Application of a recombinant protein for the serological diagnosis of canine leishmaniasis

This work reports the results obtained by a new enzyme-linked immunosorbent assay (ELISA) test developed for the serological diagnosis of canine leishmaniasis.The new ELISA is based on a recombinant protein obtained by joining different antigens of Leishmania infantum.Test performances have been evaluated through the screening 227 sera of dogs, infected and uninfected by L. infantum. The new ELISA test has been compared to the indirect immunofluorescent-antibody test (IFAT) as a reference assay of canine leishmaniasis, and to a commercial ELISA.Excluding from the total number of IFAT positive sera the 27 sera with IFAT titre 1:40 (considered doubtful), the recombinant ELISA showed 97.0% specificity, 93.9% sensitivity and 95.5% agreement with IFAT. The commercial ELISA showed 78.2% specificity, 94.9% sensitivity and 86.5% agreement with IFAT.The results demonstrate a higher performance of the new recombinant ELISA test for the detection of negative samples, with a greater agreement with the reference test (IFAT).     

稳定分泌抗羊种布鲁菌脂多糖单克隆抗体细胞株的筛选与应用

研制抗羊种布鲁菌脂多糖抗原的单克隆抗体,并应用其建立检测布病的双夹心ELISA方法。本研究采用热酚水法提纯羊种布鲁菌(16M菌株)的脂多糖抗原,并经SDS-PAGE鉴定。用脂多糖和灭活的羊种布鲁菌16M作为免疫抗原,交替免疫6～8周龄BALB/c雌鼠,第1次免疫用羊种布鲁菌标准菌16M全菌加等量弗氏完全佐剂;第2次免疫用脂多糖加等量弗氏不完全佐剂。将脂多糖作为包被抗原建立间接ELISA方法,筛选针对抗羊种布鲁菌(16M菌株)脂多糖的单克隆抗体杂交瘤细胞株。筛选出3株能稳定分泌抗羊种布鲁菌脂多糖单克隆抗体的杂交瘤细胞株,分别命名为5H3、6B8和3H7,细胞培养上清的ELISA效价在1∶1 000～1∶5 000,小鼠腹水单克隆抗体ELISA效价在1∶10 000～1∶160 000;抗体亚类鉴定表明:5H3、6B8属于IgM亚类,3H7属于IgG3亚类;特异性试验结果显示:3株杂交瘤细胞分泌的抗体不与大肠杆菌O157裂解抗原、鸡白痢沙门氏菌裂解抗原、鸭源鸡杆菌脂多糖抗原以及福氏志贺菌裂解抗原反应,仅与灭活的羊种布鲁菌(16M)发生反应。布鲁菌虎红平板凝集试验和试管凝集试验检测结果显示,获得的单抗可与标准检测抗原形成明显的颗粒凝集物和伞状凝集物。利用所建立的单克隆抗体细胞株,建立了一种检测布鲁菌的双夹心间接ELISA方法,并进行了特异性和敏感性检测。对模拟样品和临床样品进行检测,准确性均很高。     

Production and characterization of rabbit anti-idiotypic antibodies directed against a murine monoclonal anti-B. abortus antibody

The protective anti-B. abortus monoclonal antibody ISS/32 (Ab1) was used as an immunogen to induce anti-idiotypic antibodies (Ab2) in rabbits. The purpose was to produce and characterize anti-idiotypic antibodies that share conformational similarity with the corresponding bacterial epitope recognized by Ab1. The rabbit anti-IdAb so induced was isolated and affinity-purified. Its specificity for the paratope of Ab1 was determined by evaluating its ability to compete withB. abortus for binding to Ab1 in a competitive ELISA assay. The anti-idiotypic ISS/32 antibodies were able to compete withB. abortus for binding to Ab1 in a dose-dependent manner. Hence, the data indicated that the rabbit anti-Id ISS/32 antibodies reacted with or near the antigen-binding site of Ab1.Abbreviations Ab antibody - anti-Id anti-idiotypic - ELISA enzyme linked immunosorbent assay - IgG immunoglobulin - i.p. intraperitoneal - mAb monoclonal antibody - PBS phosphate-buffered saline - s.c. subcutaneously - TD PBS + 0.05% Tween 20% + 1% yeast extract     

