Competitive Inhibition Enzyme Immunoassay Research Articles

γ-Hydroxyphosphonate inducing single-chain antibodies capable of catalyzing soman hydrolysis

A γ-hydroxyphosphonate P 6 ( O 1 -methyl- O 2 -(1, 2, 2-trimethylpropyl)-2-hydroxy-5- nitrophenyl methylphosphonic acid) which is proposed to be an analog of the transition state in hydrolysis of soman was synthesized. Artificial antigens were obtained by conjugating P 6 to the carrier proteins BSA (bovine serum albumin) and LPH ( Limulus polyphenus hemocyanin). Mice were immunized with P 6 -LPH and recombinant single-chain antibody phage display library was constructed. After 4 rounds of panning against P 6 -BSA and competitive inhibition enzyme immunoassay, more than 70 strains of phage antibodies capable of binding soman were obtained and 11 of them can accelerate the hydrolysis reaction of soman. One of them (EP 6 ) was studied further. Soluble single-chain antibody was prepared and purification was performed by gel filtration and ion exchange chromatography. The kinetic experiment was carried out showing that the turnover number k cat = 198 min -1 and the rate enhancement k cat / k uncat = 122 419. When 0.16 mg mL -1 EP 6 was preincubated in vitro with 0.132 mmol L -1 (220 mg kg -1 = 1.1×LD 95 ) of soman prior to the administration to mice by subcutaneous route, all animals (19 mice) survived whereas all the control mice (14) treated with PBS and soman died within 30 min. Furthermore, EP 6 could prolong the latent time of spasm and death when mice were passively immunized with EP 6 intravenously 15 min before 1×LD 95 of soman challenge. These results demonstrate that EP 6 is able to increase the rate of soman degradation and protect against soman's toxicity, especially in vitro.

Allergy to bovine beta-lactoglobulin: specificity of human IgE to tryptic peptides.

Bovine beta-lactoglobulin (Blg) is a major cow's milk allergen. It is the main whey protein, without any counterpart in human milk. Blg chemical hydrolysates appeared to retain most of the immunoreactivity of the native protein. Allergenicity of Blg has already been shown to be associated with the four peptides derived from cyanogen bromide cleavage of Blg. To map the major allergenic epitopes (e.g. regions of the molecule able to bind IgE) on Blg using specific IgE from sera of 46 milk-allergic patients as a probe. Direct and competitive inhibition enzyme immunoassays involving immobilized native protein or purified peptides derived from Blg tryptic cleavage. Several peptides capable of specifically binding human IgEs were identified and were classified according to the intensity and frequency of the responses. The major epitopes appeared to be fragments (41-60), (102-124) and (149-162) recognized by 92, 97 and 89% of sera, respectively, whilst a second group which contained the fragments (1-8) and (25-40) was recognized by 58 and 72% of the population. A third group, comprising peptides (9-14), (84-91) and (92-100), was still detected by more than 40% of sera. Three peptides were identified as major epitopes, recognized by a large majority of human IgE antibodies. Numerous other epitopes are scattered all along the Blg sequence.

Analysis of PCBs in waste oil by enzyme immunoassay

Immunoassays constitute a powerful analytical technique for sensitive, quantitative, fast and economical determination of trace contaminants in environmental monitoring. The enzyme-linked immunosorbent assay (ELISA)-has been used extensively to detect numerous medicinal, agricultural and environmental compounds. It is an effective tool for screening samples that will be subsequently analyzed by another method, as well as for rapid, quantitative analysis of a large number of samples. A commercially available kit has been applied successfully to the screening of polychlorinated biphenyls (PCBs) in transformer oil samples. The kit uses a competitive inhibition enzyme immunoassay (EIA) for recognition of the PCB structure. The test specificity is restricted to PCBs, with high sensitivity for Aroclors 1016, 1242, 1248, 1254 and 1260. Kit performance was validated by comparison of EIA and GC results for 35 transformer oil samples.

