Change of Amino Acid Residues in Idiotypic Nanobodies Enhanced the Sensitivity of Competitive Enzyme Immunoassay for Mycotoxin Ochratoxin A in Cereals

Anti-idiotypic nanobodies, usually expressed by gene engineering protocol, has been shown as a nontoxic coating antigen for toxic compound immunoassays. We here focused on how to increase immunoassay sensitivity by changing the nanobody’s primary sequence. In the experiments, two anti-idiotype nanobodies against monoclonal antibody 1H2, which is specific to ochratoxin A, were obtained and named as nontoxic coating antigen 1 (NCA1) and nontoxic coating antigen 2 (NCA2). Three differences between the nanobodies were discovered. First, there are six amino acid residues (AAR) of changes in the complementarity determining region (CDR), which compose the antigen-binding site. One of them locates in CDR1 (I–L), two of them in CDR2 (G–D, E–K), and three of them in CDR3 (Y–H, Y–W). Second, the affinity constant of NCA1 was tested as 1.20 × 108 L mol−1, which is about 4 times lower than that of NCA2 (5.36 × 108 L mol−1). Third, the sensitivity (50% inhibition concentration) of NCA1 for OTA was shown as 0.052 ng mL−1, which was 3.5 times lower than that of nontoxic coating antigen 2 (0.015 ng mL−1). The results indicate that the AAR changes in CDR of the anti-idiotypic nanobodies, from nonpolar to polar, increasing the affinity constant may enhance the immunoassay sensitivity. In addition, by using the nontoxic coating antigen 2 to substitute the routine synthetic toxic antigen, we established an eco-friendly and green enzyme-linked immunosorbent assay (ELISA) method for rapid detection of ochratoxin A in cereals. The half-maximal inhibitory concentration (IC50) of optimized ELISA was 0.017 ng mL−1 with a limit of detection (LOD) of 0.003 ng mL−1. The optimized immunoassay showed that the average recoveries of spiked corn, rice, and wheat were between 80% and 114.8%, with the relative standard deviation (RSD) ranging from 3.1–12.3%. Therefore, we provided not only basic knowledge on how to improve the structure of anti-idiotype nanobody for increasing assay sensitivity, but also an available eco-friendly ELISA for ochratoxin A in cereals.

An efficient fiber-based ELISA for detection of antibody against fowl adenovirus serotypes 7 and 8

An outbreak of inclusion body hepatitis caused by fowl adenovirus serotype 8 (FAdV-8) has caused significant economic losses in the poultry industry worldwide. However, a rapid serology test kit specific to FAdV-8 is not available to date. We developed a fiber-based ELISA using the purified GST-fiber of FAdV-8 as coating antigen to measure antibodies against FAdV-8. Specificity analysis showed that our ELISA could react with sera against FAdV-7, -8a, and -8b, but not with sera against the other pathogens tested. Moreover, detection of positive sera with our ELISA had 83% and 94% agreement with an indirect immunofluorescence assay (IFA) and a commercial ELISA from BioChek, respectively. Our ELISA was also effective in the detection of antibodies against FAdV-8 in sera from both experimentally infected and clinically vaccinated chickens. Our FAdV-8 fiber-based ELISA can be a valuable tool to specifically and sensitively detect antibodies against FAdV-7 and/or -8 in infected or vaccinated chickens.

A one-step incubation ELISA kit for rapid determination of dibutyl phthalate in water, beverage and liquor

A one-step incubation ELISA kit based on monoclonal antibody against dibutyl phthalate (DBP) was developed. After optimizing concentrations of coating antigen, antibody and composition of the assay buffer, an inhibition curve was plotted. The IC50 is 29.6 ng·mL-1, and the detection limit for DBP is 3.6 ng·mL-1. Compared with other ELISA methods, this ELISA kit had a simpler sample preparation, costed less time for detection and could detect more types of sample. The recoveries of DBP in water, beverage and liquor samples were range from 78% to 110.4%, the range of coefficient of variations is 7.7-15.3%. The cross reactivity was very low (&1%) except that for butyl benzyl phthalate (3.9%) and the di-isobutyl phthalate (12.5%). The detection results in liquor showed good correlation with those from GC-MS. All data above indicated that this kit could be used as the fast and high-throughput screening of DBP in water, beverage and liquor.

