以合成肽为抗原检测人巨细胞病毒抗体的酶联免疫吸附试验的建立及评价

目的以合成多肽为抗原建立用于人巨细咆病毒（HCMV）抗体检测的间接酶联免疫吸附试验（ELISA）。方法以固相多肽合成技术合成了HCMV7个抗原性肽段,并对其抗原性进行了初步评价,选择高抗体反应的肽段为抗原,建立检测HCMV特异性抗体的间接ELISA。结果7个肽段中,抗体反应性较高肽段有4个;以合成多肽为抗原的间接ELISA对HCMV IgG抗体检测的敏感性和特异性分别为90．9％和100．0％,对于IgM抗体检测的敏感性和特异性分为90．0％和100．0％;与国外试剂盒测定结果有良好的符合性;该方法的精密度及稳定性良好。结论合成的HCMV多肽片段可作为抗原用于HCMV的检测,以此多肽为抗原建立的间接ELISA可用于临床HCMV特异性抗体的检测。     

精子肽的固相合成及其在免疫性不孕诊断中的应用研究

目的合成特异性精子肽，井对其抗原性进行初步评价，从而探讨检测抗精子抗体的ELISA试剂盒制备。方法应用固相多肽合成技术，以Fmoc基团保护的氨基酸为原料，合成特异性精子肽。以高效液相色谱（HPLC）技术鉴定纯化，并用ELISA检测其抗原性。结果成功合成了所需肽段，HPLC结果显示纯度达95％以上。该合成肽包被的ELISA试剂盒检测抗精子抗体的结果，与商品化试剂盒相比较符合率达90．6％。结论固相多肽合成技术可用于合成高纯度特异性的精子肽，且所合成肽段具有良好的抗原活性，包板建立的ELISA方法可以用于免疫性不孕中抗精子抗体的检测。     

巨细胞病毒抗原性肽段的固相合成及其抗原性的初步评价

目的合成巨细胞病毒结构蛋白的强抗原性表位肽段,并对其抗原性进行初步评价。方法根据巨细胞病毒结构蛋白的抗原性表位,以Fmoc基团保护的氨基酸为原料,用固相多肽合成技术(SPPS)合成了具有强抗原性的部分肽段,以高效液相色谱技术鉴定纯化,并用酶联免疫吸附剂试验对其抗原性进行了初步评价。结果成功合成了包含巨细胞病毒的结构蛋白强抗原性表位的两个肽段,纯度达95%以上,并且具有很高的抗原性。结论固相多肽合成技术可以用于制备巨细胞病毒的抗原性表位肽段,并且所合成肽段具有良好的抗原活性,可以作为抗原用于巨细胞病毒感染的检测。     

巨细胞病毒抗原性肽段的固相合成及其抗原性的初步评价

目的合成巨细胞病毒结构蛋白的强抗原性表位肽段,并对其抗原性进行初步评价.方法根据巨细胞病毒结构蛋白的抗原性表位,以Fmoc基团保护的氨基酸为原料,用固相多肽合成技术(SPPS)合成了具有强抗原性的部分肽段,以高效液相色谱技术鉴定纯化,并用酶联免疫吸附剂试验对其抗原性进行了初步评价.结果成功合成了包含巨细胞病毒的结构蛋白强抗原性表位的两个肽段,纯度达95%以上,并且具有很高的抗原性.结论固相多肽合成技术可以用于制备巨细胞病毒的抗原性表位肽段,并且所合成肽段具有良好的抗原活性,可以作为抗原用于巨细胞病毒感染的检测.     