Evaluation of monoclonal antibodies with specificity of human IgA, IgA subclasses and allotypes and secretory component results of an IUIS/WHO collaborative study

51 monoclonal antibodies (McAb) with putative specificity for human IgA, the IgA subclasses, Am allotypes or secretory component (SC) were evaluated for immunoreactivity and specificity by nine laboratories employing immunodiffusion, agglutination, immunohistological assays, immunoblotting and direct binding and competitive inhibition enzyme immunoassays. McAbs specific for IgA PAN ( n = 24), IgA1 ( n = 7), IgA2 ( n = 3), IgA2m(2) ( n = 2), non-IgA2m(2) ( n = 4) and SC or secretory IgA ( n = 5) were identified that were immunoreactive and specific in the assays employed. The McAbs identified as IgA- or SC-reactive were shown to be non-reactive to human IgG, IgM, IgD, IgE, κ and λ by direct binding and competitive inhibition immunoassays. Interestingly, no McAbs with restricted specificity for IgA2m(1) were identified. Some McAbs displayed higher affinity and/or better performance in one or several of the assay groups. The IgA- and SC-specific McAbs identified in this international collaborative study have potential as immunochemical reference reagents to identify and quantitate monomeric and polymeric IgA in human serum and secretions.

Detection of the organophosphorus nerve agent sarin by a competitive inhibition enzyme immunoassay.

Two artificial antigens, NalphaNepsilon-di(O,O-diisopropyl) phosphoryl L-lysine (DIP)- bovine serum albumin (BSA) conjugate (DIP-BSA) and DIP-KLH (keyhole limpet hemocyanin), were synthesized. Antibodies against sarin (O-isopropyl methylphosphonofluoridate) were obtained after immunization of rabbits with DIP-KLH conjugate. A competitive inhibition enzyme immunoassay (CIEIA) was developed to detect the organophosphorus nerve agent sarin. The antibody solutions could be inhibited by sarin as low as 10(-6) mol/l, and the standard curve was linear over 3 orders of magnitude. The coefficients of intraassay and interassay variation of this method were 5.4-6.2% (n = 11) and 8.0-9.5% (n = 6) at a sarin concentration range of 10(-3)-10(-6) mol/l, respectively. The recovery of sarin in water samples at the concentration of 5 x 10(-5) mol/l was in the range of 96.8-102.5%. The specificity of the antiserum was assessed by comparing the inhibition induced by sarin with soman, Vx, isopropyl alcohol and isopropyl methyl phosphonic acid. The results showed that less than 5 mmol/l soman, 2 mmol/l Vx, 16 mmol/l isopropyl alcohol and 8 mmol/l isopropyl methyl phosphonic acid did not influence the determination of sarin in water samples.

Production, characterization and application of monoclonal antibodies against the organophosphorus nerve agent Vx.

Two monoclonal antibodies (Vx-BB8 and Vx-EA11) to the chemical warfare agent Vx were produced and characterized. A competitive inhibition enzyme immunoassay was developed to detect Vx concentrations as low as 3.7 x 10(-7) - 3.7 x 10(-6) mol/l in biological samples. Vx-BB8 400 micrograms given intravenously immediately before 1 x LD95 Vx or 400 micrograms Vx-BB8 intraperitoneally 1.5 h-3 days before 1 x LD95 Vx could protect all the tested mice from death.

Determination of Procymidone in vegetables by a commercial competitive inhibition enzyme immunoassay

An ELISA commercial kit was used to quantitate residues of Procymidone at very low levels (< 20 μg/kg) in pepper samples. Samples were extracted with ethyl acetate-sodium sulphate and an aliquot is evaporated to dryness and reconstituted in 10 ml of light petroleum. Sample clean up is accomplished by aspirating 2 ml of the light petroleum extract through a silica gel solid phase disposable cartridge. Following aspiration, the sample was eluted with 2 ml of ethyl ether-petroleum ether (1:1). The eluted fraction was evaporated and dissolved with sonication in 2 ml of water before immunoassay analysis. The limit of Procymidone detection was 0.6 μg/kg. The assay logarithmic response was linear from 5 to 80 μg/kg of Procymidone residue. Intra-assay percentage coefficients of variation (%C.V.) ranged from 3.5 to 18.0 and inter-assays %C.V.s varied from 8.0 to 16.0. Comparison studies between gas chromatography with electron capture detection and ELISA analyses showed an acceptable coincidence at levels of 8 μg/kg. However, lower levels of ca. 2 μg/kg were detectable by ELISA only. No cross-reactivity problems were found in the ELISA test associated with the presence of other dichloroaniline derivatives and other pesticides studied.

