CN117024582A - 一种抗人rbp4单克隆抗体及其应用 - Google Patents
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Abstract
本发明公开了一种抗人RBP4单克隆抗体及其应用，该单克隆抗体包含重链可变区和轻链可变区，所述重链可变区和轻链可变区各自具有3个CDR区，重链可变区CDR1‑3的氨基酸序列分别如SEQ ID NO.1‑3所示，所述轻链可变区CDR1‑3的氨基酸序列分别如SEQ ID NO.4‑6所示。该抗人RBP4单克隆抗体与抗人RBP4多克隆抗体混合物用于制备胶乳比浊试剂盒，可以消除现有技术中采用多克隆抗体制备检测试剂产生的批间差。
Description
技术领域
本发明属于生物医学技术领域，尤其是一种抗人RBP4单克隆抗体及其应用
背景技术
RBP是肝细胞、小肠及其他组织细胞合成，广泛存在于血浆及其他体液中的一类蛋白。血浆中主要是RBP4，分子量大约21kD，其主要功能与前白蛋白共同和维生素A结合、转运，并保护其不被氧化。血浆中RBP4分子量小，可自由滤过肾小球，但原尿中99％的RBP4被近曲小管上皮细胞重吸收并分解，仅微量从尿排泄。
维生素A缺乏症、低蛋白血症、肝病、甲状旁腺功能亢进、吸收不良综合征等，可出现血清RBP4降低。而血清RBP4升高多见于各种原因所致肾小球滤过功能损伤，也见于过量摄入维生素A、营养过剩性脂肪肝等。
目前检测RBP4血清含量主要是采用胶乳比浊法，需要使用多克隆抗体来制备检测试剂。以兔多克隆抗体为主。但是多克隆抗体主要来源于免疫后的动物血清，因此抗体的质量容易受到动物状态，免疫工艺，免疫抗原以及佐剂等影响，具有难以控制的批间差。
发明内容
针对上述现有技术中存在的问题，本发明的目的在于提供一种抗人RBP4单克隆抗体及其应用。
本发明具体技术方案如下：
本发明第一方面提供一种抗人RBP4单克隆抗体，所述单克隆抗体包含重链可变区和轻链可变区，所述重链可变区和轻链可变区各自具有3个CDR区，其中：
所述重链可变区CDR1的氨基酸序列为：NYWMN(SEQ ID NO.1)，
所述重链可变区CDR2的氨基酸序列为：MIDPSDSETHYNQMFK(SEQ ID NO.2)，
所述重链可变区CDR3的氨基酸序列为：SPQLGDYILDY(SEQ ID NO.3)，
所述轻链可变区CDR1的氨基酸序列为：KASQDINKYIA(SEQ ID NO.4)，
所述轻链可变区CDR2的氨基酸序列为：YTSTLQP(SEQ ID NO.5)，
所述轻链可变区CDR3的氨基酸序列为：LQYDNLLWA(SEQ ID NO.6)。
进一步地，所述重链的氨基酸序列如SEQ ID NO.7所示，和/或所述轻链的氨基酸序列如SEQ ID NO.8所示。
本发明第二方面提供一种多核苷酸，所述多核苷酸包含编码所述单克隆抗体的核苷酸序列。
进一步地，编码所述单克隆抗体重链的核苷酸序列如SEQ ID NO.9所示，和/或编码所述单克隆抗体轻链的核苷酸序列如SEQ ID NO.10所示。
本发明第三方面提供所述的抗人RBP4单克隆抗体在非诊断目的的人RBP4检测中的应用，或者在制备检测人RBP4的试剂盒中的应用。
本发明第四方面提供一种检测人RBP4的胶乳比浊试剂盒，所述试剂盒包含所述的抗人RBP4单克隆抗体。
进一步地，所述试剂盒还包含试剂R1和试剂R2；
所述试剂R1包括pH7.0-8.0的4-羟乙基哌嗪乙磺酸，NaCl，聚乙二醇-8000，NaN3；
优选地，所述试剂R1包括pH7.0-8.0的4-羟乙基哌嗪乙磺酸，100-150mMNaCl，1-5g/L聚乙二醇-8000，0.9g/LNaN3；
所述试剂R2包括pH6.0-7.0的MES缓冲溶液，抗人RBP4单克隆抗体与抗人RBP4多克隆抗体包被的胶乳。
进一步地，所述试剂盒还包含校准品。
本发明的有益效果为：
