Development of an immunochromatographic assay for rapid detection of clorprenaline in pig urine

A clorprenaline (CLP) monoclonal antibody, 4G1, raised against the CLP-hapten was successfully prepared for the development of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and immunochromatographic assays for the detection of CLP in pig urine samples. The half maximal inhibitory concentration of the antibody was 0.435?ng/mL, and the limit of detection was 0.104?ng/mL. The linear range was 0.104–1.818?ng/mL, and cross-reactivity with the CLP analogue was <5%. In the spiked sample and recovery test, the recovery ranged from 81.8% to 100.5%, indicating that the ic-ELISA was suitable for CLP analysis in pig urine. The critical value of the immunochromatographic strip in pig urine was 10?ng/mL and could be used for semi-quantitative analysis of CLP. Thus, this immunochromatographic strip was suitable for quickly and sensitively detecting CLP residues in pig urine samples.     

Development of an indirect enzyme-linked immunosorbent assay and lateral-flow test strips for pefloxacin and its analogues in chicken muscle samples

An anti-pefloxacin (PEF) monoclonal antibody (mAb), an indirect competitive enzyme-linked immunosorbent assay and lateral-flow test strip methods were developed to detect fluoroquinolone (FQ) residues in chicken muscle samples. Under optimised conditions, the anti-PEF mAb showed reasonable cross-reactivity with nine FQs with a limit of detection of 0.082?ng/mL assayed in 0.01?M phosphate buffered saline (PBS) solution. The intra- and inter-assay recoveries from spiked samples were within the range of 62.42–111.47% and 63.5–113.79%, respectively. The visual cut-off values of the lateral flow test strip in 0.01 M PBS and in food matrices were within the range of 2.5–50?ng/mL and 5–100?µg/kg, respectively. These results show that the anti-PEF mAb immunoassay and lateral flow test strip methods are suitable for simultaneous detection and routine monitoring of FQ residues in food.     

Preparation of an anti-dexamethasone monoclonal antibody and its use in development of a colloidal gold immunoassay

A lateral flow colloidal gold (CG) immunoassay strip has been developed for detection of dexamethasone (DEX) residues in milk samples. For this purpose, an anti-DEX monoclonal antibody (McAb), based on a DEX succinic anhydride derivative hapten, was prepared and characterized. The McAb showed a high specificity to DEX, the half inhibitory concentration of the antibody was 0.095?ng/mL, its limit of detection (LOD) was 0.017?ng/mL, and its linear range of detection was 0.034–0.265?ng/mL. The developed CG immunoassay had a visual cut-off value of 0.3?ng/mL in phosphate buffered saline (PBS) and 0.5?ng/mL in milk samples. Each test requires 10 min. Analysis of DEX in milk indicated that the results of strip assay had a strong agreement with indirect competitive enzyme-linked immunosorbent assay. Therefore, the CG immunoassay is a sensitive screening method for semi-quantitative and qualitative detection of DEX residues in milk samples.     

Development of a monoclonal antibody assay and immunochromatographic test strip for the detection of amikacin residues in milk and eggs

An amikacin-sensitive monoclonal antibody (MAb) assay and immunochromatographic test strip were developed and applied for the detection of amikacin residues in bovine milk and chicken eggs. The immunoassay was specific to amikacin and showed no cross-reactivity with other aminoglycosides. The half maximum inhibitory concentration (IC₅₀) of the assay was 0.65?ng/mL and the results were obtained within 90?min. Recoveries from spiked food matrices were within the range of 73.55–84.61% for bovine milk and 73.70–105.75% for whole egg. The strip test results were obtained within 10?min and showed a visual detection limit of 5.0?ng/mL for both food matrices. These results show that the MAb immunoassay and strip test developed in this study are very specific to amikacin and sufficiently sensitive for detection and routine monitoring of amikacin residues in food.     

