An enzyme-linked immunosorbent assay for detection of anti-Bacillus piliformis serum antibody in rabbits

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of rabbit serum antibody directed against the causative agent of Tyzzer's disease, Bacillus piliformis. Ninety-four percent agreement was found between the ELISA and an indirect fluorescent antibody test. The sensitivity of the ELISA was 95% and its specificity was 92% as compared to the indirect fluorescent antibody test (IFAT). The rabbit origin B. piliformis isolate used in this ELISA was found to be cross-reactive by ELISA and IFAT to B. piliformis isolates of rat, gerbil and horse origin. This suggests that a single B. piliformis isolate may be used as antigen for an ELISA utilizable for multiple species.     

Production of a monoclonal antibody to cortisol: application to a direct enzyme-linked immunosorbent assay of plasma.

Cortisol mouse monoclonal antibodies were produced and characterized. Of the four clones studied, supernatant from one clone (A2), compared with other cortisol monoclonal antibodies, showed minimal cross-reactivity to other C21 steroids and was suitable for the direct determination of cortisol in plasma by enzyme-linked immunosorbent assay using a standard 96-well microtiter plate. The enzyme-linked immunosorbent assay uses the immobilized antigen approach, in which cortisol in plasma samples or standards competes with immobilized steroid for antibody-binding sites. After washing, the cortisol antibody bound to the wells of the microtiter plate is detected with antimouse immunoglobulin conjugated to horseradish peroxidase. Following further washing, o-phenylenediamine substrate is added. The enzyme-linked immunosorbent assay is robust and semiautomated. The mean +/- SD recovery from plasma was 97% +/- 6%. Precision studies on three different plasma pools showed mean coefficients of variation of 7.6% and 8.6% for within- and between-assay variation, respectively. The satisfactory performance criteria allow its use in the routine laboratory.     

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.     

Fluorometric enzyme-linked immunosorbent assay for the measurement of IgE antibody to mite Dermatophagoides farinae

We have developed a fluorometric enzyme-linked immunosorbent assay for measuring IgE antibody to Dermatophagoides farinae. Polystyrene microplates were coated with proteins extracted from the mites. The IgE antibody which attached to the solid-phase antigen was detected by anti-IgE antibody conjugated with beta-galactosidase. Four-methylumbelliferyl-beta-D-galactoside was used as the enzyme substrate and the fluorescence intensity of the reaction product was measured. The antibody levels determined by this method well correlated with those determined by the radioallergosorbent test (RAST). This method is simpler and less expensive to carry out than the RAST when dealing with a large number of serum specimens for seroepidemiological studies of asthma and nasal allergy.     

基于纳米颗粒的赭曲霉毒素A高灵敏酶联免疫检测方法的建立及应用

赭曲霉毒素(ochratoxins)主要是由青霉菌Penicillium和曲霉菌Aspergillus产生的有毒次级代谢产物,常见于发霉或发酵的农产品中,其中赭曲霉毒素A(ochratoxin A,OTA)毒性最强且最为普遍。OTA是粮食作物和饲料的重要污染物,在加工、储存或运输过程中均可产生,具有肾毒性和免疫毒性,可通过蓄积作用发挥毒性效应,对人类和动物健康造成严重威胁。本研究通过将OTA单克隆抗体包被于纳米磁珠(magnetic nanoparticles,MNPs)表面,获得具有免疫活性的磁珠抗体复合物(MNPs-Anti OTA),并制备生物素标记的偶联抗原OTA-BSA-Bio,后续采用链酶亲和素标记的纳米金颗粒(Strep-HRP-AuNPs)催化底物进行信号检测,最终建立了OTA高灵敏检测方法(MNPs-bs-AuNPs-ELISA)。在最优条件下,经计算该方法检测下限(IC10)为0.01ng/mL,检测区间(IC20-IC80)为0.02-0.73ng/mL,半数抑制率(IC50)为0.13ng/mL。与OTA类似物OTB、OTC交叉反应性为4.3%和8.1%,对其他常见真菌毒素AFB1、ZEN、FB1、DON、CIT和PAT均无交叉反应。玉米、面粉和大豆样本中的加标回收率可达85.6%-115.7%,对天然样本中OTA含量的检测结果表明,该方法与LC-MS/MS相关性良好。本研究建立的MNPs-bs-AuNPs-ELISA可满足谷物及饲料样本中OTA的快速、高灵敏度定量检测,成本较低,具有很好的应用前景。     

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.     

