Monoclonal antibody-based enzyme immunoassay for mercury(II) determination

A monoclonal antibody (K3C6) was developed against Hg(II) and applied in different enzyme immunoassay (EIA) formats to determine the test system with the highest sensitivity. A detection limit of 1. 0 microg/L Hg(II) could be achieved with a competitive format in contrast to a detection limit of 2.1 microg/L Hg(II) with a noncompetitive EIA. A competitive displacement EIA yielded the best detection limit of 0.4 microg/L Hg(II) and was well suited to measuring real samples. For this purpose different water samples were diluted at least 1:10 to avoid matrix effects and subsequently spiked with 1 microg/L HgCl(2). Recovery of the spiked samples was between 80 and 120%.     

Monoclonal antibody-based enzyme immunoassay for pediocins of Pediococcus acidilactici.

Monoclonal antibody (MAb) R2-AR against pediocin RS2 was developed. Mice were immunized for 12 weeks with pediocin RS2 conjugated to a polyacrylamide gel. Two hybridoma fusions yielded an MAb that in Western blots (immunoblots) reacted only with pediocins RS2 and AcH (3 kDa) from Pediococcus acidilactici RS2 and H, respectively, and did not react with any other bacteriocin, including sakacin A from Lactobacillus sake Lb 706, leuconocin LCM1 from Leuconostoc carnosum LM1, nisin from Lactococcus lactis ATCC 11454, and pediocin A from Pediococcus pentosaceus FBB61. Each of the bacteriocin bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels was confirmed to be biologically active by a gel overlay test performed with sensitive indicator organisms. In dot immunoblot assays, the MAb could detect a minimum of 32,000 arbitrary units of pediocin RS2 or AcH per ml. In colony immunoblot assays, the MAb was used successfully to differentiate bac+ and bac- variants of P. acidilactici RS2 strains.     

Generation of polyclonal antibodies against nisin: immunization strategies and immunoassay development.

Murine polyclonal antibodies reactive to the lantibiotic bacteriocin nisin A (nisA) have been produced by immunization with nisA-cholera toxin and nisA-keyhole limpet hemocyanin (nisA-KLH) conjugates. Mice immunized with nisA-cholera toxin developed nisA-specific antibodies with low relative affinities and poor sensitivities, while the immunization of mice with nisA-KLH conjugates resulted in the production of nisA-specific antibodies with high relative affinities and much-increased sensitivities. nisA antibodies could also be readily mass produced in less than 8 weeks in ascites fluid by using the nisA-KLH conjugate. A competitive direct enzyme-linked immunosorbent assay (ELISA) whereby nisA-horseradish peroxidase and free nisA competed for antibody binding was devised. The detection limit for nisA in the competitive direct ELISA with the nisA-KLH-generated antibodies was from 5 to 100 ng/ml, while the amount of free nisA required for 50% antibody binding inhibition ranged from 0.3 to 5 micrograms /ml. Both antisera and ascites polyclonal antibodies cross-reacted with nisZ either in the supernatant of a producer strain or with the pure lantibiotic but did not cross-react at all with non-lantibiotic-type bacteriocins. These polyclonal antibodies should find a wide usage from nisA ELISA analysis in foods and other matrices.     

Quantitative determination of factor VIII antigen with an enzyme immunoassay

We describe an enzyme-immunoassay for the determination of factor VIII antigen. After representation of the isolation of proteins the enzyme-immunoassay is presented. The principle of the method is the following: Test plasma is mixed with rabbit antibody in excess and incubated at 37 degrees C. The incubation mixture is added to polystyrene tubes, which are coated with human factor VIII. The rabbit antibody is available to adhere to factor VIII coating the tube and can be detected with an enzyme-labeled antibody to rabbit IgG. This method is sensitive to 7.8 . 10(-3) U/ml factor VIII antigen; the variation coefficient is 10.9%.     

A monoclonal antibody-based enzyme immunoassay for the detection of T-2 toxin at picogram levels

