甲状腺过氧化物酶抗体间接时间分辨荧光免疫分析法的建立及临床应用

目的建立甲状腺过氧化物酶（TPO）抗体间接时间分辨荧光免疫分析法（TRFIA）。方法用TPO抗原包板,用铕（Eu^3＋）标记兔抗人IgG做标记物,建立检测人血清TPO抗体的间接TRFIA。结果TPO抗体-TRFIA的敏感性为1.0 IU/mL;线性范围达1 000 IU/mL;批内变异系数（CV）为3.1%-3.6%,批间CV为3.3%-3.6%;平均回收率为98.89%;与电化学发光分析法（ECLIA）比对,相关系数达0.983 2;与临床诊断高度相关。结论本研究建立的TPO抗体-TRFIA是一个高灵敏和可靠的检测方法,有助于甲状腺疾病的临床诊断。     

间接包被ELISA法检测血清游离β-HCG

目的 研制生物素-亲合素间接包被ELISA法定量检测血清游离β-HCG试剂盒。 方法 用生物素化牛血清清蛋白和链霉亲合素包被微孔反应板,生物素化抗游离β-HCG抗体和酶标记抗游离β-HCG抗体检测游离β-HCG,评价试剂盒相关技术参数。 结果 本试剂盒具有较好的稳定性,灵敏度0.1 ng/ml,回收率95.9％~102.5％,批内CV 7.5％~11.1％,批间CV12.5%~18.1%,与FSH、TSH、LH和α-HCG的交叉反应率均<0.1％,检测范围0.1~500 ng/ml,与美国DRG公司试剂盒的相关系数r2=0.789 2,P<0.05。 结论 本方法研制的试剂盒灵敏度高,特异性好,操作方法简便,适于临床常规应用。     

甲状腺球蛋白抗体竞争TRFIA技术的建立及临床应用

目的建立甲状腺球蛋白抗体(TgAb)时间分辨荧光免疫分析(TRFIA)竞争检测法。方法用甲状腺球蛋白抗原包被96孔板,用铕(Eu3+)标记羊抗鼠IgG做标记物,TRFIA检测人血清中的TgAb。结果 TgAb-TRFIA的敏感性为1.0 IU/mL;批内变异系数(CV)为3.3%～4.1%,批间CV为3.7%～4.7%;平均回收率为97.9%;热稳定性好;与电化学发光分析技术(ECLIA)比对,相关系数达0.881 3;男女TgAb测定值差异无统计学意义;TgAb的正常值范围为≤110 IU/mL。结论建立的TgAb-TRFIA是一个高敏感和可靠的检测方法,有助于甲状腺疾病的临床诊断。     

IgA缺乏症献血者酶联免疫试验筛查方法的建立

目的建立适合用于大规模IgA缺乏症献血者筛选的ELISA检测方法。方法采用间接酶联免疫法,在微孔板中包被羊抗人IgA、用辣根过氧化物酶标记羊抗人IgA抗体作为酶标二抗。结果建立的ELISA测定法灵敏度为0.1μg/mL,IgA浓度为0.1、100μg/mL的批间变异系数(CV)是1.74%、3.49%,批间CV中位数为3.48%(范围:1.83%~6.96%)。检测过程约80min。结论成功建立了IgA缺乏症筛查的ELISA测定法,其灵敏度高、特异度强、省时、操作简易,可用于大规模筛选IgA缺乏症献血者及建立IgA缺乏献血者库。     

固相亲合素在游离甲状腺素免疫检测中的应用

目的：以亲合素－生物素分离技术为基础,尝试通过固化抗原（标记抗体）或固化抗体（标记抗原衍生物）两种免疫分析构型实现具有代表性的游离激素－游离甲状腺素（FT4）的固相免疫分析测定。方法：应用牛血清白蛋白－亲合素联接物制备固相亲合素,以此为基础制备固相生物素化甲状腺素衍生物或固相生物素化抗甲状腺素抗体,并分别用于标记抗体法或标记抗原衍生物法的FT4免疫分析。结果：标准曲线的四参数非线性拟合度良好;标记抗标法测定的灵敏度为0.42pmol/L,批内批间的CV％均小于7％,标记衍生物法的灵敏度为0.36pmol/L,批内批间的CV％均小于7％,两种方法相关性良好（r=0．9734,P＜0．01）。结论：通过应用固相亲合素固化抗原或固化抗体两种方式,进一步肯定了固相亲合素作为通用固相分离技术在免疫分析中的应用价值,为游离甲状腺素测定提供了实验 依据。     

