Production of antibodies and development of highly sensitive formats of enzyme immunoassay for saxitoxin analysis

In this paper the production of antibodies against saxitoxin (STX) is described, as is the optimization and comparison of two competitive ELISA formats (direct and indirect) for the detection of this toxin. Tests were performed in a 96-well microplate using the toxin-specific polyclonal antibodies produced in our laboratory, obtained from rabbits immunized with saxitoxin-keyhole limpet hemocyanin (STX-KLH). In indirect ELISA format saxitoxin, conjugated to bovine serum albumin (STX-BSA) was coated onto the microtitre plate and incubated with standard toxin and anti-STX antibody. A goat anti-rabbit IgG Peroxidase conjugate was used to enable detection. In the direct ELISA format, STX standard, STX conjugate to horseradish peroxidase (STX-HRP), and enzyme substrate/chromogen solution were sequentially added to the microplate after antibody coating.Results showed the saxitoxin detection limit to be 3 and 10 pg mL(-1) for direct and indirect ELISA formats, respectively.The suitability of the assay for quantification of saxitoxin in mussels was also studied. Samples were spiked with saxitoxin before and after sample treatment to study the extraction efficiency and matrix effect, respectively. After treatment, samples were analysed at 1:1000 v/v dilution in PBS to minimize the matrix effect and to detect the regulatory limit of 40-80 micro g saxitoxin per 100 g mussels as stipulated by the Food and Drug Administration. The efficiency of extraction of saxitoxin was from 72 to 102%. These data were confirmed by liquid chromatography coupled with fluorimetric detection, the technique currently used for quantitative determination of toxins in seafood.     

Amperometric Immunoassay of Azinphos‐Methyl in Water and Honeybees Based on Indirect Competitive ELISA

Abstract

An electrochemical immunosensor based on indirect competitive ELISA technique has been developed and tested for the detection of azinphos‐methyl in aqueous solutions and spiked honeybee extracts. The detection of the pesticide was based on competition for binding to monoclonal antibodies with an ovalbumin (OVA) conjugate, followed by the incubation with anti‐mouse IgG labeled with horseradish peroxidase, whose activity was measured amperometrically with hydroquinone as the substrate. The sensitivity of the azinphos‐methyl assay, estimated as the IC₅₀ value, was found to be 1.2 nmol L^?1 (60 min incubation), with a linear range of 0.6–500 nmol L^?1 in optimal conditions. The matrix effect on the detection of azinphos‐methyl in honeybee extract was found negligible, with the recovery values in the range 92–105%.     

Novel enzyme-linked immunosorbent assay for determination of fluvastatin in plasma at picogram level

For the first time, an enzyme-linked immunosorbent assay (ELISA) has been developed and validated for the determination of fluvastatin (FLV) in plasma samples at picogram level. The assay employed a polyclonal antibody that specifically recognizes FLV with high affinity, and FLV conjugate of bovine serum albumin (FLV-BSA) immobilized onto microplate wells as a solid-phase. The assay involved a competitive binding reaction between FLV, in plasma sample, and the immobilized FLV-BSA for the binding sites on a limited amount of the anti-FLV antibody. The bound anti-FLV antibody was quantified with horseradish peroxidase-labeled second anti-rabbit IgG antibody (HRP-IgG) and 3,3′,5,5′-tetramethylbenzidine (TMB) as a substrate for the peroxidase enzyme. The concentration of FLV in the sample was quantified by its ability to inhibit the binding of the anti-FLV antibody to the immobilized FLV-BSA and subsequently the color intensity in the assay wells. The conditions for the proposed ELISA were investigated and the optimum conditions were employed in the determination of FLV in plasma samples. The assay limit of detection was 10 pg mL⁻¹ and the effective working range at relative standard deviations (RSD) of ≤5% was 20-1000 pg mL⁻¹. Analytical recovery of FLV from spiked plasma was 97.1-102.7 ± 2.85-6.25%. The precision of the assay was satisfactory; RSD was 2.46-5.37 and 3.19-6.64% for the intra- and inter-assay precision, respectively. The analytical procedure is convenient, and one can analyze ∼200 samples per working day, facilitating the processing of large-number batch of samples. The proposed ELISA has a great value in routine analysis of FLV for its therapeutic monitoring and pharmacokinetic studies.     