Development of enzyme-linked immunosorbent assay for detection of Mycoplasma pneumoniae antigens

The variant of enzyme-linked immunosorbent assay (ELISA) for detection of Mycoplasma pneumoniae (Mp) antigens in sera of patients with respiratory infections was developed. Sensitivity of detection of soluble antigens of Mp in modeling experiment varied from 1.5 to 1.0 ng/ml (on protein). Approbation of the assay was performed using 50 serum samples obtained from patients with confirmed diagnosis of respiratory mycoplasmosis. In the ELISA test Mp antigens were detected in 96% of samples. Obtained results were confirmed by testing of these serum samples and isolated from them circulating immune complexes (CICs) in immunoblotting using polyclonal antibodies labeled by horse-radish peroxidase. Mp antigenswere detected both in free state and as components of CICs. Specific reaction was observed with proteins, which molecular mass varied from 30 to 170 kDa (30, 37, 45, 56, 58, 72, 90, 130 and 170 (160) kDa). Obtained results point to appropriateness of use of developed assay for detection Mp antigens in sera of patients with respiratory infections.     

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)     

A monoclonal antibody to scopolamine and its use for competitive enzyme-linked immunosorbent assay.

A hybridoma clone producing a monoclonal antibody (SC78.H81) against scopolamine was established. The monoclonal antibody was an IgG1 (k) antibody with high affinity (1.6 x 10(9) M-1 for methylscopolamine). The monoclonal antibody was cross-reactive with methylscopolamine and butylscopolamine, and showed weak cross-reactivity with 6 beta- and 7 beta-hydroxyhyoscyamine. The cross-reaction with L-hyoscyamine, atropine, scopine and DL-tropic acid was very weak. A competitive enzyme-linked immunosorbent assay using SC78.H81 was established to quantify scopolamine. The sensitivity of the assay allowed detection of 20 pg assay-1 (0.2 ng ml-1) of scopolamine. The assay was applied to the estimation of scopolamine content in hairy root cultures of a Duboisia hybrid.     

Application of enzyme-linked immunosorbent assay for mass examination of strongyloidiasis in okinawa, Japan

The enzyme-linked immunosorbent assay was tested to evaluate whether it could be applicable in screening for mass examination of strongyloidiasis. A total of 2906 inhabitants in three areas (858 in Gushikawa Village, 849 in Nakazato Village and 1199 in Sashiki Town) were screened by the enzymatic assay and approximately 11–30% (11.8% in Gushikawa, 17.0% in Nakazato and 27.7% in Sashiki) were considered to be antibody positive. In the parasitological follow-up examinations of those who were antibody positive, actual infection was found in more than half (51%) the subjects. The overall infection rates estimated from the results reached 5.8% in Gushikawa, 9.1% in Nakazato and 14.0% in Sashiki (mean = 10.4%). The infection rates were significantly higher than those in previous surveys conducted in the same areas. The ELISA technique was found to be useful for strongyloidiasis screening and for seroepidemiological purposes in Okinawa.     

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.     

Capillary-based enzyme-linked immunosorbent assay for highly sensitive detection of thrombin-cleaved osteopontin in plasma

In this study, a highly sensitive capillary-based enzyme-linked immunosorbent assay (ELISA) has been developed for the analysis of picomolar levels of thrombin-cleaved osteopontin (trOPN), a potential biomarker for ischemic stroke, in human plasma. Using a square capillary coated with 8.5 μg/ml anti-human trOPN capture antibody for ELISA, the linear range obtained was 2 to 16 pM trOPN antigen. This concentration range was in the detection window of trOPN antigen in plasma samples. Compared with the conventional microplate-based ELISA, the current capillary technique significantly reduced the amounts of reagent from milliliter to microliter, reduced the analysis time from 8 to 3 h, and had a better sensitivity and detection limit performance from approximately 50 pM down to 2 pM of trOPN antigen. These results indicate that this capillary-based immunoassay is a potential tool for biomarker detection and may be useful in clinical trials and medical diagnostic applications.     

Development of an enzyme-linked immunosorbent assay for detection of IgM antibodies to Babesia bigemina in cattle

A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Negative sera from cattle imported from tick-free areas, serum samples collected from infected B. bigemina cattle were used to validate the test. The specificity was 94% and sensitivity of the Elisa 87.5%. Sera from 385 cattle deriving from areas free from tick-borne diseases, which were submitted to a preimmunization process, were screened by this technique. The Elisa detected seroconversion on the 14th day post-inoculation in animals either infested with Boophilus microplus ticks (infected with B. bigemina), or inoculated with B. bigemina infected blood. Antibody titers decreased after day 33; however, all animals remained positive until the end of the experiment (124 days). The ELISA described may prove to be an appropriate serological test for the detection of IgM antibodies against B. bigemina.     

