Anti-BTX-1 mAb was highly specific to BTX-1 with the affinity of 1 1

Anti-BTX-1 mAb was highly specific to BTX-1 with the affinity of 1 1.06 108 L/mol. Author Contributions S.W., S.L. these toxins, and BTX-1, BTX-2, and BTX-3 are more common than additional kinds of brevetoxins in nature. Ingestion of brevetoxin-contaminated shellfish causes neurotoxic shellfish poisoning (NSP). Shellfish contaminated with low concentration of BTXs can cause a nonlethal form of food poisoning, and high concentration of BTXs also affect fish, birds, and marine mammals, causing massive epizootic events [4]. Since these toxins have negative effects on seafood industries and detrimental effects on human health, it has raised global consciousness to develop practical and sensitive detection methods [2,5,6]. In the past years, mouse bioassay as the traditional method often applied to assess toxicity. This means that all the toxins are first measured by subjecting them to animal bioassays [7]. Due Rabbit Polyclonal to NM23 to the animal rights concerns, the number and cost of animals required, and some additional limitations in the mouse toxicity assay method, additional useful techniques for detection of brevetoxins are needed. So far, newer methods for detection of BTXs in biological samples have been developed, such as liquid chromatography coupled with mass spectrometry and tandem mass spectrometry assay (LC-MS/MS) [8,9], practical pharmacology centered assay [1], immunoassay [10], and colloidal platinum probe-based immune chromatographic assay [11]. LC-MS centered assays and additional instrumental methods are sensitive to detect marine toxin contamination in seafood samples. However, these methods have some disadvantages, such as complex pre-treatment methods, and time-consuming, laborious, and expensive costs [12]. Consequently, the ideal method for detection of marine toxins should be simple, rapid, cost-effective, and highly sensitive. In fact, immunological methods with the advantage of high level of sensitivity, rapidity, and low-cost become as the most attractive approach for practical purposes. Monoclonal antibody (mAb) with high specificity and affinity is definitely widely used in immunological methods [13,14,15]. The enzyme-linked immune sorbent assay (ELISA) technique has been widely used for diagnostic, TAK-285 residue, and marine toxins detection, which is sensitive, economical, simple, and easy. Colloidal platinum nanoparticles (AuNPs) are visible and can become detected with the naked eye. Consequently, AuNPs conjugate with mAb to develop AuNPs-antibody become probably one of the most popular methods in immunochromatographic assay. There are some reports about mAb for BTX-2 [16] and BTX-3 [11], which belongs to BTX-B type. To our knowledge, there are still no published content articles about the detection of BTX-1 (BTX-A) based on mAb. The purpose of our study was to obtain high-specificity and -affinity mAb against BTX-1 toxin. Then, indirect competitive ELISA (ic-ELISA) and colloidal platinum strips based on the mAb were founded for the detection of BTX-1 toxin. 2. Results and Discussion 2.1. Conjugates Preparation and Animal Immunization BTX-1 is definitely a small molecular excess weight marine toxin without immunogenicity. In order to obtain a mAb against BTX-1, BTX-1 should be coupled to a carrier protein. Bovine serum albumin (BSA) and ovalbumin (OVA) are frequently used as carrier proteins for conjugate preparation, and BTX-1-BSA and BTX-1-OVA conjugates were prepared by the succinic anhydride method and isobutyl chloroformate method. The agarose gel electrophoresis results of conjugates in Number 1A,B showed the migration velocity of BTX-1-BSA and BTX-1-OVA conjugates were faster than that of the carrier protein alone, indicating that BTX-1 was successfully conjugated to the BSA and OVA carrier proteins, respectively. BTX-1-OVA conjugate as an immune antigen was used to immunize health female BALB/c mice that could generate antibody against BTX-1, while BTX-1-BSA was used as a covering antigen for determining the anti-serum titer and in subsequent ELISA experiments. Open in a separate window Number 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: TAK-285 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. The titer result showed the OD value of blood samples from BALB/c mice immunized with BTX-1-OVA was significantly higher than that of the control (Number 1C).The result further suggested that BTX-1-OVA complete antigen was successful in inducing the antibody against BTX-1. Consequently, the spleen cells of these two mice were selected to perform cell fusion experiments. 2.2. Screening and Characterization of Positive Hybridoma Cell Collection With this study, spleen cells from an immunized BALB/c mouse were fused with SP2/0 myeloma cells TAK-285 in the percentage of 10:1 by polyethylene glycol (PEG, 1450). After cell fusion, cells were cultured in HAT medium. After 9 d, the titer of tradition supernatant was measured by indirect ELISA. After three times sub-cloning, a positive cell line named 6C6 secreting mAb against BTX-1 was acquired successfully (Number.