伏安免疫法检测牛奶中氯霉素残留

为探索用于现场检测牛乳中氯霉素(CAP)残留的高灵敏度及特异性强的免疫传感器方法,本实验在制备CAP单克隆抗体的基础上,以卵清蛋白-氯霉素(OVA-CAP)偶联物为包被抗原,并将其包被到聚苯乙烯反应板上;在孵育反应中,样品中的CAP与OVA-CAP竞争结合CAP单克隆抗体,洗涤后加入碱性磷酸酶(ALP)标记的二抗,经再次孵育及洗涤后加入对硝基苯磷酸(pNPP)底物液;反应终止后用线性导数伏安法记录pNPP水解产物的氧化峰电流。实验结果表明,用免疫传感法测试CAP的灵敏度高于传统的间接竞争酶联免疫吸附法(ELISA)。该方法检测CAP的检出限为0.064μg/L;检测线性范围为0.15~600μg/L,测试牛奶样品的平均回收率为89.8%。另外,由于免疫电化学传感器体积较小,便于携带,操作简单,可实现牛乳样品中CAP残留的现场检测。     

Comparison of monomeric and polymeric horseradish peroxidase as labels in competitive ELISA for small molecule detection

We have developed a simple and sensitive competitive enzyme-linked immunosorbent assay (ELISA) to determine aflatoxin B1 (as a model small analyte) and using streptavidin-polymeric horseradish peroxidase complex (SApolyHRP) as a label for signal amplification. The performance of the assay was evaluated by comparing it with the classical indirect competitive ELISA using HRP labeled anti-mouse IgG as the tracer antibody. The results indicate that the SApolyHRP-based competitive ELISA exhibits a typically 2.4-fold steeper slope of the linear working range of the calibration curve compared to the monomeric HRP based classical ELISA, i.e., the sensitivity was increased. The SApolyHRP conjugate causes a typically 19-fold stronger signal generation in comparison to the traditional HRP labeled anti-mouse IgG at the same concentration (25 ng mL^?1). Moreover, the SApolyHRP-based assay has a much wider linear range and a 3.8-fold better signal-to-noise ratio. Considering its simplicity, sensitivity and ease of operation, this competitive ELISA is considered to be a promising tool for small molecule immunodetection.

Specific polyclonal-based immunoassays for sulfathiazole

A highly sensitive and specific enzyme-linked immunosorbent assay has been developed for detection of sulfathiazole (STZ, 4-amino-N-thiazol-2-yl-benzenesulfonamide). A set of haptens was synthesized in order to produce polyclonal antibodies against sulfonamides. Two ELISA formats (antibody-coated and conjugate-coated) were also investigated using all the serum/coating conjugate combinations that showed specific recognition. The developed ELISA succeeded in detection of STZ at concentrations as low as 0.03 ng mL^–1 over a measurable range of 0.12–6.71 ng mL^–1. Selectivity studies have demonstrated that other sulfonamides do not interfere significantly (<10%) with analysis of STZ by this immunochemical technique. Analysis of spiked bee honey samples by the developed ELISA method showed recoveries were good. The selectivity and sensitivity (IC₅₀=1.6 ng mL^–1) make it a suitable screening method for determination of low levels of STZ in food samples.     

Surface Plasmon Resonance Immunosensor for Anionic Surfactants Based on an Indirect Competitive Immunoreaction

Abstract

A highly sensitive surface plasmon resonance immunosensor for the determination of linear alkylbenzene sulfonate (LAS) was fabricated. The method is based on an indirect competitive reaction of an anti‐LAS antibody in a sample solution with LAS immobilized on a sensor chip and with LAS in the sample solution. A sensor chip immobilized with LAS was prepared by utilizing an electrostatic interaction between an LAS conjugate, LAS–horseradish peroxidase (LAS‐HRP), and a self‐assembled monolayer of 11‐amino‐1‐undecanethiol hydrochloride, which was preliminary prepared on a gold thin film of the sensor chip. The quantitative determination of LAS in the concentration range 10–1000 ppb was achieved by using the proposed immunosensor.     

