CN107153118A - 用于检测及预测氨苯砜超敏综合征的elispot诊断试剂盒 - Google Patents

用于检测及预测氨苯砜超敏综合征的elispot诊断试剂盒 Download PDF

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CN107153118A
CN107153118A CN201710332739.6A CN201710332739A CN107153118A CN 107153118 A CN107153118 A CN 107153118A CN 201710332739 A CN201710332739 A CN 201710332739A CN 107153118 A CN107153118 A CN 107153118A
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elispot
dapsone
diagnostic kits
content
predicting
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王洪生
姜海琴
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Institute of Dermatology and Skin Disease Hospital of CAMS
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Institute of Dermatology and Skin Disease Hospital of CAMS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis

Abstract

本发明公开了一种用于检测及预测氨苯砜超敏综合征的ELISPOT诊断试剂盒，包括氨苯砜。本发明作为检测及预测氨苯砜超敏综合征的试剂盒，具有操作简便、检测快捷、灵敏度高、方便携带的优点，适用于氨苯砜超敏综合征早期诊断的检测。

Description

用于检测及预测氨苯砜超敏综合征的ELISPOT诊断试剂盒
技术领域
本发明涉及一种用于检测及预测氨苯砜超敏综合征的ELISPOT诊断试剂盒。
背景技术
目前，麻风病是由麻风杆菌引起的慢性传染性疾病。麻风杆菌主要侵犯皮肤和神经，造成肢体的畸残，不仅导致患者的劳动力丧失，而且引起社会对患者的恐惧和歧视，造成患者个人、家庭等社会问题。20世纪80年代，世界卫生组织(WHO)推荐联合化疗(MDT)的方案，随着该方案的全球范围内推广应用，大多数国家和地区的麻风病发病率明显降低，我国亦然。氨苯砜(4，4’-Diamonodiphenyl sulfone，DDS)是MDT方案中必需的药物。然而氨苯砜可导致部分患者罹患严重的药物过敏---氨苯砜超敏综合征，该综合征在临床上常表现为：发热、淋巴结肿大、剥脱性皮炎、溶血和肝炎等，严重可导致患者死亡。我们研究显示：我国麻风患者中氨苯砜综合征的发病率约为2％，而死亡率可高达12.5％，该病已成为导致我国麻风病患者死亡的最主要的原因之一。另外，DDS在其他皮肤病的治疗中也有较好的疗效，如疱疹样天疱疮、类天疱疮、聚合性痤疮等；但同样因其具有可引起患者罹患氨苯砜超敏综合征的风险，而致其在临床应用上受阻。
鉴于上述原因，迫切需要用于检测氨苯砜超敏综合征的临床早期诊断试剂盒。
发明内容
本发明要解决的技术问题是提供三种用于检测及预测氨苯砜超敏综合征的ELISPOT诊断试剂盒。
对于第一种用于检测及预测氨苯砜超敏综合征的ELISPOT诊断试剂盒，本发明采用的技术方案是，包括氨苯砜。
作为优选，氨苯砜的浓度为20～40ug/ml。
对于第二种用于检测及预测氨苯砜超敏综合征的ELISPOT诊断试剂盒，本发明采用的技术方案是，由以下所列组分组成：
氨苯砜(20-40ug/mL)、Anti-Human IFN-r、ELISPOT板、生物素连接Human IFN-r、链霉素-HRP、AEC底物反应液、阳性对照(50ng/mL PMA和1ug/mL inomycin)、阴性对照(1640完全培养基)、1640完全培养基、Tween-20、PBS。
作为优选，所述氨苯砜的浓度为20-40ug/mL且含量为100uL；
Anti-Human IFN-r的含量为1mL；
ELISPOT板的规格为96孔；
生物素连接Human IFN-r的含量为1mL；
链霉素-HRP的含量为1mL；
AEC底物反应液的含量为5mL；
阳性对照PMA和inomycin各50uL；
阴性对照的含量为3mL；
1640完全培养基的含量为100mL；
Tween-20的含量为100mL；
PBS的含量为500mL。
作为优选，阳性对照为50ng/mL PMA和1ug/mL inomycin；阴性对照为1640完全培养基。
对于第三种用于检测及预测氨苯砜超敏综合征的ELISPOT诊断试剂盒，本发明采用的技术方案是，由表1所列组分及含量组成。
表1试剂盒成分
本发明的有益效果是：
作为用于检测及预测氨苯砜超敏综合征的ELISPOT诊断试剂盒，具有操作简便、检测快捷、灵敏度高、方便携带的优点，适用于氨苯砜超敏综合征早期诊断的检测。
具体实施方式
一、实验材料
1、单一核细胞：由中国医学科学院皮肤病医院麻风疾控中心提供病人单一核细胞样本，液氮保存待用。
2、选取10例病人、10例健康人细胞：
(1)单一核细胞药物刺激20小时ELISPOT检测
(2)单一核细胞用氨苯砜刺激T细胞增殖7天，ELISPOT检测。
3、试剂盒成分
表1试剂盒成分
二、实验方法
1、抗体包被与封闭：
(1)PBS稀释Anti-Human IFN-r(1∶200)，加之ELISPOT孔板中，每孔100uL，4度过夜孵育；
(2)1640完全培养基洗一遍，再加入1640完全培养基室温封闭2小时。
2、细胞刺激：
(1)1640完全培养基配置氨苯砜(1∶1000)，加入ELISPOT孔板中，每孔100uL；
(2)单一核细胞稀释，加入每孔2X 10*5；
(3)阳性对照100uL，加入每孔2X10*5细胞；
(4)阴性对照100uL，加入每孔2X 10*5细胞；
(5)37℃二氧化碳培养箱孵育16-26小时，水洗两遍，PBS洗3遍。
3、抗体检测
PBS稀释生物素连接Human IFN-r(1∶250)，加入加入ELISPOT孔板中，每孔100uL；室温孵育2小时。水洗两遍，PBS洗3遍。
4、链霉素-HRP
PBS稀释生物素连接链霉素-HRP(1∶200)，加入加入ELISPOT孔板中，每孔100uL；室温孵育1小时。水洗两遍，PBS洗3遍。
5、底物反应
加100uL AEC至每孔，室温孵育15分钟。
三、结果
选取10例病人及10例健康人单一核细胞，比较与氨苯砜共孵育T细胞反应频率，表2表明，在相同药物浓度、相同培养时间及相同培养温度条件下，10例病人及10例健康人在氨苯砜刺激及共孵育7天下T细胞反应频率。通过直观显示氨苯砜刺激及共孵育7天下T细胞反应频率，氨苯砜超敏综合征病人在本试剂盒检测下能灵敏的监测T细胞反应，其斑点形成数远远超过健康人。
表2：各中受试者在药物刺激后T细胞反应
以上所述的本发明实施方式，并不构成对本发明保护范围的限定。任何在本发明的精神和原则之内所作的修改、等同替换和改进等，均应包含在本发明的权利要求保护范围之内。

