Establishment of monoclonal antibody and scFv immuno-based assay for Cry2Aa toxin in spiked grain samples.

Bacillus thuringiensis (Bt) Cry toxins have been widely used in the development of genetically modified organisms (GMOs) for pest control. This work aimed to establish more cost effective methods for used Cry2Aa toxins. Three immunoassay methods (IC-ELISA, DAS-ELISA, and CLEIA) were successfully developed in this work. The mAb was used as the detecting antibody, for the IC-ELISA, the range of IC20 to IC80 was 1.11 µg/mL - 60.70 µg/mL, and an IC50 of 10.65 µg/mL. For the DAS-ELISA, the limit of detection (LOD) and limit of quantitation (LOQ) were 10.76 ng/mL and 20.70 ng/mL, respectively. For the CLEIA, the LOD and LOQ were 6.17 ng/mL and 7.40 ng/mL, respectively. The scFv-based detections were the most sensitive for detecting Cry2Aa. The LOD and LOQ for the DAS-ELISA were 118.75 ng/mL and 633.48 ng/mL, respectively. The LOD and LOQ for the CLEIA, read as 37.47 ng/mL and 70.23 ng/mL, respectively. The fact that Cry2Aa toxin was recovered in spiked grain samples further demonstrated that the approaches might be used to identify field samples. These methods provided good sensitivity, stability, and applicability for detecting Cry2Aa toxin, promising ultrasensitive monitoring and references for Cry toxins risk assessment.

The preparation of polyclonal antibody against chlordimeform and establishment of detection by indirect competitive ELISA.

Objective: Chlordimeform is a chemical pesticide that is highly carcinogenic and toxic. The purpose of this study was to establish an enzyme-linked immunosorbent assay (ELISA) method for the detection of chlordimeform in aquaculture and fish farming. METHODS: Chlordimeform was coupled with bovine serum albumin (BSA) and ovalbumin (OVA) as carrier proteins. A chlordimeform-BSA conjugate was used as an immunogen, and chlordimeform-OVA was used as a coating antigen. Chlordimeform-BSA was used to immunize rabbits, and a polyclonal antibody was prepared. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was established to detect chlordimeform. RESULTS: The working range of the established IC-ELISA method for chlordimeform detection was 1-20 ng/mL. The IC50 was 3.126 ng/mL, and the lower limit of detection (LOD) of chlordimeform was 0.637 ng/mL. The recovery of chlordimeform from spiked water samples ranged from 81% to 107%. CONCLUSION: An anti-chlordimeform polyclonal antibody was successfully developed, and a novel IC-ELISA was established to detect chlordimeform in aquaculture.

Preparation of Monoclonal Antibody and Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay and Fluorescence-Linked Immunosorbent Assay for Detecting 3-Amino-5-methylmorpholino-2-oxazolidinone (AMOZ) in Edible Animal Tissue.

To monitor the illegal used of furaltadone, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and fluorescence-linked immunosorbent assay (FLISA) based on a monoclonal antibody (mAb) were developed for the detection of 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), the major metabolite of furaltadone in animal tissues. The highly specific mAb, which was very sensitive to a nitrophenyl derivative of AMOZ (2-NP-AMOZ) with IC50 values of 0.11 and 0.09 ng/mL for ic-ELISA and FLISA, respectively, was selected for the development of immunoassays. For both the ic-ELISA and FLISA for AMOZ-spiked experiments, acceptable recovery rates of 81.1-105.3% and coefficients of variation of 4.7-9.8% were obtained. In addition, results from both ic-ELISA and FLISA methods for spiked samples' data showed excellent correlation coefficients ranging from 0.9652 to 0.9927. Meanwhile, the proposed ic-ELISA and FLISA for thirty spiked samples were confirmed by standard LC-MS/MS with high correlation coefficients of 0.9911 and 0.9921, respectively. These results suggest that the developed ic-ELISA and FLISA are valid and cost-effective tools for high-throughput monitoring methods for AMOZ residues in animal tissues.