本发明提供一种抗人RBP4单克隆抗体，其重链可变区和轻链可变区各自具有3个CDR区，重链可变区CDR1-3的氨基酸序列分别如SEQ ID NO.1-3所示，轻链可变区CDR1-3的氨基酸序列分别如SEQ ID NO.4-6所示。该抗人RBP4单克隆抗体与抗人RBP4多克隆抗体混合物用于制备胶乳比浊试剂盒，可以消除现有技术中采用多克隆抗体制备检测试剂产生的批间差。
附图说明
图1为单抗+多抗组与纯多抗组临床样品相关性曲线图。
具体实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1：单克隆抗体的制备和纯化
(1)免疫动物
使用6-8周龄雌性BALB/c小鼠，按照预先制定的免疫方法进行三次免疫注射。
第一次免疫用2mL注射器每只小鼠腹股沟皮下注射0.2mL乳化液(100μL重组蛋白RBP4(用1×PBS稀释)+100μL弗氏完全佐剂，含100μg抗原)。
第二次免疫间隔两周，用2mL注射器每只小鼠腹腔注射0.2mL乳化液(100μL重组蛋白RBP4(用1×PBS稀释)+100μL弗氏不完全佐剂，含100μg抗原)。
第三次免疫间隔两周，用2mL注射器每只小鼠腹股沟皮下注射注射0.2mL乳化液(100μL重组蛋白RBP4(用1×PBS稀释)+100μL弗氏不完全佐剂，含100μg抗原)。
测试ELISA效价根据融合时间安排，在第三次免疫7-10天后取血，通过常规的间接ELISA法检测小鼠血清的效价，选择效价达到1:100000的小鼠用于之后细胞融合。
在细胞融合前3天，对筛选出的小鼠进行加强免疫，小鼠腹腔注射200μL液体(含60μg抗原，溶剂为1×PBS，pH7.4)。
(2)细胞融合
采用眼球摘除放血法处死小鼠，无菌操作取出脾脏，放入带200目不锈钢网的平皿中研磨，制备细胞悬液。将准备好的同系骨髓瘤细胞SP2/0与小鼠脾细胞按一定比例混合(1:5-1:10)，并加入促融合剂聚乙二醇(50％)。采用HAT选择培养基，进行杂交瘤细胞的选择性培养和筛选。
用ELISA方法检测杂交瘤细胞培养上清：将抗原用包被液(pH9.6，0.05M碳酸盐缓冲液)稀释为2μg/mL加入96孔酶标板中，100μL/孔，放入4℃冰柜中包被过夜。弃去包被液，用PBST(磷酸盐缓冲液，pH7.4)洗板三次，加入封闭液3％BSA-PBST(加入3％牛血清白蛋白的磷酸盐缓冲液)，200μL/孔，37℃孵育1小时。弃去封闭液，PBST(磷酸盐缓冲液，pH7.4)洗板一次，甩干板子。将杂交瘤细胞培养上清加入酶标板100μL/well，同时加入PBST(磷酸盐缓冲液)作为阴性对照。37℃孵育1小时。弃上清，用洗板机PBST洗板三次，加入稀释好的anti-mouseIgG/HRP，100μL/well，37℃孵育1小时。弃上清，用洗板机PBST洗板三次，加底物TMB100μL/well，RT，避光37℃，10-15min后加入50μL终止液(2mol/L硫酸)。使用酶标仪测定，酶标仪设置为：450nm比色。和阴性对照相比，OD值为2.5倍之上即为阳性。最后筛选得到一株效价最好的抗RBP4的杂交瘤细胞株，命名为LC023。随后进一步对上述杂交瘤细胞株送测序公司进行抗体基因序列测序分析。
经测序分析，获得的抗人RBP4单克隆抗体LC023，包含重链和轻链，其中重链可变区CDR1的氨基酸序列如SEQ ID NO.1所示(NYWMN)、CDR2的氨基酸序列如SEQ ID NO.2所示(MIDPSDSETHYNQMFK)，和CDR3的氨基酸序列如SEQ ID NO.3所示
(SPQLGDYILDY)，轻链可变区CDR1的氨基酸序列如SEQ ID NO.4所示(KASQDINKYIA)、CDR2的氨基酸序列如SEQ ID NO.5所示(YTSTLQP)，和CDR3的氨基酸序列如SEQ ID NO.6所示(LQYDNLLWA)。
抗人RBP4单克隆抗体重链的氨基酸序列(SEQ ID NO.7所示)：
