CN113683688B - 抗人类免疫缺陷病毒ⅰ型p24抗原（hiv-1 p24）兔单克隆抗体及其应用 - Google Patents
抗人类免疫缺陷病毒ⅰ型p24抗原（hiv-1 p24）兔单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域，公开了兔抗人类免疫缺陷病毒Ⅰ型P24抗原(HIV‑1P24)单克隆抗体OTIR5D12和OTIR2C5,所述的抗体是利用分选特异B细胞并培养筛选及分子克隆重组产生。所述兔单克隆抗体OTIR5D12和OTIR2C5的免疫原为原核细胞大肠杆菌表达的HIV‑1P24全长蛋白，这2个兔单克隆抗体可以识别HIV‑1P24蛋白表面的不同抗原决定簇，其OTIR5D12抗体轻链(VL)的氨基酸序列如SEQIDNO.1所示；OTIR5D12抗体的重链(VH)的氨基酸序列如SEQIDNo.2所示。其OTIR2C5轻链(VL)的氨基酸序列如SEQIDNO.11所示；OTIR2C5的重链(VH)的氨基酸序列如SEQIDNo.12所示。本发明的抗人类免疫缺陷病毒Ⅰ型P24抗原(HIV‑1P24)的兔单克隆抗体可应用于生物样本中HIV‑1P24蛋白或慢病毒颗粒滴定的测定，包括但不限于双抗体夹心ELISA或化学发光法试剂盒的制备。
Description
技术领域
本发明属于生物技术领域，尤其涉及抗人类免疫缺陷病毒Ⅰ型P24抗原 (HIV-1P24)兔单克隆抗体及其在免疫检测方面应用。
背景技术
1型人类免疫缺陷病毒(HIV-1)的gag基因编码一种称为Pr55Gag的前体蛋白。病毒蛋白酶PR切割此前体产生p17、p24、p7和p6蛋白质，这些蛋白质是病毒颗粒组装所必需的。p24是一种主要的病毒核心结构蛋白，HIV包膜蛋白、gpl20、gp41或它们的前体蛋白gpl60拥有最大程度的基因变异，然而HIV-1 P24 抗原在不同的HIV类型中是最保守的蛋白质。因此，检测HIV-1 P24可以更广泛地进行HIV基因型的检测。它的检测通常被用作HIV-1感染和病毒载量的指标。
中国专利关于HIV-1 P24兔单克隆抗体的条目基乎没有，国际专利申请号 US5,173,399(Mouse monoclonal antibody to HIV-1 P24 and their use in diagnostictests)公布了2个鼠单克隆抗体，31-42-19由杂交瘤ATCC HB 9726,31-90-25由杂交瘤ATCCHB 9725产生，未提到兔单克隆抗体及抗体CDR序列。国际专利申请号US 2021/0040185 A1(Anti HIV antibodies)公布了抗HIV的四种抗体 L1A1,L1A4,L1A2,L2A1，原始来源是从捐赠者中发现，专利中抗体是通过分子克隆重组表达获得的人源化抗HIV抗体，而且专利中未提到是抗HIV-1 P24的抗体。
发明内容
本发明的目的是提供特异性好、亲和力高的抗HIV-1 P24兔单克隆抗体 OTIR5D12和OTIR2C5及其在双抗体夹心ELISA或化学发光法制备的免疫检测试剂盒中的应用，可应用于生物样本中HIV-1 P24蛋白或慢病毒颗粒滴定的细胞培养上清中HIV-1 P24抗原的定量检测。
所述兔单克隆抗体为兔单克隆抗体OTIR5D12或兔单克隆抗体OTIR2C5。
所述兔单克隆抗体OTIR5D12和兔单克隆抗体OTIR2C5以原核细胞大肠杆菌表达的全长HIV-1 P24蛋白为免疫原。通过免疫注射新西兰大白兔，取免疫兔子外周血单核细胞(PMBCs)，分选特异性B细胞培养、筛选并利用分子克隆重组技术获得。
所述兔单克隆抗体OTIR5D12，其轻链可变区(VL)含109aa，其氨基酸序列如SEQ IDNO.1所示；所述抗体的重链(VH)含115aa，其氨基酸序列如SEQ IDNo.2所示。
所述兔单克隆抗体OTIR5D12，其中VL区域包括3个抗原决定簇：CDR1、 CDR2和CDR3，抗原决定簇的区域相应地分别为27aa-34aa，52aa-54aa和 91aa-99aa。其氨基酸序列分别如SEQ ID No.3-5所示。
所述兔单克隆抗体OTIR5D12，其中VH区域包括3个抗原决定簇：CDR1、 CDR2和CDR3，抗原决定簇的区域相应地分别为25aa-32aa，50aa-56aa和 93aa-104aa。其氨基酸残基序列分别如SEQ ID No.6-8所示。
所述兔单克隆抗体OTIR2C5，其轻链可变区(VL)含106aa，其氨基酸序列如SEQ IDNO.11所示；所述抗体的重链(VH)含113aa，其氨基酸序列如SEQ IDNo.12所示。