[Rapid detection of aflatoxin B_1 in Chinese herbal medicines by indirect and direct competitive enzyme-linked immunosorbent assays: a comparative analysis].

The indirect competitive enzyme-linked immunosorbent assay(ic-ELISA) and direct competitive enzyme-linked immunosorbent assay(dc-ELISA) were performed for the rapid detection of aflatoxin B_1(AFB_1) in Astragali Radix, Zingiberis Rhizoma Recens, Jujubae Fructus, and Nelumbinis Semen with self-made antigens and antibodies. Different extraction methods were investigated to reduce the matrix effects of different medicinal parts in Chinese herbal medicines. The sensitivity of dc-ELISA method was improved by optimizing the molar ratio of AFB_1 to horseradish peroxidase(HRP). In this study, the sensitivity(IC_(50)) of ic-ELISA and dc-ELISA was 0.046 and 0.023 ng·mL~(-1), with the limit of detection(LOD) of 0.007 and 0.004 ng·mL~(-1), respectively. The detection time was 3 h and 50 min for ic-ELISA and dc-ELISA, respectively. The recovery rates were within the range of 62.96%-104.4%, with RSDs of less than 10%. Confirmed by LC-MS/MS, three positive samples of Nelumbinis Semen were detected from 53 samples. Two ELISA methods established in this study were accurate, rapid and sensitive, and can be used for rapid screening of AFB_1 in Chinese herbal medicines such as Astragali Radix, Zingiberis Rhizoma Recens, Jujubae Fructus, and Nelumbinis Semen. In addition, the advantages and limitations of the two methods were compared and discussed, which can provide a reference for the testing institutions to choose the proper method.

Development of ELISA and chemiluminescence enzyme immunoassay for quantification of histamine in drug products and food samples.

Histamine (HA) is a biogenic amine associated with allergies and food poisoning. It is an important indicator of food freshness and quality. In recent years, a series of medical negligence cases have been reported to be related to the intravenous injection of antibiotics produced via fermentation with fish peptone due to HA contamination. To detect HA efficiently, mouse monoclonal antibody was developed. An enzyme-linked immunosorbent assay (ELISA) and a chemiluminescence enzyme immunoassay (CLEIA) were developed and compared with conventional HPLC analysis. Both immunoassays showed low cross-reactivity, low 50% inhibitive concentration (IC50; 1.2 µg/mL and 1.1 µg/mL), low limits of detection (LODs, IC10; 89.0 ng/mL and 73.4 ng/mL), and appreciable recoveries in spiked foods and drugs (from 73.4 to 131.0% and from 77.0 to 119.0%, espectively), demonstrating that the developed methods are sensitive, specific, fast, and reliable for HA detection in complicated real samples. Graphical abstract.

An investigation on the prevalence of peste des petits ruminants in the camels of Sindh, Pakistan.

The present study investigated the status of peste des petits ruminants (PPR) for the first time in the camels of Pakistan. The samples were collected from the camel residing area of Sindh, Pakistan, and analyzed for breeds (Dhatti and Larri), districts (Tharparkar and Umerkot), age (young, adult, and old), and sexes (male and female). The sera samples (n = 200) were analyzed for the detection of antibodies using a competitive enzyme-linked immunosorbent assay (cELISA). Moreover, the nasal and fecal samples were screened for the PPR virus. Finally, the positive nasal and fecal samples were validated using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocapture enzyme-linked immunosorbent assay (Ic-ELISA). The cELISA results showed an overall prevalence of 8.5% PPR in the study area. The camels of Tharparkar (10.9%; 95% confidence interval (CI) 9.2-12.9) showed higher seroprevalence of PPR antibodies than those of Umerkot (5.5%; 95% CI 4.1-7.2). Moreover, the Larri breed exhibited slightly greater resistance against the disease, because the camels of Dhatti breed (9.0%; 95% CI 7.5-11.0) exhibited a numerically higher (p > 0.05) seroprevalence of PPR in comparison with those of Larri breed (7.9%; 95% CI 6.4-9.9). Furthermore, the young and old camels were more susceptible to the disease attack, because the adults (6.3%; 95% CI 5.0-7.8) exhibited significantly (p < 0.05) lower prevalence rate than the young (9.2%; 95% CI 7.6-11.1) and old (10.3%; 95% CI 8.9-11.9) camels. Finally, the results of the Ic-ELISA and HA test established the 8.3 and 8.2% prevalence of PPR antigen in nasal and fecal material samples, respectively, while the RT-PCR results validated the seropositive animals. These findings confirmed that the prevalence of PPRV infection in the camels of the Sindh province of Pakistan hence urged the need to take effective measures for prevention and control of the disease.

