A monoclonal antibody to T-2 toxin

A specific, high affinity monoclonal antibody to T-2 toxin of Fusarium has been produced. The monoclonal antibody was conjugated to horse-radish peroxidase and employed to develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for the toxin. The sensitivity of the ELISA was 10 ng/ml with a working range up to 500 ng/ml. The antibody cross-reacted with HT-2 toxin (25%) but did not bind to any other trichothecene tested.     

Human serum thyroglobulin determination with monoclonal antibody one-step assay: minimum interference of autoantibody

Combination of two thyroglobulin monoclonal antibodies (monoAbs) recognizing epitopes which are rarely recognized by an antibody enabled us to develop a rapid one-step enzyme immunoassay of serum Tg. Of 87 monoclonal antibodies, 20 were selected for the purpose. The method is a sandwich technique employing a monoAb covering microplate and horse-radish peroxidase monoAb conjugate. A combination of monoAb 7A7A solid phase and 31A2E for the conjugate gave the best results. The assay takes 60 min and the minimal detectable amount is 2 ng/ml. Intraassay variation is from 4 to 7%. Interassay variation is 5 to 12%. The recovery rate for Tg added to normal sera is between 89 and 111%. The correlation coefficient with the polyclonal antibody method in Tg hemagglutination negative sera is 0.98. The presence of autoantibody in sera up to 10 X 2(4) hemagglutination titer does not affect the recovery rate to a statistically significant extent.     

Signal amplification of microarray-based immunoassay by optimization of nanoliposome formulations

The use of microarray-based immunoassay is often limited by its sensitivity. To increase the sensitivities of such an immunoassay, liposome encapsulation was explored. Two different liposome formations and several preparation methods were examined to optimize encapsulation and signal-enhancing efficacy for enzyme-linked immunosorbent assay (ELISA) and antibody array. The signal amplification by liposome encapsulation was demonstrated through a detection for foodborne pathogenic Listeria. In plate-trapped antigen (PTA) ELISA, horseradish peroxidase (HRP)-loaded liposome increased signal 9-fold more than the control. Limits of detection (LODs) of HRP-encapsulated liposome were 6.4×10(5) and 5.5×10(6)CFU/ml in sandwich ELISA and antibody array, respectively. Furthermore, when chromogenic 4-chloro-1-naphthol (4-CN) substrate was used for signal development in the antibody array, the signal could be detected with the naked eye. These results suggest that the liposome encapsulation technique can have great potential for signal amplification and, therefore, for increasing assay sensitivity for various formats of immunoassay, especially microarray-based format.     

A monoclonal antibody to aflatoxin B1: detection of the mycotoxin by enzyme immunoassay

A monoclonal antibody (McAb) was produced after fusion of mouse (X63.Ag8.6.5.3) myeloma cells with spleen cells isolated from female Balb-c/NZB F1 hybrid mice immunized with aflatoxin B₁ (oxine)-keyhole limpet haemocyanin conjugate. The hybridoma cell line producing antibody specific for aflatoxin B₁ (AFB₁) was grown in tissue culture and as an ascites tumour. The ascitic fluid gave suitably high dilution titres (1:800 000) by enzyme immunoassay and was conjugated to horseradish peroxidase by a two-step procedure with glutaraldehyde. The conjugate was used to develop a direct competitive enzyme-linked immunosorbent (ELISA) assay for AFB₁. The sensitivity of the ELISA was 0–2 ng/ml with a working range up to 10 ng/ml for AFB₁. The specificity of the McAb was determined and it was shown not to cross-react significantly with any of the metabolites tested. This McAb and the direct competitive ELISA described may prove of use in the detection of AFB₁ in foods and feeds.     

成人腹泻轮状病毒ELISA方法的建立和应用

本文通过特异性试验、阻断试验、交叉试验、敏感度试验和重复性试验,建立了成人腹泻轮状病毒一酶联免疫吸附试验法(ADRV—ELISA)。应用此法检测了全国20多个省区202份病人腹泻标本,检出率为91％。采用本ELISA、核酸电泳、电镜三种方法对48份病人腹泻标本进行了双盲法检测比较,结果三种方法的阳性检出率分别为100％、85.4％、56.25％(P<0.05)。实验结果表明,本ELISA应用于检测成人腹泻轮状病毒(ADRV),具有敏感度高。特异性强等优点。     

Enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxins in foods.