所述兔单克隆抗体OTIR2C5，其中VL区域包括3个抗原决定簇：CDR1、 CDR2和CDR3，抗原决定簇的区域相应地分别为27aa-32aa，50aa-52aa和 89aa-96aa。其氨基酸序列分别如SEQ ID No.13-15所示。
所述兔单克隆抗体OTIR2C5，其中VH区域包括3个抗原决定簇：CDR1、 CDR2和CDR3，抗原决定簇的区域相应地分别为25aa-32aa，50aa-56aa和 93aa-102aa。其氨基酸残基序列分别如SEQ ID No.16-18所示。
所述的兔单克隆抗体包括兔单克隆抗体OTIR5D12或兔单克隆抗体 OTIR2C5可以与HIV-1 P24高特异性结合，可通过本领域技术人员所公知的的方法，制备成检测HIV-1P24的各种免疫检测试剂盒。尤其是，应用于双抗体夹心ELISA或化学发光法制备的免疫检测试剂盒中。双抗体夹心ELISA检测HIV-1 P24标准品，检测灵敏度能达到1.61pg/ml。
附图说明
图1为兔单克隆抗体OTIR5D12和OTIR2C5重链和轻链全长扩增产物的电泳图，M为DNA分子量Marker。
图2为双抗体夹心ELISA法检测HIV-1 P24标准曲线，横坐标为HIV-1 P24 蛋白浓度，纵坐标为检测的OD值。标准曲线的R2＝0.9983，线性检测范围 0-200pg/mL，推出样品浓度计算公式：y＝0.01591x+0.0254。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件、实验室手册中所述的条件或按照制造厂商所建议的条件。
实施例1:抗HIV-1 P24兔单克隆抗体的制备
一、HIV-1 P24重组蛋白的制备
从Genebank中选调HIV-1 P24基因，该基因编码HIV-1 P24全长231个氨基酸(aa)。根据核苷酸序列合成HIV-1 P24基因，设计两条引物并分别引入酶切位点SgfI和MluI，克隆入表达载体pET23a-N-His，构建重组HIV-1 P24表达质粒。采用本领域技术人员所知的技术方法将HIV-1 P24重组表达质粒转染 E.coli细胞，裂解离心获得可溶性蛋白，经镍柱亲和层析柱纯化，获得纯化的 HIV-1 P24重组蛋白。SDS-PAGE电泳鉴定纯度。电泳后从凝胶上观察到分子量为26kDa左右的目的蛋白条带，纯度在85％以上，达到了制备单抗的纯度要求。
二、动物免疫
上述纯化的HIV-1 P24重组蛋白以完全弗氏佐剂乳化，采用皮下注射方法免疫2kg左右的新西兰大白兔，免疫剂量为500μg/只，间隔两周后进行第二次免疫，以不完全弗氏佐剂乳化，免疫剂量为250μg/只。免疫两次后取尾血以 ELISA法梯度稀释测定血清效价；依据ELISA效价128000时的OD450大于 1.0为标准，根据结果判定是否收集PBMCs或是继续免疫，选取抗体效价最高的兔子进行PBMCs收集。
三、PBMCs分离、特异性B细胞分选、克隆重组将兔仰卧固定在手术台上，将心脏部位被毛减掉，用酒精消毒皮肤，选择心搏最明显处用50ml注射器穿刺，针头刺入心脏后即有血液涌入注射器，取得所需血量后迅速将针头拔出，将注射器中的全血转入无菌50ml管中，与等量的PBS混匀后逐滴缓慢的加入到淋巴细胞分离液上方，室温400×g离心30分钟，离心后，液面由上至下分为四层：黄色血浆层、白色薄膜层即单个核细胞层、分离液层及红细胞层，小心吸取单个核细胞层并用PBS洗涤去除血小板和淋巴细胞分离液后即可获得兔PBMCs。从兔PBMCs中继续分选抗原特异性的B细胞进行培养，培养后的B 细胞上清用抗原包被的ELISA板筛选阳性克隆。阳性克隆的细胞收集裂解后提取RNA并反转录成cDNA,天然配对的兔单克隆抗体轻重链全长序列从对应阳性克隆的cDNA中被扩增出来，通过克隆重组方法构建兔单克隆抗体表达载体，并经测序确定序列。扩增的全长PCR产物结果如图1。
四、单克隆抗体的制备和纯化为了获得多株识别HIV-1 P24蛋白的兔单克隆抗体，本发明将兔单克隆抗体重链、轻链基因装载在表达载体上，将质粒转染 KEK293细胞；转染120-144小时获得培养上清中含有重组的识别HIV-1 P24蛋白的兔单克隆抗体。收取细胞悬液，离心取上清，亲和层析法进行抗体纯化。以BCA法测定纯化后的单抗浓度，再分装、冻干。
实施例2:抗HIV-1 P24兔单克隆抗体OTIR5D12或OTIR2C5的鉴定
一、兔单克隆抗体的效价
采用倍比稀释法稀释兔单克隆抗体OTIR5D12或OTIR2C5，用间接ELISA 测抗体效价，结果显示本发明涉及的兔单克隆抗体OTIR5D12或OTIR2C5效价分别为6.6×106或3.3×106。