[Preparation of highly sensitive monoclonal antibody against aflatoxin B_1 and its application in rapid detection of contamination in Ziziphi Spinosae Semen].

A highly sensitive monoclonal antibody against aflatoxin B_1(AFB_1) was prepared and an indirect competition enzyme-linked immunosorbent assay(ic-ELISA) was established based on the antibody which was used for high-throughput and rapid screening of AFB_1 contamination in Chinese herbal medicines to ensure the safety of medication. In this study, the structure of AFB_1 was modified by improved oxime method, and the carrier protein was coupled by EDC-NHS method to obtain the complete antigen of AFB_1, which was more convenient and environmental friendly. The Balb/c female mice were immunized using increasing the immunization dose and various ways of injection, and finally the AFB_1 monoclonal antibody was prepared. The AFB_1 monoclonal antibody belongs to IgG_(2 b) immunoglobulin by identifying its immunological characteristics, and its sensitivity(IC_(50)) can reach 0.15 µg·L~(-1), and the affi-nity is 2.81×10~8 L·mol~(-1). The cross-reaction rates of AFB_2, AFG_1, and AFG_2 were 35.07%, 8.75%, and 1.15%, respectively, and there was almost no cross-reactivity with other mycotoxins. Based on the high sensitivity and specificity of the antibody, an ic-ELISA method was established and applied to the determination of AFB_1 contamination in Ziziphi Spinosae Semen. According to the matrix matching standard curve, the linear concentration range for AFB_1 was 0.05-0.58 µg·L~(-1)(R~2=0.992), the recoveries were 88.00%-119.0%, and the detection limit was 1.69 µg·kg~(-1). The AFB_1 in 33 batches of Ziziphi Spinosae Semen samples was determined by ic-ELISA, and the contamination level was 3.62-206.58 µg·kg~(-1). The linear correlation coefficient between the detection results of ic-ELISA and UHPLC-MS/MS was 0.996, and there were no false positive and false negative cases. It indicates that the established ic-ELISA is accurate and reliable, and could provide a simple and effective technique for fast screening of AFB_1 contamination in Ziziphi Spinosae Semen, and also could be considered as the reference for the detection and monitoring of AFB_1 contamination in other Chinese herbal medicines.

Preparation of an anti-isoprocarb monoclonal antibody and its application in developing an immunochromatographic strip assay.

In this study, a carboxyl group was introduced into the isoprocarb molecule to obtain an isoprocarb hapten, which was then coupled with a protein to obtain an artificial antigen. Three monoclonal antibody cell lines, 1D11, 6E6 and 1B5, were finally obtained by mouse immunization, cell fusion and subcloning, and the antibody produced by cell line 1B5 had the best affinity and sensitivity. The monoclonal antibody was highly sensitive and specific for isoprocarb, with an IC50 of 2.09 ng/ml and a cross-reactivity rate of <0.21%. By optimizing the indirect competitive (ic)-ELISA, the optimal conditions were determined to be pH 7.4, 0% methanol and 0.8% NaCl, the limit of detection value was 0.23 ng/ml, and the linear range of the ic-ELISA was 0.46-9.62 ng/ml. The recovery rate of the isoprocarb cucumber sample was 97-99% for the ic-ELISA method. In addition, we successfully developed an immunochromatographic test strip for the detection of isoprocarb residues. The cutoff values in phosphate-buffered saline and cucumber extract were 10 and 25 ng/ml, respectively. Both methods met the requirements for isoprocarb residue detection in agricultural products, and can be used for semiquantitative and qualitative analysis of isoprocarb in vegetables.