QVQLQQPGAELVRPGASVKLSCKASGYTFTNYWMNWVKQRPGQGLRWIGMIDPSDSETHYNQMFKDKATLTVDKSSSTANMQLSSLTSEDSAVYYCARSPQLGDYILDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWPNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
抗人RBP4单克隆抗体轻链的氨基酸序列(SEQ ID NO.8所示)：
DIQMTQSPSSLSASLGGKVTITCKASQDINKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDFATYYCLQYDNLLWAFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNLYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
抗人RBP4单克隆抗体重链的核苷酸序列(SEQ ID NO.9所示)：
CAAGTGCAACTCCAACAGCCCGGTGCCGAGCTCGTGCGACCCGGGGCCTCCGTGAAACTGAGCTGTAAAGCATCAGGCTATACCTTCACCAACTATTGGATGAACTGGGTCAAGCAGAGGCCCGGACAAGGCTTGAGGTGGATTGGGATGATAGACCCTTCTGATTCAGAGACGCACTACAACCAGATGTTTAAAGACAAGGCCACACTGACAGTCGACAAAAGCTCTAGCACCGCCAACATGCAACTGTCATCACTGACTTCAGAAGATAGTGCTGTGTACTACTGCGCAAGGAGCCCTCAATTGGGTGATTATATCCTCGACTACTGGGGTCAAGGAACAAGTGTGACCGTGTCTTCAGCAAAGACAACTCCACCATCCGTGTATCCTTTGGCTCCTGGATCCGCAGCGCAGACCAACAGCATGGTCACACTGGGCTGTCTGGTAAAAGGGTATTTTCCAGAACCCGTCACCGTGACGTGGAATTCCGGCAGCCTCAGCTCCGGGGTTCACACGTTCCCTGCGGTTCTGCAGTCTGATCTCTACACGCTGTCCTCTTCCGTTACCGTCCCTAGCAGTACCTGGCCCAGCGAGACTGTGACCTGCAATGTAGCCCACCCCGCCTCAAGCACGAAGGTGGATAAAAAGATTGTCCCAAGGGATTGCGGTTGCAAACCCTGTATATGTACAGTGCCCGAGGTCTCTTCCGTCTTCATCTTCCCGCCCAAACCCAAGGACGTGTTGACTATCACGCTGACGCCGAAGGTCACTTGCGTAGTGGTGGATATCTCAAAGGACGACCCCGAGGTCCAATTCTCATGGTTTGTCGACGATGTCGAAGTACACACCGCCCAGACGCAGCCCAGAGAAGAGCAGTTCAATTCTACCTTCCGGAGCGTTAGCGAGCTGCCCATTATGCACCAGGACTGGCCAAATGGCAAAGAGTTTAAATGCAGAGTTAATTCCGCTGCCTTCCCAGCCCCAATTGAAAAAACTATCAGCAAGACAAAGGGCCGCCCAAAGGCTCCACAGGTTTACACTATCCCCCCACCAAAAGAACAGATGGCCAAAGACAAGGTCAGTCTGACATGCATGATCACAGACTTCTTCCCCGAGGACATCACAGTGGAATGGCAATGGAACGGACAGCCAGCAGAAAACTACAAGAACACCCAGCCTATCATGAATACCAATGGGAGCTACTTCGTTTACTCCAAGCTCAACGTCCAGAAGAGCAACTGGGAGGCCGGAAATACATTCACGTGTTCCGTCCTGCACGAAGGGCTGCACAACCATCATACTGAGAAGAGTCTGAGCCACAGCCCCGGGAAG