二、抗体配对
为了挑选包被抗体和检测抗体的最佳组合，采用棋盘组合，将获得的多株 HIV-1P24兔单克隆抗体相互配对。基本步骤：多株单克隆抗体分别包被酶标板， 4℃过夜。第二天取出酶标板，PBST洗涤一次，1％BSA溶液37℃封闭2小时， PBST洗涤3次；每孔加入HIV-1P24蛋白100μl，浓度分别为500、250、125、 62.5、31.25、15.625、7.8125、3.90625和0pg/ml，37℃孵育1小时；孵育完成后取出酶标板，PBST洗涤3次，分别加入生物素标记的上述多株抗体作为检测抗体，37℃孵育1小时；孵育完成后取出酶标板，PBST洗涤3次，分别加入 HRP标记的Avidin，37℃孵育30min。PBST洗涤5次，加入TMB底物，37℃显色10min。取出后加终止液，在酶标仪上测定OD450读数。根据样品的OD 值与阴性对照的背景值，选出最为理想的兔单克隆抗体对，配对筛选结果见表1。本发明涉及的抗体OTIR5D12作为包被抗体，抗体OTIR2C5作为检测抗体最佳。抗人类免疫缺陷病毒Ⅰ型P24抗原(HIV-1 P24)兔单克隆抗体OTIR5D12和 OTIR2C5可以识别人类免疫缺陷病毒Ⅰ型P24蛋白表面的不同抗原决定簇。
表1抗体配对实验筛选结果
实施例3:兔单克隆抗体OTIR5D12或OTIR2C5可变区基因与氨基酸序列分析
分别以OTIR5D12或OTIR2C5抗体的重组质粒为DNA模板，根据模板上轻链及重链5'端载体序列设计轻链可变区及重链可变区测序引物，采用测序仪 ABI3730进行测序。经测序获得兔单克隆抗体OTIR5D12和OTIR2C5轻链及重链可变区的核苷酸序列。
利用互联网，在http://www.imgt.org上使用IMGT/V-QUEST分析软件，分别将轻链与重链的核苷酸序列进行测序结果数据分析，得到兔单克隆抗体 OTIR5D12的轻链氨基酸序列如SEQ ID NO.1所示，重链氨基酸序列如 SEQ ID NO.2所示。VL全长为109个氨基酸，其FR的4个结构域氨基酸数分别为26、17、36和10，CDR的3个结构域氨基酸数分别为8、3和9，CDR1、 CDR2和CDR3的区域相应地分别为27aa-34aa，52aa-54aa和91aa-99aa，其氨基酸序列分别:QSVYNSNW、KAS、QGGYNNGD。
通过分析，所述兔单克隆抗体OTIR5D12VH全长为115个氨基酸，其FR 的4个结构域氨基酸数分别为24、17、36和11，CDR的3个结构域氨基酸数分别为8、7和12，CDR1、CDR2和CDR3分别为25aa-32aa，50aa-56aa和 93aa-104aa，其氨基酸序列分别为GFSLNSYY、LDTSGST、ARGAYYSAYGDA。
通过分析，所述兔单克隆抗体OTIR2C5的轻链氨基酸序列如SEQ ID NO.11 所示，重链氨基酸序列如SEQ ID NO.12所示。VL全长为106个氨基酸，其FR 的4个结构域氨基酸数分别为26、17、36和10，CDR的3个结构域氨基酸数分别为6、3和8，CDR1、CDR2和CDR3的区域相应地分别为27aa-32aa，50aa-52aa 和89aa-96aa，其氨基酸序列分别:QSISSW、SAS、QSYYGGNY。
通过分析，所述兔单克隆抗体OTIR2C5 VH全长为113个氨基酸，其FR 的4个结构域氨基酸数分别为24、17、36和11，CDR的3个结构域氨基酸数分别为8、7和10，CDR1、CDR2和CDR3分别为25aa-32aa，50aa-56aa和 93aa-102aa，其氨基酸序列分别为GIDLSSNV、SSLNGGT、ARGDGSAYYL。
实施例4:抗HIV-1 P24兔单克隆抗体OTIR5D12或OTIR2C5用于制备双抗体夹心ELISA检测试剂盒
采用本领域技术人员所公知的基于ELISA双抗体夹心法原理的检测技术，制作HIV-1 P24检测试剂盒，用于生物样本中HIV-1 P24蛋白或慢病毒颗粒滴定的测定。
一、试剂盒组成
1.包被OTIR5D12抗体的板条：用PBS缓冲液稀释抗体至5μg/ml，包被到微孔板上，每孔100μl，4℃孵育过夜，以PBST洗涤3次，甩干；加含有1％ BSA、5％蔗糖、0.05％Proclin300的PBS进行封闭，37℃反应2h，弃去孔内封闭液，甩干；将包被板置于37℃烘箱2h，即完成包被，以铝箔袋密封，存放于 4℃保存备用。
2.试剂配制