Taxane-Specific Monoclonal Antibodies: Measurement of Taxol, Baccatin III, and "Total Taxanes" in Taxus brevifolia Extracts by Enzyme Immunoassay

Three monoclonal antibodies with either specificity to taxol or baccatin III, or cross-reactivity with several common taxanes have been prepared and used to develop sensitive competitive-inhibition enzyme immunoassays. The hybridomas producing these monoclonal antibodies were obtained by fusing P3X63Ag8.653 plasmacytoma cells and splenocytes from mice hyperimmunized with keyhole limpet hemocyanin-7-succinyltaxol or -7-succinylbaccatin III conjugates. Direct and indirect competitive inhibition enzyme immunoassays were developed with these monoclonal antibodies and microtiter plates coated with bovine serum albumin conjugates of the complementary hapten. Detection limits for the direct competitive inhibition enzyme immunoassays, conducted in buffer containing 10% MeOH, were 0.6 nM taxol for 3C6 (anti-taxol); 1.1 nM baccatin III for 3H5 (anti-baccatin III); and 0.6 nM taxol or baccatin III for 8A10 (anti-taxane). The immunoassays accurately detected taxol, baccatin III, and "total taxanes" in crude MeOH extracts of Taxus brevifolia bark and in hplc fractions of these extracts.

Rapid Analysis of PCBs in Soil by Enzyme Immunoassay

Abstract A commercially available kit has been applied successfully to screening for polychlorinated biphenyls (PCB's) in soil samples. The kit uses a competitive inhibition Enzyme ImmunoAssay (EIA) for recognition of the PCB structure. Test specificity is restricted to PCB's, primarily Aroclors 1016, 1242, 1248, 1254, and 1260. Soil sample preparation and analysis can be performed in the field or lab using disposable kit components. Screening of unidentified Aroclors at 4 levels from 1 to 50 μg/g in soil is possible using the calibrators in the kit. This screening use has been reviewed by the USEPA Office of Solid Waste and will be proposed for inclusion in the 4000 series of screening methods in the next SW846 update. Conventional analysis (Soxhlet/GC-ECD) and the EIA kit were compared by Manitoba Hydro using 112 field samples over one year. EIA technology was found to be an effective screening tool for determining PCB concentration at contaminated sites. Routine use of these kits in conjunction with conventional Soxhlet extraction procedures has increased the lab's testing capability and reduced the amount of samples requiring conventional testing, providing substantial savings to the corporation.

Estrogen and progesterone receptors in breast cancer microsamples simultaneously quantified by enzyme-ligand immunoassay

A new method (enzyme-ligand immunoassay, ELIA) is described for the estimation of estrogen (ER) and progesterone (PR) receptors in microsamples of human breast cancer tissue. The technique, based on the nonisotopic measurement of receptor-bound estradiol and progesterone, involves three steps: (a) simultaneous saturation of active receptors with their respective authentic ligands, (b) heat treatment of the cytosol to release the steroids from their cognate receptors before or after absorption with dextran-coated charcoal, and (c) measurement of both steroids present in the cytosol by a modified competitive-inhibition enzyme immunoassay. The useful range of the method was 10-4000 pmol/L for ER and 6.5-1000 pmol/L for PR. The correlation coefficient (r) between the one-point and Scatchard plot analysis was 0.95 for ER and 0.99 for PR. Comparison of the one-point ELIA and expected values with the radioligand binding assay (RLBA) results for EORTC samples gave r = 0.88 and 0.99 for ER and PR, respectively. Further comparison of the one-point ELIA with RLBA and with a commercial enzyme immunoassay, in blind testing of cancer tissue microsamples from 70 patients, gave good agreement for ER with r = 0.95-0.97 and concordance of 92.9-94.4% (cutoff, 15 pmol/g protein) against the other two methods. The results were more disperse in all three methods for PR estimation, the assay correlating perhaps better with the enzyme immunoassay (r = 0.90) at a concordance of 89.4% (same cutoff value).