双抗体夹心ELISA检测水牛γ-干扰素方法的建立及初步应用

纯化2株针对水牛γ-干扰素的单克隆抗体,其中1株进行HRP标记作为检测抗体,另1株单抗作为捕获抗体,建立检测水牛γ-干扰素的双抗体夹心ELISA(DAS-ELISA)。通过对封闭液、抗体稀释液等条件进行优化,经过方阵滴定试验确定捕获抗体的最佳浓度为625 ng/mL,检测抗体的工作效价为1∶100;所建立的DAS-ELISA方法检测灵敏度25 ng/mL,批内变异系数为0.52%～6.86%,批间变异系数为5.6%～7.07%。采集60头单次皮内注射结核菌素的水牛肝素钠抗凝全血,利用牛结核杆菌特异性抗原rHIS-CFP10/ESAT-6融合蛋白体外刺激外周血淋巴细胞释放IFN-γ,用所建立的夹心ELISA检测所有样本,与皮内变态反应试验比较,结果显示,自制夹心ELISA方法检测水牛结核病的敏感性为62%,特异性为87%,符合率为75%,表明可应用于水牛结核病的检测。     

阪崎肠杆菌单克隆抗体制备及ELISA检测方法的建立

试验旨在制备抗阪崎肠杆菌的单克隆抗体,初步建立其ELISA检测方法。以灭活的阪崎肠杆菌全菌体为抗原免疫BALB/c小鼠,筛选血清效价高的小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,制备杂交瘤细胞,并用间接ELISA法选取阳性杂交瘤细胞,扩大培养后测定单克隆抗体的效价,进行特异性及抗体间的配对,使用mAb亚类检测试剂盒鉴定单克隆抗体的亚型,并利用得到的抗体建立双抗体夹心ELISA检测方法。本试验得到3株具有良好特异性能稳定分泌单克隆抗体的阳性细胞株5C10、2B6和Ab02,经两两配对,据阳性D_{450 nm}值及P/N值选择1：20000稀释的5C10作为包被抗体,1：40000稀释的Ab02作为酶标二抗,建立ELISA检测法,应用建立的方法与荧光定量PCR方法检测动物实验室保存的20份进出口送检奶粉样品,结果显示试验结果一致。本试验成功制备阪崎肠杆菌的单克隆抗体并建立其ELISA检测法,为大批量快速检测阪崎杆菌奠定了基础。     

Management factors associated with Babesia bovis seroprevalence in cattle from eastern Yucatán, Mexico

The effect of management on the seroprevalence of Babesia bovis was studied in 399 Bos indicus cattle 1–2 years old from 92 farms in the eastern Yucatán, México. The management factors studied were: farm-type, production system, herd size, farm size, stocking density, vector control, dipping interval, type of dipping, type of acaricide and cattle introduction to the farm. A cross-sectional study was carried out (2-stage cluster sampling). The number of serum samples was proportionally distributed according to the number of farms in the nine locations of eastern Yucatán, México (399 animals from 92 farms). Antibody activity to B. bovis was tested using an indirect ELISA. The farms with a seroprevalence ≤75% were considered as cases and those with seroprevalence >75% were considered as controls. The variables with p ≤ 0.20 were included in fixed effects logistic regression. The seroprevalence of the zone was 73.8% (66.3–81.3%). The following risk factors were found: Stocking density (<1 head/ha, OR = 4.04, CI (OR) = 1.20–13.62) and dipping interval (>60 days, OR = 5.07 CI (OR) = 1.26–20.48).     

Seroprevalence and risk factors of caprine toxoplasmosis in Minas Gerais, Brazil

The objective of this work was to carry out a study on caprine toxoplasmosis in the state of Minas Gerais, Brazil. To determine the prevalence of toxoplasmosis in goats in Minas Gerais, 767 sera from goats were tested by ELISA (enzyme-linked immunosorbent assay) and IFAT (indirect fluorescence antibody test). The prevalence of antibodies to Toxoplasma gondii was 43.0% and 46.0% by ELISA and IFAT, respectively. It was observed that 26.8% of the goats show low-avidity IgG to T. gondii. These results suggest the presence of animals in recent phase of toxoplasmosis in Minas Gerais. The risk factors for toxoplasmosis in goats were: age over 36 months (OR = 1.21; IC 95% 1.02–1.44), use of pen (OR = 1.83; IC 95%1.01–3.31) and pure breed animals (OR = 2.49; IC 95% 1.11–5.59).     