β-人绒毛膜促性腺激素光激化学发光免疫分析方法的建立

目的建立一种检测人血清β-人绒毛膜促性腺激素（β-HCG）浓度的光激化学发光免疫分析方法。方法采用2株针对不同抗原表位的抗β-HCG抗体,其中一株抗体用来包被发光微粒,另一株抗体标记生物素,以双抗体夹心法检测人血清中的β-HCG浓度,并与Beckman-Coulter公司试剂（化学发光法）进行比较。结果发光微粒浓度为100μg/mL、生物素标记抗体浓度为7μg/mL时,检测范围为0~1 000 mIU/mL,灵敏度为0.16 mIU/mL,平均回收率为100.3%,批内变异系数（CV）为0.80%~3.99%,批间CV为2.25%,与TSH、FSH和LH的交叉反应率低,稳定性较好,与化学发光法的符合率好（r=0.99）。结论光激化学发光免疫分析方法测定β-HCG具有较高灵敏度、精密度和准确性,与化学发光法的符合率较好,适用于临床测定。     

肺炎衣原体抗体酶免疫诊断试剂盒的研制及应用

目的建立酶免(间接)法检测人血清中肺炎衣原体抗体。方法用HEP-2细胞培养肺炎衣原体AR-39株,获得肺炎衣原体抗原后,利用该抗原与人血清中特异性抗体发生反应及辣根过氧化物酶标记的抗抗体催化TM B显色来指示人血清中抗肺炎衣原体抗体的存在。结果抗原稀释度1μg/m l,血清稀释度为1∶50,目测效果较好。门诊随机血清阳性检出率高达8.78%,此法特异性94.70%,灵敏度90.50%,试剂盒批内差CV=8.87%,批间差CV=6.92%。结论该方法简便快捷,特异性、灵敏度、准确度较好,可以在医院大力推广。     

狂犬病毒核蛋白抗原ELISA定量法的建立及初步验证

目的建立检测狂犬病疫苗各工序产品中核蛋白抗原含量的双抗体夹心ELISA法。方法以兔抗狂犬病毒多抗为包被抗体,辣根过氧化物酶标记的抗狂犬病毒核蛋白单抗作为酶标记抗体,对狂犬病毒核蛋白抗原含量进行定量测定,并对该方法进行初步验证。结果此方法线性相关系数大于0.97;最佳线性范围为0.000625～0.01IU/ml,定量限度为0.000625IU/ml;准确性为102%～109%;变异系数(CV)为7.2%～9.4%;与小牛血清、牛血清清蛋白(BSA)、卵清蛋白(OVA)、流感疫苗纯化液、乙脑疫苗纯化液、甲肝疫苗纯化液等均无交叉反应。结论该方法特异性强,灵敏度高,准确性、重复性和稳定性好,可用于狂犬病疫苗各工序产品中核蛋白抗原的定量检测。     

链霉亲和素—生物素—ELISA方法的建立及其在HBsAg测定中的应用

根据链霉亲和素（ＳＡ）与生物素（Ｂ）之间有很强亲和力的原理，建立了ＥＬＩＳＡ板先用ＳＡ包被再与生物素化抗体结合的间接包被方法。通过试验，选择了生物素化白蛋白（Ｂ－ＢＳＡ）预包被，再连接ＳＡ以制备ＳＡ酶标板。应用生物素化的ＨＢｓ单抗及酶标记的抗ＨＢｓ单抗，建立了检测ＨＢｓＡｇ的ＳＡＢ－ＥＬＩＳＡ方法，得到良好的效果。     