酶联免疫吸附试验检测人疱疹病毒7型抗体的研究

目的建立一种简便、快速、可靠的检测人疱疹病毒7型(HHV 7)特异性IgG和IgM酶联免疫吸附试验(ELISA)。方法应用HHV 7标准株G lasgow感染SupT1细胞,制备并纯化细胞工程抗原和重组pp85抗原;对325份血清HHV 7抗体进行检测,建立HHV 7特异性IgM和IgG间接ELISA,并与Q iagen公司ELISA试剂盒检测结果进行比较。结果采用细胞工程抗原和重组抗原制备的HHV 7 IgG和IgM ELISA诊断试剂敏感性、特异性、稳定性及重复性[变异系数(CV)<10%]较好;以Q iagen公司ELISA试剂盒为参比,重组抗原IgG ELISA检测上述标本的敏感性、特异性和符合率分别为98.1%、94.1%和97.8%,IgM ELISA分别为84.6%、99.7%和99.1%;与细胞工程抗原相比,重组抗原诊断试剂的特异性高而敏感性略低。结论自制HHV 7 IgG和IgMELISA检测试剂敏感特异,可用于临床HHV 7感染的诊断及流行病学调查。     

多肽抗原检测心肌肌球蛋白重链抗体方法研究

目的建立抗肌球蛋白重链(AMHC)自身抗体的间接酶联免疫吸附法(ELISA),并探讨此抗体与病毒性心脏病的关系。方法以合成肽作为抗原,建立ELISA方法,检测心肌炎40例,扩张型心肌病48例和30名正常人血清中AMHC自身抗体。结果阳性血清批内和批间变异系数分别为0.086与0.127,用抗原吸收后,吸光度(A)值下降了62.9%,慢性病毒性心肌炎,扩张型心肌病患者AMHC自身抗体阳性率分别为42.1%和47.9%,显著高于急性病毒性心肌炎组(9.5%)和正常对照组(3.3%)。结论用多肽抗原代替天然肌球蛋白检测AMHC自身抗体,特异性和敏感性高,操作简便,为临床开展心肌炎、心肌病的免疫学监测提供了新的检测方法。     

双抗原夹心ELISA检测抗HCV总抗体

目的建立检测抗HCV抗体的双抗原夹心ELISA法,评价其可行性。方法将与His或GST融合表达的HCV基因工程抗原,分别用作ELISA的包被和酶标记抗原,建立用于抗HCV总抗体检测的双抗原夹心ELISA。用此方法检测1 968份临床血清标本,并以间接ELISA(北京万泰试剂)与之对照;此外,用套式RT-PCR检测部分血清的HCV RNA。结果有1 761份血清2种ELISA检测均为阴性,有190份血清均为阳性,两种方法符合率为99.1%;有17份血清的检测结果不相符,间接法阳性而本法阴性的14份,其中HCV RT-PCR阳性1份;本法阳性而间接ELISA阴性的3份,其中RT-PCR阳性2份。双抗原夹心ELISA与间接ELISA的敏感性分别为99.48%、98.96%,特异性分别为99.94%、99.27%。结论新研制的检测抗HCV总抗体的双抗原夹心ELISA具有较高的敏感性和特异性,值得作进一步的深入研究。     

结核分枝杆菌抗原优势肽段融合抗原38kD-ESAT6-CFP10的构建与抗原性初步检测

目的为了提高结核病诊断试剂的特异性和敏感性,构建了结核分枝杆菌抗原优势肽段融合抗原38kD-ESAT6-CFP10,并且采用双抗原夹心ELISA方法初步分析了该融合抗原的抗原性。方法运用生物学软件分析抗原优势肽段并模拟其串联后的三维结构,以确保串联后仍保持良好的抗原性。通过PCR方法分别扩增38kD、ESAT-6和CFP40抗原优势肽段基因并将其进行连接,然后将融合基因分别连接入pBVIL1和pGEX-4T-2表达载体,在大肠杆菌菌株DH5α中进行表达。将两种不同载体表达的38kD-KSAT6-CFP10融合抗原分别作为包被抗原和标记抗原,以双抗原夹心ELISA方法初步评价其抗原性。结果获得了38kD、ESAT-6和CFP10的抗原优势肽段融合抗原38kD-ESAT6-CIW10,并在大肠杆菌中进行了高效表达,初步验证所获得的抗原具有良好的抗原性。结论该种抗原优势肽段融合抗原有望成为结核病血清学诊断的重要候选抗原,并且双抗原夹心ELITSA方法有助于提高检测的特异性和敏感性。     