Immunoassay based on peroxidase silver conjugate for the determination of human immunoglobulins against tick-borne borreliosis (lyme disease) by voltammetry and spectrophotometry

In this work two strategies for the synthesis of peroxidase silver conjugates for the qualitative and quantitative determination of immunoglobulins (IgG) to ixodid tick-borne borreliosis (ITBB) (Lyme disease) in human serum were proposed. The first approach for Ab-HRP@AgNP conjugate synthesis involved silver nanoparticles (Ag NPs) capped with a commercial peroxidase conjugate (Ab-HRP) by passive adsorption. The second strategy was based on the initial coupling of Ag NPs with human anti-species antibodies (Ab) by passive adsorption followed by the introduction of horseradish peroxidase (HRP) enzyme into the reaction mixture as a blocking reagent for Ab@AgNP@HRP conjugate synthesis. The formation of peroxidase silver conjugates was proved by UV/Vis spectroscopy and Transmission Electron Microscopy (TEM). The catalytic activity of Ab-HRP@AgNP and Ab@AgNP@HRP conjugates was evaluated by Michaelis-Menten kinetics. A commercially available 96-well microtiter plate with recombinant antigens to ITBB was used as a platform for immobilization of analyzed IgG. The HRP in Ab-HRP@AgNP conjugate was found to retain a sufficient level of activity for interaction with the H₂O₂ substrate to form an intensely colored reaction product. Therefore Ab-HRP@AgNP conjugate can be used in enzyme-linked immunosorbent assay (ELISA) with spectrophotometric detection of 3,3’,5,5’-Tetramethylbenzidine (TMB Ox) for quantitative determination of IgG to ITBB in human serum in the concentration range 12.5–800 ng ml⁻¹ with LOD 2 ng ml⁻¹. Ab@AgNP@HRP conjugate is recommended for the electrochemical determination of IgG to ITBB in human serum at LOD 3 ng ml⁻¹ with registration of silver oxidation by linear sweep anodic stripping voltammetry (LSASV). Ag NPs in Ab-HRP@AgNP and Ab@AgNP@HRP conjugates do not change electrochemical activity during storage and can be used as an electrochemical label in LSASV method in case of HRP inactivation. The immunoassay based on peroxidase silver conjugates expands the analytical potential for the determination of IgG to ITBB especially during the period of increasing incidence.     

Production and analytical characterization of neopterin immunoreagents for biosensor developments

Neopterin is a valuable biomarker of cellular immunity associated with various pathological situations such as viral and bacterial infections, autoimmune, cardiovascular, neurodegenerative and malignant disorders. To produce specific antibodies against neopterin for a rapid multi-biomarker-based diagnosis, a novel hapten derivative was synthesized and attached to carrier proteins. The thoroughly characterized conjugates were used for immunization of BALB/c mice and rabbits. The produced monoclonal antibody reached in both direct and indirect enzyme-linked immunosorbent assay (ELISA) format LoD of 0.18 and 0.45 μg L⁻¹, respectively, and was a superior immunoreagent for further biosensor developments with regard to assay sensitivity and material availability. The best polyclonal antibody was somewhat more sensitive in direct ELISA with LoD of 0.05 μg L⁻¹. The optimized ELISA method was evaluated with blood samples collected from patients with renal insufficiency, patients with sepsis, patients without confirmed clinical diagnosis, and healthy volunteers. In plasma samples, neopterin concentrations ranging from 3.2 to 103 μg L⁻¹ could be determined with the monoclonal ELISA whereas twofold lower results were obtained with the polyclonal ELISA. A satisfactory correlation of results was found between the polyclonal ELISA and IBL Neopterin ELISA kit within the concentration range 0.5–16 μg L⁻¹ (R = 0.874; n = 40), and slightly lower correlation was found for monoclonal-based ELISA (R = 0.819; n = 40). These data show that the generated antibodies may be used as functional analytical reagents for the integration into multianalyte biochip detection systems.     