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of staphylococcal enterotoxins in foods. The "double-antibody sandwich" protocol combines parts of several procedures reported previously. Horseradish peroxidase was conjugated to antibody specific for an enterotoxin, and the antibody-enzyme conjugate was assayed with a 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid)-H2O2 substrate solution. Enterotoxins were added to a variety of foods that were representative of those implicated in staphylococcal food poisoning outbreaks. Extracts of the foods were assayed by the ELISA and radioimmunoassay. Enterotoxin levels below 1 ng/g of food were consistently detectable by the ELISA. These results compared favorably with those of the radioimmunoassay. Experiments confirmed the interference of protein A in double-antibody sandwich ELISAs. Although protein A interference has not been demonstrated to be a problem in food extracts, we suggest a screen for protein A interference in which immunoglobulin G from nonimmunized rabbits is used. All of the known staphylococcal enterotoxins could be detected by this method. Analysis of a food product for entertoxin by the ELISA can be completed in an 8-h working day.     

Detection of pathogenic bacteria in food samples using highly-dispersed carbon particles

There is an unmet need for detection methods that can rapidly and sensitively detect food borne pathogens. A flow through immunoassay system utilizing highly dispersed carbon particles and an amperometric technique has been developed and optimized. A sandwich immunoassay format was utilized in which pathogenic cells were captured by antibodies immobilized onto activated carbon particles, and labeled with horseradish peroxidase (HRP) conjugated antibodies. Flow of the peroxidase substrates resulted in an amperometric signal that is proportional to the number of captured cells. Factors influencing the analytical performance of the system, such as the quantity of carbon particles and concentrations of capture antibody, enzyme labeled antibody, and enzyme substrates, were investigated and optimized. Detection and quantification of Escherichia coli, Listeria monocytogenes and Campylobacter jejuni were demonstrated with low detection limits of 50, 10, and 50 cells/ml, respectively, and an overall assay time of 30 min. Milk and chicken extract samples were spiked with various concentrations of these pathogens and were used to challenge the system. The system design is flexible enough to allow its application to the detection of viruses and proteins.     

The first clinical trial for determination of alpha 1 fetoprotein by means of Sevatest-ELISA AFP Kit (micro I)

At the Institute of Sera and Vaccines, Praha, was invented and tested on clinical samples a kit for detection and quantification of alpha 1 fetoprotein in human serum. It is a heterogeneous EIA on the "sandwich" principle. Rabbit antibody to alpha 1 fetoprotein (further AFP) was used for coating the solid surface and goat horse-radish peroxidase labelled antibody to AFP was used as the tracer. Microtitration plate of Czechoslovak manufacture (KOH-I-NOOR, Dalecín) type P with 96 wells was used as the solid phase. The range of an approximately linear part of the calibration curve was intentionally chosen between 10 and 400 ng/ml, since in this way it fills the detection gap in AFP determination between 10 and 200 ng/ml, which is, on the one hand, a physiological value of AFP in human serum and, on the other hand, the bottom limit of sensitivity of counter immunoelectrophoresis (CIEP). Attention was devoted both to reproducibility of the method, i.e. results of intra- and interassays, and comparability with other foreign ELISA Kits. According to the correlation analysis, the kit was ascertained to be very well comparable with kits of foreign provenance. The coefficient of variation (CV) for the interassays varied between 11 and 16% and for intraassays it equalled 15%.     

Monoclonal Antibodies for Detection and Serotyping of Cucumber Mosaic Virus

Two monoclonal antibodies (MAbs) to cucumber mosaic virus (CMV) were selected from a panel of MAbs for use in the direct DAS (double antibody sandwich)-ELISA. Two different test procedures were developed: an ELISA with polyclonal and monoclonal antibodies (mixed ELISA) for the routine detection of CMV and a MAb-ELISA with two MAbs directed against different epitopes for the specific detection of the N serotype which is prevalent in GDR. The conventional two-step incubation of plates precoated with IgG was compared with simultaneous incubation of test sample and labelled antibody (one-step incubation). The mixed ELISA proved to be more sensitive than the direct DAS-ELISA with polyclonal antisera in detecting CMV in crude sap of infected plants. On the other hand, the MAb-ELISA could be used for serotyping of CMV isolates which is important in epidemiological investigations and in resistance breeding. Both the two-step and the one-step procedures gave similar results with some advantages of the latter procedure. One-step incubation is not only time-saving but seems to be also more sensitive with regard to the detection limit. However, care must be taken to circumvent the hook-effect occurring at high virus concentrations.     