Production and characterization of a biotinylated single-chain variable fragment antibody for detection of parathion-methyl.

In this article, we reported the development of a biotinylated single-chain variable fragment (scFv) antibody based indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for parathion-methyl (PM) detection. Firstly, a phage display library was generated using a pre-immunized BALB/C mouse against a specific hapten of PM. After four rounds of panning, the scFv gene fragments were transferred into a secreted expression vector. Then, the scFv antibodies were secreted expressed and screened by IC-ELISA against PM. The selected scFv antibody was fused with a biotin acceptor domain (BAD) and inserted into pET-28a(+) vector for high-level expression in Escherichia coli BL2 (DE3). After optimizing expression conditions, the scFv-BAD antibody was expressed as a soluble protein and biotinylated in vitro by the E. coli biotin ligase (BirA). Subsequently, the biotinylated scFv-BAD antibody was purified with a high yield of 59.2 ± 3.7 mg/L of culture, and was characterized by SDS-PAGE and western blotting. Finally, based on the biotinylated scFv-BAD, a sensitive IC-ELISA for detection of PM was developed, and the 50% inhibition value (IC50) of PM was determined as 14.5 ng/mL, with a limit of detection (LOD, IC10) of 0.9 ng/mL. Cross-reactivity (CR) studies revealed that the scFv antibody showed desirable specificity for PM.

The first report of peste des petits ruminants (PPR) in camels (Camelus dromedarius) in Iran.

In mid-July 2013, an outbreak of peste des petits ruminants (PPR) was observed in a herd of camels after they were imported from Kuwait to the Khuzestan province in southwest of Iran. The clinical signs of the affected animals included sudden death, fever, oral erosion, and ecthyma like lesions, yellowish diarrhea, pneumonia and respiratory distress, enlargement of lymph node, severe dehydration, dermatitis, ulcerative keratitis, and conjunctivitis. Necropsy findings included keratoconjunctivitis, congestion and consolidation of the lung, paleness of the liver, and enlargement and edema of lymph nodes. Histopathological exam revealed degeneration and acute hyperemia of the lungs, fatty change and necrotic foci in the liver, tubular necrosis in the kidneys, and necrotic dermatitis. We used immunocapture enzyme linked immunosorbent assay (ELISA) to confirm peste des petits ruminants virus (PPRV) and differentiate it from rinderpest virus. Then virus genome was studied by molecular analysis for detecting of strain and substrain of the virus. Immunocapture ELISA of all specimens reacted positively against PPRV antigens. Also, reverse transcription polymerase chain reaction (RT-PCR) results in the lung and lymph nodes of the dead camels consolidated the cause of disease to be PPRV. The present study is the first report of the PPRV outbreak in camels in Iran.

Establishment of enzyme-linked immunosorbent assay for aristolochic acid.

