Direct enzyme immunoassay in microtitration plate and test strip format for the detection of saxitoxin in shellfish

Saxitoxin was coupled to horseradish peroxidase via a novel adaptation of the periodate reaction. Based on polyclonal antibodies against saxitoxin, this conjugate was used for the development of two formats of direct enzyme immunoassay (EIA)–a microtitration enzyme-linked immunosorbent assay (ELISA) and a test strip EIA. The detection of saxitoxin without instrumentation by visual evaluation of the test strip EIA is described. The detection limits for saxitoxin were 7 pg/ml (0·35 pg/assay) in the ELISA and 200 pg/ml in the test strip EIA using visual evaluation. Employing a simple procedure of sample preparation, both ELISA and test strip EIA were applied to the analysis of shellfish. The detection limits for saxitoxin in shellfish tissue of the ELISA and the test strip assay were 3 and 4 ng/g, respectively.     

Enzyme immunoassay for the measurement of 17alpha-estradiol 17-N-acetylglucosaminide in rabbit urine.

17alpha-estradiol 17-N-acetylglucosaminide (17alphaE2 17NAG) is an estrogen metabolite hitherto obtained only in rabbits. To gain insight into this unique conjugate, an enzyme immunoassay (EIA) was established by using antiserum elicited against 3-[3-(1-carboxypropyl)] ether of 17alphaE2 17NAG-bovine serum albumin conjugate; horseradish peroxidase, as a label; and 3,3',5,5'-tetramethylbenzidine, as a chromogen. The method proved to be specific, and the detection range of the assay was 0.20-10.00 ng/ml. A proposed double conjugate, 3-glucuronide of 17alphaE2 17NAG, was synthesized to validate the EIA. The EIA was applied to the determination of the urinary level of 17alphaE2 17NAG in male and female (pregnant and non-pregnant) rabbits with and without beta-glucuronidase-sulfatase preparation from Helix pomatia. The results showed that 17alphaE2 17NAG was mainly excreted as a double conjugate (17alphaE2 17NAG 3-glucuronide and/or 3-sulfate) and that its level varies during pregnancy.     

Enzyme immunoassays of JH III and JH III diol using acetylcholinesterase as tracer: An alternative to radioimmunoassay

Immunogens were prepared by coupling JH III and JH III diol to human serum albumin. Specific and sensitive antibodies were obtained by immunizing rabbits. Pure acetylcholinesterase (EC 3.1.1.7) from electric eel (Electrophorus electricus) was covalently coupled to the acids from hydrolyzing JH III or JH III diol. The enzyme immunoassays (EIA) were performed in 96-well microtiter plates coated with second antibody (pig anti-rabbit).The JH III EIA performance was equivalent to or better than radioimmunoassay using an iodinated tracer: the sensitivity was 0.91 ng/ml at 50% B/B₀, the detection limit was 0.2 ng/ml; cross-reactivity was less than 1% for the diols of JH I, JH II and JH III, and 30% for JH III acid. For the JH III diol assay, the EIA sensitivity was 1.9 ng/ml at 50% B/B₀ and the detection limit was 0.2 ng/ml; cross-reactivity was less than 1% for JH III and JH III acid, and 14 and 10% for JH I diol and JH II diol. Finally, use of semi-automatic apparatus allowed rapid and easy EIA analyses in which the enzyme label is a long-lived reagent and handling of radioactive compounds is avoided.     

Preparation and characterization of monoclonal antibodies to the cholera toxin

Monoclonal antibodies to cholera toxin were obtained. They do not cross-react with the termolabile toxin (LT) of Escherichia coli, ricin, diphtherial toxin, staphylococcus enterotoxins of SEA, SEB, SEI, SEG, or the lethal factor and protective antigen of the anthrax toxin. Pairs of antibodies for the quantitative measurement of the cholera toxin in sandwich enzyme immunoassay (EIA) were selected. The detection limit of the toxin is 0.2 ng/ml for plate EIA and 0.44 ng/ml for microchip EIA. The presence of milk, broth, or surface water in the toxin samples does not reduce the sensitivity of EIA.     