Use of a monoclonal antibody to estrone-3-glucuronide in an enzyme-linked immunosorbent assay (ELISA)

A direct urinary ELISA for estrone-3-glucuronide has been produced following cloning and characterisation of a monoclonal antibody to the above estrogen metabolite. The ELISA follows our established pattern of absorbing a thyroglobulin conjugate, to which estrone-3-glucuronide has been coupled, to the wells of a microtitre plate using guanidine hydrochloride. A competition reaction between either standards/samples and the adsorbed hormone compete for antibody combining sites. The assay is completed by addition of an anti-mouse Ig-peroxidase complex and read at 492 nm following additions of O-phenylenediamine substrate in under 4 h. The correlation between urinary "total estradiol" and "total estrone and estradiol" is very good and, in conjunction with our ELISA for pregnanediol glucuronide, has allowed for the improved clinical management of infertile and subfertile women.     

A comparative study on the efficacy of a pest-specific and prey-marking enzyme-linked immunosorbent assay for detection of predation

The efficacy of two different antigen–antibody combinations to detect predation on eggs of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) was compared. The first method was an indirect enzyme‐linked immunosorbent assay (ELISA) using monoclonal antibody‐based gut content analysis that detects H. armigera egg protein. The second method was a sandwich ELISA that detects an exotic protein [rabbit immunoglobulin G (IgG)] applied as an external marker to H. armigera eggs. The target predators were the predatory beetles Dicranolaius bellulus (Guerin‐Meneville) (Coleoptera: Melyridae) and Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae). Beetles were fed with H. armigera eggs that had been marked with rabbit IgG and then held at various intervals after prey consumption. Each individual beetle was then assayed by both ELISA techniques to identify the prey remains in their guts. The two ELISA methods were further tested on field‐collected predators. Specifically, protein‐marked egg masses were strategically placed in a cotton field. Then, predators from surrounding cotton plants were collected at various time intervals after the marked eggs were exposed and assayed by both ELISAs to detect the frequency of predation on the marked eggs. The rabbit IgG‐specific sandwich ELISA had a higher detection rate than the H. armigera‐specific indirect ELISA under controlled and field conditions for both predator species. Moreover, a greater proportion of field‐collected D. bellulus tested positive for predation than H. variegata. The advantages and disadvantages of using prey‐marking ELISAs instead of pest‐specific ELISA assays are discussed.     

A background-free assay for cells forming antibody to glucose oxidase coupled with an enzyme-linked immunosorbent assay (ELISA) to quantitate antibody secretion

We describe an immunocytochemical assay for cells forming antibody to glucose oxidase (GO). The method is specific in that only cells containing intracytoplasmic antibody capable of binding the immunogen (GO binding cells; GOBC) are stained. The method is sensitive because there is no GO activity in mammalian tissues. This lack of background readily permits detection of one GOBC among 10(6) nucleated lymphohemopoietic cells. The technique is reliable because purified chemicals are used. Although it is not possible to determine the Ig class of antibody formed by an individual cell, as can be done with the hemolytic plaque assay, the amount and class of secreted antibody to GO can be quantitated by an indirect enzyme-linked immunosorbent assay (ELISA), which is also described. GO is immunogenic and stimulates the formation of large numbers of GOBC in the popliteal lymph nodes after injection with adjuvant into the footpads of mice, but 1-mg doses injected IV or IP are lethal because of its enzymatic activity, which causes hypoglycemia and methemoglobinemia.     

Comparison of an enzyme-linked immunosorbent assay, an immunofluorescence assay and a hemagglutination inhibition assay for detection of antibodies to K-papovavirus in mice

The sensitivity of a newly developed enzyme-linked immunosorbent assay (ELISA) for detection of antibody to K virus was compared with the sensitivities of an immunofluorescence assay (IFA) and a hemagglutination inhibition assay (HIA). Specific pathogen-free BALB/c RIVM mice, 5 weeks old, were inoculated intraperitoneally with a mouse organ suspension containing 10(4.5) TCID50 of K virus per dose. Control animals were inoculated with a control mouse organ suspension. No clinical signs were observed during the 7 weeks they were followed for the development of serum antibody. The ELISA proved to be the most sensitive of the three assays and demonstrated K virus-specific antibodies as early as 3 days after infection.     

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。     

Description of an enzyme-linked immunosorbent assay for the detection of protein tyrosine kinase

A solid immunoassay for the detection of protein tyrosine kinases has been developed. It is based on the binding of the synthetic polypeptide poly(Glu.Na,Tyr) 4:1 to microELISA wells, where the phosphorylation reaction takes place in the presence of ATP and enzyme. The phosphorylated tyrosine residues produced in the reaction are finally detected, in the same well, by means of an ELISA using monoclonal antiphosphotyrosine antibody, peroxidase-labeled goat anti-mouse IgG antibody, and substrate. The amount of protein tyrosine kinase activity present in the sample is proportional to the color at 492 nm developed in each well.