In recent years, the nephrotoxicity and carcinogenicity of aristolochic acid have attracted worldwide attention, and the traditional Chinese medicine containing this ingredient has been banned in many places, affecting the TCM industry. To meet this challenge, researchers have developed various detection methods, such as high-performance liquid chromatography, gas chromatography-mass spectrometry and thin-layer chromatography. A rapid detection method must therefore be developed to ensure safety. A polyclonal antibody capable of recognizing aristolochic acid was prepared, and an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established to detect the amount of aristolochic acid in the sample to be measured. Methods Using 1-(4-chlorophenyl) cyclobutylamine as a hapten, immunogens and coating antigens were obtained by coupling with bovine serum albumin (BSA) and chicken ovalbumin (OVA) using the active ester method. UV scanning confirmed the successful coupling of the conjugate, and New Zealand white rabbits were immunized. The obtained antibody serum was screened for the best antibody by ic-ELISA detection. Use the chessboard method to determine three optimal combinations of original coating concentration and antibody dilution ratio, establish a standard curve for each combination to obtain the best combination, and establish a rapid detection method. Finally, the standard aristolochic acid A was added to the purchased apple vinegar and canned coffee for recycling experiments to verify the detection method.By changing the antigen antibody concentration, the antibody showed the highest sensitivity to aristolochic acid standard at the original coating, 1000-fold dilution, IC50 of 24.88 ng/mL, limit of detection IC10 of 3.19 ng/mL, and detection range IC20-IC80 of 6.81-90.91 ng/mL. The recovery experiments under this conditions yielded a recovery rate of 92%-105%, within reasonable limits, indicating the success of the ELISA rapid detection method. Conclusion The enzyme-linked immunoassay method established in this paper can quickly detect the content of aristolochic acid in the sample to be tested, and the antibody prepared by this method has good broad-spectrum and can detect other aristolochic acid, such as aristolochic acid A, aristolochic acid B, aristolochic acid C, and aristolochic acid D.

Preparation of monoclonal antibodies recognizing pharmacologically active metabolites of metamizole based on rational hapten design and their application in the detection of animal-derived food.

Metamizole (MET) is an antipyretic and analgesic drug, the illegal use of which can result in residues of MET metabolites in edible tissues of animals. In this study, a computational chemistry-assisted hapten screening strategy was used to screen for the optimal immunogenic hapten-A and the optimal coating antigen hapten-G-OVA. A monoclonal antibody capable of recognizing two pharmacologically active metabolites of MET, 4-methylamidinoantipyrine (MAA) and 4-aminoantipyrine (AA), was prepared from the hapten-A. The antibody showed excellent specificity for MAA and AA and almost no cross-reactivity with the pharmacologically inactive metabolites 4-formamidinoantipyrine (FAA) and 4-acetamidinoantipyrine (AAA). An ic-ELISA was developed for the simultaneous detection of MAA and AA in animal-derived food, the limits of detection for MAA ranged from 0.93 to 1.18 µg/kg, while those for AA ranged from 1.74 to 4.61 µg/kg. The recovery rate fell within the range of 82 %-110 %, with a coefficient of variation less than 16.39 %.

Development of a Sensitive Monoclonal Antibody-Based Colloidal Gold Immunochromatographic Strip for Lomefloxacin Detection in Meat Products.

Lomefloxacin (LOM), an antibiotic crucial for preventing various animal diseases in animal husbandry, can pose serious health risks when found in excessive amounts in meat products. The development of highly specific and sensitive colloidal gold immunochromatographic test strips is essential for the accurate detection of this class of antibiotics. Our study utilized a monoclonal antibody (mAb) assay and immunochromatographic strips to detect lomefloxacin residues in meat products. The results showed minimal cross-reactivity with other structural analogs, with a maximum half inhibitory concentration (IC50) of 0.93 ng/mL and a linear range of 0.38 to 2.3 ng/mL for the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). The recovery of LOM was 80% to 120%, with an average coefficient of variation below 5%. The immunochromatographic strip test results showed a visual detection limit of 2.5 ng/g, meeting the market requirements for the test. This study highlights the significance of specific and sensitive testing methods for detecting lomefloxacin, ensuring consumers' safety and health.

Development of a Monoclonal Antibody-Based Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Rapid Detection of Gallic Acid.