Detection of rotavirus in faecal specimens with a monoclonal antibody enzyme-linked immunosorbent assay: Comparison with polyclonal antibody enzyme immuno-assays and a latex agglutination test

Monoclonal antibodies have been produced against the 81/36F strain of rotavirus. One of them, was chosen as diagnostic reagent: it showed high ELISA reactivity with all the bovine, human and porcine rotavirus strains tested and reacted with VP6, structural protein product known to support the common rotavirus antigen.

A sandwich ELISA procedure using the chosen monoclonal as “capture and detecting” antibody was performed to detect rotavirus in faecal samples from experimentally inoculated newborn calves: it always gave a negative response with meconium and a positive response for the stool specimens which rotavirus have been isolated. This assay was compared with Enzygnost and Slidex Rota Kit tests and with a non-commercial sandwich ELISA test using polyclonal antibodies: it showed more sensitivity than the agglutination test and was as sensitive as the other two tests to detect rotavirus in routine diagnostic material. The test evaluated showed no equivocal results.     

Development of a monoclonal antibody against deoxynivalenol for magnetic nanoparticle-based extraction and an enzyme-linked immunosorbent assay

Monoclonal antibody (mAb, NVRQS-DON) against deoxynivalenol (DON) was prepared. DON-Ag coated enzyme linked immunosorbent assay (ELISA) and DON-Ab coated ELISA were prepared by coating the DON-BSA and DON mAb. Quantitative DON calculation ranged from 50 to 4,000 ng/mL for DON-Ab coated ELISA and from 25 to 500 ng/mL for DON-Ag coated ELISA. 50% of inhibitory concentration values of DON, HT-2, 15-acetyl-DON, and nivalenol were 23.44, 22,545, 5,518 and 5,976 ng/mL based on the DON-Ab coated ELISA. Cross-reactivity levels of the mAb to HT-2, 15-acetyl-DON, and nivalenol were 0.1, 0.42, and 0.40%. The intra- and interassay precision coefficient variation (CV) were both <10%. In the mAb-coated ELISA, mean DON recovery rates in animal feed (0 to 1,000 µg/kg) ranged from 68.34 to 95.49% (CV; 4.10 to 13.38%). DON in a buffer solution (250, 500 and 1,000 ng/mL) was isolated using 300 µg of NVRQS-DON and 3 mg of magnetic nanoparticles (MNPs). The mean recovery rates of DON using this mAb-MNP system were 75.2, 96.9, and 88.1% in a buffer solution spiked with DON (250, 500, and 1,000 ng/mL). Conclusively we developed competitive ELISAs for detecting DON in animal feed and created a new tool for DON extraction using mAb-coupled MNPs.     

小反刍兽疫病毒核蛋白单克隆抗体的制备与初步应用

针对小反刍兽疫病毒核蛋白制备特异性的单克隆抗体,并对其进行生物学特性鉴定和初步应用。以纯化的Bacmid-PPRV-N重组蛋白为抗原免疫BALB/c小鼠,取免疫小鼠的致敏脾细胞与SP2/0骨髓瘤细胞在PEG作用下融合,获得单克隆抗体,并通过染色体技术等方法研究其生物学特性,将其作为竞争单抗,Bacmid-PPRV-N重组蛋白作为检测抗原建立竞争ELISA检测方法。结果表明:经克隆和间接ELISA筛选,获得了2株能稳定分泌抗小反刍兽疫病毒N蛋白抗体的杂交瘤细胞株,分别命名为5B11和3H10-3B8。生物学特性鉴定试验表明:5B11和3H10-3B8抗体类型和亚类均为IgG2b;5B11单抗腹水的效价达1∶819 200,3H10-3B8达1∶12 800;血清学试验证明2株单抗均能与Bacmid-PPRV-N重组蛋白抗原结合,具有高度的特异性;相加ELISA试验结果显示,5B11和3H10-3B8 2株单克隆抗体分别识别N蛋白上不同的抗原位点;2株杂交瘤细胞的染色体均为99～104。应用建立的c-ELISA检测方法对222份血清样品进行PPRV抗体的检测,与参考试剂盒比较得到98.20%的符合率。本研究获得了2株能稳定分泌抗PPRV N蛋白单克隆抗体的杂交瘤细胞株,以单抗5B11作为竞争抗体建立了PPRV的c-ELISA检测方法。     

IgG, IgM and IgA in the serum of cattle naturally infected with Mycobacterium paratuberculosis

Serum IgG, IgM and IgA antibody response in 20 cattle naturally infected with Mycobacterium paratuberculosis and in 15 non-infected cattle were measured by enzyme-linked immunosorbent assay. A strong IgG response was detected in 16 (80%) of the infected animals. Diagnostic levels of IgM were detectable in all of the infected animals as well as in 8 (53%) of the non-infected animals. Animals with paratuberculosis had a very weak specific serum IgA response and this appears to be of little value in detection of infection in these animals.     