Thirteen monoclonal antibodies reactive with HT-2 were prepared by using a HT-2 hemisuccinate coupled to human serum albumin as antigen for the immunization of BALB/c mice. In a competitive enzyme immunoassay on a double antibody solid phase using HT-2 hemisuccinate coupled to horseradish peroxidase as enzyme linked toxin all antibodies reacted much better with T-2 toxin and acetyl T-2 than with HT-2. Eleven antibodies showed almost the same sensitivity and specificity, and one of these, designated 3E2, is extensively described. Its cross-reactivities with HT-2, T-2 toxin, acetyl T-2, iso T-2, T-2 tetraol tetraacetate and T-2 triol were 1·0, 140·2, 161·2, 0·32, 0·14 and 0·016, respectively. Two other antibodies, designated 2A4 and 2A5, behaved quite differently. The cross-reactivities of antibody 2A4 with these toxins were: 1·0, 113·9, 374·4, 1·35, 0·34 and 0·023, respectively; for antibody 2A5 they were 1·0, 46·1, 155·4, 8·31, 0·9 and 0·08, respectively. All antibodies proved to be IgGl. By using the antibody 3E2 a highly sensitive and very specific enzymc immunoassay for the detection of T-2 toxin was developed. The detection limit for T-2 toxin was 5 pg/ml (0·25 pg/assay).     

Detection of Aspergillus fumigatus mycotoxins: immunogen synthesis and immunoassay development

Immunological detection of secreted low molecular weight toxins represents a potentially novel means of diagnosing infection by the fungus Aspergillus fumigatus. Two such metabolites, gliotoxin and helvolic acid, were selected and conjugated to thyroglobulin for antisera generation in rabbits. Gliotoxin was initially activated using N-[p-maleimidophenyl] isocyanate (PMPI) and subsequently conjugated to S-acetyl thioglycolic acid N-hydroxysuccinimide-activated thyroglobulin, whereas helvolic acid was activated with N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in the presence of thyroglobulin prior to immunisation. To facilitate subsequent antisera evaluation, both toxins were similarly conjugated to bovine serum albumin (BSA). Matrix-Assisted Laser Desorption Ionisation-Time Of Flight (MALDI-TOF) mass spectrometry and SDS-PAGE analysis confirmed covalent attachment of toxins to BSA in the ratios of 15 and 2.4 mol per mol BSA for gliotoxin and helvolic acid, respectively. Resultant high titer antisera were capable of detecting both BSA-conjugated toxins (inhibitory concentration (IC)(50): 4-5 microg/ml). Free toxins were also detectable by competitive immunoassay, whereby 10 microg/ml free gliotoxin (30 microM) and helvolic acid (17 microM), respectively, inhibited antibody binding to cognate toxin-BSA previously immobilised on microwells. This work confirms that sensitive and specific antisera can be raised against fungal toxins and may have an application in diagnosing fungal infection.     

Enzyme immunoassay for the determination of hexestrol in meat

An indirect competitive enzyme-linked immunosorbent assay (ELISA) of hexestrol (HES), an anabolic hormone forbidden for use in livestock farming, has been developed. Conditions of ELISA have been optimized by varying the concentrations of the coating conjugate (HES-ovalbumin), anti-HES antiserum, casein, and Tween 20. In the absence of Tween 20 in the reaction mixture, the detection limit (IC₁₀) equaled 0.01 ng/ml, IC₅₀ equaled 0.17 ng/ml, and the working range (IC₂₀–IC₈₀) equaled 0.03–0.86 ng/ml, while, in the presence of 0.05% Tween 20, these values equaled 0.05, 2.9, and 0.26–32.0 ng/ml, respectively. Standard deviation of the analysis results did not exceed 5.4%. If ELISA was performed in the absence of detergents, the recovery value upon HES determination in spiked beef samples ranged from 74 to 147%.     

Enzyme immunoassay for the determination of the glycopeptide antibiotic eremomycin

An indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed using rabbit polyclonal antibodies against the eremomycin-glucose oxidase conjugated antigen. This technique allows the glycopeptide antibiotic eremomycin to be determined both in aqueous solutions (with a sensitivity as high as 0.1 ng/ml) and in blood plasma. The cross-reactivity of the antibodies with vancomycin was 0.4% of that for eremomycin, while teicoplanin was almost not recognized. Experiments with blood plasma samples diluted 1: 10 showed that the assay was linear over the concentration range 1–30 ng/ml and that the variation coefficient did not exceed 10%. The high sensitivity and selectivity of this test make it suitable for pharmacokinetic studies and drug monitoring analysis.     

A doubly amplified electrochemical immunoassay for carcinoembryonic antigen

An ultrasensitive electrochemical immunoassay (EIA) for the detection of carcinoembryonic antigen (CEA) is described in this report. The assay involves utilizing enzyme-catalyzed deposition of a redox polymer and electrocatalytic oxidation of ascorbic acid (AA) by the deposited redox polymer, a dual-amplification scheme to enhance analytical signals. Briefly, CEA capturing antibody and redox polymer anchoring agent were covalently immobilized on a gold electrode. After incubating with CEA, the electrode was treated in detection antibody-glucose oxidase conjugate solution. Thereafter, it was dipped into the redox polymer solution. Upon the addition of glucose, the redox polymer was enzymatically reduced and deposited on the electrode surface. The deposited redox polymer exhibits excellent electrocatalytic activity towards the oxidation of AA. Consequently, CEA could be quantified amperometrically. This electrochemical immunoassay combines the specificity of the immunological reaction with the sensitivity of the doubly amplified electrochemical detection.     