Production of ultrasensitive antibodies against aflatoxin B1

AIMS: To produce specific antibodies against the haptenic fungal toxin aflatoxin B1 (AFB1) and apply these antibodies in immunochemical assays for aflatoxins. METHODS AND RESULTS: Rabbits were immunized using an AFB1-bovine serum albumin conjugate and serum titres determined by double-antibody enzyme immunoassay. High titres of antibodies with very high affinity for AFB1 were obtained 15 and 4 weeks after the initial immunization and the first booster immunization respectively. The antibodies were employed in enzyme immunoassay (EIA) and immunoaffinity chromatography (IAC) methods for aflatoxins. With a detection limit of 15.8 pg ml(-1) for AFB1, the EIA employing these antibodies is the most sensitive test for AFB1 described so far. In IAC columns, these antibodies provided high binding capacity for all major aflatoxins, including AFB1, AFB2, AFG1 and AFG2. CONCLUSION: The antibodies described here are useful for the analysis of trace levels of aflatoxins. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyclonal antibody-based EIA and IAC methods for aflatoxin analysis offer a suitable alternative to the more expensive monoclonal antibody-based methods.     

Enzyme immunoassay of ampicillin in milk

An indirect immunoassay for quantitative determination of ampicillin (range, 10-1000 ng/ml) in buffer or milk has been developed. Polyclonal antibodies were obtained against ampicillin conjugated with bovine serum albumin; the conjugate was synthesized by direct condensation using carbodiimide. The antibodies were specific for ampicillin and exhibited low cross-reactivity to other penicillins (azlocillin, 17%; penicillin G, 10%; piperacillin, 5%; and carbenicillin, 4%). Matrix effects were minimized by combining the use of a casein-supplemented buffer (content of casein, 1%) with sample dilution. The threshold of ampicillin detection in milk (diluted tenfold) was equal to 5.0 ng/ml (which corresponded to 50 ng/ml of the original sample).     

A direct competitive enzyme-linked immunosorbent assay by antibody coated for diethyl phthalate analysis

A direct competitive enzyme-linked immunosorbent assay (ELISA) has been developed for detection of diethyl phthalate (DEP). Protein-hapten conjugate was synthesized to produce polyclonal antibodies against DEP. Experimental parameters were optimized, including immunoreaction conditions, the dilution ratio of horseradish peroxidase (HRP)-antigen conjugate, time of the antibody coated, effect of pH, and ionic strength. The limit of detection was 0.096 ng/ml, and the linear range was 0.1-3500 ng/ml with a regression coefficient (R²) of 0.9957. Recoveries were between 96.4 and 106.2%. The cross-reactivities of the anti-DEP antibody to six structurally related phthalate esters were less than 9%. The method was successfully applied to the determination of DEP in tap water, river water (Yangtze River), and leachate from plastic drinking bottles. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for DEP monitoring. The results obtained were compared with those obtained using the high-performance liquid chromatography method.     

Enzyme-Linked Immunosorbent Assay of Ampicillin in Milk

An indirect immunoassay for quantitative determination of ampicillin (range, 10–1000 ng/ml) in buffer or milk has been developed. Polyclonal antibodies were obtained against ampicillin conjugated with bovine serum albumin; the conjugate was synthesized by direct condensation using carbodiimide. The antibodies were specific for ampicillin and exhibited low cross-reactivity to other penicillins (azlocillin, 17%; penicillin G, 10%; piperacillin, 5%; and carbenicillin, 4%). Matrix effects were minimized by combining the use of a casein-supplemented buffer (content of casein, 1%) with sample dilution. Limit of detection for ampicillin in milk (diluted tenfold) was equal to 5.0 ng/ml (which corresponded to 50 ng/ml of the original sample).     

Development of radioimmunoassay and enzyme immunoassays for luteinizing hormone in dromedaries (Camelus dromedarius).

Polyclonal antibodies against luteinizing hormone in dromedary (camLH) were raised in a rabbit and enabled the development of homologous immunoassays (radioimmunoassay, competitive enzymeimmunoassay (EIA) and sandwich EIA) for the measurement of circulating camLH in plasma. These assays were highly specific for camLH since neither dromedary follicle-stimulating hormone, growth hormone nor prolactin cross-reacted significantly. The lowest detection limit (0.08 ng/ml) was obtained with the sandwich EIA. In addition to its high specificity and sensitivity, this method does not require radiolabelled molecules or expensive laboratory facilities. It can be performed in the field using a portable, battery-powered plate reader.     