酶联免疫吸附试验检测人疱疹病毒7型抗体的研究

目的 建立一种简便、快速、可靠的检测人疱疹病毒7型（HHV7）特异性IgG和IgM酶联免疫吸附试验（ELISA）。方法 应用HHV7标准株Glasgow感染SupT1细胞,制备并纯化细胞工程抗原和重组ppS5抗原;对325份血清HHV7抗体进行检测,建立HHV7特异性IgM和IgG间接ELISA,并与Qiagen公司ELISA试剂盒检测结果进行比较。结果 采用细胞工程抗原和重组抗原制备的HHV7 IgG和IgM ELISA诊断试剂敏感性、特异性、稳定性及重复性[变异系数（CV）〈10％]较好;以Qiagen公司ELISA试剂盒为参比,重组抗原IgG ELISA检测上述标本的敏感性、特异性和符合率分别为98．1％、94．1％和97．8％,IgM ELISA分别为84．6％、99．7％和99．1％;与细胞工程抗原相比,重组抗原诊断试剂的特异性高而敏感性略低。结论 自制HHV7 IgG和IgM ELISA检测试剂敏感特异,可用于临床HHV7感染的诊断及流行病学调查。     

双抗原夹心酶联免疫技术检测丙型肝炎病毒抗体

1993年3月，国家卫生部下发文件要求对献血员进行抗HCV抗体的检测，随后有10几家检测HCV抗体的ELISA试剂上市，并进行批批检定，但全是间接ELISA法。由于间接法技术自身的缺陷及各生产厂家的产品存在一定的质量问题，导致了一些误诊和漏检。为此，应从根本上解决试剂本身的质量问题，建立双抗原夹心ELISA技术检测抗HCV抗体可大大提高检测试剂的特异性和敏感性。目前双抗原夹心法大多用于检测抗HIV和Tp抗体。此类试剂的敏感性和特异性均优于标记抗人免疫球蛋白的间接法。而丙型肝炎病毒抗体的检测仍采用间接ELISA技术，目前已有快速金标试剂采用双抗原夹心法原理，通过免疫层析检测抗HCV抗体。但灵敏度较低，不能用于献血员的筛选。我们通过对基因工程抗原进行改造，以适应标记辣根过氧化物酶，建立双抗原夹心ELISA法检测抗HCV抗体。     

Improved serologic detection of hepatitis C virus with a paramagnetic microparticle assay using multiple antigenic sequences

A paramagnetic microparticle (MP) assay for antibody to hepatitis C virus (anti-HCV) was developed, in which the probe for antibody consisted of synthetic peptides corresponding to HCV capsid and nonstructural c-100 regions, as well as a recombinant protein corresponding to the nonstructural c33c region. Assay performance was evaluated by testing serum from 108 geographically diverse patients with non-A, non-B hepatitis (NANBH). The frequency of anti-HCV reactivity detected with the MP assay and with an enzyme-linked immunosorbent assay (ELISA) for c-100 was 91 and 70 percent, respectively. All c-100 HCV ELISA-reactive specimens also reacted on the MP assay. In addition, anti-HCV seroconversion in three plasma donors was detected one to two blood collection dates earlier by the MP assay than by the c-100 HCV ELISA and at similar blood collection dates by the MP assay and a second-generation anti-HCV ELISA. Serologic responses to the three distinct antigenic regions of HCV in NANBH patients varied: reactivity to all three antigens was most common (49%), reactivity to both capsid and c33c (40%) was next most common, and single-antigen reactivity was rare (4%). MP assay reactivity of 825 volunteer donors was 0.1 percent. These results demonstrate both the utility of additional HCV antigens for an effective anti-HCV screening assay and the application of paramagnetic MP technology to serologic testing for HCV infection.     