Hapten synthesis for enzyme-linked immunoassay of the insecticide triazophos

Two haptens of the insecticide triazophos (O,O-diethyl O-[1-phenyl-1H-1,2,4-triazol-3-yl] phosphorothioate) were synthesized by introducing appropriate spacers in the O-ethyl site of the analyte molecular structure. First, thiophosphoryl chloride (PSCl(3)) reacts with methanol at low temperature to give O-ethyl dichlorothiophosphate. After reacting with 1-phenyl-3H-1,2,4-triazol, the O-ethyl dichlorothiophosphate was transformed into the intermediate O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) chlorothiophosphate. Then the intermediate reacts with 4-aminobutyric acid and 6-aminobutyric acid to produce hapten I and hapten II, respectively. The molecule structures of the two haptens were identified by (1)H nuclear magnetic resonance spectrum and mass spectrum. An enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody was also developed to evaluate the two haptens. Results showed that the monoclonal antibodies with high titers were obtained after immunizing with protein conjugates of these haptens and that the immunoassay has high affinity and specificity to triazophos. These results suggested that the haptens were synthesized successfully and could be used for immunoassay for the rapid screening and sensitive determination of this insecticide.     

Fluorescein isothiocyanate-hapten immunoassay for determination of peptide-cell interactions

We have developed a fluorescein isothiocyanate (FITC)-hapten immunoassay, where a FITC-labeled peptide binding to a cell is assayed as the amount of immunoreactive fluorescein present in a cell lysate. An antifluorescein-horseradish peroxidase conjugate binds to either a fluoresceinated peptide in the lysate or a fluorescein attached to the wells of a microtiter plate in a competitive fashion. After washing, solid-phase peroxidase activity is measured and inversely related to the amount of FITC-labeled peptide present. To demonstrate the assay, the interaction of a FITC-labeled bombesin-like peptide with the gastrin-releasing peptide receptor on PC-3 and HT-29 cells was investigated. Using PC-3 cells, we obtained similar displacement curves and numbers of binding sites per cell by both the FITC-hapten immunoassay and a reference radioreceptor assay. The FITC-hapten immunoassay is a sensitive and versatile method, since the same commercially available reagents can be used to assess interactions between any peptide and any receptor. In addition, the FITC-labeled peptide can be used to visualize receptors in fluorescent-activated cell sorting or fluorescent microscopy.     

A monoclonal antibody-based immunoradiometric assay for detection of circulating antigen in Bancroftian filariasis

A monoclonal antibody designated Gib 13 has been used in an immunoradiometric assay (IRMA) to detect circulating antigen in the sera of Wuchereria bancrofti-infected subjects from an endemic area of Papua New Guinea. A clear association between the presence of patent infection and the Gib 13 target epitope in serum was established because 93% of microfilaremic individuals were antigen-positive. Moreover, there was a significant correlation between levels of serum antigen and blood microfilarial counts. Detection of circulating antigen in amicrofilaremic subjects with acute symptoms of lymphatic filariasis, and 53% of asymptomatic amicrofilaremic subjects, but not in nonendemic controls, suggests that the Gib 13 IRMA will also be of value in the diagnosis of occult filariasis. However, as in all IRMA based on detection of potentially immunogenic molecules in man, antibodies can be expected to be the major contributor to reduced sensitivity of the assay.     

Enzyme immunoassay for determination of sulfamethoxypyridazine in honey

An enzyme immunoassay technique for the detection of sulfamethoxypyridazine in honey, developed using rabbit polyclonal antibodies raised against N-sulfonyl-4-aminobutyric acid, which contains a structural group characteristic of sulfonamides, is proposed. Under the optimized conditions, the sulfamethoxypyridazine detection limit was 0.05 ng/ml, with the entire analysis procedure taking 2 h. In total, 24 honey samples were tested using the protocol based on tenfold dilutions of samples without their preliminary treatment.     

