Article

Development of highly sensitive chemiluminescence enzyme immunoassay based on the anti-recombinant H(C) subunit of botulinum neurotoxin type A monoclonal antibodies.

Department of Immunology, The Fourth Military Medical University, No. 169 Changle West Road, Xi'an 710032, Shaanxi Province, People's Republic of China.
Analytica chimica acta (Impact Factor: 4.31). 07/2012; 735:23-30. DOI: 10.1016/j.aca.2012.05.008
Source: PubMed

ABSTRACT Botulinum neurotoxins (BoNTs) are the most poisonous substances ever known. The early detection of these toxins could bear more time for appropriate medical intervention. The standard method for detecting BoNTs is the mouse bioassay, which is time consuming (up to 4 days) and requires a large number of laboratory animals. The immunologic detection methods could detect the toxins within a day, but most of these methods are less sensitive compared with the mouse bioassay due to the lack of high-affinity antibodies. Recently, the recombinant H(C) subunit of botulinum neurotoxin type A (rAH(C)) was expressed as an effective vaccine against botulism, indicating that the rAH(C) could be an effective immunogen that raises the monoclonal antibody (mAb) for detecting BoNT/A. After immunized BALB/c mice with rAH(C), 56 mAbs were generated. Two of these mAbs were selected to establish a highly sensitive sandwich chemiluminescence enzyme immunoassay (CLEIA), in which FMMU-BTA-49 and FMMU-BTA-22 were used as capture antibody and detection antibody, respectively. The calculated limit of detection (LOD) based on molecular weight of rAH(C) and BoNT/A reached 0.45 pg mL(-1). This CLEIA can be used in the detection of BoNT/A in matrices such as milk and beef extract. This method has 20-40 fold lower LOD than that of the mouse bioassay and takes only 3 h to complete the detection, indicating that it can be used as a valuable method to detect and quantify BoNT/A.

0 Bookmarks
 · 
86 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: In this study, detection of staphylococcal enterotoxin A (SEA) in multi-matrices using a highly sensitive and specific microplate chemiluminescence enzyme immunoassay (CLEIA) has been established. A pair of monoclonal antibodies (mAbs) was selected from 37 anti-SEA mAbs by pairwise analysis, and the experimental conditions of the CLEIA were optimized. This CLEIA exhibited high performance with a wide dynamic range from 6.4pgmL(-1) to 1600pgmL(-1), and the measured low limit of detection (LOD) was 3.2pgmL(-1). No cross-reactivity was observed when this method was applied to test SEB, SEC1, and SED. It has also been successfully applied for analyzing SEA in a variety of environmental, biological, and clinical matrices, such as sewage, tap water, river water, roast beef, peanut butter, cured ham, 10% nonfat dry milk, milk, orange juice, human urine, and serum. Thus, the highly sensitive and SEA-specific CLEIA should make it attractive for quantifying SEA in public health and diagnosis in near future.
    Analytica chimica acta 09/2013; 796:14-9. · 4.31 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Specific screening methods for complex food matrices are needed that enable unambiguous and sensitive detection of bio threat -agents (BTAs) such as Bacillus anthracis spores and microbial toxins (e.g. Staphylococcal enterotoxin B (SEB), Clostridial Botulinum neurotoxins (BoNTs). The present study describes an image-based 96 well Meso Scales Discovery (MSD) electochemiluminescent (ECL) assay for simultaneous detection of BTAs in dairy milk products. The limit of detection of this ECL assay is 40 pg/mL for BoNT/A complex, 10 pg/mL for SEB, and 40,000 CFU/mL for Bacillus anthracis spores in dairy milk products. The ECL assay was successfully applied to screen type A Clostridium botulinum outbreak strains. The results of this study indicate that this ECL assay is very sensitive, rapid (< 6 h), and multiplex in nature. The ECL assay has potential for use as an in vitro screening method for BTAs over other comparable immunoassays.
    Journal of the Science of Food and Agriculture 07/2013; · 1.76 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Botulinum neurotoxin A (BoNT/A), the most acutely poisonous substance to humans known, cleave its SNAP-25 substrate with high specificity. Based on the endopeptidase activity, different methods have been developed to detect BoNT/A, but most lack ideal reproducibility or sensitivity, or suffer from long-term or unwanted interferences. In this study, we developed a simple method to detect and quantitate trace amounts of botulinum neurotoxin A using the IgY antibody against a linear-peptide substrate. The effects of reaction buffer, time, and temperature were analyzed and optimized. When the optimized assay was used to detect BoNT/A, the limit of detection of the assay was 0.01 mouse LD50 (0.04 pg), and the limit of quantitation was 0.12 mouse LD50/ml (0.48 pg). The findings also showed favorable specificity of detecting BoNT/A. When used to detect BoNT/A in milk or human serum, the proposed assay exhibited good quantitative accuracy (88% < recovery < 111%; inter- and intra-assay CVs < 18%). This method of detection took less than 3 h to complete, indicating that it can be a valuable method of detecting BoNT/A in food or clinical diagnosis.
    PLoS ONE 01/2013; 8(3):e58908. · 3.73 Impact Factor