Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.     

Detection of Clostridium botulinum type G toxin by enzyme-linked immunosorbent assay.

Clostridium botulinum type G toxin was detected and quantified readily with the enzyme-linked immunosorbent assay. With the double-sandwich technique and alkaline phosphatase as the enzyme indicator, C. botulinum toxin type G was detected in quantities equaling those required for one mouse intraperitoneal median lethal dose. The time required for the procedure was approximately 6.5 h, but this requirement could have been reduced to 5.5 h or less with the use of precoated plates stored at -70 degrees C. Cross-reactions did not occur with culture extracts of C. sporogenes of C. botulinum types B, C, D, E, and F. Acidic preparations of C. botulinum type A exhibited nonspecific reactivity. Likewise, 50% of the C. subterminale isolates tested were cross-reactive in the assay. These latter isolates express similar metabolic and physiological characteristics with C. botulinum type G.     

Enzyme linked immunosorbent assay (ELISA) for detection of Clostridium botulinum type B toxin.

The enzyme-linked immunosorbent assay using different techniques has been applied to determine botulinum type B toxin. With the so-called "sandwich" technique, about 5,000 mouse ip LD50 of type B toxin can be detected. With the "double-sandwich" technique, about 400 mouse ip LD50 of toxin is detected and different commerical antisera are useful. For accurate quantification of botulinum toxins in culture filtrates, addition of EDTA to samples seems to be necessary. Cross-reactivity of the assay depends on the specificity of the antisera against botulinum type B toxin used and is almost eliminated with antiserum prepared against the toxic component of type B toxin.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum toxin type A.

The enzyme linked immunosorbent assay using the so-called "double-sandwich technique" has been applied to determine botulinum toxin type A. By this assay, 50-100 mouse ip LD50 of toxin type A can be detected. No cross-reaction occurs with botulinum toxins of other types tested. In all probability this is due to the high specificity of the antiserum prepared against the toxic component of type A toxin.     

Characterization of monoclonal antibodies against altered (T103I) amidase from Pseudomonas aeruginosa

Monoclonal antibodies (MAbs) against mutant (T103I) amidase from Pseudomonas aeruginosa were raised by hybridoma technology. To select MAbs suitable for immunoaffinity chromatography, hybridoma clones secreting polyol-responsive MAbs (PR-MAbs) were screened that bind antigen tightly but release under mild and nondenaturing elution conditions. It was found that about 10% of enzyme-linked immunosorbent assay (ELISA)-positive hybridoma produce these MAbs as their ag-ab complex can be disrupted by propylene glycol in the presence of a suitable salt. Two of these hybridoma clones (F6G7 and E2A6) secreting PR-MAbs against mutant amidase were selected for optimization of experimental conditions for elution of amidase by using ELISA elution assay. These hybridoma cell lines secreted MAbs of IgM class that were purified in a single step by gel filtration chromatography, which revealed a single protein band on native polyacrylamide gel electrophoresis (PAGE). Specificity studies of this MAb revealed that it recognized specifically a common epitope on mutant and wild-type amidases as determined by direct ELISA. This MAb exhibited a higher affinity for denatured forms of wild-type and mutant amidases than for native forms as revealed by affinity constants (K), suggesting that it recognizes a cryptic epitope on an amidase molecule. Furthermore, MAb E2A6 inhibited about 60% of wild-type amidase activity, whereas it activated about 60% of mutant amidase (T103I) activity. The data presented in this work suggest that this MAb acts as a very useful probe to detect conformational changes in native and denatured amidases as well as to differentiate wild-type and mutant (T103I) amidases.     