Development of ic-ELISA and lateral-flow immunochromatographic assay strip for the detection of vancomycin in raw milk and animal feed

A highly sensitive monoclonal antibody (mAb) 3H4 against vancomycin (VAN) was prepared. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and lateral-flow immunochromatographic assay (ICA) were developed based on the mAb. The 50% inhibition concentration (IC₅₀) value and limit of detection (LOD) value of ic-ELISA method for vancomycin were 0.59 and 0.06?ng/mL, and for norvancomycin were 1.51 and 0.13?ng/mL under optimized conditions as pH 7.4, 0.4% (m/v) NaCl, and 5% (v/v) acetonitrile. In lateral-flow ICA, the visual limit of detection (vLOD) value and cut-off values for vancomycin were 1 and 2.5?ng/mL, and for norvancomycin were 5 and 10?ng/mL under optimized conditions as pH 8.6 with 1?mg/mL coating antigen and 1?µg/mL gold nanoparticle-labeled mAb. In raw milk and animal feed samples, recovery rates from ic-ELISA ranged from 89.2% to 121.6%. The vLOD and cut-off value were 5–10?ng/g and 100–200?μg/kg, respectively. Therefore, both methods were sensitive, rapid, and effective for the on-site detection and rapid mass screening of samples.     

Development of indirect competitive ELISA and lateral-flow immunochromatographic assay strip for the detection of sterigmatocystin in cereal products

Sensitive and specific anti-sterigmatocystin (STG) monoclonal antibody (mAb) 4G10 was obtained by immunization and cell fusion. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method and lateral-flow immunochromatographic assay (ICA) strip method were developed for the detection of STG in cereal products based on this mAb. The 50% inhibition concentration and limit of detection for the ic-ELISA method were 0.092 and 0.015?ng/mL, respectively. The visual limit of detection (vLOD) and cut-off value for the lateral-flow ICA strip method were 0.1 and 0.5?ng/mL, respectively. From the analysis of different cereal samples (wheat, maize and rice), the recovery rates ranged from 78.3% to 122.0% for the ic-ELISA method. For the lateral-flow ICA strip, the vLODs were 3, 1.2 and 3?ng/g, and the cut-off values were 12, 6 and 6?ng/g for wheat, maize and rice, respectively. Therefore, both of the developed methods are suitable for the on-site detection and rapid screening of numerous samples.     

Residues determination of carbofuran in vegetables based on sensitive time-resolved fluorescence immunoassay

Using direct competitive time-resolved fluorescence immunoassay (TRFIA), a rapid, highly selective and sensitive method was developed for the determination of carbofuran residues in lettuce and carrot. The method was based on a direct competitive immunoassay using europium-labelled anti-carbofuran monoclonal antibody and carbofuran-ovalbumin as coated antigen. The sensitivity, estimated as the I ₅₀ value, was 34.54 ng/mL, with a detection limit (I ₁₀) of 1.94 ng/mL and a practical working range between 1.33 and 341.6 ng/mL. The average recoveries of carbofuran from lettuce and carrot were 81.26–108.0% and 113.9–124.8%, respectively. Eventually, confirmation test between TRFIA and enzyme-linked immunoassay (ELISA) was performed. It confirmed that the results given by the TRFIA method were in agreement with those of the ELISA method.     

Development of an indirect competitive enzyme-linked immunosorbent assay and immunochromatographic assay for hydrocortisone residues in milk

An anti-hydrocortisone (HDS) monoclonal antibody, 2G8, based on a HDS succinic anhydride derivative hapten, was prepared and used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and an immunochromatographic assay for the detection of HDS in milk samples. The half inhibitory concentration (IC₅₀) of the antibody was 0.095?ng/mL, its limit of detection was 0.013?ng/mL, its linear range of detection was 0.026–0.356?ng/mL, and its cross-reactivity with HDS analogs was <5%. In spiked samples and a recovery test, the recovery rates ranged from 92% to 98.5%, indicating the suitability of this ic-ELISA for the analysis of HDS in milk. The immunochromatographic strip had a cutoff value of 2?ng/mL in milk and could be used for the semiquantitative analysis of HDS. When milk samples were added to the sample pad of the strip, a bright test line indicated <0.2?ng/mL HDS, a weak test line indicated 0.2–2?ng/mL HDS, and no test line indicated ≥2?ng/mL HDS. Analysis of HDS in milk samples showed that results acquired by the immunochromatographic assay agreed well with results acquired by ic-ELISA. Thus, the ic-ELISA and strip assay developed in this study rapidly and sensitively detect HDS residues in milk samples.     