抗人RBP4单克隆抗体轻链的核苷酸序列(SEQ ID NO.10所示)：
GATATACAGATGACGCAAAGCCCTTCCTCCCTCTCCGCTTCTCTCGGCGGGAAGGTCACTATCACTTGCAAAGCATCACAAGACATTAACAAATACATCGCCTGGTACCAGCACAAGCCTGGTAAGGGACCCAGACTGCTTATCCACTATACTAGCACCCTGCAACCTGGCATCCCTAGCAGATTCTCAGGCTCTGGAAGTGGCAGGGACTACAGTTTTTCCATCAGCAATCTGGAGCCTGAAGATTTCGCCACATACTACTGCCTGCAGTATGATAATCTCCTGTGGGCCTTCGGAGGCGGAACAAAACTTGAGATTAAGAGAGCTGACGCAGCCCCCACAGTGAGCATTTTCCCACCATCCTCTGAACAGCTTACTTCCGGAGGCGCCTCCGTTGTATGTTTCCTGAACAACCTGTATCCCAAGGACATTAATGTGAAATGGAAGATAGACGGAAGTGAGCGCCAGAACGGGGTCCTTAACTCATGGACGGATCAGGACTCAAAAGACAGCACCTATAGTATGTCTTCAACCCTGACGCTGACAAAGGACGAGTACGAACGGCACAATAGTTATACCTGTGAGGCCACCCACAAGACTTCCACCAGCCCCATTGTGAAGAGCTTCAATCGGAATGAATGC
(3)单克隆抗体的制备和纯化
将LC023抗体重链和轻链基因序列构建入pcDNATM 3.4TOPOTM TA(InvitrogenTM)，然后采用PEI瞬时转染expi293F细胞表达LC023抗体。转染后在37℃，8％二氧化碳，120rpm条件下培养7天，3500rpm离心收集上清，采用proteinA亲和纯化获得抗体LC023，抗体经过浓缩后浓度定到4mg/mL。
实施例2：抗人RBP4单克隆抗体用于胶乳比浊试剂盒的制备
1、胶乳比浊试剂盒
本实施例提供的胶乳比浊试剂盒包含试剂R1、试剂R2、校准品。
试剂R1的配方为：pH7.0-8.0的100mM4-羟乙基哌嗪乙磺酸，100-150mMNaCl，1-5g/L聚乙二醇-8000，0.9g/LNaN3。
试剂R2包括pH6.0-7.0的MES缓冲溶液，抗人RBP4单克隆抗体与兔抗人RBP4多克隆抗体包被的胶乳，稳定剂。在一个具体实施方案中，试剂R2通过如下方法制备：
1)取90nm羧基微球(成都鸬鹚生物技术有限公司)1mL，加入250μL的pH6-7的MES缓冲液稀释，37℃预热，并快速搅拌。称取20μgEDC，溶于1mLpH6-7的MES缓冲液。将溶解后的EDC0.09mL加入不断搅拌的微球溶液中，37℃孵育30min。
2)取1800μg实施例1制备的抗人RBP4单克隆抗体与1800μg兔抗人RBP4多克隆抗体(贵州鸬鹚生物技术有限公司)，加入2mL包被缓冲液稀释待用。
3)向步骤1)活化完成的微球溶液中加入6mL包被缓冲液，并快速搅拌，然后缓慢滴入步骤2)混合好的抗体溶液。抗体滴加完后，继续在37℃搅拌2小时。加入封闭剂和稳定剂，充分搅拌混匀，即得到试剂R2。
本实施例胶乳比浊试剂盒在用于RBP4检测时，仪器：日立7180生化分析仪；测试波长：600nm；测试方法：终点法。具体步骤如下：
(1)在37℃下，试剂R1与校准品或待测血清样本混合3-5分钟，然后加入试剂R2，开始反应10s后测量A1。在一个具体实施方案中，试剂R1:血清样本:试剂R2(体积比)＝300:3:100。反应5分钟后测量A2。计算△A，△A＝A2-A1。