Claims (10)

1.用于检测及预测氨苯砜超敏综合征的ELISPOT诊断试剂盒，其特征在于，包括氨苯砜。
2.根据权利要求1所述的ELISPOT诊断试剂盒，其特征在于，所述氨苯砜的浓度为20-40ug/mL。
3.用于检测及预测氨苯砜超敏综合征的ELISPOT诊断试剂盒，其特征在于，由以下所列组分组成：
氨苯砜、Anti-Human IFN-r、ELISPOT板、生物素连接Human IFN-r、链霉素-HRP、AEC底物反应液、阳性对照PMA和inomycin、阴性对照、1640完全培养基、Tween-20、PBS。
4.根据权利要求3所述的ELISPOT诊断试剂盒，其特征在于：
所述氨苯砜的浓度为20-40ug/mL且含量为100uL；
所述Anti-Human IFN-r的含量为1mL；
所述ELISPOT板的规格为96孔；
所述生物素连接Human IFN-r的含量为1mL；
所述链霉素-HRP的含量为1mL；
所述AEC底物反应液的含量为5mL；
所述阳性对照PMA和inomycin各50uL；
所述阴性对照的含量为3mL；
所述1640完全培养基的含量为100mL；
所述Tween-20的含量为100mL；
所述PBS的含量为500mL。
5.根据权利要求3或4所述的ELISPOT诊断试剂盒，其特征在于：所述阳性对照为PMA和inomycin。
6.根据权利要求5所述的ELISPOT诊断试剂盒，其特征在于：所述PMA的浓度为50ng/mL，所述inomycin的浓度为1ug/mL。
7.根据权利要求3或4所述的ELISPOT诊断试剂盒，其特征在于：所述阴性对照为1640完全培养基。
8.用于检测及预测氨苯砜超敏综合征的ELISPOT诊断试剂盒，其特征在于，由表1所列组分及含量组成。
表1 试剂盒成分
9.根据权利要求8所述的ELISPOT诊断试剂盒，其特征在于：所述阳性对照为50ng/mLPMA和1ug/mL inomycin。
10.根据权利要求8所述的ELISPOT诊断试剂盒，其特征在于：所述阴性对照为1640完全培养基。
CN201710332739.6A 2017-05-11 2017-05-11 用于检测及预测氨苯砜超敏综合征的elispot诊断试剂盒 Pending CN107153118A (zh)

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CN111234005A (zh) * 2020-03-13 2020-06-05 中国医学科学院皮肤病医院(中国医学科学院皮肤病研究所) 氨苯砜药物超敏反应性t细胞受体及其用途

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