应用病毒感染细胞酶联免疫吸附试验检测肾综合征出血热病毒抗原和抗体

应用病毒感染细胞酶联免疫吸附试验(VIC-ELISA)检测肾综合征出血热病毒(HFRSV)感染性滴度比双抗体间接ELISA和间接免疫荧光法(IFA)分别敏感10倍和100倍。VIC-ELISA检测兔抗HFRSV抗体的滴度比双抗体间接夹心ELISA和IFA分别敏感1.6倍和8倍。VIC-ELISA能敏感、快速、有效地检测HFRSV抗原和抗体。     

Development and evaluation of various enzyme-linked immunosorbent assays for the detection of Clostridium perfringensβ anti-toxins

The aim of our work was to develop an enzyme-linked immunosorbent assay for the detection of antibodies against the Clostridium perfringens beta toxin. For this purpose, five different ways of performing an enzyme-linked immunosorbent assay were investigated. Positive and negative sera of different animals and partially purified beta toxin were used. In all enzyme-linked immunosorbent assay tests, microplates were first coated with monoclonal antibodies against the C. perfringens beta toxin. Actually, the first three ways of performing enzyme-linked immunosorbent assay proved to be an inhibition or a blocking enzyme-linked immunosorbent assay. In the first of these modifications, the examined serum was added on a microplate after the toxin. In the second two tests, they were added simultaneously after they were incubated together (60 min at room temperature or overnight at 4 degrees C, respectively). An anti-toxin conjugate was used for the detection. It was also used in a competitive enzyme-linked immunosorbent assay, where it was added together with the examined serum on the microplate, to which the toxin was already bound. The fifth way of performing an enzyme-linked immunosorbent assay differed from others by the use of conjugated anti-species immunoglobulin for the detection. The biggest differences in absorbances between positive and negative sera were found in the blocking enzyme-linked immunosorbent assay, where the mixture of the toxin and the examined serum were previously incubated overnight at 4 degrees C. The smallest differences in absorbance were found when anti-species conjugates were used.     

Application of an enzyme-linked immunosorbent assay for screening of T-2 toxin-producing Fusarium spp

Culture filtrates of Fusarium species were subjected without clean-up procedures to an indirect competitive enzyme-linked immunosorbent assay with anti-T-2 toxin monoclonal antibody. Fusarium sporotrichioides, F. poae, F. tricinctum, and F. culmorum strains were positive for T-2 toxin, with a minimum detection limit of 5 pg per assay (100 pg/ml of culture filtrate), and the assay data correlated well with the gas-liquid chromatographic data.     

Application of an enzyme-linked immunosorbent assay for screening of T-2 toxin-producing Fusarium spp.

Culture filtrates of Fusarium species were subjected without clean-up procedures to an indirect competitive enzyme-linked immunosorbent assay with anti-T-2 toxin monoclonal antibody. Fusarium sporotrichioides, F. poae, F. tricinctum, and F. culmorum strains were positive for T-2 toxin, with a minimum detection limit of 5 pg per assay (100 pg/ml of culture filtrate), and the assay data correlated well with the gas-liquid chromatographic data.     

Monoclonal antibodies recognising serum immunoglobulins and surface immunoglobulin-positive cells of puffer fish, torafugu (Takifugu rubripes)

Immunoglobulin of the torafugu, Takifugu rubripes, was purified by a combination of precipitation by low ionic strength dialysis and gel filtration. The Ig was used to immunise mice for the production of monoclonal antibody (MAb). Supernatants of hybridoma cultures were screened by enzyme-linked immunosorbent assay using purified-torafugu Ig-coated plates, and two stable hybridomas producing MAbs against torafugu Ig were obtained. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions and Western blotting indicated that one MAb (16F3) was specific for the deglycosylated heavy chain of torafugu, and the other MAb (4H5) did not bind to the reduced Ig, suggesting that 4H5 recognised the higher-order structure of Ig. Under non-reduced conditions, both MAbs recognised mainly a 750 kDa band and also minor bands of 672, 410 and 205 kDa. MAb 16F3- and 4H5-primed magnetic beads (Dynabeads) adsorbed 84.9+/-3.3% and 63.6+/-4.4% of the torafugu Ig, respectively. The Ig adsorbed by MAb 16F3-primed Dynabeads was reactive to 4H5 on immunoblotting, and vice versa, indicating that the epitopes for both MAbs are held on the same Ig molecule. Both of these MAbs cross-reacted extensively with the Ig of other Takifugu species, but not with other genus. The MAbs were used to identify surface Ig-positive lymphocytes in the spleen, pronephros, peripheral blood and thymocytes of torafugu by flow cytometry. Flow cytometric analysis of the cells in the lymphocyte-enriched fraction revealed that 50.2+/-6.9% in the PBL, 11.8+/-1.7% in the mesonephros, 13.3+/-2.1% in the pronephros, 42.5+/-4.3% in the spleen and 3.2+/-0.6% in thymus were reactive to 4H5 or 16F3.     