Effectiveness of methods of isolating specific class-G rabbit antibodies for the immunoenzyme determination of human placental alkaline phosphatase

Four different chromatographic methods of IgG isolation from rabbit antisera to placental alkaline phosphatase (HPAP) have been compared. The antibodies were obtained by ion-exchange chromatography and affinity chromatography on protein-A-sepharose, on the sepharose with immobilized antigen. IgG samples were characterized by the content of specific antibodies to HPAP and checked in enzyme immunoassay (EIA). IgG purified on immobilized antigen were found to be the optimal both from the point of view of the specific antibodies content and EIA sensitivity, but satisfactory results could be also obtained with ion-exchange and protein-A-chromatography purified IgG. The last two isolation methods are simpler and provide 3-10 ng/ml sensitivity of HPAP detection, which is lower, as compared with the test employing affinity antibodies (1 ng/ml), but allows the detection of HPAP in serum samples.     

Use of monoclonal antibodies against horse immunoglobulin in an enzyme immunoassay of bacterial toxins and toxoids

Immunization of BALB/c mice by horse antiserum against diphtheria made it possible to obtain IgG₁ monoclonal antibodies (MoAbs) 2B7E4 specific for light chains of horse immunoglobulin (Ig). Unlike commercial preparations of anti-horse immunoglobulin antibodies, which are specific for the whole Ig molecule or its Fc-fragment, the peroxidase (HRP) conjugate of the MoAb, 2B7E4-HRP did not interact with human, mouse, rabbit, and sheep Igs, or horse albumin. The conjugate obtained was used with MoAbs against bacterial toxins and commercial horse antitoxins, as a universal reagent in sandwich enzyme immunoassay (ELISA) for bacterial toxins and toxoids. The detection sensitivity of diphtheria toxin/toxoid equaled 0.0005 Lf/ml; tetanus toxin and toxoid were detected with sensitivities of 20 LD₅₀/ml and 0.005 UI/ml, respectively. A similar sandwich ELISA for botulinum toxoids (group measurement) allowed types A, B, and E to be detected at 0.02, 0.002, and 0.001 UI/ml, respectively; selective measurement was only possible in the case of type E toxoid (0.001 UI/ml).     

Development and testing of radio and enzyme immunoassays for acidic fibroblast growth factor (aFGF)

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor from bovine brain stimulate growth in a variety of tissues in several species. Despite the 55% amino acid sequence homology of the two forms of FGF, a specific immunoassay of aFGF has been developed using a polyclonal antibody raised in a rabbit. Two immunoassays were compared: a radioimmunoassay (RIA) using 125I aFGF and an enzyme immunoassay (EIA) using aFGF coupled to the tetrameric form of acetylcholinesterase (aFGF-AchE) as tracer. With EIA, the detection limit was 1.5 ng/ml, versus 2.2 ng/ml with RIA, while the dose at 50% was 5.9 ng/ml for EIA and 9.6 ng/ml for RIA. Using a modified EIA procedure where aFGF-AchE was added 2 h after the other reagents, the dose at 50% binding was 1.5 ng/ml. Examples of the performance of both immunoassays are presented for various brain extracts of different species including human. The aFGF content obtained by these methods correlates (CR = 0.987) with the values obtained by biological assay.     

A quantitative HPLC–MS method for the simultaneous determination of testosterone, 11-ketotestosterone and 11-β hydroxyandrostenedione in fish serum

A simple and novel HPLC–MS method for the simultaneous quantification of testosterone, 11-ketotestosterone, and 11β-hydroxyandrostenedione in fish serum was developed and validated. Separation was achieved on a C-18 column using a water–acetonitrile mobile-phase with a cycle time of 12 min. Ion detection was performed using ESI positive SIM at [M+H] (m/z 303, 303, 289). The linear ranges (0.2–50 ng/ml), limits of detection (0.1–0.2 ng/ml) and quantification (0.2–0.5 ng/ml) were established. The method was validated by measuring the three androgens in goldfish sera, displaying comparable values to those reported by other analytical techniques (RIA, EIA).     