化学发光磁微粒免疫分析法定量检测HBsAg

摘要：目的：建立定量检测HBsAg的化学发光磁微粒免疫分析法。 方法：2株抗HBs单克隆抗体分别标记吖啶衍生物和生物素化,生物素化的抗HBs通过链霉亲合素包被到磁珠上形成致敏磁珠。将待测标本加入吖啶标记的抗HBs（吖啶-抗HBs）与致敏磁珠的体系中,形成致敏磁珠-HBsAg-吖啶标记抗HBs夹心复合物,磁性分离清洗。重复加样1次,磁性捕获分离后激发吖啶衍生物发光,用化学发光分析仪检测光强度,计算标本中HBsAg的浓度。 结果：该法线性方程为Y=0.919X+4.28,相关系数r²为0.99,线性范围为0.019 7~250.00 IU/mL。灵敏度可达0.020 7 IU/mL。0.1、3.2和40 IU/mL样本各重复测定10次的变异系数（CV）值分别为2.70%、4.40%和2.70%。与Abbott公司HBsAg定量检测试剂盒(化学发光微粒子免疫检测法) 检测40份标本浓度的线性相关方程为Y=0.980 8X+0.209 9,r²=0.99,两法结果差异无统计学意义（t=1.519,P＞0.05）。以10、50、100和200 IU/mL作为检定点,各检定点相对偏差分别为0.18%、1.50%、1.71 %和1.81%。 结论: 建立的方法有较高的灵敏度、合适的线性,同时具备良好的可重复性,与Abbott公司试剂盒检测结果高度相关。     

液相蛋白质芯片检测血清C反应蛋白方法的建立

目的 建立液相蛋白质芯片检测血清C反应蛋白（CRP）的方法,探讨该方法诊断冠心痛的临床应用价值。方法 用聚苯乙烯荧光微珠,将抗CRP单克隆抗体包被在微珠上,将另一针对CRP不同抗原表位的单克隆抗体生物素化;包被好的微珠加入到96孔板中,将待测血清分别加入各孔,用CRP标准品建立标准曲线,各孔中加入生物素化抗CRP单抗和PE荧光素标记的链霉亲合素。在Bio-Plex液相蛋白质芯片分析仪上检测296份血清样品。同一血清标本同时用免疫比浊法测定。比较两种方法对CRP检测的敏感性和特异性。结果 液相蛋白质芯片检测CRP方法对诊断冠心病的敏感性（78,3％）和特异性（87．1％）较免疫比浊法的敏感性（63．0％）和特异性（86．5％）高,χ^2=28,94,P〈0．001。结论 建立了一种检测血清CRP的新方法,对诊断冠心痛有临床应用价值。     

Generation of recombinant, enzymatically active human thyroid peroxidase and its recognition by antibodies in the sera of patients with Hashimoto''s thyroiditis.

A full-length cDNA clone for human thyroid peroxidase (TPO) inserted into the mammalian cell expression vector pECE was stably transfected into Chinese hamster ovary (CHO) cells. Clones were assayed for human TPO mRNA, TPO protein, and TPO enzymatic activity. One subclone, expressing the highest TPO enzymatic activity, was used in further studies. FACS analysis of these cells preincubated in Hashimoto's serum revealed approximately 100-fold greater fluorescence compared with controls, indicating that recombinant TPO is expressed on the cell surface. Particulate antigen was extracted from these cells and studied by Western blot analysis using a panel of Hashimoto's sera of known antimicrosomal antibody (anti-MSA) titer. Under nonreducing conditions a broad, immunoreactive band of approximately 200 kD was observed, as well as a doublet of approximately 110 kD. All of the 36 Hashimoto's sera tested reacted with these bands, most in proportion to their anti-MSA titer. Six normal sera tested against this antigen(s) were nonreactive, as were the Hashimoto's sera tested against nontransfected CHO cells. Western blots under reducing conditions revealed a considerably diminished signal, with some of the sera of lower anti-MSA titer becoming negative, the loss of the 200-kD broad band, and the apparent conversion of the 110-kD doublet into a single band. Preincubation of cells in tunicamycin revealed no decrease in TPO immunoreactivity. In conclusion, we expressed enzymatically active human TPO in nonthyroidal eukaryotic cells. Our data prove that functionally active TPO is a major component of the thyroid microsomal antigen.     

