CN106996972B - 适于麻风病早期诊断的elispot试剂盒 - Google Patents

适于麻风病早期诊断的elispot试剂盒 Download PDF

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CN106996972B
CN106996972B CN201710426251.XA CN201710426251A CN106996972B CN 106996972 B CN106996972 B CN 106996972B CN 201710426251 A CN201710426251 A CN 201710426251A CN 106996972 B CN106996972 B CN 106996972B
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early diagnosis
leprosy
elispot
disease early
kit
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CN106996972A (zh
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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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    • 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
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Abstract

本发明公开了一种适于麻风病早期诊断的ELISPOT试剂盒，包括MMP‑2、NDO‑BSA和LID‑1。本发明作为检测及预测麻风病的试剂盒，具有操作简便、检测快捷、灵敏度高、方便携带的优点，适用于麻风病早期诊断的检测。

Description

适于麻风病早期诊断的ELISPOT试剂盒
技术领域
本发明涉及适于麻风病早期诊断的ELISPOT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，主要病变在皮肤和周围神经。临床表现为麻木性皮肤损害，神经粗大，严重者甚至肢端残废。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。本病在世界上流行甚广，我国则流行于广东、广西、四川、云南以及青海等省、自治区。20世纪80年代，世界卫生组织(WHO)推荐联合化疗的方案，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，但是我国每年仍有近900例新发病人被发现，鉴于上述原因，迫切需要用于检测麻风病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适于麻风病早期诊断的ELISPOT试剂盒。
对于第一种适于麻风病早期诊断的ELISPOT试剂盒，本发明采用的技术方案是，包括MMP-2、NDO-BSA和LID-1。
作为优选，MMP-2，NDO-BSA，LID-1的浓度均为20～40ug/mL。
对于第二种适于麻风病早期诊断的ELISPOT试剂盒，由以下所列组分组成：
MMP-2，NDO-BSA，LID-1，Anti-Human IFN-γ，ELISPOT板，生物素连接Anti-HumanIFN-γ，链霉素-HRP，AEC底物反应液，阳性对照PMA和ionomycin，阴性对照，1640完全培养基，Tween-20，PBS。
作为优选：
MMP-2、NDO-BSA、LID-1的浓度均为20～40ug/mL且含量均为100uL；
Anti-Human IFN-γ的含量为1mL；
ELISPOT板的规格为96孔；
生物素连接Anti-Human IFN-γ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ionomycin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。
对于第三种适于麻风病早期诊断的ELISPOT试剂盒，由表1所列组分及含量组成。
表1试剂盒成分
作为优选，阳性对照为50ng/mL PMA和1ug/mL ionomycin。
作为优选，阴性对照为1640完全培养基。
本发明的有益效果是：
作为检测及预测麻风病试剂盒，具有操作简便、检测快捷、灵敏度高、方便携带的优点，适用于麻风病早期诊断的检测。
具体实施方式
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例瘤型病人、10例结核样型病人、10例健康人细胞：单一核细胞药物刺激20小时ELISPOT检测。
3、试剂盒成分(见表1)
表1试剂盒成分
二、实验方法
1、抗体包被与封闭：
(1)以PBS按200∶1的体积比例稀释Anti-Human IFN-γ，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)以1640完全培养基按1000∶1的体积比例分别配置MMP-2、NDO-BSA和LID-1，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2X 10*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
以PBS按250∶1的体积比例稀释生物素连接Anti-Human IFN-γ，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
以PBS按200∶1的体积比例稀释生物素连接链霉素-HRP，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、结果与讨论
选取10例瘤型病人、10例结核样型病人及10例健康人的单一核细胞，比较与MMP-2，NDO-BSA，LID-1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例瘤型病人、10例结核样型病人及10例健康人在MMP-2，NDO-BSA，LID-1分别刺激下T细胞反应频率。通过直观显示MMP-2，NDO-BSA，LID-1刺激下T细胞反应频率，麻风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
注：表1中所有受试者的细胞类型均为单一细胞核。
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 (2)

1.适于麻风病早期诊断的ELISPOT试剂盒，其特征在于：
表1 试剂盒成分
所述的ELISPOT试剂盒由表1所列组分及含量组成。
2.根据权利要求1所述的ELISPOT试剂盒，其特征在于：所述阳性对照为50ng/mL PMA和1ug/mL ionomycin。
CN201710426251.XA 2017-06-07 2017-06-07 适于麻风病早期诊断的elispot试剂盒 Active CN106996972B (zh)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Differential Interferon-g Production Characterizes the Cytokine Responses to Leishmania and Mycobacterium leprae Antigens in Concomitant Mucocutaneous Leishmaniasis and Lepromatous Leprosy;Denise S et al;《Clinical Infectious Diseases》;20041222;第40卷;摘要，e8页右栏最后一段至e8页左栏第一段 *
New Biomarkers with Relevance to Leprosy Diagnosis Applicable in Areas Hyperendemic for Leprosy;Annemieke Geluk et al;《J Immunol》;20120413;第188卷(第10期);摘要，4783页右栏最后一段至4784页左栏第一段，4788页左栏 *
Postgenomic Mycobacterium leprae antigens for cellular and serological diagnosis of M. leprae exposure, infection and leprosy disease;Annemieke Geluk et al;《Lepr Rev》;20111231;第82卷;416页最后一段至417页第3段 *
Synergistic antigen combinations for the development of interferon gamma release assays for paucibacillary leprosy;Oliveira RM et al;《Eur J Clin Microbiol Infect Dis.》;20140312;第33卷(第8期);1415-24 *

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