CN1053276C - 一种检测胰岛细胞抗体的方法 - Google Patents
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Abstract
本发明涉及一种检测胰岛细胞抗体的方法，它是采用猪的胰腺切片做抗原，检测人的胰岛细胞抗体，来预测、诊断和鉴别胰岛素依赖型糖尿病，为临床大夫提供重要依据。本发明方法由于猪胰腺来源广泛，价格低廉，而为广泛性进行ICA的检测开辟了一条新的途径。
Description
本发明涉及一种检测胰岛细胞抗体的方法。
糖尿病是一种严重危害人民健康的常见、多发的内分泌代谢病。糖尿病发生率日益增多，据报告目前全世界糖尿病人数高达6000万人，另外，还有很多患者未被发现，估计约有1.2亿以上的糖尿病患者。在我国，目前估计有糖尿病患者1500-2000万人。随着我国改革开放，国民经济的繁荣昌盛，人的寿命在不断延长，人口老龄化日趋显著，此病势必将进一步增多。更为严重的是糖尿病的慢性并发症，如视力障碍、晚期肾脏病、动脉粥样硬化、糖尿病酮症酸中毒等等，不仅病人家庭和社会要为之耗费大量的医疗费用，也是造成很多此类病人致残致死的原因，给国家和社会带来了巨大的压力和冲击。
糖尿病临床过程多种多样，使临床分型有时变得十分困难。目前已知胰岛细胞抗体(ICA)是检测胰岛素依赖型糖尿病(insulin-dependentdiabetes mellitus IDDM)的重要免疫学指标之一。
自Bottazzo等在1974年应用间接免疫荧光法，首次发现胰岛素依赖型糖尿病患者血清中有胰岛细胞抗体(ICA)之后，各国学者又对之进行了不同程度的改进。但因免疫荧光法荧光易猝灭，背景中非特异性荧光较多，常导致结果判定困难，后来，又有学者将免疫酶标技术用于ICA的检测中，如ABC法、PAP法、POPA法等。因这些方法敏感性高，特异性强，大大推动了ICA测定的实际应用。但是，由于血型抗体的干扰，常规的ICA测定均需用“O”型血人正常胰腺做抗原。因人胰腺来源十分困难，“O”型血人胰腺来源则更难，所以，目前ICA的检测一直未能成为糖尿病诊断中的常规检测指标。
本发明目的在于改进现有技术之缺点，而拓宽抗原来源提供一种采用猪的胰腺切片做抗原，检测人的胰岛细胞抗体，来予测、诊断和鉴别胰岛素依赖型糖尿病，为临床大夫提供重要依据。
本发明目的是这样测定并实现的，取10头健康猪的胰腺切片作抗原；用“O”型血正常人胰腺做抗原测定的ICA及谷氨酸脱氢酶(GAD，由澳大利亚国际糖尿病研究所测定)抗体测定均为阳性并已确诊为IDDM患者的血清20份；非糖尿病患者阴性血清10份；同时设阴性对照切片(用磷酸缓冲液PBS代替血清)。经过多次重复实验，用“O”型血正常人胰腺和健康猪胰腺切片检测ICA，20例IDDM均为阳性，10例非糖尿病患者和阴性对照组均为阴性。因此我们认为用猪胰腺切片替代“O”型血正常人胰腺切片检测胰岛素依赖型糖尿病患者血清中的ICA是可行的。具体步骤为，取猪的新鲜胰腺组织置于波英氏(Bouin’s)液中，在1℃-6℃的条件下放置40-56小时，然后脱水，浸蜡、石蜡包埋，制成1-8微米厚的切片。
本发明方法由于猪胰腺来源广泛，价格低廉，而为广泛性进行ICA的检测开辟了一条新的途径。
下面引用实施例对本发明方法详述如下。
图1为猪胰腺胰岛细胞阳性照片
图2为人胰腺胰岛细胞阳性照片
图3为猪胰腺胰岛细胞阴性照片
实施例
1、取健康猪之新鲜胰腺组织，固定于波英氏(Bouin’s)液中，在4℃冰箱固定48小时，常规脱水，浸蜡、石蜡包埋，制成5微米厚的切片。
2、取待查患者血清；
3、POPA法检测ICA
(1)石蜡切片常规脱蜡，流水冲洗；
(2)0.01M PBS PH7.4洗5′×3次；
(3)分别滴加各稀释度的血清湿盒内，置室温60′或4℃过液；
(4)PBS洗5′×3次；
(5)滴加POPA液30；
(6)PBS洗5′×3次；
(7)0.05M Tris-Hcl buffer液洗5′×2次；
(8)0.025％3′3-2氨基联苯胺液显色1′-5；
(9)封片观察；
(10)结果：
如IDDM患者血清滴加猪胰腺切片或“O”型血正常人胰腺切片，胰岛细胞浆内均有清晰的棕黄色阳性颗粒沉着，背影浅淡清晰，如图1、图2所示(见附件照片1、2)；如滴加非糖尿病患者的血清，其猪胰腺切片胰岛细胞均未着染如图3所示(见附见照片3)，和阴性对照组的人胰腺切片一样，呈阴性反应。
Claims (2)
1、一种检测胰岛细胞抗体的方法，是利用免疫测定法检测胰岛细胞抗体，其特征在于：采用猪的胰腺切片做抗原。
2、根据权利要求1所述的一种检测胰岛细胞抗体的方法，其特征在于：取猪的新鲜胰腺组织置于波英氏 液中，在1℃-6℃的条件下放置40-56小时，然后脱水，浸蜡、石蜡包埋，制成1-8微米厚的切片。
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| CN1193110A CN1193110A (zh) | 1998-09-16 |
| CN1053276C true CN1053276C (zh) | 2000-06-07 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100439920C (zh) * | 2001-11-28 | 2008-12-03 | Rsr有限公司 | 检测与胰腺胰岛细胞抗原分子和/或胰岛素反应的自身抗体 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100439920C (zh) * | 2001-11-28 | 2008-12-03 | Rsr有限公司 | 检测与胰腺胰岛细胞抗原分子和/或胰岛素反应的自身抗体 |
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