Preparation and characterization of a polyclonal antibody from rabbit for detection of trinitrotoluene by a surface plasmon resonance biosensor

A polyclonal antibody against trinitrophenyl (TNP) derivatives was raised in rabbit, and the antibody was applied to detection of trinitrotoluene (TNT) using a surface plasmon resonance (SPR) biosensor. TNP-keyhole limpet hemocyanine (TNP-KLH) conjugate was injected into a rabbit, and a polyclonal anti-TNP antibody was realized after purification of the sera using protein G. Aspects of the anti-TNP antibody against various nitroaromatic compounds, such as cross-reactivities and affinities, were characterized. The temperature dependence of the affinity between the anti-TNP antibody and TNT was also evaluated. The quantification of TNT was based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-β-alanine-ovalbumin (TNP-β-ala-OVA) and anti-TNP antibody was inhibited in the presence of free TNT in solution. TNP-β-ala-OVA was immobilized to the dextran matrix on the Au surface by amine coupling. The addition of a mixture of free TNT to the anti-TNP antibody was found to decrease the incidence angle shift due to the inhibitory effect of TNT. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range of 3 × 10⁻¹¹ to 3 × 10⁻⁷ g/ml. To increase the sensitivity of the sensor, anti-rabbit IgG antibody was used. After flowing the mixture of free TNT and anti-TNP antibody, anti-rabbit IgG antibody was injected, and the incidence angle shift was measured. Amplification of the signal was observed and the detection limit was improved to 1 × 10⁻¹¹ g/ml.     

Development of an enzyme-linked immunosorbent assay for metolcarb residue analysis and investigation of matrix effects from different agricultural products

The development of a direct competitive enzyme-linked immunosorbent assay based on polyclonal antibodies for N-methylcarbamate insecticide metolcarb is described. Two new haptens for the metolcarb were designed and synthesized. Both haptens were conjugated with keyhole limpet hemocyanin to form the immunogens. Four rabbits were immunized with the immunogens for production of polyclonal antibodies against metolcarb. Antisera titers were tested on the homologous coating antigens using a noncompetitive indirect enzyme-linked immunosorbent assay. The high titer antisera were used to develop the direct competitive enzyme-linked immunosorbent assay for the detection of metolcarb. The antibody–antigen combination with the highest selectivity for metolcarb was further optimized and its tolerance to changes in chemical conditions (ionic strength, pH value, and organic solvent) was studied. Under optimum conditions, the sensitivity and the limit of detection were determined to be 22 μg L⁻¹ and 1.2 μg L⁻¹ respectively. Determination of metolcarb in fruit juices and vegetables was accomplished by simple, rapid, and efficient extraction methods. Recoveries of metolcarb from spiked samples ranged from 80.5% to 109.5%. Validation of the developed immunosorbent assay was conducted by comparison of results from high-performance liquid chromatography. The correlation between the data obtained using developed immunosorbent assay and high-performance liquid chromatography was high (R ² = 0.9884). Therefore, the developed immunosorbent assay in this study was suitable for the rapid quantitative determination of metolcarb in agricultural products.     

Effect of hapten structures on specific and sensitive enzyme-linked immunosorbent assays for N-methylcarbamate insecticide metolcarb

Five different haptens of the N-methylcarbamate insecticide metolcarb were designed and synthesized. All of the haptens were conjugated with ovalbumin (OVA) for the coating antigen, and one hapten containing all of the structure of metolcarb was conjugated with bovine serum albumin (BSA) for the immunogen. Two polyclonal antisera were raised against the BSA conjugate, and ten antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for metolcarb. A class-specific combination was found, with the I₅₀ of the assay ranged from 0.64 to 20.98 μg mL⁻¹ for seven tested N-methylcarbamate insecticides except for pirimicarb. Considering titer, I₅₀ and cross-reactivity of all combinations of antibody/coating conjugate, a competitive indirect enzyme-linked immunosorbent assay (ELISA) in a homologous system, whose limit of detection (LoD) reached 1.4 ng mL⁻¹, was presented. The results of competitive ELISAs indicated that coating hapten structure can significantly affect not only assay sensitivity but also its specificity.     