Utility of B-cell epitopes based peptides of RD1 and RD2 antigens for immunodiagnosis of pulmonary tuberculosis

Tuberculosis (TB) continues to be a major health problem due to lack of accurate, rapid, and cost-effective diagnostic tests. Serodiagnostic tests incorporating highly specific region of difference (RD) antigens (early secretory antigenic target 6 [ESAT-6], culture filtrate protein 10 [CFP-10], culture filtrate protein 21 [CFP-21], and mycobacterial protein from species tuberculosis 64 [MPT-64]) have recently been shown to be promising for specific diagnosis of TB in our lab. However, only few studies have reported the use of synthetic peptides of RD antigens, and none has used them to differentiate TB from sarcoidosis, a close mimic of smear-negative pulmonary TB (PTB) with entirely different management. The present study was conducted with an aim to study the utility of B-cell epitopes based peptides of RD1 (ESAT-6, CFP-10) and RD2 (CFP-21, MPT-64) antigens for immunodiagnosis of PTB for which sputum smear-positive PTB patients, sputum smear-negative PTB patients, sarcoidosis patients, and healthy controls (n = 24/group) were recruited. Bioinformatic software Bcepred was used to predict linear B-cell epitopes, using physico-chemical properties on a non-redundant dataset. Seven peptides as representative B-cell epitopes of ESAT-6, CFP-10, CFP-21, and MPT-64 were evaluated as targets of the antibody responses in TB patients and controls by enzyme-linked immunosorbent assay (ELISA). The current study showed sensitivity with individual peptides ranging from 37.5% to 83.3% for smear positive, 25% to 58.3% for smear negative as compared to 4.16% to 20.8% for sarcoidosis. Four out of 7 peptides that showed higher reactivity with TB patients and better discrimination from sarcoidosis patients representing ESAT-6, CFP-10, CFP-21, and MPT-64 were selected for multiepitope ELISA. The combination of peptides yielded 83.3% sensitivity for smear positive, 62.5% for smear negative, and only 4.16% for sarcoidosis. The specificity, however, for all the peptides/combination was 100%. Combination of peptides has proven to be better than individual peptides as per the latest criteria of the World Health Organization according to which a test that can replace smear microscopy with sensitivity of >90% for smear-positive patients and >65% for smear-negative TB patients with a specificity >95%, and thus, the present study suggests that a test based on combination of peptides selected from mycobacterial RD1 and RD2 antigens could be important for promoting an early diagnosis and management of otherwise difficult to diagnose smear-negative PTB patients. Moreover, it can also be used to discriminate sarcoidosis from PTB, thus preventing the misdiagnosis and mismanagement.     

Potent epitopes derived from Fasciola gigantica cathepsin L1 in peptide-based immunoassay for the serodiagnosis of human fascioliasis

A peptide-based enzyme-linked immunosorbent assay (ELISA) was developed and evaluated for its diagnostic ability to detect human IgG antibodies against Fasciola gigantica cathepsin L1. Two previously identified B-cell epitopes of cathepsin L1 were synthesized as single synthetic peptides (acetyl-DKIDWRESGYVTEVKDQGNC-carboxamide and acetyl-DKIDWRESGYVTELKDQGNC-carboxamide) and their diagnostic potential was evaluated. The peptide-based ELISA was compared with an indirect ELISA with crude excretory-secretory products or with partially purified specific 27-kDa (FG27) antigen from adult F. gigantica. In an analysis of the sera of 13 patients infected with F. gigantica, 212 patients with other parasitic infections, 32 patients with cholangiocarcinoma, and 57 healthy controls, the sensitivity, specificity, accuracy, and positive and negative predictive values of this peptide-based ELISA with both peptides had the same performance and were shown to be 100%, 97.3%, 97.5%, 61.9%, and 100%, respectively. When 4 different ELISAs were compared, the results revealed that the specificity, sensitivity, accuracy, and negative predictive values of all antigens were similar except for the positive predictive value that was highest in the ELISA with the FG27 antigen. These results demonstrated that peptide antigens can be used in the serodiagnosis of human fascioliasis with the additional advantage that they are relatively cheap and easy to produce. This rapid, highly sensitive and specific peptide-ELISA has the potential to be used in future large-scale prevalence surveys throughout Southeast Asia.     