Detection of immunoreactive peptides of Fasciola hepatica, with sera from infected subjects, by enzyme immunoelectrotransfer

In order to observe the immunoreactive peptides in a crude extract of adult Fasciola hepatica specimens, proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose paper. Next, sera from 14 human cases of F. hepatica infection, parasitologically confirmed, which presented high titers of specific antibodies against F. hepatica detected by ELISA, were reacted with the blotted peptides and immunodetected by an anti-human IgG conjugated with horse-radish peroxidase. SDS-PAGE showed at least 18 bands ranging 96 to 14 Kd. Groups of peptides weighing 94-66 Kd, 43-36 Kd and 35-14 Kd reacted with serum antibodies of 12 fascioliasis patients. In the remaining two cases, reactive peptides were not clearly observed. The 94-66 Kd components were immunoreactive with 12 out of the 14 serum samples. On the other hand, 43-36 Kd peptides reacted with 4 of the 14 sera and only 3 out of the 14 sera of infected individuals showed reaction with 30-14 Kd. F. hepatica infection induces in humans diverse antibody responses, being 94-66 Kd bands the most immunoreactive peptides and would be potential serodiagnostic antigens.     

单克隆抗体与多克隆抗体配对ELISA方法比较

以人绒毛膜促性腺激素(HCG)为抗原,制备出对HCG的多克隆抗体和特异性单克隆抗体,并进行抗体纯化和特性分析,利用辣根过氧化物酶(HRP)分别对其进行了标记.采用双抗夹心ELISA试验,探讨了多克隆抗体与单克隆抗体配对的若干事项.结果表明,利用单克隆抗体和酶标多克隆抗体配对,并用含动物血清的稀释液稀释酶标抗体,可实现对检测原的高特异性和高灵敏度检测.     

Specific determination of influenza H7N2 virus based on biotinylated single-domain antibody from a phage-displayed library

The unpredicted spread of avian influenza virus subtype H7N2 in the world is threatening animals and humans. Specific and effective diagnosis and supervision are required to control the influenza. However, the existing detecting methods are laborious, are time-consuming, and require appropriate laboratory facilities. To tackle this problem, we isolated VHH antibodies against the H7N2 avian influenza virus (AIV) and performed an enzyme-linked immunosorbent assay (ELISA) to detect the H7N2 virus. To obtain VHH antibodies with high affinity and specificity, a camel was immunized. A VHH antibody library was constructed in a phage display vector pMECS with diversity of 2.8 × 10⁹. Based on phage display technology and periplasmic extraction ELISA, H7N2-specific VHH antibodies were successfully isolated. According to a pairing test, two VHH antibodies (Nb79 and Nb95) with good thermal stability and specificity can recognize different epitopes of H7N2 virus. The capture antibody (Nb79) was biotinylated in vivo, and the detection antibody (Nb95) was coupled with horseradish peroxidase (HRP). Based on biotin–streptavidin interaction, a novel sandwich immune ELISA was performed to detect H7N2. The immunoassay exhibited a linear range from 5 to 100 ng/ml. Given the above, the newly developed VHH antibody-based double sandwich ELISA (DAS–ELISA) offers an attractive alternative to other diagnostic approaches for the specific detection of H7N2 virus.     

Comparison of Polyclonal Antisera in the Detection of Tomato Spotted Wilt Virus Using the Double Antibody Sandwich and Cocktail ELISA

Different polyclonal antisera and enzyme-linked immunosorbent assay (ELISA) procedures have been tested for their potential to detect tomato spotted wilt virus (TSWV). The virus could efficiently be detected in high dilutions of sap from infected plants, and at low concentrations of purified virus and nucleocapsid protein preparations in the cocktail ELISA and the double antibody sandwich ELISA (DAS-ELISA). Amounts of 1 to 3 ng of virus protein still gave positive readings using purified preparations, while sap could be diluted approximately 100,000 times. Differences in the detection level were observed using nucleocapsid protein antiserum (anti-N-serum) and the antiserum against intact virus particles (anti-TSWV-serum), but both antisera showed to be powerful sera for the detection of TSWV. Using anti-N-serum, TSWV could be detected in highly diluted extracts of different hosts, and also in leaf extracts or intact tissues stored for 30 days under different conditions. These results indicate that the TSWV nucleocapsid protein remains antigenic for long periods.     