Generation and characterization of polyclonal antibodies against microcystins-Application to immunoassays and immunoaffinity sample preparation prior to analysis by liquid chromatography and UV detection

Polyclonal antibodies against microcystin-LR (MC-LR), a cyclic heptapeptide toxin, were generated in rabbits using MC-LR-BSA. An enzyme-linked immunosorbent assay (ELISA) was developed for the characterization of the antibodies and their potential use for analytical purposes. The concentration of MC-LR that inhibits 50% of antibody-antigen binding (IC₅₀) was 0.5 μg L⁻¹ for the indirect ELISA format and 0.9 μg L⁻¹ for the direct ELISA, using MC-LR-horseradish peroxidase conjugate. The limit of detection corresponding to IC₈₀ was found to be 0.06 μg L⁻¹, well below the Word Health Organization level for drinking water of 1 μg L⁻¹. The direct competitive ELISA was applied to water samples and was shown useful for screening purposes. The developed anti-microcystin antibodies were immobilized on solid supports for use in selective solid phase extraction (SPE) systems, prior to liquid chromatography (LC) quantification. An immunoaffinity cartridge (IAC), a Sepharose^®-based cartridge incorporating 2 mg of antibodies allowed the selective and quantitative recovery of a mixture of 0.2 μg of MCs showing potential use in sample preparation of real matrices. When applied to water and green algae samples, average recoveries from Sepharose^®-based cartridges were in the range of 86-113% for water samples and 85-92% for blue-green algae samples. Selectivity of the IAC clean-up was proven by comparison with non-specific solid phase extraction using octadecylsilica (ODS) sorbent. Results obtained using LC/UV after IAC clean-up agreed well with results obtained using liquid chromatography and mass spectrometry detection (LC/MS and LC/MS/MS) after SPE-C18 clean-up, allowing therefore to validate the resulting technique.     

Synthesis and Characterization of Artificial Antigens for Copper and Application for Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay

Copper was conjugated to two carrier proteins using penicillin G sodium salt to synthesize artificial antigens for copper. The antigens were identified by ultraviolet spectrometry, circular dichroism, and inductively coupled plasma–optical emission spectrometry. The results of ultraviolet spectrometry showed characteristic absorption peak shifts between haptens and carrier proteins. Circular dichroism showed that the secondary structure of the conjugates was an α-helix. These results suggested that the artificial antigens for copper were synthesized successfully. The polyclonal antibodies of copper were produced by immunizing New Zealand white rabbits with copper antigens. An indirect competitive enzyme-linked immunosorbent assay based on the polyclonal antibodies was developed. The concentration of copper ion was quantified based on the ability of its ethylenediaminetetraacetic acid complexes to inhibit the binding of the goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase and, subsequently, color formation in the assay. The assay showed an IC₅₀ value of 0.475 microgram per milliliter with a detection limit of 0.007 microgram per milliliter. The antibody showed high affinity with copper and low cross-reactivity with other metals. The recovery of copper from fortified water was between 78.0 and 122 percent. The results indicate that the assay is a convenient supplemental analytical tool for monitoring copper ions in aquatic environments.     

