Microcystin-LR (MC-LR) is a type of biotoxin that pollutes the ecological environment and food. The study aimed to obtain new nanobodies from phage nanobody library for determination of MC-LR. The toxin was conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), respectively, then the conjugates were used as coated antigens for enrichment (coated MC-LR-KLH) and screening (coated MC-LR-BSA) of MC-LR phage nanobodies from an alpaca phage display nanobody library. The antigen-specific phage particles were enriched effectively with four rounds of biopanning. At the last round of enrichment, total 20 positive monoclonal phage nanobodies were obtained from the library, which were analyzed after monoclonal phage enzyme linked immunosorbent assay (ELISA), colony PCR and DNA sequencing. The most three positive nanobody genes, ANAb12, ANAb9 and ANAb7 were cloned into pET26b vector, then the nanobodies were expressed in Escherichia coli BL21 respectively. After being purified, the molecular weight (M.W.) of all nanobodies were approximate 15 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified nanobodies, ANAb12, ANAb9 and ANAb7 were used to establish the indirect competitive ELISA (IC-ELISA) for MC-LR, and their half-maximum inhibition concentrations (IC₅₀) were 0.87, 1.17 and 1.47 μg/L, their detection limits (IC₁₀) were 0.06, 0.08 and 0.12 μg/L, respectively. All of them showed strong cross-reactivity (CRs) of 82.7–116.9% for MC-RR, MC-YR and MC-WR, and weak CRs of less than 4.56% for MC-LW, less than 0.1% for MC-LY and MC-LF. It was found that all the IC-ELISAs for MC-LR spiked in tap water samples detection were with good accuracy, stability and repeatability, their recoveries were 84.0–106.5%, coefficient of variations (CVs) were 3.4–10.6%. These results showed that IC-ELISA based on the nanobodies from the alpaca phage display antibody library were promising for high sensitive determination of multiple MCs.

微囊藻毒素-LR（MC-LR）是一种污染生态环境和食物的生物毒素。该研究旨在从噬菌体纳米抗体库中获得新的纳米抗体，用于测定MC-LR。将该毒素分别与匙孔血蓝蛋白（KLH）和牛血清白蛋白（BSA）偶联，然后将该偶联物用作包被抗原用于富集（包被的MC-LR-KLH）和筛选（包被的MC-LR-BSA）来自羊驼噬菌体的MC-LR噬菌体纳米抗体展示了纳米抗体库。抗原特异性噬菌体颗粒通过四轮生物淘选有效富集。在最后一轮富集中，从文库中获得了总共20个阳性单克隆噬菌体纳米抗体，并通过单克隆噬菌体酶联免疫吸附测定（ELISA），菌落PCR和DNA测序进行了分析。最多的三个阳性纳米抗体基因ANAb12，大肠杆菌BL21。纯化后，十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）显示所有纳米抗体的分子量（MW）约为15 kDa。纯化的纳米抗体ANAb12，ANAb9和ANAb7用于建立MC-LR间接竞争ELISA（IC-ELISA），其半数最大抑制浓度（IC ₅₀）为0.87、1.17和1.47μg / L，检测极限（IC ₁₀）分别为0.06、0.08和0.12μg/ L。它们均显示出MC-RR，MC-YR和MC-WR的强交叉反应性（CR）为82.7-116.9％，MC-LW的弱交叉反应性小于4.56％，MC-LY的交叉反应性小于0.1％和MC-LF。结果发现，自来水样品检测中所有加标MC-LR的IC-ELISA都具有良好的准确性，稳定性和可重复性，其回收率为84.0–106.5％，变异系数（CV）为3.4–10.6％。这些结果表明，基于羊驼噬菌体展示抗体文库的纳米抗体的IC-ELISA对高灵敏度测定多种MC很有希望。