流行性乙型脑炎病毒IgG抗体捕获酶联免疫吸附试验检测方法的建立

目的利用流行性乙型脑炎(乙脑)病毒特异性单克隆抗体建立检测人血清中乙脑病毒IgG抗体的捕获ELISA方法。方法将乙脑病毒特异性单克隆抗体包被在固相载体上,加入灭活并经过纯化的乙脑病毒抗原,再加入待测血清,经洗涤、显色等判定最终检测结果。采用免疫荧光法作为对照。结果与免疫荧光法相比较,本方法的灵敏度为95.6%,特异度为85.7%,一致性为91.3%;本方法的灵敏度和特异度高于市售的两种检测试剂。结论本研究所建立的乙脑病毒IgG抗体捕获ELISA方法具有良好的检测性能,可以应用于正常人群血清中乙脑病毒IgG抗体的筛查。     

Highly sensitive assays of autoantibodies to thyroglobulin and to thyroid peroxidase

These highly sensitive assays are based on the interaction between thyroid autoantibodies and 125I-labeled autoantigens. Serum samples are incubated with labeled thyroid peroxidase (TPO) or thyroglobulin (Tg) to allow the formation of antibody-labeled antigen complexes. The complexes are then precipitated by addition of solid-phase Protein A. In the presence of high concentrations of TPO antibody or Tg antibody, more than 50% of the respective labeled antigen was precipitated, whereas only 1-2% was precipitated in the absence of autoantibody. Interassay CVs were 3.2% and 5.7%, respectively, for the anti-TPO and anti-Tg assays. There was no cross-reactivity between Tg antibody and TPO antibody. Results correlated highly significantly with results from other assay systems based on antigen-coated cells or plastic supports, but the assays described here were considerably more sensitive. Scatchard analysis of the assay data provided information on the affinity and serum concentration of TPO autoantibodies (ka approximately 10(9) L/mol and concentrations up to 1 g/L) and Tg autoantibodies (ka approximately 4 x 10(10) L/mol and concentrations up to 1 g/L). Overall, these assays provide a sensitive, precise, and convenient system for measuring and investigating the properties of thyroid autoantibodies.     

Isolation of a complementary DNA clone for thyroid microsomal antigen. Homology with the gene for thyroid peroxidase.

The thyroid microsomal antigen (MSA) in autoimmune thyroid disease is a protein of approximately 107 kD. We screened a human thyroid cDNA library constructed in the expression vector lambda gt11 with anti-107-kD monoclonal antibodies. Of five clones obtained, the recombinant beta-galactosidase fusion protein from one clone (PM-5) was confirmed to react with the monoclonal antiserum. The complementary DNA (cDNA) insert from PM-5 (0.8 kb) was used as a probe on Northern blot analysis to estimate the size of the mRNA coding for the MSA. The 2.9-kb messenger RNA (mRNA) species observed was the same size as that coding for human thyroid peroxidase (TPO). The probe did not bind to human liver mRNA, indicating the thyroid-specific nature of the PM-5-related mRNA. The nucleotide sequence of PM-5 (842 bp) was determined and consisted of a single open reading frame. Comparison of the nucleotide sequence of PM-5 with that presently available for pig TPO indicates 84% homology. In conclusion, a cDNA clone representing part of the microsomal antigen has been isolated. Sequence homology with porcine TPO, as well as identity in the size of the mRNA species for both the microsomal antigen and TPO, indicate that the microsomal antigen is, at least in part, TPO.     

血小板膜受体检测方法的建立及细胞因子的作用研究

目的建立流式细胞术检测血小板膜蛋白GMP-140的实验方法,分析血小板生成素(TPO)与GM-CSF因子对血小板膜糖蛋白GMP-140表达的影响。方法以生理盐水、TPO溶液及GM-CSF溶液为对照和实验条件与血小板孵育,加入荧光标记的单克隆抗体,应用流式细胞术检测血小板膜蛋白GMP-140的表达率。结果125ng/mlTPO实验组GMP-140表达率与对照组相比有显著性差别(P<0.001);100ng/mlGM-CSF实验组与对照组相比无显著性差别(P>0.5)。结论成功建立了流式细胞术检测血小板膜蛋白GMP-140的实验方法。TPO在一定浓度下对血小板有刺激激活作用,100ng/ml浓度的GM-CSF在体外直接作用于血小板对其无明显刺激作用。     