Development of indirect competitive enzyme-linked immunosorbent and immunochromatographic strip assays for carbofuran detection in fruits and vegetables

Carbofuran is a highly toxic pesticide used in fruits and vegetables. In this study, we produced a specific and sensitive monoclonal antibody (mAb) which was prepared based on a hapten that was derivatized with benzofuranol against carbofuran. Following mice immunization and cell fusion, we obtained three monoclonal cell lines: 6D5, 3H2, and 3C6. The cell line 3H2 generated mAb with the highest affinity and sensitivity. The half maximal inhibitory concentration was 0.3?ng/mL, and the cross-reactivity was <1%. Based on this mAb, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and immunochromatographic test strip (ICS) assay were developed to detect carbofuran residues in cucumbers and apples. The working range of ic-ELISA was 0.1–1?ng/mL, and the cutoff value of ICS was 1?ng/mL. The analytical recovery of carbofuran in cucumber and apple samples ranged from 81% to 97%. Both methods represent rapid screening tools for carbofuran detection in fruits and vegetables.     

Development of an immunochromatographic test strip for the detection of papaverine in pure ginger powder

A leteral-flow immunochromatographic assay was developed for the detection of papaverine (PAPA) in pure ginger powder samples. We produced a sensitive monoclonal antibody againsts PAPA (anti-PAPA mAb) by immunizing BALB/c mice with a well-characterized PAPA-keyhole limpet hemocyanin conjugate, produced in our laboratory. The coating antigen (PAPA-ovalbumin) and goat anti-mouse IgG antibody were used as the capture reagent in the control line of the test strip. Under optimized conditions, the cut-off limits of the test strip was 1?ng/mL in 0.01?M PBS (pH 7.4) and 5?ng/mL in pure ginger powder. The results were obtained within 5 min. The results revealed that the developed method is a sensitive, rapid, and simple tool for the detection of PAPA in pure ginger powder.     

Dual-labelled immunoassay with goldmag nanoparticles and quantum dots for quantification of casein in milk

A dual-labelled immunoassay using goldmag nanoparticles (GMNPs) and CdTe quantum dots (QDs) was applied for the quantification of casein in milk. In this method, anti-casein monoclonal antibody bound to GMNPs was used as capture probe, and the anti-casein polyclonal antibody, labelled with CdTe QDs, was employed as detection probe. This dual-labelled immunoassay was optimized and applied in the testing of casein content of three brands of commercial milks. The results showed a good linear tendency between casein concentrations and fluorescent intensities in the range of 4.617–289.9?ng/mL, and the half inhibition concentration (IC₅₀) was 36.59?ng/mL. Besides, the recovery rates of this developed immunoassay were in accordance with those in traditional enzyme-linked immunosorbent assay for rapid detection of casein content in commercial milks. It suggested that this dual-labelled immunoassay was reliable, and could provide important theoretical value and practical significance in the identification and quantification of milk allergens.     

Development of a monoclonal antibody assay and a lateral flow strip test for the detection of paromomycin residues in food matrices

A monoclonal antibody (MAb) was produced against paromomycin and used in the development of an immunoassay and a lateral flow strip test for the detection of paromomycin residues in food matrices. The MAb had 25.1% cross-reactivity with neomycin, but was insensitive to other aminoglycosides. Tested under optimized conditions in 0.01 M phosphate-buffered saline, the half maximum inhibitory concentration (IC₅₀) of the MAb was 0.61?ng/ml for paromomycin and 2.43?ng/ml for neomycin, with results obtained within 90?min. The mean recoveries from spiked food matrices were within the range of 64.56–105.85% for paromomycin and 54.08–100.55% for neomycin. The strip test results for different food matrices were obtained within 5?min and showed visual detection limits of 2.5–20?ng/ml (µg/kg) for paromomycin and 10–30?ng/ml for neomycin. Therefore, the developed immunoassay and strip test can be used in food analysis for routinely monitoring not only paromomycin but also neomycin residues.     

