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. 2018 Feb 8;10(2):75.
doi: 10.3390/toxins10020075.

Preparation of Monoclonal Antibody for Brevetoxin 1 and Development of Ic-ELISA and Colloidal Gold Strip to Detect Brevetoxin 1

Sumei Ling  1 , Shiwei Xiao  2 , Chengjie Xie  3 , Rongzhi Wang  4 , Linmao Zeng  5 , Ke Wang  6 , Danping Zhang  7 , Xiulan Li  8 , Shihua Wang  1
Affiliations

Preparation of Monoclonal Antibody for Brevetoxin 1 and Development of Ic-ELISA and Colloidal Gold Strip to Detect Brevetoxin 1

Sumei Ling et al. Toxins (Basel). .

Abstract

Brevetoxin-1 (BTX-1), a marine toxin mostly produced by the dinoflagellatae Karenia brevis, has caused the death of marine organisms and has had numerous toxicological effects on human health. Hence, it is very necessary to develop a rapid, economical, and reliable immunoassay method for BTX-1 detection. In this study, two kinds of complete antigen were synthesized using the succinic anhydride and isobutyl chloroformate two-step methods. Conjugate BTX-1-OVA was used as an antigen for mice immunization, and BTX-1-BSA for measuring the titer of the produced antibodies. A hybridoma cell line 6C6 stably secreting monoclonal antibody (mAb) against BTX-1 was obtained by fusing SP2/0 myeloma cells with the spleen cells from the immunized mouse. The hybridoma 6C6 was injected into the abdomen of BALB/c mice to obtain ascites, and the anti-BTX-1 mAb was harvested from ascites by precipitation with caprylic acid/ammonium sulfate (CA-AS). The anti-BTX-1 mAb was identified as an IgG1 subtype, and the cross-reactivity results showed that anti-BTX-1 mAb was highly specific to BTX-1 with the affinity of 1.06 × 10⁸ L/mol. The indirect competitive ELISA results indicated that the linear range for BTX-1 detection was 14-263 ng/mL with IC50 of 60 ng/mL, and a detection limit of 14 ng/mL. The average recovery rate from the spiked samples was 88 ± 2% in intra-assay and 89 ± 2% in inter-assay. The limit of detection (LOD) using the colloidal gold strip was 200 ng/mL with high specificity. Therefore, the anti-BTX-1 mAb can be used to detect BTX-1 in shellfish and other related samples.

Keywords: Brevetoxin-1; ELISA; colloidal gold strip; monoclonal antibody.

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Conflict of interest statement

All authors declare that they have no conflict of interest statement.

Figures

Figure 1
Figure 1
Identification of the conjugates and anti-serum titer. (A,B) Analysis of conjugates and carrier protein by non-denaturing agarose electrophoris. (A) Lane 1: BTX-1-BSA conjugates sample, Lane 2: BSA; (B) Lane 1: BTX-1-OVA conjugates sample, Lane 2: OVA. (C) Titer of mice anti-serum measured by indirect ELISA. Mouse 1 and 2 were immunized with BTX-1-OVA conjugate, and the control was immunized with only adjuvant and PBS.
Figure 2
Figure 2
Screening and characterization of positive hybridoma cell against BTX-1. (A) The supernatant titer of the positive cell line 6C6 is 1.5, and the supernatant of SP2/0 is selected as a negative control; (B) the number of positive hybridoma cell chromosomes is 104; (C) the subtype of antibody is IgG1.
Figure 3
Figure 3
Purification and titer of the anti-BTX-1 mAb. (A) SDS-PAGE of the purified anti-BTX-1 mAb. Lane M: maker, Lane 1: ascites, Lane 2: the purified antibody; (B) The titer of the purified anti-BTX-1 mAb was identified by indirect ELISA. (C) The purified anti-BTX-1 mAb was saved in −20 °C and the titer was identified by indirect ELISA at different times.
Figure 4
Figure 4
The affinity, specificity, and the cross-reactivity of the anti-BTX-1 mAb were tested by ELISA. (A) Affinity was determined by indirect ELISA. Different concentrations of coating antigen was used to determine the affinity constant by indirect ELISA, and the affinity constant is 1.055 × 108 L/mol; (B) The specificity analysis of the purified antibody. Different kinds of complete antigen were coated, and the antibody did not react with other antigens; (C) cross-reactivity of anti-BTX-1 mAb to other toxins was determined by ic-ELISA. The antibody did not have the cross-reaction with other marine toxins.
Figure 5
Figure 5
Development of detection method based on ic-ELISA. (A) A standard curve was made by competitive inhibition ELISA. The logistic equation was y = 0.08779 + (0.098917 − 0.08779)/[1 + (x/58.00982)1.60278], with a correlation coefficient (R2) of 0.98078. The half inhibitory concentration (IC50) of BTX-1 binding to anti-BTX-1 mAb was 60 ng/mL; (B) the linear equation is y = 47.197x − 34.411, with a correlation coefficient (R2) of 0.0.9719.
Figure 6
Figure 6
Construction and characterization of colloidal gold strip test; (A) the description of strip test results. In the absence of BTX-1 in the sample solution, both two line exits on the control and test zone, indicating negative. Only one control line stands positive for enough toxin binding to the anti-BTX-1-BSA McAb. If no lines, or only the test line was red, it indicated invalid results; (B) cross-reactivity of the test strip with other toxins, such as OA, DA, BTX-2, BTX-3, TTX, CTX, STX; (C) the detection limit of colloidal gold strip test for BTX-1; (D) the real sample solution detection of colloidal gold strip for BTX-1.
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