Fluorescent competitive aptasensor for detection of aflatoxin B1

Aflatoxin B1 (AFB₁) is one of the most commonly found mycotoxins in food commodities, particularly cereals, oilseeds, spices and tree nuts. In the past decade, aptamers have come into limelight and emerged as a new biosensing element replacing antibodies in various detection formats. Herein we report a faster, more sensitive, high throughput method for the detection of AFB₁ using AFB₁‐specific aptamers. The assay format was based on a competitive reaction of the fluorescent tagged aptamer specific to AFB₁ with the aflatoxin conjugate. Under optimal conditions, a linear range of detection (50 ng to 50 pg) was achieved with a limit of detection (LOD) of 10 pg/mL in the buffer system. Results of inter‐ and intra‐assay revealed that the assay was repeatable with standard deviation in acceptable range. The assay was also validated in food samples such as dried red chilies, groundnut and whole pepper with recovery in the range of 92 to 102% at 10 ng/mL and 100 pg/mL levels. The aptasensor assay was also compared with standard analytical method of HPLC and was found to be more sensitive. This detection technique has the potential to be developed into a biosensor platform for AFB₁ detection.     

Development of Enzyme Immunoassays for the Herbicide Chlorsulfuron

Antibodies against the herbicide chlorsulfuron have been raised and characterized. Enzyme immunoassays (EIAs) for chlorsulfuron, involving labeled antigen or labeled antibodies, have been developed. The kinetics of antigen–antibody interactions in the EIA systems developed has been studied. Both systems exhibit equal sensitivity (1 ng/ml). The values of the coefficient of variation (CV), determined within the range of chlorsulfuron concentrations to be measured by the systems (1–100 ng/ml), are not in excess of 8%. The possibility of using glucose oxidase as a label in EIAs for chlorsulfuron has been demonstrated. Lack of cross-reactivity with a series of sulfonyl-/arylurea derivatives and triazines makes it possible to recommend the EIA systems developed for chlorsulfuron determination in the environment.     

Competitive immunoassay for analysis of vitamin B(12)

In the current work, direct competitive enzyme-linked immunosorbent assay (ELISA) was developed for derivatized vitamin B₁₂ by generating chicken egg yolk immunoglobulins (IgY) against derivatized vitamin B₁₂ and purified using affinity chromatography. Checkerboard assay was performed with vitamin B₁₂ antibody and vitamin B₁₂–alkaline phosphatase conjugate followed by its conjugate characterization using ultraviolet (UV) spectroscopy and high-performance liquid chromatography (HPLC). The limit of detection was 10 ng/ml with a linear working range of 10 to 10,000 ng/ml. The affinity constant (K_a) of the vitamin B₁₂ antibody was found to be 4.23 × 10⁸ L/mol. Cross-reactivity with other water-soluble vitamins was found to be less than 0.01% except for analogs of vitamin B₁₂ that showed 12% to 35%. The intra- and interassay coefficients of variation were found to be in the ranges from 0.0005% to 1.2% and 0.009% to 1.03%, respectively. The assay was validated with the HPLC method in terms of sensitivity, specificity, precision, and recovery of vitamin B₁₂ with spiked multivitamin injections, tablets, capsules, and chocolates. The HPLC method had a detection limit of 500 ng/ml with a linear working range of 1000 to 10,000 ng/ml. After extraction of vitamin B₁₂ using Amberlite XAD, the developed ELISA method correlated well with the established HPLC method with a correlation coefficient of 0.90.     

Quantum dot-based fluorescence immunosorbent assay for the rapid detection of bacitracin zinc in feed samples

Bacitracin zinc (BAC), a polypeptide antibiotic, is utilized as a feed additive due to its ability to promote growth in animals. However, the abuse of BAC can lead to a great threat to food safety. Therefore, there is an urgent need to develop a rapid and sensitive detection method. In this study, a monoclonal antibody (mAb) against BAC with excellent sensitivity and specificity was obtained. For the first time, quantum dots (QDs) were conjugated with the prepared mAb against BAC and rabbit anti-mouse antibody to fabricate a direct and an indirect competitive fluorescence-linked immunosorbent assay (dc-FLISA and ic-FLISA) to detect BAC. The IC₅₀ of dc-FLISA and ic-FLISA were 0.28 ng/ml and 0.17 ng/ml, respectively. The limits of detection were 0.0016 ng/ml and 0.001 ng/ml, respectively, and the detection ranges were 0.0016–46.50 ng/ml and 0.001–35.65 ng/ml, respectively. In addition, the recovery rate of the two methods ranged from 93.5% to 112.0%, and the coefficient of variation (CV) was less than 10%. Therefore, the methods developed in this work have the merits of low cost, simple operation, and high sensitivity, which provide an effective analytical tool for BAC residue detection in feed samples.     