1.生物素结合物配制采用Thermo Scientific的EZ-LinkTM Sulfo-NHS-LC-Biotin标记抗HIV-1 P24兔单克隆抗体OTIR2C5，滴定工作浓度，配制成1:20的浓缩液，稀释液为含1％BSA、5％甘油、0.05％Proclin 300的PBS，过滤除菌。
2.洗涤缓冲液为常规的pH7.4的PBST，含0.05％Proclin300，配制成20 倍浓缩液。
3.HRP标记的Avidin配制成1:20的浓缩液，稀释液为含1％BSA、5％甘油、0.05％Proclin 300的PBS，过滤除菌。
4.酶的底物液(显色液)单组分TMB(从市场采购)。
5.样本稀释液含1％BSA、0.05％Proclin 300的PBS，过滤除菌。
6.终止液用10ml HCl(36-38％)加入110ml蒸馏水中，混合过程需缓慢进行，配置成1N的HCl。
7.标准品HIV-1 P24重组蛋白，以含1％BSA、5％蔗糖、10％甘油、 0.05％Proclin300的PBS为稀释液，将样品稀释为20μg/ml，过滤除菌，无菌分装。
二、检测操作要点
1、为保证检测结果的准确性，建议标准品及样本均设双孔测定。每次检测均需做标准曲线。
2、如标本中待测物质含量过高，请先用样本稀释液进行稀释，以使样本符合试剂盒的检测范围，最后计算时再乘以相应的稀释倍数。
3、加样：加样时，请使用一次性的洁净吸头，避免交叉污染。加样时应尽量轻缓，避免起泡，将样本加于酶标板孔底部，切勿沿孔壁加样。
4、温育：为防止样本蒸发或污染，温育过程中酶标板必须覆上板贴，实验过程中酶标板应避免处于干燥的状态。温育过程中应随时观察温箱温度是否恒定于37℃，及时调整。温育过程中，温箱不易开启太多次，以免影响温度平衡。
5、洗涤：洗涤过程非常重要，不充分的洗涤易造成假阳性。
6、显色：为保证实验结果的准确性，底物反应时间到后应尽快加入终止液。可在加入底物溶液后每隔一段时间观察一下显色情况以控制反应时间(比如每隔 10分钟)。当肉眼可见标准品前3-4孔有明显梯度蓝色，后3-4孔显色不明显时，即可加入终止液终止反应，此时蓝色立刻变为黄色。终止液的加入顺序应尽量与底物溶液的加入顺序相同。
7、底物溶液应为浅蓝色或无色，如果颜色严重变深则必须弃用。底物溶液易受污染，请避光妥善保存。
三、对慢病毒滴度的测定
上述制备的检测HIV-1 p24蛋白双抗体夹心ELISA检测试剂盒从4度冰箱取出，平衡到室温。将标准品用PBS从20μg/ml稀释到200pg/ml，制备成0、 12.5、25、50、100、200pg/ml共6个系列浓度标准品。2个灭活的慢病毒颗粒样品，用空培养基分别稀释2500倍、5000倍，以OTIR5D12抗体为包被抗体、 OTIR2C5抗体为检测抗体，用上述检测方法检测2个慢病毒颗粒样本，检测结果见表2。本发明所述的兔单克隆抗体可对慢病毒颗粒滴定的细胞培养上清中 HIV-1 p24蛋白进行定量检测。依据表2制作HIV-1 P24检测标准曲线，标准曲线的R2＝0.9983，线性检测范围0-200pg/mL，推出样品浓度计算公式：y＝0.01591x +0.0254，检测灵敏度能达到1.61pg/ml，见图2。根据标准曲线，计算灭活的慢病毒颗粒样品中HIV-1P24的浓度分别为106384pg/ml、97358pg/ml，对应的慢病毒滴度分别为1.06x107TU/mL、0.97x107TU/mL(用公式107TU/mL＝1x105 pg/mL计算)。
表2.双抗体夹心ELISA法检测灭活的慢病毒颗粒样品
OTIR5D12序列表
<110> 无锡傲锐东源生物科技有限公司
<120> 抗人类免疫缺陷病毒Ⅰ型P24抗原（HIV-1 P24）兔单克隆抗体及其应用
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1 5 10 15
Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Tyr Asn Ser
20 25 30
Asn Trp Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu
35 40 45
Leu Ile Tyr Lys Ala Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe
50 55 60
Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Val
65 70 75 80
Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Gly Tyr Asn Asn
85 90 95
Gly Asp Gly Phe Gly Gly Gly Thr Glu Val Val Val Lys
100 105
<210> 2
<211> 115
<212> PRT
<213> Oryctolagus cuniculus (rabbit)
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1 5 10 15
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20 25 30
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35 40 45
Ile Leu Asp Thr Ser Gly Ser Thr Asp Tyr Ala Ser Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Val Thr
65 70 75 80
Ser Pro Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Ala
85 90 95
Tyr Tyr Ser Ala Tyr Gly Asp Ala Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 3
<211> 8
<212> PRT
<213> Oryctolagus cuniculus (rabbit)
<400> 3
Gln Ser Val Tyr Asn Ser Asn Trp
1 5
<210> 4
<211> 3
<212> PRT
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Lys Ala Ser
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<210> 5
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Gln Gly Gly Tyr Asn Asn Gly Asp Gly
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Gly Phe Ser Leu Asn Ser Tyr Tyr
1 5
<210> 7
<211> 7
<212> PRT
<213> Oryctolagus cuniculus (rabbit)
<400> 7
Leu Asp Thr Ser Gly Ser Thr
1 5
<210> 8
<211> 12
<212> PRT
<213> Oryctolagus cuniculus (rabbit)
<400> 8
Ala Arg Gly Ala Tyr Tyr Ser Ala Tyr Gly Asp Ala
1 5 10
OTIR2C5序列表
<110> 无锡傲锐东源生物科技有限公司
<120> 抗人类免疫缺陷病毒Ⅰ型P24抗原（HIV-1 P24）兔单克隆抗体及其应用
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Ala Phe Glu Leu Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser Trp
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys
65 70 75 80
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Tyr Gly Gly Asn Tyr
85 90 95
Phe Gly Gly Gly Thr Glu Val Val Val Lys
100 105
<210> 12
<211> 113
<212> PRT
<213> Oryctolagus cuniculus (rabbit)
<400> 12
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Gly Ser
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Asn Val
20 25 30
Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45
