Development of highly sensitive chemiluminescence enzyme immunoassay based on the anti-recombinant HC subunit of botulinum neurotoxin type A monoclonal antibodies
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.
- SourceAvailable from: Chiaki Nagai-Okatani[Show abstract] [Hide abstract]
ABSTRACT: Atrial natriuretic peptide (ANP) is a peptide hormone that is synthesized and secreted by cardiac tissues and plays a pivotal role in maintaining cardiovascular homeostasis. Clinically, ANP is used as a marker of cardiovascular diseases, including heart failure. Although multiple ANP assays are currently available, a more sensitive assay is required for the direct measurement of plasma ANP where there is limited plasma availability, especially in mouse experiments. In the present study, we developed a plate-based sandwich chemiluminescent enzyme immunoassay for the measurement of plasma ANP in rats and mice without the need for prior extraction. To minimize non-specific binding, we performed a single-step PEGylation procedure targeting the immobilized antibody, which markedly improved the assay's sensitivity and linearity. The linear range was 0.1-250 pM, and the minimum detection limit was 0.13 pM, five-fold lower than the lowest value of the commercially available kits. ANP was directly measured in plasma samples without detectable cross-reactivity with B-type and C-type natriuretic peptides. The accuracy of the assay was confirmed by spike recovery tests and dilution tests and by comparison with a conventional radioimmunoassay. Based on the species cross-reactivity, this assay can be used to measure human ANP.Analytical Biochemistry 06/2014; 461. DOI:10.1016/j.ab.2014.05.022 · 2.31 Impact Factor
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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 03/2013; 8(3):e58908. DOI:10.1371/journal.pone.0058908 · 3.53 Impact Factor
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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. DOI:10.1016/j.aca.2013.07.044 · 4.52 Impact Factor