附试验检测血清HMGB-1水平在胃癌患者中的临床意义

目的应用双抗体夹心酶联免疫吸附试验(ELl SA法),分析胃癌患者血清HMGB-1(高迁移率族蛋白B-1)水平的临床意义。方法使用ELISA法对98例胃癌患者血清HMGB-1水平进行检测,用电化学发光法对血清癌胚抗原(CEA)含量进行测定,并和40例胃良性病变者及40例正常者进行比较。结果胃癌组患者血清中HMGB-1与CEA水平均明显高于正常对照组与胃良性病变组(P0.01)。和传统肿瘤标志物CEA对比,血清中HMGB-1检测在胃癌早期患者中有良好敏感性(70.6%)与准确性(74.0%),并具有统计学意义(P0.05)。结论 HMGB-1和胃癌的发生发展密切相关,检测血清中HMGB-1的含量可以提高早期胃癌的诊断水平。     

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.     

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 indirect enzyme-linked immunosorbent assay for the detection of diacetoxyscirpenol in wheat and corn

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for quantitation of diacetoxyscirpenol (DAS) in wheat flour and corn meal. Diacetoxyscirpenol was first converted to its “b”-carboxymethoxyl oxime and then conjugated to bovine serum albumin (BSA) via a water-soluble carbodiimide method. This conjugate was then coated to a microtiter plate and incubated with rabbit anti-DAS antibody and sample extract. The amount of anti-DAS antibody bound to the plate was then determined by reaction of goat anti-rabbit IgG-peroxidase complex, and subsequent reaction with substrate. Samples spiked with DAS were extracted with acetone and subjected to a simple cleanup procedure by passing them through a reversed-phase Sep-Pak C-18 cartridge. The minimum detection level for DAS was 5 picograms per assay. Average recoveries from samples of wheat flour spiked with DAS in the 1 – 100 ppb range were 97.5 ± 17.8% for one gram samples, and 97.2 ± 19.9 % for fifty gram samples. Average recoveries from corn meal samples spiked in the same range were 98.8 ± 22.6 % for one gram samples, and 99.7 ± 17.3 % for fifty gram samples.     

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.     

A sandwich enzyme-linked immunosorbent assay for human serum paraoxonase concentration

Serum paraoxonase (PON) is associated with plasma high density lipoproteins, and prevents the oxidative modification of low density lipoproteins. We have developed a sensitive sandwich enzyme-linked immunosorbent assay (ELISA), using two monoclonal antibodies against PON, to measure serum PON concentration. The concentration of PON in healthy Japanese subjects was 59.3 +/- 1.3 microgram/mL (mean +/- SEM; n = 87). Serum PON concentrations in relation to the PON 192 genetic polymorphism were: 69.5 +/- 2.9 microgram/mL in the QQ genotype; 63.0 +/- 1.9 microgram/mL in the QR genotype; and 52.8 +/- 1.7 microgram/mL in the RR genotype. Concentrations were significantly lower in the RR than in the QQ genotype (P < 0.01). Serum paraoxonase specific activity was higher in RR than in QQ subjects (18.6 +/- 0.40 vs. 2. 56 +/- 0.05 nmol/min/microgram, P < 0.01), but arylesterase specific activity was unrelated to genotype. PON concentration was positively associated (P < 0.001) with both serum arylesterase activity and, after adjusting for the effect of the position 192 polymorphism, with serum paraoxonase activity. Subjects with angiographically verified coronary heart disease had significantly lower PON concentrations than the healthy controls (52.0 +/- 2.3 microgram/mL; n = 35, P < 0.01). This association was independent of the position 192 genotype. Our new ELISA should be of value for epidemiologic and clinical studies of serum PON concentration. immunosorbent assay for human serum paraoxonase concentration.     

An enzyme-linked immunosorbent assay for the detection of canine antibodies to canine adenoviruses.

An enzyme-linked immunosorbent assay was used to detect canine immunoglobulin G antibodies specific for infectious canine hepatitis virus and the serologically related canine adenovirus Type 2. The sequential development of homologous and heterologous antibodies was measured by the enzyme-linked immunosorbent assay and serum neutralization tests in two groups of dogs which were experimentally infected with either infectious canine hepatitis virus or canine adenovirus Type 2. Both tests were comparable in their abilities to detect the development of homologous and heterologous antibodies. Homologous antibodies were detected earlier and to a higher titer in both tests. There was a 98% agreement between the serum neutralization test and the enzyme-linked immunosorbent assay when sera from 224 random-source dogs were examined for infectious canine hepatitis virus antibodies. The enzyme-linked immunosorbent assay was found to be a highly efficient and rapid test to determine the immune status of dogs to infectious canine hepatitis virus and canine adenovirus Type 2.     

An enzyme-linked immunosorbent assay for detection of chronic subclinical Pasteurella pneumotropica infection in mice.

Serum samples from seventy-five, 3- to 12-week-old and 16 retired breeder male Swiss mice from a conventional colony with enzootic chronic subclinical Pasteurella pneumotropica infection were tested by enzyme-linked immunosorbent assay (ELISA) and Western blots for IgG antibodies to whole cell (WC) and lipooligosaccharide (LOS) antigens of P. pneumotropica. In 3- to 12-week-old mice, serum antibody levels to LOS exceeded those to the WC preparation. Western blots of sera from mice in this age group substantiated that a major component of the early IgG antibody response was directed against LOS antigens. Higher antibody levels to both antigen preparations in 3-week-old mice compared to mice 4 and 6 weeks old were interpreted as reflecting a decline in antibodies acquired from the dam. Active immunity indicative of infection was first detected at 8 weeks of age. Serum samples from retired breeder mice (28 weeks of age) also had substantial antibody titers to LOS but, in contrast to sera from mice in the younger age groups, retired breeders had significantly greater IgG reactivity to WC preparations than to LOS antigens. The superior specificity of the LOS antigen compared to the WC preparation in the ELISA was demonstrated by testing serum samples from retired breeder mice against WC and LOS antigens from P. ureae, P. multocida, and P. hemolytica. The reactivity of IgG against LOS antigens from these organisms was negligible, whereas substantial titers were evident to WC antigens. This ELISA, using LOS preparations as antigen, is a useful serologic assay for the detection of subclinical P. pneumotropica infection in mice.     

Monoclonal antibody based enzyme-linked immunosorbent assay for aminoglycoside antibiotic kanamycin in foodstuff

Monoclonal antibodies to aminoglycoside antibiotic kanamycin (KM) were raised as a result of mice complex immunization with glutaraldehyde conjugates BSA with KM, tobramycin (TM) and gentamicin. Using antibodies an indirect competitive enzyme-linked immunosorbent assay was developed. This method allows to determine antibiotic up to 1.2 ng/ml in water solutions, milk and eggs and up to 2.5 ng/ml in honey. The recovery rate from these products spiked with KM was 83, 84 and 96% respectively. The assay of KM based on homologous and heterologous solid-phase conjugates were estimated. The cross-reactivity with TM could vary from 7 to 54%. The same indexes for of amikacin were more constant and reached 7-8%. The other aminoglycosides showed no inhibitory activity.