Gallic acid (GA) is closely related to the quality of herbal medicines and other agricultural products. In order to facilitate the rapid detection of GA, we developed a monoclonal antibody-based ic-ELISA method. Antigens with and without connecting arms were prepared. It was found that the introduction of connecting arms (linear carbon chain) was beneficial for immune response. By utilizing hybridoma technology, a specific mAb (anti-GA-M702) was screened and identified, which exhibited a 1:40,500 antibody titer and IgG2b antibody subtype. The ic-ELISA assay was established based on anti-GA-M702. The optimal working concentrations of the encapsulated antigen and antibody were 0.5 µg/mL and 0.67 µg/mL, respectively. The ic-ELISA method showed a linear detection range of 297.17-2426.61 ng/mL for GA with a sensitivity of 849.18 ng/mL. It displayed a good applicability for the determination of GA in Galla chinensis. In conclusion, the ic-ELISA method provides an efficient approach to the rapid detection of GA in products.

Hapten synthesis, monoclonal antibody production and immunoassay development for analyzing spiropidion residues.

Spiropidion developed by Syngenta shows high insecticidal and acaricidal activity against a wide range of sucking pests. In this study, according to the structure of spiropidion, two haptens were synthesized by introducing carboxyl groups from the ester group. After cell fusion, a monoclonal antibody (mAb 8B5) of spiropidion was obtained. The IC50 of the established heterologous indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was 7.36 ng/mL, and its working range was 1.75-34.92 ng/mL. The average recoveries were 76.05-124.78% in the Yangtze River and citrus samples. Moreover, the ic-ELISA results of 15 citrus samples agreed well with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Overall, the established ic-ELISA could be applied for the spiropidion residue monitor in food and agricultural samples.

Establishment of Indirect Competitive Enzyme-linked Immunosorbent Assay (ic-ELISA) for Copper ion (Cu2+) in Raw Meat Products.

Adding an appropriate amount of copper to feed can promote the growth and development of livestock; however, a large amount of heavy metal copper can accumulate in livestock through the enrichment effect, which poses a serious threat to human health. Traditional Cu2+ detection relies heavily on complex and expensive instruments, such as inductively coupled plasma-optical emission spectrometry (ICP-OES) and inductively coupled plasma-mass spectrometry (ICP-MS); thus, convenient and simple rapid detection technologies are urgently needed. In this paper, synthesized copper antigens were used to immunize mice and highly specific anticopper monoclonal antibodies were obtained, which were verified to exhibit high affinity and specificity. Based on the above antibodies, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established for the rapid detection of copper content in pork. The standard inhibition curve of the method was obtained by antigen-antibody working concentration screening, in which the half inhibitory concentration (IC50) was 11.888 ng/mL, the limit of detection (LOD) was 0.841 ng/mL and the correlation coefficient R2 of the curve was 0.998. In the additive recovery experiment, the recovery rate ranged from 90% to 110%, and the coefficient of variation (CV) was less than 10%, indicating that the method achieved high accuracy and precision. Finally, the results of ic-ELISA combined with Bland-Altman analysis showed a high correlation with ICP-MS, and the correlation coefficient (R2) reached 0.990 when the copper concentration was less than 200 ng/mL. Thus, the ic-ELISA method exhibits high reliability.

Generation of a highly specific recombinant full-length antibody for detecting ethirimol in fruit and environmental water.

High-performance antibodies are core reagents for highly sensitive immunoassays. Herein, based on a novel hapten, a hybridoma secreting the high-affinity anti-ethirimol monoclonal antibody (mAb-14G5F6) was isolated with an IC50 value of 1.35 µg/L and cross-reactivity below 0.20% for 13 analogs. To further address the challenge of hybridoma preservation and antibody immortalization, a recombinant full-length antibody (rAb-14G5F6) was expressed using the HEK293(F) expression system based on the mAb-14G5F6 gene. The affinity, specificity, and tolerance of rAb-14G5F6, as characterized by indirect competitive enzyme-linked immunosorbent assay and noncompetitive surface plasmon resonance, exhibited high concordance with those of mAb-14G5F6. Further immunoassays based on rAb-14G5F6 were developed for irrigation water and strawberry fruit with limits of detection of 0.0066 and 0.036 mg/kg, respectively, recoveries of 80100%, and coefficients of variation below 10%. Furthermore, homology simulation and molecular docking revealed that GLU(L40), GLY(L107), GLY(H108), and ASP(H114) play important roles in forming hydrogen bonds and pi-anion ionic bonds between rAb-14G5F6 and ethirimol, resulting in the high specificity and affinity of rAb-14G5F6 for ethirimol, with a KD of 5.71 × 10-10 mol/L. Overall, a rAb specific for ethirimol was expressed successfully in this study, laying the groundwork for rAb-based immunoassays for monitoring fungicide residues in agricultural products and the environment.