Development of IC-ELISA and immunochromatographic strip assay for the detection of flunixin meglumine in milk

Flunixin meglumine (FM) is a novel nonsteroidal anti-inflammatory drug for animals, which has antipyretic, analgesic, and anti-inflammatory effects. The drug, which was originally used to relieve inflammation in horses, musculoskeletal disorders, and pain, has been approved for use in endotoxemia, infectious diseases in swine, etc. A sensitive anti-FM monoclonal antibody 2H4 was prepared and used to develop an indirect competitive enzyme-linked immunosorbent assay and immunochromatographic strip assay for the detection of FM in milk. The complete antigen and coating antigen were conjugated with bovine serum albumin and ovalbumin, respectively. The monoclonal antibody 2H4, with a half inhibition concentration of 0.29?ng/mL, had a limit of detection of 0.432?ng/mL and a linear range of detection of 0.08664–0.97226?ng/mL. A sensitive and convenient immunochromatographic strip assay was developed with an FM cutoff value of 0.29?ng/mL. The developed methods were suitable for the detection of FM in milk.     

Development of an icELISA and immunochromatographic strip for detection of norfloxacin and its analogs in milk

We developed a monoclonal antibody (mAb) to detect norfloxacin (NOR) using a novel hapten by introducing an amino group via amido linkage into the piperazine ring of NOR. The sensitivity of an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this mAb showed an improved IC₅₀ value from 1.18 to 0.12?ng/mL and the limit of detection from 0.37 to 0.05?ng/mL by using a heterologous coating (pazufloxacin coupled to ovalbumin). The recovery rate for NOR in milk ranged from 77.3% to 117.8%. Cross-reactivity (CR) experiments showed good CR with nine common fluoroquinolone analogs: pefloxacin, lomfloxacin, ciprofloxacin, enrofloxacin, dafloxacin, sparfloxacin, ofloxacin, marbofloxacin and flumequine. Furthermore, based on this antigen–antibody combination, an immunochromatographic strip with cut-off value of 1?ng/mL was developed. Given their good sensitivity and CR, the developed icELISA and immunochromatographic strip could be useful for detection of NOR and its analogs in milk.     

Development of an immunochromatographic lateral flow test strip for detection of beta-adrenergic agonist Clenbuterol residues

A rapid immunochromatographic lateral flow test strip was developed in a competitive format with the gold-conjugated monoclonal antibody to specifically determine the residues of Clenbuterol (CL), a beta-adrenergic agonist. The test strip is made up of a sample pad, a conjugate reagent pad, a test membrane containing a control line and a test line, and an absorbent pad. CL standard samples of 0, 0.1, 0.3, 0.9, 2.7, 8.1 ng/ml in swine urine were determined by the test strip. It was shown that detection limit of the test strip was as low as 0.1 ng/ml of CL and that the half of maximal inhibition concentration (IC50) in relative optical density was calculated to be 1.78+/-0.17 ng/ml under an optical density scanner. The sensitivity by eye measurement was 1.0 ng/ml. It takes 10 min to accomplish a test. Parallel analysis of urine samples from pigs fed with CL showed comparable results obtained from the test strip and GC-MS. Therefore, the test strip is very useful as a screening method for quantitative, semi-quantitative or qualitative detection of CL residues in swine urine.     

Development of an immunochromatographic test strip and ic-ELISA for tetrabromobisphenol: a detection in lake water and rice pudding samples

A sensitive and selective indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and an immunochromatographic test strip were developed for the detection of tetrabromobisphenol A (TBBPA), an important brominated flame retardant. A monoclonal antibody (mAb) against TBBPA was produced and the half inhibition concentration (IC50) was 0.44?ng/ml, with the linear range 0.15–1.32?ng/ml. TBBPA recovery by ic-ELISA was 70.7–108.8% from rice pudding samples and 110.2–119.8% from lake water samples. A sensitive and rapid immunochromatographic test strip was developed using this specific mAb, with a good cut-off value of 25 and 50?ng/ml in lake water and rice pudding samples, respectively. The results of this study revealed that the ic-ELISA and immunochromatographic test strip represent useful methods of TBBPA detection in lake water and rice pudding samples.     

