摘要
目的用纯化的强直性脊柱炎（AS）患者血清IgG从噬菌体随机12肽库中筛选AS患者特异性血清标志物。方法用健康人纯化血清免疫球蛋白IgG作为固相配基吸附非特异性噬菌体，以纯化AS患者血清免疫球蛋白IgG包被，按吸附、洗脱、扩增的淘洗过程对上述处理过的噬菌体液进行3轮筛选，随机挑取噬菌体克隆，并用phage-ELISA法检测其与AS血清的结合情况，同时对部分克隆测序。用phage—ELISA检测阳性克隆与50例AS患者、30例SLE患者、30例类风湿关节炎（RA）患者、30例骨关节炎（OA）患者以及50名健康对照者的血清IgG的结合情况。比较AS患者ESR、C反应蛋白（CRP）和与阳性噬菌体克隆反应的吸光度（A）值之间的相关性。结果用AS患者的阳性血清IgG作为配基筛选与纯化血清IgG未结合的噬菌体，3轮筛选的回收率从2．2×10。％增加至1．9×10。％。对从第3轮洗脱物中挑选出的20个克隆进行结合试验，发现17个克隆与As患者纯化血清IgG结合阳性。测序表明有7个克隆源于同一克隆，其外源插入肽为LALPPIJAPNHHH（ASl）。用该阳性克隆检测AS组A值为（0．767±0．250），SLE组为（0．491±0．250），RA组为（0．445±0．194），OA组（0．302±0．113），健康对照组为（0．294±0．150），5组A值整体比较（F=42．292，P〈0．01）以及各对照组与AS组比较差异均具有统计学意义（P〈0．01）。AS组、SLE组、RA组、OA组及健康对照组的阳性率分别为92．0％、56．7％、50．0％、13．3％和14．0％，AS组高于对照组（χ^2=77．418，P〈0．01）。噬菌体阳性克隆AS1诊断AS患者的敏感度为100％，特异度为80％，阳性预测值为83．3％，阴性预测值为100％，准确性为90％，且AS患者血清与AS1反应的A值与ESR、CRP之间为正相关关系（R2=0．165，P=0．448；R。=0．259，P=0．270）。结论用纯化的AS患者血清IgG从噬菌体随机12肽库
Objective To screen the specific serum biomarker of ankylosing spondylitis （AS）from phage random peptide library of 12 amino acids with the serum IgG of AS patients. Methods Phage random peptide library was first immunoscreened with purified IgG from healthy individuals to adsorb non-specific phages, and microtiter wells were coated with purified IgG of AS patients, then the above-treated phages were screened 3 rounds according to the process of adsorption, elution and amplification. Positive clones obtained were selected and detected by phage-ELISA and some of them were sequenced. The binding test of positive clones with the serum sample from 50 AS patients, 30 systemic lupus erythematosus （SLE） patients,30 rheumatoid arthritis （RA） patients, 30 osteoarthritis （OA） as well as 50 healthy individuals were detected using phage-ELISA. The correlations were compared among the absorbance （A） value, erythrocyte sedimentation rate （ESR） and C-reactive protein （CRP） in AS patients. Results When serum IgG of AS patients was used as ligand for screening of the phages which were not bound to healthy individuals, the radio of output to input increase from 2. 2 ~ 10-5% to 1.9 ~ 10-2% after 3 rounds screening. At the third round of screening, 20 clones were selected for reacting with antibodies from AS patients. 17 of them were proved to specifically react with the sera of AS patients. The results of sequences indicated that these 7 clones sequenced came from the same one, and the sequence inserted in the coat protein III was deduced to be LALPPLAPNHHH named AS1. When AS1 reacted with the serum samples from 50 AS patients and 140 controls by ELISA, the A value was 0. 767 ± 0. 250 in AS group, 0. 491 ± 0. 250 in SLE group, 0. 445 ±0. 194 in RA group, 0. 302±0. 113 in OA group, 0. 294 ±0. 150 in healthy control group. There were statistical significances of A values among 5 groups （ P 〈 0. 01 ） or between the AS group and the control group （P 〈0. 01 ）. And positive rates of the positi
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2009年第9期1019-1024,共6页
Chinese Journal of Laboratory Medicine
关键词
脊柱炎
强直性
肽库
免疫球蛋白G
生物学标记
酶联免疫吸附测定
Spondylitis, ankylosing
Peptide library
Immunoglobulin G
Biological markers
Enzyme-linked immunosorbent assay