Free taxanes and the release of bound compounds having taxane antibody reactivity by xylanase in female, haploid-derived cell suspension cultures ofTaxus brevifolia

Haploid cells derived from the female gametophyte ofTaxus brevifolia produce multinucleate egglike cells that have grown as cell suspensions over a 5-yr period. Two new media are formulated, one without nitrates, to select for haploid cell lines and to enhance free taxane production when cell lines are artificially aged by withholding subculture. The cell suspensions produce bound taxanes that are detectable with anti-taxane monoclonal antibodies on cell surfaces and on particles released into the culture medium. Recovery and detection of bound taxanes are based on a competitive inhibition enzyme immunoassay (CIEIA). Tissues extracted exhaustively with methanol then treated with xylanase release CIEIA-detectable taxanes. Approximately 300% more taxanes were detected in the xylanase-treated tissue culture material versus the control. Wood (winter bark) taken fromT. brevifolia and treated in the same way with xylanase release 20% more taxanes than could be detected in the control.

Determination of Methyl 2-Benzimidazolecarbamate in Bulk Fruit Juice Concentrates by Competitive-Inhibition Enzyme Immunoassay

A polyclonal enzyme immunoassay (EIA) was used to quantitate methyl 2-benzimidazolecarbamate (MBC or carbendazim), a degradation product of benomyl, in bulk fruit juice concentrates. These concentrates are used by industrial producers to prepare juice or juice concentrates sold in supermarkets. Total sample analysis time was less than 18 min without cleanup or 35 min with cleanup. As many as 8 samples can be analyzed simultaneously, with a limit of quantitation of 10 ppb. The assay's dynamic range ran from 0.5 to 20 ppb MBC but was best from 0.5 to 10 ppb. Intra-assay coefficients of variation (CVs) varied from 4.0 to 13% for standards and from 4.1 to 26% for samples. Interassay CVs varied from 4.5 to 47% for standards and 5.6 to 22% for samples. Average recovery of several juice concentrates spiked at 10 to 290 ppb was 97%. A total of 140 juice concentrates comprising 20 different kinds of juice were analyzed by 2 EIA methods and one liquid chromatographic (LC) procedure. MBC-positive samples gave the following correlation coefficients: 0.954 for EIA without cleanup vs LC, 0.956 for EIA with cleanup vs LC, and 0.978 for EIA with cleanup vs EIA without cleanup. MBC concentrations in MBC-positive juice samples ranged from 5 to 2960 ppb.

Determination of Thiabendazole in Fruits and Vegetables by Competitive-Inhibition Enzyme Immunoassay

An enzyme immunoassay (EIA) was developed for analysis of thiabendazole (TBZ) in fruits and vegetables. A commercial kit using a polyclonal antibody for benomyl-carbendazim was used. Total analysis time, including sample preparation, was 35 min. As many as 8 samples can be analyzed simultaneously, with a limit of quantitation of 9 ppb. The assay's dynamic range ran from 9 to 304 ppb TBZ. Intra-assay coefficients of variation (CVs) ranged from 5.0 to 9.6% for standards and 11 to 30% for samples. Interassay CVs varied from 4.4 to 15% for standards and from 10 to 24% for samples. Average recovery from 29 samples spiked at 50-50,000 ppb was 116%. A total of 107 food products comprising 12 different fruits and vegetables and their processed products were analyzed for their TBZ content by EIA and liquid chromatography (LC). Of these samples, 84 were positive for TBZ by both methods, with a correlation coefficient (r) of 0.989. Eight samples had none detected by either technique, and 15 were positive for carbendazim. Concentrations of TBZ ranged from 11 to 94,000 ppb. The immunoassay with methanol sonication shows promise as a rapid screening method for TBZ in foods.