抗主要组织相容性相关蛋白MICA / B 的单克隆抗体制备及应用

目的:制备抗主要组织相容性相关蛋白A/ B(MICA/ B)的单克隆抗体并对其特异性进行验证。方法:以重组MICA*012 蛋白免疫小鼠后将其脾细胞与SP2/0 细胞进行融合和筛选得杂交瘤细胞株,并用ELISA 对其中9B10 细胞株产生的单克隆抗体进行效价检测以及通过Western blot、Luminex、免疫荧光对其特异性进行检测。结果:获得6 株杂交瘤细胞株,其中9B10 细胞株产生单克隆抗体(mAb)9B10 的最低反应浓度为0.02 ng/ μl,且mAb 9B10 可应用于Western blot、Luminex 和免疫组化荧光试验对MICA 和MICB 分子的检测。结论:成功获得可同时检测MICA 和MICB 表达的单克隆抗体9B10。     

Sensitive double antibody sandwich ELISA for the quantification of phosvitin

An effective double antibody sandwich ELISA (DAS-ELISA) method based on monoclonal (mAb) and chicken egg yolk IgY antibodies was developed to determine phosvitin (PV) content in therapeutic and functional products. Leghorn laying hens were immunized with purified PV to produce anti-PV IgY antibody in the egg yolk. High anti-PV IgY titer obtained from the egg yolks collected during 4–10 weeks of the immunization period contained approximately 6.2% of specific anti-PV IgY in total IgY. The PV detection range of the DAS-ELISA and biotinylated DAS-ELISA was 16.8–90 and 7.5–40?ng/mL, respectively. However, biotinylated DAS-ELISA was the better method for PV quantification in terms of accuracy and sensitivity. This highly efficient PV detection method may recuperate the performance of the existing protein assay methods as well as facilitate future research on PV bioactivities and applications.     

Quantitative double antibody sandwich ELISA for the determination of gliadin

A sensitive double antibody sandwich ELISA (DAS-ELISA) based on chicken anti-gliadin IgY and biotinylated monoclonal antibody (mAb) was developed for the quantification of gliadin in foods. The anti-gliadin IgY and mAb specifically detected gliadin in wheat, barley, and rye by indirect ELISA and Western-blot assay. Using anti-gliadin IgY as capture antibody and biotinylated mAb as detecting antibody, the sensitivity of DAS-ELISA has a linear standard range of 4-40?ng/mL, showing that the limit of detection (LOD) corresponds to 4?ng/mL gliadin in assay buffer, equivalent to 0.8?ppm in foods. The intra-assay expressed as percentage of coefficients of variation (%CV) was 7.25% average of six food samples. The interassay precision was 9.51% in food samples. The combination of anti-gliadin IgY and biotinylated mAb in the DAS-ELISA provides a reliable, sensitive, and inexpensive tool for the detection of gliadin in gluten-free and gluten-containing food products.     

Goldmag-based enzyme-linked immunosorbent assay for determination of α-lactalbumin in milk

In this work, a goldmag-based enzyme-linked immunosorbent assay was developed for determination of α-lactalbumin (α-LA) in milk. The magnetic nanoparticle functionalized with polyetherimide was synthesized by one-pot method and coated with two layers of gold nanoparticles on the surface to synthesize goldmag nanoparticles. Anti-α-LA monoclonal antibody, prepared by hybridoma cell lines via cell fusion, was then bound to this goldmag nanoparticle to develop a capture nanoprobe. The results showed that this developed immunoassay had a good linear range of 2.33–127.1?ng/mL with IC₅₀ of 17.2?ng/mL. Besides, recovery rates for α-LA in four commercial milks were from 86.7% to 109.8%. The coefficients of variation in intra-assay and inter-assay were 3.9–6.8% and 5.5–9.8%, separately, which could meet the requirements to quantify α-LA content in milk. This goldmag-based immunoassay might have considerable potentials in the detection of food allergies.