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An automated chemiluminescent immunoassay (CLIA) based on acridinium ester-magnetic microspheres was established for the detection of ochratoxin A (OTA) in milk. Anti-mouse IgG antibodies were conjugated to magnetic microspheres and the OTA-BSA conjugates were labeled with acridinium ester. Different CLIA experimental conditions, including magnetic microsphere concentration, OTA antibody concentration, labeled OTA-BSA conjugate concentration, OTA antibody and labeled OTA-BSA dilution, reaction time, and sample pre-treatment conditions, were optimized. Under the established optimal conditions, the following results were obtained using the CLIA method: a limit of detection of 6.38 ng/kg, a half-maximal inhibitory concentration (IC50) of 31.40 ng/kg, a linear range of 11.23 to 99.20 ng/kg, and intra- and inter-assay coefficients of variation <8%. When determining OTA in milk samples, the limit of detection was 25.52 ng/kg, the IC50 was 125.60 ng/kg, the linear range was 44.92 to 396.80 ng/kg, and the addition recovery was 95.08% to 105.83%. The cross-reactivities with ochratoxin B and ochratoxin C were 6.52% and 24.57%, respectively, and there was no obvious cross-reaction with other mycotoxins. Milk sample detection values obtained using the CLIA method were consistent with those obtained using high-performance liquid chromatography-tandem mass spectrometry. Moreover, the reagents used for the CLIA method could be stored stably for more than 9 months at 2~8 °C. In conclusion, the CLIA method established in this study is sensitive, accurate, rapid, and easy to operate. The detection time is only 30 min, and the method is particularly suitable for the rapid screening of OTA residues in large-scale milk samples. [ABSTRACT FROM AUTHOR]

In this study, a specific monoclonal antibody against bongkrekic acid (BA) was produced. The antibody was conjugated to colloidal gold as a tracer to develop a colloidal gold immunochromatography assay (GICA). The effect of working conditions, including pH value of labeling, mass concentrations of antibody and antigen, ionic strength, surfactant as well as sample pretreatment on GICA performance were studied. Under the optimal condition, the developed GICA showed a quantitative limit of detection of 1.2 µg/kg, a detection range of 1.8-7.2 µg/kg, and a qualitative detection limit of 16.0 µg/kg, respectively. The proposed assay has shown good specificity, and exhibited no cross-reaction with other toxins commonly found in food. Recovery test was done by using tremella, agaric, and rice flour samples and ranged from 80.2% to 101.2% with the relative standard deviation (RSD) less than 6.4%. Besides, the results had good agreement with that of liquid chromatography-tandem mass spectrometry (LC-MS/MS). In conclusion, the proposed assay can meet the demand for mass screening of bongkrekic acid in food in an accurate, sensitive, convenient, rapid, and low-cost manner. [ABSTRACT FROM AUTHOR]

Acetylcholinesterase (AChE) plays a key role in the pesticide determination. However, the extraction of AChE from natural materials has the disadvantages of low yield, complex purification and poor stability. Therefore, the preparation of recombinant AChE with high performance becomes the hot topic of researchers in recent years. In this article we summarize the progress in the expression of recombinant AChE and the improvement of its analytical characteristic. Finally, we point out that the directed evolution strategy combined with surface display technology is the future trend on improving recombinant AChE activity.

Objective: To establish a new, real time, dynamic and direct optical detection method for mast cell degranulation caused by anaphylactoid reaction., Method: A CD63-GFP plasmid was constructed and introduced steadily into rat basophilic leukemia (RBL-2H3) cells. The movements of CD63-GFP, which was located on both the granule membranes and the plasma membranes of RBL cells stimulated by Compound 48/80, were studied by confocal laser scanning microscope (CLSM) and total internal reflection fluorescence microscope (TIRFM) both inside and on the surface of living RBL-2H3 cells., Result: Before antigen stimulation, most granules with CD63-GFP hardly moved in RBL cells. However, after antigen stimulation, the granules moved dramatically. They reached the plasma membranes in a few minutes and fused with them instantaneously. The velocity of the granule movement toward the plasma membranes on antigen stimulation was calculated to be 0.05 micron x s(-1)., Conclusion: Analysis of the movement of each granule provided a new insight into the elementary process of degranulation. The method is rapid, sensitive and reliable, which could be used as a new detection method for anaphylactoid reaction in vitro.

To construct the recombinant vector pBV220-scFv and express anti-clenbuterol (CBL) scFv antibody in Escherichia coli, we amplified the scFv gene using plasmid pCANTABSE-CBL as a template, recombined it with pPICZalphaA, then amplified the scFv-His-tag gene from plasmid pPICZalphaA-scFv and linked it with expression plasmid pBV220. We identified the recombinant plasmid by restrictive enzyme digestion, PCR amplification and sequence analysis. Finally, we transformed the recombinant vector into E. coli DH5alpha that was temperature-induced and expressed recombinant protein. We identified the recombinant protein by SDS-PAGE, Western blotting and indirect competitive ELISA. The results show that recombinant plasmid pBV220-scFv contained the inserted fragment with highest homology about 99.8%. The expression of scFv induced by temperature show 37 kD Mw and anti-His-tag mAb recognized-activity by SDS-PAGE and Western blotting respectively, and could competitively combine with CBL, the IC50 is 4.55 ng/mL. The recombinant plasmid pBV220-scFv is constructed and expresses the scFv gene of CBL in E. coli successfully. This study suggests the corresponding immunoassay methods could be set up by the recombinant scFv.

Objective: To compare the immunogenity of the TTX artificial antigen with two different carrier proteins, bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), and antiserum against each immunogen were well investigated., Methods: The hapten Tetrodotoxins (TTX) were linked to carrier protein BSA or KLH as immunogen, and linked ovalbumin (OVA) as coating antigen by the Mannich method. Then the 6-8 weeks Babl/c mice were immunized with TTX-BSA or TTX-KLH. And the TTX-BSA also was another coating antigen in the detection of TTX-KLH antiserum. The antiserum was detected by indirect enzyme-linked immunosorbent assay (iELISA) and indirect competitive enzyme-linked immunosorbent assay (icELISA)., Results: The high titer of antiserum was 1:64000 after immunizing mouse. In the detection of TTX-KLH antiserum, the A450 value with the TTX-OVA coating antigens was a little higher than the TTX-BSA coating antigen, and IC50 was 10-20 ng/ml by icELISA., Conclusion: The artificial antigen was successfully synthesized, and the conjugate of TTX-KLH was testified to have a better immunogenity in comparison with TTX-BSA, this investigation will benefit development of TTX immunoassay.