New highly sensitive enzyme immunoassay for the determination of pravastatin in human plasma

New highly sensitive enzyme immunoassay (EIA) has been developed and validated for the determination of pravastatin (PRV) in human plasma samples. PRV was coupled to keyhole limpt hemocyanin (KLH) and bovine serum albumin (BSA) via its terminal carboxylic acid group by carbodiimide reagent. PRV-KLH conjugate was used as an immunogen for raising anti-PRV polyclonal antibody in rabbits. The generated anti-PRV antibody recognized PRV with high affinity and selectivity. PRV-BSA conjugate was immobilized onto microwell plates and used as a solid phase. The assay involved a competitive binding reaction between PRV, in plasma sample, and the immobilized PRV-BSA for the binding sites on a limited amount of the anti-PRV antibody. The anti-PRV antibody bound to the plate wells was quantified with horseradish peroxidase-labeled anti-immunoglobulin second anti-rabbit IgG antibody and 3,3′,5,5′-tetramethylbenzidine as a substrate for the peroxidase enzyme. The concentration of PRV in the sample was quantified by its ability to inhibit the binding of the anti-PRV antibody to the immobilized PRV-BSA and subsequently the color development in the assay wells. The conditions of the proposed EIA were investigated and the optimum conditions were employed in the determination of PRV in plasma samples. The assay limit of detection was 0.2 ng mL⁻¹ and the effective working range at relative standard deviation (RSD) of ≤5% was 0.5-20 ng mL⁻¹. The mean analytical recovery of PRV from spiked plasma was 100.9 ± 2.98%. The precision of the assay was satisfactory; RSD was 2.61-3.70 and 3.96-4.17% for intra- and inter-assay precision, respectively. The analytical procedure is convenient, and one can analyze ∼200 samples per working day, facilitating the processing of large-number batch of samples. The proposed EIA has a great value in the routine analysis of PRV in plasma samples for its therapeutic monitoring and pharmacokinetic studies.     

Determination of Estradiol by Biotin‐Avidin‐Amplified Electrochemical Enzyme Immunoassay

Abstract

A simple, sensitive biotin‐avidin‐amplified electrochemical enzyme‐linked immunosorbent assay (ELISA) for the determination of estradiol (E₂) was proposed in this paper. The complex of biotinylated anti‐E₂ antibody and horseradish peroxidase‐labeled avidin (HRP‐avidin) were regarded as a probe in this system. The activity of labeled enzyme was measured with electrochemical methods using o‐phenylenediamine as substrate. Coupled with the plate‐coated antigen, indirect ELISA format using E₂‐ovalbumin, the electrochemical detection was performed for E₂ with the detection limit of 21.0 pg/ml, and the linear range of determination of 50.0–500.0 pg/ml. The proposed method has been used for the determination of E₂ in river water with satisfactory results. Compared with the traditionally spectrophotometric ELISA detection, this method shows greatly heightened sensitivity. The limit of detection improved by about two orders of magnitude, which is very suitable for the conditions with extremely low concentration of analyte or very small volumes of sample present.     

Immunoenzyme assay of nonylphenol: study of selectivity and detection of alkylphenolic non-ionic surfactants in water samples

Immunoenzyme assay (ELISA) is proposed and characterized for determination of alkylphenol ethoxylates, a primary class of manufactured non-ionic surfactants. The assay is based on the obtained polyclonal antibodies against nonylphenol (NP), the main stable intermediate of the decomposition of nonylphenol ethoxylates. A mixture of non-modified branched isomers of NP was applied as hapten coupled to protein carriers by Mannich reaction with the use of formaldehyde. The proposed ELISA format is based on immobilized NP-(soybean trypsin inhibitor) conjugate as a competitor of antigen molecules contained in the tested sample for binding with specific antibodies indirectly labeled via an anti-species immunoperoxidase conjugate. The developed ELISA allows to reveal NP with the limit of detection about 10 ng ml⁻¹ and NP-related compounds such as octylphenol, alkylphenoletoxylates, alkylphenolcarboxylates and their halogenated derivatives. The ELISA was applied for assaying polluted water samples, namely influents and effluents from different wastewater treatment plants (WWTP) and tap water. ELISA and chromatographic data demonstrate good correlation (r = 0.94), while ELISA gives higher values. Due to endocrine disrupting and other toxic activities of some metabolites of alkylphenolic non-ionic surfactants, the developed assay may be effectively used in ecological monitoring and sanitary control.     