Immobilized antibody-based flow type enzyme immunosensor for determination of human serum albumin

A flow-type enzyme immunosensor was prepared for the electrochemical determination of human serum albumin (HSA). The immunosensor was constructed from the immobilized antibody (anti-HSA IgG) reactor and an oxygen electrode. The immunochemical reaction of catalase-labelled antibody with HSA was completed with 30 min. After the immunochemical reaction, hydrogen peroxide solution was injected into the system and a peak current was obtained within 2 min. A linear relationship was observed between the current increase and the logarithm of HSA concentration in the range 10⁻⁸-10⁻⁶ g ml⁻¹. The minimum measurable concentration was 10⁻⁸ g ml⁻¹. The current increase was reproducible with 10% of the relative errors when a sample solution containing 10⁻⁷ g ml⁻¹ of HSA was used. The minimum measurable concentration increased to 10⁻⁹ g ml⁻¹ when hydrogen peroxide was recycled for 5 min in the reactor system. The immobilized antibody reactor could be reused. HSA in human serum was determined by the system proposed.     

Development of an antibody-based assay for determination of baculovirus titers in 10 hours

The baculovirus expression system has been used to produce large amounts of biologically active proteins by infecting insect cells with a recombinant baculovirus expressing the target protein. For an efficient expression of the target protein, it is necessary to infect insect cells with an adequate amount of virus. However, current methods are time-consuming and either have technical difficulties or are limited as a result of virus expression mechanism using a reporter gene. A novel method is developed to yield virus titers in 10 h that is easy to perform using 96-well plates and applicable to both any Autographa californica nucleopolyhyderovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV)-based recombinant baculovirus. This assay uses an antibody to a DNA-binding protein to detect the infected cells via immunostaining. The titer is determined by counting foci produced as a result of infection of the virus under a fluorescent microscope. The required incubation period was shortened considerably because infected cells expressed viral antigens at the post-infection time of 4 h. Therefore, 10 h was enough to estimate the virus titer including virus infection time, insect cell culture, and estimation of virus titer. Titers determined using this immunological assay are comparable, both in value and validity, to those obtained using a traditional method, provided that the stocks have titers above 10(3) pfu/mL.     

Monoclonal antibody-based immunoassay of vitellogenin in the blood of male channel catfish (Ictalurus punctatus).

1. A monoclonal antibody to vitellogenin of channel catfish (Ictalurus punctatus) was made, and its specificity was demonstrated using Western blots of serum from female fish, estradiol-treated male fish, untreated male fish, vitellogenin purified by three different methods and egg extracts. 2. An enzyme-linked immunosorbent assay (ELISA), using this monoclonal antibody, detected vitellogenin in the plasma of 59 out of 60 untreated 17-24-month-old male channel catfish with a mean concentration of 338 micrograms/ml and a maximum concentration of 4240 micrograms/ml. 3. Vitellogenin levels in male channel catfish were unrelated to testicular stage, gonadosomatic index and month.     

Development of a competitive enzyme immunoassay for factor VIII-related antigen

An enzyme immunoassay system basing on a competitive method has been developed to measure factor VIII related antigen (F. VIII R:Ag). A sufficient discrimination at low F. VIII R:Ag concentrations was gained. This method appears to be sensitive to 7,8 X 10(-3) U/ml F. VIII R:Ag showing an intraassay coefficient of variation (CV) of 0,11. In comparison to the commonly used Laurell electroimmunodiffusion assay for factor VIII significant less antisera per sample for the enzyme immunoassay technique is necessary.     

Rapid and sensitive quantitative immunoassay for the large simian virus 40 T antigen

A quantitative, enzyme-linked immunoadsorbent assay has been developed for the simian virus 40 large T antigen. When hamster anti-simian virus 40 tumor serum was used, this method permitted specific identification of large T antigen and its analog, the D2 hybrid protein, a molecule with the same C-terminal approximately 600 amino acids as large T antigen. The sensitivity limit of this test was 0.63 ng of protein. The slopes of the regression lines of the enzyme-linked immunosorbent assay titrations performed with highly purified D2 or simian virus 40 large T antigen and with crude extracts of simian virus 40-infected monkey and transformed human cells were identical. Thus, the curve generated with a purified protein, such as D2, can serve as a quantitative standard for the measurement of large T antigen in a wide variety of extracts. Furthermore, solutions containing high salt concentrations and buffers containing up to 0.1% Nonidet P-40 did not interfere with the assay, making it applicable to the measurement of large T antigen in a variety of chromatographic fractions. The enzyme-linked immunosorbent assay was three times more sensitive, was significantly faster to perform, and was quantitatively valid over a much broader large-T-antigen concentration range than the complement fixation test. As such, it should be useful in future studies of the structure and function of this protein.