Detection of ochratoxin A in porcine kidneys by a monoclonal antibody-based radioimmunoassay

By using an indirect enzyme-linked immunosorbent assay, eight monoclonal antibodies (MAbs) were selected. Mice were immunized with ochratoxin A that was conjugated to bovine serum albumin. The hybridoma cell line designated 10G2 was grown in tissue culture and as an ascites tumor. The MAb was characterized to be specific to ochratoxin A and of the immunoglobulin G (IgG) class. Subsequently, the ascites fluid of this hybridoma was used in a competitive solid-phase IgG radioimmunoassay on protein A-Sepharose CL-4B, with [14C]ochratoxin A as tracer. Porcine kidneys were extracted with 0.5% phosphoric acid in chloroform. A two-step cleanup was achieved on a Sep-Pak C18 cartridge and a Sep-Pak silica cartridge. Radioimmunoassay with MAbs coupled to protein A-Sepharose CL-4B allowed the detection of ochratoxin A in porcine kidneys at a concentration as low as 0.2 ng/g.     

A new type sandwich immunoassay for microcystin: production of monoclonal antibodies specific to the immune complex formed by microcystin and an anti-microcystin monoclonal antibody.

To develop an ultrasensitive immunoassay for microcystins (MCs), a group of heptapeptide hepatotoxins produced by cyanobacteria, we produced monoclonal antibodies (MAbs) which specifically recognize the immune complex (IC) formed by an anti-MC MAb (MC MAb) and MCs. The use of the anti-IC MAb (IC MAb) as the secondary antibody made it possible to develop a sandwich type immunoassay, which is theoretically superior to the widely used competitive immunoassay in sensitivity as well as accuracy. A MC MAb mixed with microcystin-LR (MCLR) to form the IC was immunized to mice. Three IC MAbs were obtained, all of which specifically reacted with the IC, but almost never reacted to MC MAb or MCLR in enzyme-linked immunosorbent assays (ELISAs). Binding kinetics study of one of the IC MAbs, 3F7, by a BIAcore biosensor technique revealed that 3F7 IC MAb could associate with free MC MAb as well as the IC, but the binding to free MC MAb was much more easily dissociated than that to the IC, thus resulting in about 300-fold higher affinity of 3F7 for the IC than for MC MAb alone (1.8 x 10(9) M(-1) and 4.6 x 10(6) M(-1) for the IC and MC MAb, respectively). Finally, 3F7 IC MAb was shown to react with the IC formed by the addition of MCLR to MC MAb-coated plates in a dose-dependent manner. Therefore, a new type sandwich immunoassay, anti-immune complex ELISA (IC ELISA) for MCs, was indeed established. The detection limit of the IC ELISA was 2 pg of MCLR ml(-1) (50 fg per assay), making it the most sensitive of all the methods for detecting MCs reported to date.     

Production and characteristics of monoclonal antibodies to the diphtheria toxin

Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.     

Detection of T-2 toxin in Fusarium sporotrichioides-infected corn by enzyme-linked immunosorbent assay.

A competitive enzyme-linked immunosorbent assay was used to screen for T-2 toxin in Fusarium sporotrichioides -infected corn. The assay detected T-2 toxin in diluted methanol extracts of corn samples at concentrations of 0.05 ng/ml. In infected corn samples, enzyme-linked immunosorbent assay and gas-liquid chromatography estimations of T-2 toxin concentrations were similar.     

