Development of a Polyclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assay for the Detection of Spores of Alicyclobacillus acidoterrestris in Various Fruit Juices.
Development of a Polyclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assay for the Detection of Spores of Alicyclobacillus acidoterrestris in Various Fruit Juices.
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DOI：
10.1021/acs.jafc.5b03841
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发表时间：
2016-01
影响因子：
6.1
通讯作者：
Sophia Mast;R. Dietrich;A. Didier;E. Märtlbauer
中科院分区：
文献类型：
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作者：
Sophia Mast;R. Dietrich;A. Didier;E. Märtlbauer
建立了一种快速、特异检测嗜酸耐热脂环酸芽孢杆菌孢子的兔多克隆抗体夹心ELISA方法。通过免疫荧光证实了抗血清与孢子的反应性。为了对ELISA进行全面评价，选择脂环酸芽孢杆菌属的61株和分离株。的特征在于其愈创木酚生产能力和遗传变异性。ELISA具有高度灵敏度，检测限取决于分离株，范围为2.1 × 10（3）- 3.8 × 10（4）个孢子/mL，但观察到一个分离株的灵敏度略低（5 × 10（5）个孢子/mL）。包容性试验表明，ELISA与所有测试的A反应。与30株芽孢杆菌孢子无交叉反应。和梭菌属（Clostridium spp.）观察了此外，用橙子、苹果（澄清和未过滤）、番茄、粉红葡萄柚、梨和白色葡萄汁测试测定适用性。用1或10个孢子/mL的A.酸土生菌富集48 h后，建立的ELISA方法可对污染样品进行可靠、重复性好的检测。富集的样品可直接用于测定，强调了开发的ELISA方法的耐用性。
A polyclonal rabbit antibody-based sandwich ELISA for the rapid and specific detection of spores of Alicyclobacillus acidoterrestris was established. The reactivity of the antisera with spores was confirmed by immunofluorescence. For a thorough evaluation of the ELISA, 61 strains and isolates of Alicyclobacillus spp. were characterized regarding their guaiacol production ability and genetic variability. The ELISA was highly sensitive, the detection limits were isolate-dependent and ranged from 2.1 × 10(3) - 3.8 × 10(4) spores/mL, except for one isolate, for which a slightly lower sensitivity (5 × 10(5) spores/mL) was observed. Inclusivity tests revealed that the ELISA reacts with all tested A. acidoterrestris, while no cross-reactions with spores of 30 strains of Bacillus spp. and Clostridium spp. were observed. Further on, the assay applicability was tested with orange, apple (clear and unfiltered), tomato, pink grapefruit, pear, and white grape juices. Juices were inoculated with 1 or 10 spores/mL of A. acidoterrestris. After enrichment for 48 h, the established ELISA enabled the reliable and reproducible detection of contaminated samples. The enriched samples could be applied directly to the assay, underlining the robustness of the developed ELISA method.