Determination of human thrombin-antithrombin III complex by enzyme immunoassay

We have developed a specific and sensitive ELISA for the measurement of the TAT in human plasma. The assay follows the sandwich principle and uses two different antibodies directed against human thrombin and human antithrombin III, respectively. The anti-thrombin antibody population used for coating was purified by immunoadsorption on immobilized prothrombin and thrombin, respectively. Antithrombin III antibodies were conjugated with peroxidase. Plasma samples containing TAT were incubated in polystyrene tubes coated with anti-thrombin antibodies; after washing, peroxidase-conjugated antithrombin III antibodies were added and bound enzyme activity was subsequently measured using o-phenylenediamine. The assay was calibrated with definite concentrations (2.0 to 60 micrograms/l) of preformed purified TAT added to TAT-poor plasma. Plots of absorbance at 492 nm against TAT concentrations revealed a linear correlation (r = 0.98). A reference range from 0.85 to 3.0 micrograms/l was calculated from TAT concentration in plasma samples from 88 healthy donors (mean value +/- SD: 1.45 +/- 0.4 micrograms/l). In patients with deep vein thrombosis confirmed by phlebography (n = 15), TAT was found up to 7-13 micrograms/l. Patients with septicemia associated with a consumption coagulopathy (n = 10) showed markedly increased TAT values (greater than or equal to 10 micrograms/l). From these data it can be concluded that measurement of TAT might be a parameter for detection of a latent clotting pathway activation.     

Detection of Piscirickettsia salmonis in fish tissues by an enzyme-linked immunosorbent assay using specific monoclonal antibodies

An enzyme-linked immunosorbent assay (ELISA) for the detection of Piscirickettsia salmonis in fish tissue samples was developed. The test uses a combination of different monoclonal antibodies specific against P. salmonis in the capture step of the assay. The antibodies 7G4, 6E2 and 2C1 chosen for the capture step are bound to the solid support with an adhesive protein purified from a bivalve mollusc, resulting in a high yield of adsorption and binding stability. The monoclonal antibody 7G4, used as a second antibody, is conjugated to horseradish peroxidase. The resulting ELISA test detected 7 different isolates of P. salmonis and does not cross-react with several other fish pathogens, revealing a high specificity and sensitivity. The test also detects P. salmonis in kidney tissue of infected coho salmon with 98% correlation with the immunofluorescence assay.     

Sensitivity and specificity of Czechoslovak ELISA HBsAg Sevac tests: comparison with other third-generation tests and counter-immunoelectrophoresis

Two variants of sandwich-type ELISA (Enzyme Linked Immunosorbent Assay) kits for HBsAg detection (Sevatest ELISA HBsAg Macro I and Sevatest ELISA HBsAg Micro I) in human sera and plasmas were developed. As the solid phase, the ELISA Macro kit and ELISA Micro kit make use of polystyrene microtubes, and polystyrene microtitration plates, respectively, of Czechoslovak production (Koh-i-noor, Dalecín). Capture anti HBs antibody for adsorption to solid phase and rabbit anti HBs antibody for labelling with horse-radish peroxidase were prepared for both tests. The sensitivity of both ELISA kits for HBsAg, equal to approx. 2 ng/ml, was determined by titrating six selected HBsAg-positive sera and the WHO Agk 76 panel of HBsAg-positive sera and the results were compared with those obtained by ELISA, RIA (Radioimmunoassay) and RPHA (Reverse passive hemagglutination) kits of different producers and by counter-immunoelectrophoresis (CIEP). The sensitivity of the new ELISA kits was comparable to that of other producers' ELISA kits, higher than that of RPHA kits and only a little lower than that of RIA kits. A set of sera of patients hospitalised with different diagnoses was tested for HBsAg. The detection rate by ELISA Macro kit 2.8 and 1.5 times higher than by CIEP and RPHA (Raphadex B), respectively, and 1.1 time lower than by RIA (Austria II).     