Monoclonal Antibody-Based Serological Detection Methods for Wheat Dwarf Virus

Wheat dwarf disease caused by wheat dwarf virus (WDV) is currently present in wheat growing regions in China and causes serious losses in wheat yield. To develop reliable and effective serological detection methods for WDV, the coat protein (CP) gene of WDV was cloned and expressed in Escherichia coli. The purified recombinant CP protein was immunized to BALB/c mice, and four hybridoma cell lines (i.e. 18G10, 9G4, 23F4 and 22A10) secreting anti-WDV monoclonal antibodies (MAbs) were obtained through the hybridoma technique. Using the prepared MAbs, an antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) and a dot-ELISA were established for detecting WDV in wheat samples. The most sensitive ACP-ELISA based on MAb 23F4 or 22A10 was able to detect WDV in 1:163,840 (w/v, g/mL) diluted WDV-infected wheat plant crude extracts. The dot-ELISA based on MAb 23F4 was the most sensitive and able to detect the virus in 1:5,120 (w/v, g/mL) diluted wheat plant crude extracts. A total of 128 wheat samples were collected from wheat growing regions in the Shaanxi and Qinghai provinces, China, and were screened for the presence of WDV using two developed serological assays. Results from the survey showed that approximately 62% of the samples were infected with WDV. PCR followed by DNA sequencing and sequence alignment validated the results from the two serological assays. Therefore, we consider that these two serological detection methods can be significantly useful for the control of WDV in China.     

Monoclonal antibody to human lung angiotensin-converting enzyme

The hybridoma producing monoclonal antibody (IgG1) to human angiotensin-converting enzyme (ACE) has been prepared by fusion of murine myeloma P3O1 with spleen cells of BALB/c mice immunized with a purified human lung ACE preparation. A high specificity of monoclonal antibody (MAb) binding to immobilized ACE has been demonstrated by enzyme-linked immunosorbent assay and that of soluble ACE by an immunoadsorption test. The latter technique permits the use of impure ACE preparations for the screening procedure. This MAb did not affect ACE activity. We believe this antibody will be useful not only for immunoassay and immunopurification of ACE, but also as a tool for the investigation of the tissue distribution of the enzyme as well as for the study of the structure and mechanism of action of ACE.     

Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure.

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.     

庆大霉素单克隆抗体的制备及试剂盒的配制

目的建立庆大霉素直接竞争酶联免疫吸附分析方法。方法应用戊二醛法制备庆大霉素完全抗原,通过杂交瘤技术筛选分泌特异性庆大霉素抗体的杂交瘤细胞株,并建立庆大霉素竞争酶联免疫吸附分析检测方法。结果获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,建立了庆大霉素竞争酶联免疫吸附分析检测方法,该方法操作简单具有良好的线性、特异性和精密度;庆大霉素质量浓度在1.5625～50.0000 ng/mL范围内,呈现良好的线性,r2=0.9913,50%抑制浓度为(IC50)为7.37 ng/mL,检测限(LOD)为1.54 ng/mL,该试剂盒与链霉素等8种药物无交叉反应。结论获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,研制的庆大霉素竞争ELISA检测试剂盒具有良好的线性、特异性和精密度。     

A practical method for measuring deoxynivalenol, nivalenol, and T-2 + HT-2 toxin in foods by an enzyme-linked immunosorbent assay using monoclonal antibodies

We have developed and tested an enzyme-linked immunosorbent assay system for individual measurement of deoxynivalenol, nivalenol, and T-2 + HT-2 toxin using monoclonal antibodies for 3,4,15-triacetyl-nivalenol, for both 3,4,15-triacetyl-nivalenol and 3,15-diacetyl-deoxynivalenol, and for acetyl-T-2 toxin. The assay system comprised three kits (desinated the DON + NIV kit, the NIV kit, and the T-2 + HT-2 kit). The practical performance of the enzyme-linked immunosorbent assay system was assessed by assaying trichothecene mycotoxins in wheat kernels. The enzyme-linked immunosorbent assay system meets all the requirements for use in a routine assay in terms of sensitivity (detection limit: deoxynivalenol 80 ng/g, nivalenol 80 ng/g, T-2 toxin 30 ng/g), reproducibility (total coefficient of variation: 1.9-6.2%), accuracy (recovery: 93.8-112.0%), simplicity and rapidity (time required: <2 h), mass handling (>42 samples/assay), and a good correlation with gas chromatography-mass spectrometry (r=0.9146-0.9991). Components derived from the wheat extract did not interfere with the assay kits. The enzyme-linked immunosorbent assay system is a useful alternative method to gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, or liquid chromatography-ultraviolet absorption for screening cereals and foods for trichothecene mycotoxin contamination.