A disposable immunosensor for detection of 17beta-estradiol in non-extracted bovine serum

This paper reports the assembly of a disposable immunosensor based on the direct competitive enzyme-linked immunosorbent assay (ELISA), for simple and fast measurement of 17β-estradiol (17β-E₂) in bovine serum, using screen-printed electrodes (SPEs) and a Palm-Sens portable electrochemical detector. The immunosensor strip was assembled immobilising, by passive adsorption, anti-rabbit IgG onto the surface of the working SPE electrode. After the interaction between anti-rabbit IgG and rabbit anti-17β-E₂ polyclonal antibodies (PAb), the competition was performed using 17β-estradiol-alkaline phosphatase conjugate (17β-E₂-AP) synthesised in our laboratory. The enzymatic substrate used for signal generation was 1-naphthylphosphate and its conversion to an electroactive product (1-naphthol) was measured using differential pulse voltammetry (DPV). To develop a prototype for field measurements, the entire competitive protocol has been optimised directly in a blank non-extracted bovine serum.According to the new EU criteria established by the Commission Decision 2002/657/EC for qualitative and quantitative screening methods, the detection capability (CCβ), was determined. The CCβ value resulted below the action limit (40 pg mL⁻¹) fixed for 17β-E_2.Spiked and real samples were analysed using the electrochemical immunostrips obtaining precision values (relative standard deviation, R.S.D.%) ranging from 8.6 to 17.0% and a recovery (R%) from 88.2 to 120.0%.Results obtained on real samples were confirmed by liquid chromatography coupled on-line with tandem mass spectrometry (LC-MS/MS) using an atmospheric pressure chemical ionisation (APCI) source and a heated nebulizer (HN) interface; this is the method currently used to confirm illegal hormone administration for regulatory purposes. The disposable immunosensor appears suitable as a screening tool for field analysis of bovine serum estradiol.     

An Enzyme Immunoassay for the Determination of Methabenzthiazuron

Abstract

A sensitive enzyme immunoassay is described for the determination of the urea herbicide methabenzthiazuron. The assay is carried out with polyclonal antibodies, which were raised in rabbits by immunization with a methabenzthiazuron-BSA conjugate containing five methabenzthiazuron residues per molecule. The ELISA was optimized on microtiter plates with a peroxidase-methabenzthiazuron tracer. The middle of the test (50% B/B₀) was found at 1.0 μg/l. The lower detection limit of methabenzthiazuron is c. 0.05 μg/l. Samples can be measured up to 10 μg/l methabenzthiazuron (upper detection limit). The assay does not require concentration or clean-up steps for drinking or ground water samples. Validation experiments showed a good accuracy and precision. Work with monoclonal antibodies is in progress.     

Enzyme-Linked Immunosorbent Assay for Hyodeoxycholic Acid in Pharmaceutical Products Using a Monoclonal Antibody

Herein is reported an immunochemical approach to determine hyodeoxycholic acid using hybridoma-secreting monoclonal antibodies. The hyodeoxycholic acid-specific antibody was produced by fusing splenocytes immunized with a hyodeoxycholic acid-bovine serum albumin conjugate with a hypoxanthine–aminopterin–thymidine-sensitive mouse myeloma cell line (SP2/0). The antibody was highly specific for hyodeoxycholic acid, with less than 0.05 percent cross-reactivity to over fifty structurally related compounds. The antihyodeoxycholic acid monoclonal antibody was then used to develop a rapid, specific, and sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) for the determination of hyodeoxycholic acid in pharmaceutical compounds. The linear dynamic range was from 0.48 to 62.5 nanograms per milliliter with an IC₅₀ value of 8 nanograms per milliliter. The icELISA results correlated well with a conventional high-performance liquid chromatography method for the determination of hyodeoxycholic acid (R² = 0.9982). This study shows that the icELISA method was successfully applied to the quantification of hyodeoxycholic acid in pharmaceutical products.