(2)使用校准品在全自动生化分析仪上通过内置曲线拟合模式拟合校准曲线，根据△A计算出待测血清样本中RBP4的含量。
2、效果检测
本实施例在进行效果检测时，设置单抗+多抗组、纯多抗组。单抗+多抗组、纯多抗组的区别在于试剂R2的配方不同，在制备试剂R2时，步骤2)区别如下：
单抗+多抗组：取1800μg抗人RBP4单克隆抗体与1800μg兔抗人RBP4多克隆抗体混合，加入2mL包被缓冲液稀释待用。
纯多抗组：取3600μg兔抗人RBP4多克隆抗体，加入2mL包被缓冲液。
采用的试剂R1的配方为：pH7.5的100mM4-羟乙基哌嗪乙磺酸，120mMNaCl，3g/L聚乙二醇-8000，0.9g/LNaN3。
I校准曲线测试和稳定性分析
将试剂在37℃热破处理，热破处理后与未热破的试剂同时测试试剂性能。具体步骤如下：
在37℃下，300μL试剂R1与3μL校准品混合5分钟，然后加入不同的100μL试剂R2，开始反应10s后测量A1。反应5分钟后测量A2。△A＝A2-A1。根据校准品的浓度和测得的△A值分别拟合出校准曲线。
各组△A检测结果如表1所示，单抗+加多抗组的胶乳比浊试剂性能比纯多抗组更好，稳定性也达到要求，与多抗一致。
表1
II临床样品的比对测试
采用单抗+多抗组、纯多抗组的试剂R2分别测定不同浓度的临床样品的△A值，然后在I中拟合的校准曲线上分别得出对应的测定浓度。检测结果如图1所示，单抗加多抗组测试结果与纯多抗组临床相关性一致，R2＝0.9917。表明单抗+多抗组可以完全替代纯多抗组用于临床测试。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。
Claims (8)
1.一种抗人RBP4单克隆抗体，其特征在于，所述单克隆抗体包含重链可变区和轻链可变区，所述重链可变区和轻链可变区各自具有3个CDR区，其中：
所述重链可变区CDR1的氨基酸序列为：NYWMN(SEQ ID NO.1)，
所述重链可变区CDR2的氨基酸序列为：MIDPSDSETHYNQMFK(SEQ ID NO.2)，
所述重链可变区CDR3的氨基酸序列为：SPQLGDYILDY(SEQ ID NO.3)，
所述轻链可变区CDR1的氨基酸序列为：KASQDINKYIA(SEQ ID NO.4)，
所述轻链可变区CDR2的氨基酸序列为：YTSTLQP(SEQ ID NO.5)，
所述轻链可变区CDR3的氨基酸序列为：LQYDNLLWA(SEQ ID NO.6)。
2.根据权利要求1所述的抗人RBP4单克隆抗体，其特征在于，所述重链的氨基酸序列如SEQ ID NO.7所示，和/或所述轻链的氨基酸序列如SEQ ID NO.8所示。
3.一种多核苷酸，其特征在于，所述多核苷酸包含编码权利要求1或2所述单克隆抗体的核苷酸序列。
4.根据权利要求3所述的多核苷酸，其特征在于，编码所述单克隆抗体重链的核苷酸序列如SEQ ID NO.9所示，和/或编码所述单克隆抗体轻链的核苷酸序列如SEQ ID NO.10所示。
5.权利要求1或2所述的抗人RBP4单克隆抗体在非诊断目的的人RBP4检测中的应用，或者在制备检测人RBP4的试剂盒中的应用。
6.一种检测人RBP4的胶乳比浊试剂盒，其特征在于，所述试剂盒包含权利要求1或2所述的抗人RBP4单克隆抗体。
7.根据权利要求6所示的胶乳比浊试剂盒，其特征在于，所述试剂盒还包含试剂R1和试剂R2；
所述试剂R1包括pH7.0-8.0的4-羟乙基哌嗪乙磺酸，NaCl，聚乙二醇-8000，NaN3；
所述试剂R2包括pH6.0-7.0的MES缓冲溶液，抗人RBP4单克隆抗体与抗人RBP4多克隆抗体包被的胶乳。
8.根据权利要求6所示的胶乳比浊试剂盒，其特征在于，所述试剂盒还包含校准品。
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