Fine specificity and neutralizing activity of human serum antibodies directed to the major antigenic region on gp 116 of human cytomegalovirus

The human antibody response to the conserved neutralization-related site on the gp 116 of human cytomegalovirus (HCMV) was investigated in healthy blood donors by the use of synthetic peptides. Anti-HCMV positive sera investigated in ELISA gave a reactivity of 48–56% with the peptide T7-13 (amino acids (aa) 67–86). Though epitope mapping revealed several individual fine specificities within this region, the average reactivity pattern was similar to that of the human monoclonal antibody (MAb) ITC88, the binding of which has been localized to aa 69–80. By the use of superparamagnetic Dynabeads coated with the peptide T7-13, serum antibodies were affinity isolated and the neutralizing activity was investigated. A clear reduction in infectivity was seen only with antibodies from one out of four sera and this serum exhibited a fine specificity nearly identical to that of MAb ITC88. A complete adsorption of antibodies to this site was not achieved, yet the results imply that antibodies against this region do not constitute a major part of the HCMV-neutralizing activity in human serum. The potent complement-independent neutralizing activity of antibodies directed to this site nevertheless suggests that it will contribute beneficially to a subunit vaccine.     

Anti-smooth muscle antibodies (ASMAs) and anti-cytoskeleton antibodies (ACTAs) in liver diseases: a comparison of classical indirect immunofluorescence with ELISA

In the diagnosis of autoimmune hepatitis type I (AIH-I), the routine assay of indirect immunofluorescence (IFL), used for the detection of anti-smooth muscle antibodies (ASMAs), has a low predictive value. On the other hand, the enzyme-linked immunosorbent assay (ELISA), which detects anti-cytoskeleton antibodies (ACTAs), presents contradictory results concerning their specific antigenic target. In this study, we first looked for the immunological properties (isotypes and antigenic targets) of autoantibodies in AIH-I and two other control liver diseases: primary biliary cirrhosis (PBC) and viral hepatitis (VH), using ELISA based on cytoskeleton proteins: F-actin, G-actin, myosin, tropomyosin, troponin, desmin, vimentin, keratin, and an extract of HEp-2 carcinoma cells. We also compared the diagnostic value of IFL and ELISA. In contrast to previous studies, we found that actin was not specific for AIH-I. No autoantigen and no antibody class or subclass discriminated AIH-I from the control diseases. IFL is more suitable for AIH-I diagnosis, as 97% of AIH-I sera but only 22% of PBC sera were ASMA-positive. Additionally, 96% of ASMA-positive, and all ASMA-negative sera from all three liver diseases were ACTA-positive. ASMA were mainly IgG, while >50% of ACTA also contained IgA and IgM. These data suggest that ACTAs recognize additional epitopes as compared to ASMAs, and they frequently occur in all liver diseases.     

用噬菌体模拟抗原检测白念珠菌菌丝蛋白抗体的研究

目的 用噬菌体展示文库技术制备白念珠菌菌丝蛋白模拟抗原，建立测定白念珠菌抗体的酶联免疫吸附测定法（ELISA）。方法 用抗白念珠菌菌丝蛋白P47的单克隆抗体从噬菌体随机12肽库中筛选能与之免疫学结合的肽，即模拟抗原表位。将该模拟抗原包被微孔板，用于酶联免疫吸附测定法测定病人血清白念珠菌菌丝蛋白抗体。结果 用噬菌体模拟抗原建立ELISA，其敏感性、特异性及重复性良好，测定了10份确诊白念珠菌侵袭性感染的病人血清，全部阳性，与用纯化的菌丝蛋白抗原P47测定结果一致；服用免疫抑制剂患者血清阳性率33％，健康人群阳性率为2％。结论 用抗白念珠菌菌丝蛋白单克隆抗体从噬菌体展示随机肽库筛到的噬菌体模拟抗原可代替白念珠菌P47抗原进行抗体测定。     

Development of an ultrasensitive polymerase chain reaction-amplified immunoassay based on mycobacterial RD antigens: implications for the serodiagnosis of tuberculosis