Quantitative Determination of Acetamiprid in Pollen Based on a Sensitive Enzyme-Linked Immunosorbent Assay

A sensitive biotinylated indirect competitive enzyme-linked immunosorbent assay (Bic-ELISA) was developed to detect acetamiprid pesticides in pollen, based on the heterogeneous coating antigen and biotinylated anti-acetamiprid monoclonal antibody. Under optimized experimental conditions, the detection limit for the Bic-ELISA was 0.17 ng/mL and the linear range was 0.25–25 ng/mL. The cross-reactivities could be regarded as negligible for the biotinylated antibodies with their analogues except for thiacloprid (1.66%). Analyte recoveries for extracts of spiked pollen (camellia pollen, lotus pollen, rape pollen) ranged from 81.1% to 108.0%, with intra-day relative standard deviations (RSDs) of 4.8% to 10.9%, and the average reproducibility was 85.4% to 110.9% with inter-assay and inter-assay RSDs of 6.1% to 11.7%. The results of Bic-ELISA methods for the Taobao’s website samples were largely consistent with HPLC-MS/MS. Therefore, the established Bic-ELISA methods would be conducive to the monitoring of acetamiprid in pollen.

Evaluation of LipL32 ELISA for detection of bovine leptospirosis in West Java

The current diagnosis of leptospirosis, micro Agglutination Test (MAT) and isolation, is expensive, impractical and technically demanding. This study was aimed at developing an ELISA based on recombinant LipL32 as a practical, inexpensive test for Leptospirosis. The DNA encoding LipL32 was isolated from Leptospira pomona, inserted into pRSET-C plasmid then expressed in E.coli BL21 as a poly-histidine-tagged protein. The amount of LipL32 protein, which was purified from the supernatant of lysed cells by a Ni-NTA column, was 1mg/l culture. This purified LipL32 was used as the coating antigen at 5µg/ml. The accuracy of ELISA was evaluated based on ROC analysis, by comparing the ELISA and MAT results of 517 bovine sera. Result in this study showed that the area under curve (AUC) was 0.853, which categorised the LipL32 ELISA as a “moderately accurate” test and indicates that the ELISA was able to differentiate positive and negative Leptospirosis serum. The result also showed ELISA LipL32 could detect serum positive MAT to Hardjo, Grippotyphosa, Tarrasovi, Rachmati and Bataviae. The optimal cut off for OD ELISA determined based on ROC curve was 0.504, and it showed sensitivity and specificity of ELISA LipL32 relative to MAT were 86.0% and 69.5%, respectively. Overall, the result in this study showed that ELISA LipL32 can be used as a rapid test for identification of anti-Leptospira antibodies in bovine.

A highly sensitive and quantitative detection method for bisphenol A (BPA) by competitive immunoassay based on surface-enhanced Raman spectroscopy

Purpose In this study, a highly sensitive and quantitative analysis method using surface-enhanced Raman scattering (SERS)-labeled immunoassay is adopted for bisphenol A bisphenol A (BPA) detection in water samples. Design/methodology/approach Primarily, an excellent SERS immuno-nanoprobe is prepared, which relays on Au/Ag core-shell nanoparticles tagged 4-mercaptobenzoic acid (4MBA) and labeled with specific antibody against BPA. Second, the coating antigen of 4,4-Bis(4-hydroxyphenol) valeric acid (BVA) coupling poly-L-lysine (PLL) conjugate (BVA-PLL) is fastened on the substrate. Based on competitive immunoassay, the antibody labeled on SERS immuno-nanoprobe will bind with the free BPA and BVA-PLL competitively. Findings A calibration curve was obtained by plotting the intensity of SERS signal of 4MBA at 1007 cm−1 versus the concentration of BPA. The results indicated that the limit of detection (LOD) for BPA is 1 ng/mL and present a great capacity for higher sensitivity. Furthermore, the method was able to quantitatively detect BPA in water samples, which was validated by high performance liquid chromatography (HPLC). Originality/value The method was developed based on competitive immunoassay, and the conjugate (BVA-PLL) was chosen as the coating antigen. Au/Ag core-shell nanoparticles played as the SERS active substrate and were labeled with Raman reporter. The value of this paper is supplying a wide potential for analysis of target analytes in the environmental monitoring and food safety.

An effective strategy for preparation of a polyclonal antibody with an addition of carbon chain of ciprofloxacin

In this study, we synthesized amino propyl ciprofloxacin (CPLX-NH2) as a ciprofloxacin (CPLX) derivative. Moreover, the immune antigen CPLX-NH2-BSA and coating antigen CPLX-NH2-OVA were prepared via CPLX-NH2 coupling with bovine serum albumin (BSA) and ovalbumin (OVA), respectively. Subsequently, the Kunming mice were immunized with immune antigen to obtain the polyclonal antibody with high titer. The regression equation of CPLX-NH2 antibody was y = –17.395x + 89.331 (R2 = 0.9961); IC50 and limit of detection (LOD) were 182.39 and 20.09 ng/mL, respectively. These results were superior to that of CPLX antibody. Meanwhile, the CPLX-NH2 antibody showed cross-reactivity to fluoroquinolones (FQNs) residues. The results of the study indicated that the proper modification of the drug, namely, the addition of a suitable spacer arm between the drug and the carrier protein will improve the efficacy of the antibody, which is a favorable concept for preparation of antibody.