Bovine brucellosis: evaluation of field sera by a competitive and superimposable ELISA utilising a monoclonal antibody against Brucella abortus lipopolysaccharide

With the aid of a horseradish peroxidase (HRP) tagged monoclonal antibody against smooth lipopolysaccharide from Brucella abortus (Bruce 1), a competitive and superimposable ELISA test procedure for bovine brucellosis has been evaluated for its ability to discriminate between Strain 19-vaccinated (S19-Vacc) and Biotype 1-infected (B1-Inf) cattle. In the competitive assay, all sera from S19-Vacc animals competed effectively against HRP-Bruce 1 (low HRP activity), while 10 out of 40 B1-Inf animals competed less effectively with Bruce 1 (high HRP activity). Successful competition by cattle antibodies would result in an increased proportion of cattle Igs binding to the assay antigen. This was confirmed by superimposing an alkaline phosphatase conjugated rabbit anti-cattle Ig after the competitive ELISA had been completed. With the superimposable assay, alkaline phosphatase activity was correspondingly high for S19-Vacc animals, and low for 36 out of 40 B1-Inf animals. The superimposable ELISA had therefore improved the discriminatory capabilities of the assay procedure from 75% to 90%.     

1种检测弓形虫急性感染的夹心ELISA方法

弓形虫（Toxoplasma gondii）是一种人畜共患机会性致病原虫，其急性感染可导致宿主产生明显的临床症状和严重的病理损伤。弓形虫致密颗粒蛋白1（dense granuleprotein 1，GRA1）是一种良好的诊断抗原，也是弓形虫急性感染的标志物循环抗原（circulating antigen，CAg）的重要组分。本研究利用TgGRA1单克隆抗体建立双抗体夹心ELISA方法，为急性弓形虫感染的检测提供依据。将免疫GRA1-His的小鼠脾细胞与SP2/0进行融合，筛选出能稳定分泌抗体的杂交瘤细胞。选择其中一种单抗与HRP标记后的鼠源GRA1多抗配对，建立1种双抗体夹心ELISA方法，检测人工感染弓形虫的猪和小鼠血清样品，并将检测效果与巢式PCR（nest PCR，nPCR）和商品化试剂盒进行比较。结果筛选到4株杂交瘤细胞，腹水效价为10⁶~10⁷，亚型均为IgG1；IFA和Western blot结果显示，4株单抗均具有良好的反应性和特异性。选择1G2单抗和HRP标记多抗配对，建立了循环抗原双抗体夹心ELISA方法，最低能够检测到血清中1.563 ng·mL^-1 GRA1抗原，或者100 ng·mL^-1 ESA。该方法与nPCR相比具有较高的一致性，较市售商品化试剂盒更为准确可靠。本研究第1次将GRA1抗原作为急性弓形虫感染的诊断指标，建立相应的检测方法，成功地在人工感染样品中检测到弓形虫急性感染，可为弓形虫急性感染的诊断提供参考，对临床上急性弓形虫病的治疗有指导意义。     