Immuno-polymerase chain reaction (I-PCR) combines the versatility of enzyme-linked immunosorbent assay (ELISA) with the exponential amplification power of PCR. The present study was designed to detect antibodies to Mycobacterium tuberculosis complex-specific region of difference (RD) antigens, i.e., early secretory antigenic target-6, culture filtrate protein-10, culture filtrate protein-21, and mycobacterial protein from species tuberculosis-64, as well as antigens in pulmonary tuberculosis patients by I-PCR assay. We could detect ESAT-6 and other RD antigens up to 0.1 fg by I-PCR assay, thus resulting in 10⁷ times higher sensitivity than that observed with ELISA. With paired sample analysis based on the detection of antibodies in serum and antigens in sputum of the same individual, the sensitivity of RD multi-antigen cocktail-based I-PCR assay was 72% in smear-negative cases and 91% in smear-positive cases of pulmonary tuberculosis with high specificity values. In extrapulmonary tuberculosis patients, higher sensitivity was observed by detecting cocktail of antigens by I-PCR assay as compared to sensitivity earlier observed in the same samples by ELISA.     

Analysis of a high-throughput HLA antibody screening assay for use with platelet donors

BACKGROUND: Passive infusion of HLA antibodies has been implicated in transfusion reactions. A rapid, inexpensive method of screening blood donors for HLA antibodies might reduce the incidence of reactions. A high-throughput microbead-flow analyzer HLA antibody detection technique was compared with an enzyme-linked immunosorbent assay (ELISA) method. STUDY DESIGN AND METHODS: Ninety-six apheresis platelet (PLT) donors were tested for antibodies to Class I and II HLA antigens with mixed-antigen microbead-flow analyzer and ELISAs. For both assays, samples reactive in the mixed-antigen assay were tested with a panel-reactive antibody (PRA) assay. Samples reactive in both the mixed-antigen and the PRA assays were considered positive. RESULTS: In the mixed-antigen microbead assay, 46 (48%) samples were reactive to Class I antigens and 20 (21%) to Class II. Further testing in the microbead PRA assay revealed that 34 (35%) had antibodies to Class I antigens, 18 (19%) to Class II, and 42 (44%) to either Class I or Class II. Class I antibodies were present in 56 percent of females and 36 percent of males. In the mixed-antigen ELISA, 4 samples were reactive with Class I antigens, 4 with Class II antigens, and 5 with Class I or Class II. All 5 reactive samples were also reactive in the ELISA PRA assay and were from females. CONCLUSION: The microbead assay was more sensitive than the ELISA and detected antibodies in a large proportion of donors. Samples reactive in the mixed-antigen microbead assay should be confirmed by a second assay before concluding that antibodies are present.     

Synthetic peptide derived from the epstein-barr virus encoded early diffuse antigen (ea-d) reactive with human antibodies

Primary infection with Epstein-Barr virus (EBV) and reactivation of latent virus are associated with increased antibody titers against the diffuse early antigen (EA-D). In order to better define the antigenic epitopes recognized by antibodies from patients with infectious mononucleosis (IM) and with other disease states, a series of synthetic peptides were prepared based on the DNA sequence encoding the EA-D molecule. One synthetic peptide (K7b) was reactive with the majority of sera from patients with acute IM. Anti-K7b activity was most readily detected among IgM and IgA antibodies and to a lesser extent among IgG antibodies. In contrast, significant elevations of anti-K7b activity were observed in less than 5% of healthy adults. Serial analysis of samples from individuals prior to and after exposure to EBV demonstrated increased anti-K7b reactivity associated with the symptoms of acute IM. Elevated anti-peptide K7b titers also were found in sera of patients with nasopharyngeal carcinoma and with Sjogren's syndrome (an autoimmune disease involving the salivary glands). Four different synthetic peptides from other regions of the EA-D molecule were not reactive with antibodies from these patients nor from IM patients. These results suggest that peptide K7b defines an antigenic epitope recognized during primary EBV infection and during viral reactivation occurring in patients with autoimmune and neoplastic disease.