Determination of Methyl 2-Benzimidazolecarbamate in Soil by Competitive Inhibition Enzyme Immunoassay

Soil was analyzed for the fungicide or fungicide degradation product methyl 2-benzimidazolecarbamate(MBC) or carbendazim by employing commercially available polyclonal enzyme immunoassay (EIA) kits. MBC extraction from soil was done either by an overnight shaking with methanol-water (80:20) or by a 10-min 2 M ammonium chloride-ethanol shake followed by partitioning into methylene chloride. Intraassay and interassay percent coefficients of variation (% CVs) ranged from 2.2 to 13 for the standards and from 7 to 41 for the samples. A total of 101 soil samples obtained from Maine, Florida, and Switzerland were analyzed for MBC using EIA and liquid chromatography (LC)

Immunorecognition of ring skeleton of taxanes by chicken egg yolk antibodies.

Anti-10 deacetylbaccatin III (DAB) antibodies (IgY) were elicited in hens immunized with a succinyl-DAB/BSA conjugate and extracted from egg yolk. As shown by indirect competitive inhibition enzyme immunoassay (CIEIA), the addition of free-DAB competitively inhibited the binding of affinity purified anti-DAB IgY to DAB/BSA solid phase conjugated antigen. The assay enabled the detection of DAB in concentrations as low as 7.5ng/ml (13.7 nM DAB), whereas anti-DAB IgY did not react with taxol even at a concentration a thousand times higher. The structural requirements of the diterpenoid nucleus for binding to IgY were considered on the basis of the levels of cross-reaction found with 10 authentic taxanes. The results indicate that anti-DAB IgY represents the first high affinity antibody produced capable of recognizing the ring skeleton of taxol precursors.

Determination of Methyl 2-Benzimidazolecarbamate in Wine by Competitive Inhibition Enzyme Immunoassay

A benomyl polyclonal enzyme immunoassay (EIA) commercial kit was used to quantitate methyl 2-benzimidazolecarbamate (MBC), a degradation product of benomyl in wine. Total analysis time, including sample preparation, was 30 min. As many as 8 samples can be analyzed simultaneously with a limit of quantitation of 5 ppb. The assay logarithmic response was linear from 0.4 to 26 ppb MBC. Intra-assay percent coefficients of variation (%CVs) ranged from 2.4 to 13 for standards and from 7.4 to 21 for actual wine samples. Interassay %CVs varied from 2.6 to 15 for the standards and from 6.9 to 23 for the samples. Average recovery from samples spiked at 10-10,000 ppb was 93% for evaporated red and white wines. MBC was determined in 134 different wines by immunoassay and liquid chromatography (LC). Of these samples, 98 were positive for MBC by both methods with a correlation coefficient (r) of 0.986. The other 36 samples had MBC levels that either were not detectable by either procedure or were below the 10 ppb limit of quantitation for LC. Concentrations of MBC in wine ranged from 5 to 1329 ppb, with the majority ranging from 10 to 300 ppb. Also, a mini-study was conducted using the plate EIA format.

An enzyme immunoassay for the determination of taxol and taxanes in Taxus sp. tissues and human plasma

A rapid and sensitive indirect competitive inhibition enzyme immunoassay (CIEIA) method has been developed for quantitating taxanes in extracts of Taxus brevifolia Nutt. tissue and in human plasma. High titer rabbit polyclonal antibodies (pAb) were raised against a conjugate of 7-succinyltaxol and keyhole limpet hemocyanin. The presence of taxane analyte competitively inhibited the binding of the rabbit anti-taxane pAbs to a 7-succinyltaxol-bovine serum albumin solid phase coating antigen. The CIEIA detected taxol and cephalomannine in concentrations as low as 0.3 ng/ml (3.5 × 10 −4 μM), but did not detect baccatin III or 10-deacetylbaccatin III at concentrations below 1000 ng/ml (1.7 μM and 1.8 μM, respectively). This method is useful for estimating the taxane content of T. brevifolia extracts and may be useful for monitoring the taxol plasma level of taxol-treated patients.