Establishment of Double-antibody Sandwich ELISA to Diagnose Acute Toxoplasmosis

The zoonotic protozoa Toxoplasma gondii is an opportunistic pathogen and distributes worldwide. Acute Toxoplasma infection causes serious pathological damages. Dense granule protein 1(GRA1) secreted by dense granule is an important component of Circulating antigen, which is an indication of acute toxoplasmosis. We aimed to use a monoclonal antibody against TgGRA1 to establish an enzyme-linked immunosorbent assay that targets antigen GRA1 in serum for acute toxoplasmosis diagnosis. First, the spleens of TgGRA1-His immunized mice were fused with SP2/0 cells,then we screened hybridomas that can constantly secret monoclonal antibody to the supernatant and injected them into mice to produce a large amount of mAbs. After the identification and purification of ascites, we choose one mAb as a capture antibody, HRP conjugated mouse anti-TgGRA1 polyclonal antibody as a detection antibody to develop sandwich ELISA. This method was used to detect samples from swine and mice artificially infected with Toxoplasma gondii. Besides, the results were compared with that of nPCR and two commercial kits to evaluate the efficiency of sandwich ELISA. We successfully got 4 mAbs with ascitic titers of 10⁶-10⁷, their subtypes are IgG1. Indirect fluorescent assay and Western blot showed that all of them can react specifically with TgGRA1.1G2 mAb and HRP conjugated mouse anti-TgGRA1 polyclonal antibody were used subsequently to establish sandwich ELISA for diagnosing acute infection. After optimization, sandwich ELISA can specifically detect 1.563 ng·mL^-1 GRA1 or 100 ng·mL^-1 ESA in serum. When detecting experimental animal samples, the sandwich ELISA exhibited the high consistency with the results of nPCR and showed higher efficiency than the commercial kits. In summary, we established a sandwich ELISA for acute toxoplasmosis diagnosis that captures one certain toxoplasma antigen GRA1, samples of artificially infected animals can be detected by this method, which makes acute toxoplasmosis diagnosis more reliable. It has guiding significance for clinical treatment of acute toxoplasmosis.     

Estimation of sensitivity, specificity and predictive values of two serologic tests for the detection of antibodies against Actinobacillus pleuropneumoniae serotype 2 in the absence of a reference test (gold standard)

Latent-class models were used to determine the sensitivity, specificity and predictive values of a polyclonal blocking enzyme-linked immunosorbent assay (ELISA) and a modified complement-fixation test (CFT) when there was no reference test. The tests were used for detection of antibodies against Actinobacillus pleuropneumoniae serotype 2 in a survey of respiratory diseases in Danish finishing pigs. The estimates were obtained by maximum-likelihood and also by a Bayesian method (implemented with Gibbs sampling). Possible dependence of diagnostic errors was investigated by comparing models where independence was assumed to models allowing for conditional dependence, given the true disease status.No strong evidence of conditional dependence in either test sensitivity or specificity was found. Assuming independence, maximum-likelihood estimates and 95% confidence intervals of the sensitivity and specificity of the ELISA were 100% and 92.8% (90.1–95.5%) and the corresponding values of the CFT were 90.6% (85.8–95.4%) and 98.6% (98.0–99.3%), respectively. Bayesian estimates and posterior 95% credible intervals of the sensitivity and specificity of the ELISA were 99.7% (98.7–100%) and 92.7% (89.9–95.3%) and of the CFT were 90.6% (86.0–95.3%) and 98.7% (98.0–99.3%). The sensitivity and specificity of a combined test, where the CFT is subsequently applied to the pig sera that test positive in the ELISA, were estimated at 90.2% (85.6–95.0%) and 99.9% (99.8–100%), respectively. The cost of the combined test was less than the cost of the use of the CFT alone, at prevalences <54%. Prevalences and predictive values and their 95% limits were estimated in six sub-samples of data. The estimates of sensitivity and specificity obtained in the present investigation generally validate those reported from other sources.     

Development of a novel enzyme-linked immunosorbent assay to detect anti-IgG against swine hepatitis E virus

Swine hepatitis E virus (HEV) is widespread throughout pigs in both developing and industrialized countries. This virus is an important zoonotic agent and a public concern worldwide. Infected pigs are asymptomatic, so diagnosing swine HEV relies on detection of the virus or antibodies against the virus. However, several obstacles need to be overcome for effective and practical serological diagnosis. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) that used a purified recombinant capsid protein of swine HEV. The potential clinical use of this assay was evaluated by comparing it with a commercial kit (Genelabs Technologies, Diagnostics, Singapore). Results of the ELISA were highly correlated with those of the commercial kit with a sensitivity of 97% and specificity of 95%. ROC (receiving operator characteristic) analysis of the ELISA data produced a value of 0.987 (95% CI, 0.977~0.998, p < 0.01). The cut-off value for the ELISA was also determined using negative pig sera. In summary, the HEV-specific ELISA developed in the present study appears to be both practical and economical.     