A 20–24 H MICROCOLONY-IMMUNOBLOT TECHNIQUE to DIRECTLY DETECT and ENUMERATE LISTERIA MONOCYTOGENES INOCULATED INTO FOODS

A microcolony-immunoblot technique (MCIBI) was developed to directly enumerate, in less than 24 h, very low numbers of Listeria monocytogenes (8–12 colony forming units: CFU/g or mL) inoculated into foods. Four meat and poultry and two dairy products were artificially inoculated with L. monocytogenes V7 diluted and plated on Oxford agar medium. Each plate was overlaid with an Immobilon-P membrane and incubated for 18–20 h at 37C. Blot-transferred colonies on these membranes were probed with C11E9 monoclonal antibody (MAb) and developed using peroxidase conjugated goat antimo use Ig G and a water insoluble substrate (3,3-diaminobenzidin tetrahydmchloride; (DAB-HCI), Nickel chloride and H₂O₂). the MCIBT gave L. monocytogenes counts that were not significantly lower than direct colony counts on selective agars. This technique allowed the recovery of 94–100% of L. monocytogenes cells inoculated into foods containing natural background flora counts of 3 × 10⁴ to 8 × 10⁶ CFU/g or mL. Using a 2 h resuscitation period on nonselective agar before overlay with Oxford media, the MCIBT allowed detection of sublethally heat injured cells of strain V7.     

Enzyme-linked immunosorbent assay for detection of chitooligosaccharides

In order to detect chitooligosaccharides (COS), an enzyme-linked immunosorbent assay (ELISA) was developed. A chitooligosaccharide mixture (COSM) conjugated to bovine serum albumin was used to immunize rabbits to produce an anti-COS polyclonal antibody. By use of specific antibody and COSM-horseradish peroxidase conjugate, we established a competitive direct ELISA (cdELISA) the detection limit of which was about 0.1 microg/ml. In the cdELISA, the cross-reactivities of the specific antibody toward glucosamine, chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose were 0.27, 27, 75, 75, 144, and 100%, respectively, and those toward N-acetylchitobiose, N-acetylchitotriose, N-acetylchitotetraose, N-acetylchitopentaose, and N-acetylchitohexaose were 1.58, 0.005, 1.08, 0.05, and 0.40%, respectively.     

Enzyme-Linked Immunosorbent Assay for Detection of Chitooligosaccharides

In order to detect chitooligosaccharides (COS), an enzyme-linked immunosorbent assay (ELISA) was developed. A chitooligosaccharide mixture (COSM) conjugated to bovine serum albumin was used to immunize rabbits to produce an anti-COS polyclonal antibody. By use of specific antibody and COSM-horseradish peroxidase conjugate, we established a competitive direct ELISA (cdELISA) the detection limit of which was about 0.1 μg/ml. In the cdELISA, the cross-reactivities of the specific antibody toward glucosamine, chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose were 0.27, 27, 75, 75, 144, and 100%, respectively, and those toward N-acetylchitobiose, N-acetylchitotriose, N-acetylchitotetraose, N-acetylchitopentaose, and N-acetylchitohexaose were 1.58, 0.005, 1.08, 0.05, and 0.40%, respectively.     

Identification of an Antigenic Determinant on Anionic Peanut Peroxidase by Monoclonal Antibodies

Two monoclonal antibodies (46–12-C12 and 23–6-C12)raised against anionic peanut peroxidase were found to haveindependent epitope sites. These topographic sites were foundto be located within a tryptic glycopeptide (Atgp) from theanionic isozyme by both indirect and non-competive ELISA andWestern blotting. The Atgp has a M_r equal to 11 000 of which 70% is carbohydrateand the peptide is probably highly hydrophobic as determinedby its high R_F (0.83) value and the amino acid composition.McAb 23–6-C12 recognized a contiguous epitope which encompassedalso the sole N-linked oligosaccharide on the anionic isozyme.That the monoclonal antibody also recognized the oligosaccharideon the -amylase, ß-glucosidase, acid phosphatase,and horse-radish peroxidase may be related to similarities insugars. Sugar removal from the Atgp or from the cross-reactivepeptide of enzymes caused loss of antibody affinity. The monoclonal antibody 46–12-C12 recognized specificallya conformational epitope near the region of the cysteine, tryptophaneand methionine residue on Atgp. Digestion of the anionic isozymeby trypsin resulted in a 40-fold loss of affinity with thismonoclonal antibody. Moreover, treatment of the Atgp with performicacid or trifluoromethane sulphonic acid caused a loss of affinitybetween the treated Atgp and this monoclonal antibody. Key words: Monoclonal antibodies, peanut, anionic peroxidase, glycopeptide, trypsin digest