摘要
目的建立血友病A患者血浆FⅧ同种抗体的ELISA检测方法,以评估经替代治疗的血友病A患者血浆FⅧ同种抗体的发生率.方法选取血友病A患者140例,其中重型84例,中型34例,轻型22例,所有患者均已经过FⅧ替代治疗;同时选取健康对照者80名.以本室制备的FⅧ单克隆抗体包被酶标板,封闭后加入重组人FⅧ,洗涤后加入待测稀释血浆,再次洗涤后加入辣根过氧化物酶标记的羊抗人IgG,邻苯二胺显色,记录490 nm处的吸光度（A490）值.所有患者血浆样本同时采用改良的Nijmegen方法检测抑制物活性.结果通过ELISA法检测,血友病A患者产生血浆FⅧ同种抗体阳性率为40.0%（56/140）,重型、中轻型患者产生抗体的阳性率分别为47.6%（40/84）、28.5%（16/56）,两组患者产生同种抗体的阳性率差异有统计学意义（x2=5.079,P＜0.05）;采用改良的Nijmegen方法检测,血友病A患者产生FⅧ抑制物阳性率为24.3%（34/140）,重型、中轻型患者产生抑制物的阳性率分别为33.3%（28/84）、10.7%（6/56）,两组患者产生抑制物的阳性率差异有统计学意义（x2=9.349,P＜0.05）.其中,25例通过ELISA法检测出同种抗体阳性的患者未被检出抑制物活性.ELISA法与改良的Nijmegen法检出的阳性率分别为40.0%（56/140）、24.3%（34/140）,差异有统计学意义（x2=15.75,P＜0.01）,具有相关性（rn=0.59,P＜0.01）;且两种方法检出结果存在一致性（Kappa=0.55,P＜0.01）.结论 ELISA法能够提高FⅧ同种抗体的检出率,血友病A患者经替代治疗后,血浆FⅧ同种抗体发生率并不低,重型患者产生抗体的阳性率高于中轻型患者.
Objective An ELISA-based assay for detecting alloantibodies against FⅧ was established to estimate the incidence of alloantibodies against FⅧ in treated patients with hemophilia A. Methods One hundred and forty patients with hemophilia A and 80 healthy controls were enrolled. Among hemophilia A patients, 84, 34 and 22 patients were in severe, moderate and mild conditions respectively. All patients were treated with plasma-derived FⅧ concentrates before. The titer wells were coated with MoAb against FⅧ which was developed in our laboratory. Then human recombinant FⅧ concentrates were applied. After incubation in room temperature for 2 hours, diluted plasma samples and HRP-conjugated goat anti-human IgG were added successively. Finally Absorbance （A490） were measured and recorded. Inhibitor activity against FⅧ for all plasma samples was measured by a modified Nijmegen assay simultaneously. Results The results showed that alloantibodies against FⅧ were found in 40.0% （56/140） patients by ELISA. And the alloantibody incidences in the severe and non-severe patients were 47.6% （40/84） and 28.5% （16/56）respectively. There was statistical significance between these two categories （x2 = 5.079, P 〈 0.05 ）. The FⅧ inhibitor activity was detected in 24.3% （34/140） patients by modified Nijmegen assay. The inhibitor incidences in the severe and non-severe patients were 33.3% （28/84） and 10.7% （6/56） respectively.There was statistical significance （x2 = 9.349, P 〈 0.05）. Twenty-five patients were positive for FⅧ alloantibodies by ELISA but had no FⅧ inhibitor activity by the modified Nijmegen assay. The positive rates of FⅧ alloantibodies and inhibitor activity were 40.0% （56/140） and 24.3% （34/140） respectively,which had significant difference （x2 = 15.75, P 〈 0.01 ） and strong positive correlation （ rn = 0.59, P 〈0.01 ）. Meanwhile the results deduced from these two tests shared a high consistency rate （ Kappa = 0.55,P 〈0.01 ）. Conclusions The detection rate for alloantibodies against F Ⅷ is enhanced by our newlydeveloped ELISA. Our results suggest that the occurrence of the alloantibodies against F Ⅷ in Chinese hemophilia A patients is not rare and the alloantibody incidence is preponderant in the patients with severe hemophilia A compared with non-severe hemophilia A patients.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2010年第10期967-971,共5页
Chinese Journal of Laboratory Medicine
基金
卫生部血栓与止血重点实验室开放课题资助项目（KLTH-2009-02）
关键词
血友病A
因子Ⅷ
同种抗体
酶联免疫吸附测定
Hemophilia A
Factor Ⅷ
Isoantibodies
Enzyme-linked immunosorbent assay