Immunoaffinity chromatography and a biotin-streptavidin amplified enzymeimmunoassay for sensitive and specific estimation of estradiol-17β

A sensitive test system has been developed for estimation ofestradiol-17β (E₂) in bovine plasma. Plasma extracts are first purified by a selective immunoaffinity chromatography (IAC) using an antibody raised against estradiol-6-carboxymethyloxime-bovine serum albumin and immobilized to Sepharose. The eluate was analysed by a competitive enzyme immunoassay (EIA) on microtitration plates. For the assay the wells of microtitration plates were coated with affinity purified sheep IgG (antirabbit IgG) that binds the hormone specific antibody raised in rabbits against estradiol-17-hemisuccinate-bovine serum albumin. E₂ is estimated by displacement of biocytinyl-E₂, that was produced by ligation of estradiol-17β, d-glucuronic acid and biocytin. Bound biocytinyl-E₂ is detected after binding of streptavidin-peroxidase and colour production by the enzyme. A very high amplification was possible with this technique and the absolute detection limit amounted to ≈120fg/well at 94% relative binding. By combination of IAC and EIA the following levels of E₂ were found in bovine plasma: male or female calves <2.7pg/ml, cycling cow 0.5–7 pg/ml, cow during last month of pregnancy 9–310 pg/ml, mature bull 5–30 pg/ml. However, up to 1110 pg E₂/ml were found in plasma of a calf after treatment with an illicit hormone preparation used for growth promotion; after 21 days levels declined to 6 pg/ml which is hardly different from controls. In conclusion, the IAC/EIA can be used for sentitive estimation ofestradiol-17β in plasma from all type of cattle and for control of improper use of E₂ after commitment of a threshhold level.     

An Immunochromatographic Assay of 2,4-Dichlorophenoxyacetic Acid and Simazine Using Monoclonal Antibodies Labeled with Colloidal Gold

A method of the competitive immunochromatographic assay of the pesticides 2,4-D (2,4-dichlorophenoxyacetic acid) and simazine (2-chloro-4,6-bis(N-ethylamino)-1,3,5-triazine) in aqueous samples was developed. Monoclonal antibodies to these pesticides labeled with colloidal gold were used to visualize the results. The sensitivity of the 2,4-D and simazine assay is 12 ng/ml, and the time of analysis is 3–7 min. The method does not differ in sensitivity from the competitive EIA using conjugates of monoclonal antibodies to the pesticides with horseradish peroxidase; however, the time of the EIA is 1.5 h. The immunochromatographic method of the pesticide detection is available and simple and may be recommended for the development of assays of any other low-molecular compounds.     

A signal-amplified electrochemical immunosensor for aflatoxin B1 determination in rice

A sensitive and simple electrochemical immunosensor based on enzymatic silver deposition amplification was constructed for the detection of aflatoxin B₁ (AFB₁) in rice. The immunosensor was based on an indirect competitive format between free AFB₁ and aflatoxin B₁-bovine serum albumin (AFB₁-BSA) conjugate immobilized on the electrode surface for binding to a fixed amount of anti-AFB₁ antibody. Then the alkaline phosphatase (ALP)-labeled anti-mouse immunoglobulin G (IgG) secondary antibody was bound to the electrode surface through reaction with primary antibody. Finally, ALP catalyzed the substrate, ascorbic acid 2-phosphate, into ascorbic acid that reduced silver ions in solution to metal silver deposited onto the electrode surface. Linear sweep voltammetry was carried out to quantify the metal silver, which indirectly reflected the amount of the analyte. The experimental parameters, such as the dilution ratio of antibody and the concentration of AFB₁-BSA conjugate, have been evaluated and optimized. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.1 to 10 ng/ml with a detection limit of 0.06 ng/ml. Good recoveries were obtained for the detection of spiked rice samples. So, the proposed method in this article could find a good use for screening AFB₁ in real samples.