Ile Ser Ser Leu Asn Gly Gly Thr Tyr Tyr Ala Ser Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Glu Leu Lys Ile Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Asp
85 90 95
Gly Ser Ala Tyr Tyr Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser
<210> 13
<211> 6
<212> PRT
<213> Oryctolagus cuniculus (rabbit)
<400> 13
Gln Ser Ile Ser Ser Trp
1 5
<210> 14
<211> 3
<212> PRT
<213> Oryctolagus cuniculus (rabbit)
<400> 14
Ser Ala Ser
1
<210> 15
<211> 8
<212> PRT
<213> Oryctolagus cuniculus (rabbit)
<400> 15
Gln Ser Tyr Tyr Gly Gly Asn Tyr
1 5
<210> 16
<211> 8
<212> PRT
<213> Oryctolagus cuniculus (rabbit)
<400> 16
Gly Ile Asp Leu Ser Ser Asn Val
1 5
<210> 17
<211> 7
<212> PRT
<213> Oryctolagus cuniculus (rabbit)
<400> 17
Ser Ser Leu Asn Gly Gly Thr
1 5
<210> 18
<211> 10
<212> PRT
<213> Oryctolagus cuniculus (rabbit)
<400> 18
Ala Arg Gly Asp Gly Ser Ala Tyr Tyr Leu
1 5 10
Claims (5)
1. 抗人类免疫缺陷病毒Ⅰ型P24抗原（HIV-1 P24）的兔单克隆抗体，其特征在于：所述兔单克隆抗体为兔单克隆抗体OTIR5D12或兔单克隆抗体OTIR2C5；
所述兔单克隆抗体OTIR5D12包含重链可变区和轻链可变区，其中所述重链可变区包含分别如SEQ ID No.6-8所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ IDNo.3-5所示的LCDR1、LCDR2和LCDR3；
所述兔单克隆抗体OTIR2C5包含重链可变区和轻链可变区，其中所述重链可变区包含分别如SEQ ID No.16-18所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ IDNo.13-15所示的LCDR1、LCDR2和LCDR3。
2. 根据权利要求1所述的抗人类免疫缺陷病毒Ⅰ型P24抗原（HIV-1 P24）兔单克隆抗体，其特征在于：所述兔单克隆抗体以原核细胞大肠杆菌表达的全长HIV-1 P24蛋白为免疫原，通过免疫注射新西兰大白兔，取免疫兔子外周血单核细胞，分选特异性B细胞培养、筛选并利用分子克隆重组技术获得。
3. 根据权利要求1所述的抗人类免疫缺陷病毒Ⅰ型P24抗原（HIV-1 P24）兔单克隆抗体，其特征在于：所述兔单克隆抗体为兔单克隆抗体OTIR5D12，其轻链可变区氨基酸序列如SEQ ID NO.1所示，其重链可变区氨基酸序列如 SEQ ID No.2所示。
4. 根据权利要求1所述的抗人类免疫缺陷病毒Ⅰ型P24抗原（HIV-1 P24）兔单克隆抗体，其特征在于：所述兔单克隆抗体为兔单克隆抗体OTIR2C5，其轻链可变区氨基酸序列如SEQ ID NO.11所示，其重链可变区氨基酸序列如 SEQ ID No.12所示。
5.权利要求1所述的抗人类免疫缺陷病毒Ⅰ型P24抗原（HIV-1 P24）兔单克隆抗体在制备用于人类免疫缺陷病毒Ⅰ型P24抗原（HIV-1 P24）免疫检测试剂盒中的应用。
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| 四种HIV抗体确证试剂盒用于HIV感染检测性能的比较研究;夏燕等;中华实验和临床病毒学杂志;第35卷(第2期);全文 * |
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