Highly selective and sensitive immunoassays for flurogestone acetate analysis in goat milk: From rational hapten design and antibody production to assay development.

The preparation of high specificity and affinity antibodies is challenging due to limited information on characteristic groups of haptens in traditional design strategy. In this study, we first predicted characteristic groups of flurogestone acetate (FGA) using quantitative analysis of molecular surface combined with atomic charge distribution. Subsequently, FGA haptens were rationally designed to expose these identified characteristic groups fully. As a result, seven monoclonal antibodies were obtained with satisfactory performance, exhibiting IC50 values from 0.17 to 0.45 µg/L and negligible cross-reactivities below 1% to other 18 hormones. The antibody recognition mechanism further confirmed hydrogen bonds and hydrophobic interactions involving predicted FGA characteristic groups and specific amino acids in the antibodies contributed to their high specificity and affinity. Finally, one selective and sensitive ic-ELISA was developed for FGA determination with a detection limit as low as 0.12 µg/L, providing an efficient tool for timely monitoring of FGA in goat milk samples.

Development of Indirect Competitive ELISA and Colloidal Gold Immunochromatographic Strip for Endosulfan Detection Based on a Monoclonal Antibody.

Endosulfan, as an effective broad-spectrum insecticide, has been banned in agricultural areas because of the potential harmful effects on human health. This study aimed to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold immunochromatographic (ICA) strip based on a prepared monoclonal antibody (mAb) for quantitative and qualitative detection of endosulfan. A new mAb with high sensitivity and affinity was designed and screened. The ic-ELISA showed a 50% inhibition concentration (IC50) value of 5.16 ng/mL for endosulfan. Under optimum conditions, the limit of detection (LOD) was determined to be 1.14 ng/mL. The average recoveries of endosulfan in spiked pear and apple samples ranged from 91.48-113.45% and 92.39-106.12% with an average coefficient of variation (CV) of less than 7%, respectively. The analysis of colloidal gold ICA strip could be completed within 15 min by naked eye and the visual limit of detection (vLOD) was both 40 ng/mL in pear and apple samples. In conclusion, both developed immunological methods were suitable and reliable for the on-site detection of endosulfan in real samples at trace levels.

Preparation of spirodiclofen monoclonal antibody and establishment of indirect competitive enzyme-linked immunosorbent assay.

Spirodiclofen, a spirocyclic tetronic acid derivative, has excellent acaricidal effect and is used worldwide to control the majority of important mite species. For monitoring its residue in food and environmental samples, two haptens containing different spacer arms were synthesized, a monoclonal antibody (mAb 5A4) against spirodiclofen was prepared, and a heterologous indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established. The 50% inhibition concentration (IC50) of ic-ELISA was 25.46 ng/mL, and the working range was 5.59-133.85 ng/mL. The ic-ELISA showed no cross-reactivity with structural analogs of spirodiclofen and other commonly-used acaricides. The average recoveries from Shiranui citrus samples and Yangtze River water were 85.62%-97.74% and 85.95%-99.30%, respectively. In the analysis of 12 citrus samples, the results of the ic-ELISA were quite similar to those of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Hence, the new immunosorbent assay provides a substitute method for the qualitative and quantitative of spirodiclofen in food and environmental samples.