猪戊型肝炎病毒ORF2主要抗原表位区间接ELISA方法的初步建立

应用原核表达系统表达猪戊型肝炎病毒ORF2蛋白C端和N端的主要抗原表位区,重组蛋白命名为ORF2-C和ORF2-N,初步建立间接ELISA诊断方法。用重组蛋白ORF2-C和ORF2-N作为诊断抗原,对反应条件进行优化,初步建立ELISA诊断方法。抗原最适包被浓度ORF2-C为8 μg/mL、ORF2-N为12 μg/mL;血清最适稀释度均为1∶50,ORF2-C作用时间为50 min、ORF2-N作用时间为90 min;酶标抗体最适稀释度为1∶5000;ORF2-C判定标准：D450 nm值≥0.348为阳性,D450 nm值<0.348为阴性;ORF2-N判定标准：D450 nm值≥0.397为阳性,D450 nm值<0.397为阴性。与戊型肝炎病毒诊断试剂盒检测结果相比,阳性符合率分别为93.3%、86.7%。利用重组蛋白建立的ELISA方法特异性、敏感性和重复性均较好,且ORF2-C的检测效率明显高于ORF2-N,该方法的初步建立为进一步完善猪戊型肝炎病毒诊断方法奠定基础。     

胃蛋白酶原Ⅱ单抗制备及夹心ELISA的初步建立

制备配对胃蛋白酶原Ⅱ(PGⅡ)单抗,建立人血清中胃蛋白酶原Ⅱ夹心ELISA检测方法。用胃蛋白酶原Ⅱ免疫Balb/c小鼠,制备免疫脾细胞,与SP2/0融合,用HAT培养基进行筛选培养,间接ELISA检测阳性克隆,对阳性孔进行多次单克隆化,选出效价高、分泌性能稳定的杂交瘤细胞,制备腹水并进行纯化。进行单抗配对,建立胃蛋白酶原Ⅱ双抗体夹心检测方法。获得1D8、1C6、2H11、2E3等4株杂交瘤,经配对试验,确定1D8、2H11可作为夹心ELISA检测PGⅡ的单抗。成功制备出配对单抗,初步建立了PGⅡ双抗体夹心ELISA方法。     

猪戊型肝炎病毒的流行病学调查

利用双抗原夹心ELISA对2009-2011年间采自广东省各地区的若干猪场的484份血清样品,进行血清流行病学调查;对69份病猪胆汁样品进行分子流行病学调查,分离得到猪源戊型肝炎病毒(swine hepatitis E virus, swHEV),并基于ORF2部分基因序列进行核苷酸序列比对、相似性分析和遗传进化树分析,以期初步了解广东各地猪群戊型肝炎(hepatitis E, HE)的感染情况和流行特点。试验结果显示,全部被检查猪的swHEV抗体平均阳性率为71.9%(348/484),分离的猪源HEV毒株均属于基因Ⅳ型,阳性率为71.0%(49/69)。调查结果表明基因Ⅳ型swHEV在广东地区普遍流行。     

检测牛乳κ-酪蛋白的间接竞争ELISA法与间接ELISA法比较

试验旨在建立一种快速简单检测牛乳κ-酪蛋白（κ-CN）含量的酶联免疫吸附（ELISA）方法,为解决牛乳蛋白掺假问题提供一定的技术支持。以牛乳κ-CN为包被抗原,以酶标抗体（HRP-IgG）为检测抗体分别建立间接竞争ELISA法和间接ELISA法,并对两种检测方法进行分析比较。结果表明：对于间接竞争ELISA法,κ-CN抗原包被浓度为2.5 μg/mL,线性范围为62.5~1 000.0 ng/mL,变异系数< 2%,回收率在98.46%~101.68%;对于间接ELISA法,κ-CN抗原包被浓度为1.56 μg/mL,线性范围为0.098~3.125 μg/mL,变异系数< 1%,回收率在99.10%~101.06%。比较两种检测方法,间接竞争ELISA法检测耗时较短,抗体应用量较少,而间接法不需要包被成本较高的目标标准蛋白,相关系数也较高,但其检测体系组成成分较多,且需包被待测样品,较难组成ELISA试剂盒体系,不宜用于现场检测。因此,大规模制备ELISA试剂盒体系用于快速检测蛋白含量时可采用间接竞争法,不仅抗体应用量少、节约成本且快速、简单,可更好地用于科研实践。