目的 建立一种快速、便捷且可靠的免疫学检测方法，用于家畜肠道寄生虫性腹泻的现场筛查与早期检测，以满足基层诊断和防控工作的实际需求。方法 分别扩增微小隐孢子虫cgd2-420、蓝氏贾第鞭毛虫α-15贾第蛋白（α-15 giardin）和牛艾美球虫热休克蛋白90（hsp90）等特异性抗原基因片段，并构建重组质粒。在大肠埃希菌中表达重组抗原蛋白并纯化，将纯化蛋白免疫BALB/c小鼠，制备单克隆抗体，并进行胶体金标记。将特异性单克隆抗体和羊抗鼠IgG抗体固定于硝酸纤维素膜上，分别作为检测线和质控线，构建胶体金免疫层析试纸条。通过优化胶体金标记条件、抗体标记量以及检测线和质控线的包被浓度，对试纸条的检测性能进行系统评价，包括灵敏度、特异性、重复性及稳定性。结果 构建的重组质粒表达了微小隐孢子虫cgd2-420、蓝氏贾第鞭毛虫α-15 giardin和牛艾美球虫HSP90重组蛋白，其相对分子质量分别约为40 000、33 000和50 000，与预期结果一致，且均具有良好的免疫原性。经BALB/c小鼠免疫、细胞融合与筛选，分别获得针对3种病原的4株、5株和5株特异性单克隆抗体，并进一步筛选出反应性能最优的单克隆抗体克隆株HP2、CG5和JD5，用于试纸条构建。经反应条件优化，确定胶体金标记体系在加入5 μL、浓度为0.2 mol/L的K₂CO₃时稳定性最佳，抗体的最佳标记量为20 μg，检测线和质控线的最优包被浓度分别为2 mg/mL和1.5 mg/mL。性能评价结果显示，该免疫层析试纸条的阳性样品最低可检稀释倍数为1∶64，对非靶标病原体未出现交叉反应，批内及批间检测结果一致，在4 ℃干燥条件下保存60 d后仍可保持稳定的检测性能。对80份临床样品进行检测表明，试纸条检测结果与PCR方法具有较高一致性，其中牛艾美球虫、微小隐孢子虫和蓝氏贾第鞭毛虫的符合率分别为93.75%、96.25%和96.25%。结论 成功建立了一种基于胶体金免疫层析技术的家畜肠道原虫快速检测方法，该方法操作简便、检测快速且性能稳定，适用于微小隐孢子虫、蓝氏贾第鞭毛虫和牛艾美球虫的现场筛查与早期诊断，为家畜寄生虫性腹泻的早期识别和防控提供了有力的技术支持。

Objective To establish a rapid, convenient, and reliable immunological test for field screening and early detection of intestinal parasitic diarrhea in livestock, so as to address the practical needs of grassroots disease diagnosis and control. Methods The specific antigen genes of Cryptosporidium parvum cgd2-420, Giardia lamblia α-15 giardin, and Eimeria bovis heat shock protein 90 (hsp90) were amplified and the recombinant plasmids were constructed. The recombinant antigen proteins were expressed in Escherichia coli and subsequently purified. BALB/c mice were immunized with purified proteins to generate monoclonal antibodies, which were subsequently conjugated with colloidal gold nanoparticles. Specific monoclonal antibodies and goat anti-mouse IgG antibody were striped onto nitrocellulose membranes as test lines and control lines to generate a colloidal gold immunochromatographic test strip. The diagnostic performance of the test strip was systematically evaluated following optimization of colloidal gold labeling conditions, antibody labeling amounts, and coating concentrations of test and control lines, including sensitivity, specificity, repeatability, and stability. Results Recombinant C. parvum cgd2-420, G. lamblia α-15 giardin, and E. bovis HSP90 proteins were successfully constructed and expressed, with relative molecular masses of approximately 40 000, 33 000, and 50 000, respectively, which were consistent with their theoretical molecular masses. All these recombinant proteins exhibited good immunogenicity. Following immunization of BALB/c mice, and cell fusion, and screening, four, five, and five specific monoclonal antibodies were obtained targeting the three pathogens, respectively, and monoclonal antibodyclones HP2, CG5, and JD5 with optimal performance were screened for preparation of the test strip. Following optimization of the reaction condition, the best stability of the colloidal gold labeling system was achieved with addition of 5 μL of 0.2 mol/L K₂CO₃, and the optimal antibody labeling amount was 20 μg, while the optimal coating concentrations for the test and control lines were 2 mg/mL and 1.5 mg/mL, respectively. The immunochromatographic test strip exhibited a minimum detectable dilution of 1∶64, and showed no cross-reactivity with non-target pathogens, which produced consistent intra-batch and inter-batch detection results and maintained stable detection performance after storage under dry conditions at 4 ℃ for 60 days. This test strip showed high consistency with PCR assay for detection of 80 clinical samples, with coincidence rates of 93.75% for E. bovis, 96.25% for C. parvum, and 96.25% for G. lamblia, respectivelys. Conclusion A simple, rapid, and stable colloidal gold-based immunochromatographic assay has been successfully established for rapid detection of intestinal protozoa in livestock, which is suitable for field screening and early diagnosis of C. parvum, G. lamblia, and E. bovis. This assay provides a powerful technical support for early identification and control of parasitic diarrhea in livestock.