ELISA assay for colon cancer

The high mortality rate for patients with colon cancer results largely from the inability to diagnose the condition in its early stages. The assay described in this report may be useful in early detection of colon cancer. In the study we use the cotton-top tamarin, Saguinus oedipus, as an animal model for studying human colon cancer. This New World monkey develops a high incidence of colitis and colon cancer. Antibodies that recognize adenocarcinoma cells and antigens from these cells can be detected in the sera of tamarins with colon cancer. The antibodies are not found in sera of healthy animals. In one procedure, tamarin adenocarcinoma cells are used to coat the wells of a microtiter plate. In an alternate procedure, a glycoprotein adenocarcinoma antigen is used. The antigen is extracted from a cultured tamarin colon cancer line by detergent solubilization followed by affinity chromatography. Test sera are examined by adding biotinylated immunoglobulin serum fractions to the coated wells. The wells are washed and an avidin-enzyme conjugate is added. Positive sera are identified by the addition of the substrate. The presence of the autoantibodies in the sera may be taken as evidence that the animal has colon cancer.     

Characterization of the thyroid microsomal antigen, and its relationship to thyroid peroxidase, using monoclonal antibodies.

MAb directed to the thyroid microsomal antigen have been developed. All bound to 101- and 107-kD bands in Western blot analysis using thyroid microsomal fraction as antigen. The MAb also bound to microsomal proteins immunoprecipitated by serum from patients having a high titer of anti-microsomal antibody but no antibodies to thyroglobulin or thyrotropin-stimulating hormone receptor. The pattern of binding was related to the amount of reducing agent. The 101- and 107-kD bands were increased by addition of dithiothreitol whereas, in its absence, numerous bands of higher molecular weight were present, suggesting a multimeric protein structure. Despite the inability to immunoprecipitate thyroid peroxidase (TPO) enzymatic activity, the MAb bound intensively in Western blot to denatured purified hog TPO and to denatured immunopurified human TPO. Trypsin digestion of the 101-107-kD antigen produced a doublet of 84-88 kD that was still immunoreactive with MAb. One of five polyclonal sera tested (with a microsomal antibody titer greater than 1/20,480 measured by the tanned red cell hemagglutination technique) also recognized the 84-88 kD trypsin fragments. Addition of V8 protease led to a disappearance of the 107-kD protein, but not the 101-kD protein, proving that this antigen is formed by two different polypeptides. The MAb bound strongly to thyroid epithelium, whereas binding to papillary carcinoma was absent or low and moderate for follicular and Hurthle cell carcinoma. This study indicates that the thyroid microsomal antigen and TPO are identical and are constituted of two different polypeptides. On SDS-PAGE the antigen appears as two contiguous bands which share common epitopes but are not identical, as proven by their size and difference in susceptibility to proteolytic digestion. The immunoreactivity of the molecule is highly dependent on a trypsin-sensitive site, which appears important in the recognition of the antigen by polyclonal sera and may have biological importance. The expression of microsomal antigenicity is variable among various thyroid carcinomas.     

Europium nanoparticles and time-resolved fluorescence for ultrasensitive detection of prostate-specific antigen

Background: Nanoparticle-based detection technologies have the potential to improve detection sensitivity in miniature as well as in conventional biochemical assays. We introduce a detection technology that relies on the use of europium(III) nanoparticles and time-resolved fluorometry to improve the detection limit of biochemical assays and to visualize individual molecules in a microtiter plate format. METHODS: Streptavidin was covalently coated on 107-nm nanoparticles containing >30 000 europium molecules entrapped with beta-diketones. In a model assay system, these nanoparticles were used to trace biotinylated prostate-specific antigen (PSA) in a microtiter plate format. RESULTS: The detection limit (mean + 3 SD of the zero calibrator) of biotinylated PSA was 0.38 ng/L, corresponding to 10 fmol/L or 60 zeptomoles (60 x 10(-21) moles) of PSA. Moreover, single nanoparticles, representing individual PSA molecules, were visualized in the same microtiter wells with a time-resolved fluorescence microscope using a x10 objective. Single nanoparticles, possessing high specific activity, were also detected in solution by a standard time-resolved plate fluorometer. CONCLUSIONS: The universal streptavidin-coated europium(III) nanoparticle label is suitable for detection of any biotinylated molecule either in solution or on a solid phase. The europium(III) nanoparticle labeling technology is applicable to many areas of modern biochemical analysis, such as immunochemical and multianalyte DNA-chip assays as well as histo- and cytochemistry to improve detection sensitivities.