Anticardiolipin antibodies in NZW x BXSB F1 mice. A model of antiphospholipid syndrome.

NZW x BXSB F1 (W/B F1) male mice develop systemic lupus-like disease, and several autoantibodies, circulating immune complexes, and lupus nephritis become apparent. The abnormally high incidence of degenerative coronary vascular disease with myocardial infarction and thrombocytopenia due to the presence of both platelet-associated antibodies and circulating antiplatelet antibodies in this animal has been reported. We found that W/B F1 male mice produced autoantibodies against cardiolipin (aCL) and that the titer of aCL increases with age. aCL from W/B F1 male mice were mainly IgG and binding activity to cardiolipin was aCL-cofactor (beta 2-glycoprotein I (beta 2-GPI)) dependent. We developed monoclonal aCL from these animals and examined specificity of the autoantibodies. All the mAb used reacted with the negatively charged phospholipids, cardiolipin, phosphatidylserine, and phosphatidylinositol, and some reacted with platelets and DNA. The addition of human or mouse beta 2-GPI enhanced the titer for monoclonal aCL from the W/B F1 mice. From the results of competitive inhibition enzyme immunoassay with monoclonal aCL and purified beta 2-GPI, aCL from the W/B F1 mice recognized the complex of CL and beta 2-GPI. The W/B F1 male mouse may be an appropriate model for use in studies on the pathologic significance of aCL in patients with antiphospholipid syndrome.

Determination of Methyl 2-Benzimidazolecarbamate in Blueberries by Competitive Inhibition Enzyme Immunoassay

Abstract A polyclonal enzyme Immunoassay (EIA) method was developed that will quantitate methyl 2- benzlmidazolecarbamate (MBC), the degradation product of benomyl, In blueberries. The entire analysis Is completed within 25 min, and up to 8 samples can be analyzed simultaneously with a detection limit of 18 ppb. The assay response was linear from 0.69 to 22 ppb MBC. Cross-reactivity was confined to the benzimidazole and dicarboxlm- Ide type pesticides. Intra-assay percent coefficients of variation (% CVs) ranged from 3.4 to 18.4 for the standards, and from 3.2 to 20.6 for the samples. Interassay % CVs varied from 7.5 to 22.3 for the standards, and from 7.5 to 22.0 for the samples. Correlation coefficient between immunoassay and liquid chromatography was 0.87 for the methanol extract and 0.98 for the methylene chloride partition of the methanol extract containing 100 mL 1% sodium chloride. None of the 40 field samples analyzed approached the tolerance of 15 ppm (mean of 20 positive samples was 55 ppb). The ability of the EIA to monitor MBC easily and Inexpensively at concentrations far below the tolerance has major implications for current dietary risk assessment methods.

Monoclonal antibodies against soman: characterization of soman stereoisomers

Hybridomas were produced which expressed monoclonal anti-soman antibodies as determined by microtiter enzyme-linked-antibody immunoassay (EIA). Each of these antibodies was titrated using a competitive inhibition enzyme immunoassay (CIEIA) with a variety of test ligands. The ligands used included soman (a racemic mixture), sarin, tabun, and each of the four stereoisomers of soman (C+ P+, C+ P-, C-P+ and C-P-). In all cases the antibodies tested exhibited IC50 values of 10(-4)-5 x 10(-6) M for soman. When sarin or tabun was used as a ligand, the antibodies exhibited no cross reactivity. All of the antibodies cross reacted with the four soman stereoisomers. A second group of hybridomas were produced which expressed monoclonal antibodies against CsPs-soman. These antibodies were used to make preliminary absolute chiral assignments to the four soman stereoisomers.