相思子毒素免疫层析胶体金试纸条的研制
摘要
将相思子毒素杂交瘤细胞株4G1、3C2扩大培养,制备单克隆抗体。纯化后抗体非特异带消失,抗体效价为1×10~6以上,亲和常数介于2.43×10~6～5.3×10~7M~(-1),抗体IgG含量在0.424mg/mL～1.323mg/mL,纯度在83.2%～93.2%。用相思子毒素制备多克隆抗体,抗体纯化后SDS-PAGE显示,多抗片段大小与预期相符,相思子毒素抗体效价为1∶25600,纯化后抗体蛋白含量为11.32 mg/mL,IgG含量为10.35mg/mL,且纯度达到91.59%。
采用胶体金免疫层析法,制备相思子毒素胶体金试纸,最低检测限在0.1μg/mL～0.5μg/mL之间,与蓖麻毒素、蓖麻凝集素和相思子凝集素不发生交叉反应。模拟样本水样的检测限为0.1μg/mL,土样浊液为0.5μg/mL,且重复性好,37℃破坏试验能保存11天半,相当于2-8℃保存17个月左右。用本实验组装的试纸进行检测,结果19份为阳性,符合率为95%。12份不同浓度的相思子毒素经过处理以后,分别用平板凝集试验(PAT)和试纸条进行检测。结果与PAT符合率为100%。
In this study,Immunoglobulin G(IgG)antibodies against abrins were prepared and purifed,the optimal factors of label were selected,and the colloid gold dipsticks for abrins developed through immunochromatomatographic colloid gold method.
The hybridoma cell strain of abrins 4G1 and 3C2 were expand-raised enable the cell to be 5.0×10~6,the Balb/C mouse were immunized to produce the monoclonal antibody ascites.The antibodies were purifed with caprylic acid-ammonium sulfate, then further purified with HiTrap Protein GHP affinity column.
The titer of the obtaind antibodies were above 1.0×10~(5.5),the affinity constants were among2.43×10~6～5.3×10~7,antibodies IgG contents were among 0.242 mg/mL～1.323 mg/mL and purity was 91.59%.The Multi-clone antibodies were purified.SDS-PAGE showed the same size of fragment as the anticipated it,abrins antibodies titer was 1:25600,content of post-purified antibodies' protein was 11.32 mg/mL,concentration of IgG was 10.35mg/mL,and the purity was 91.59%.
The colloid gold particles were prepared using the citric acid reducing process. The mean diameters of colloid gold particles were determined 15nm,25nm,30nm and 40nm by electron microscope and ultraviolet scanning,the panicle sizes were consistent,and well-distributed.Choosing 4G1,as the golden sign antibodies and taking Multi-clone,3C2 as the coating antibodies,using the colloid gold immunity chromatographic assay prepared colloid gold dipsticks,and its sensitivity,specificity, repeat ability and stability were tested,the optimal coating density was 30μg/mL abrin 4G1,and the optimal coating pH value was 8.8,the optimal blocking protein was BSA,the optimal labelling time was 25 min,and the the optimal temperature was 25℃.
The colloid gold dipsticks were prepared.The lowest testing limit of the dispicks was among 0.1μg/mL-0.5μg/mL.No cross reaction was abserved among ricin,ricinus agglutinin and abrus agglutinin,detection limit of water samples was 0.1μg/mL,the detection limit of soil samples was 0.5μg/mL,and the its repeat ability was satisfactory.12 abrins samples diluted with PBS were detected with plate agglutination test and the strip respectively,the results showed that 12 the coincidence positive of rate was 95%,The plate agglutination test(PAT)and the dipsticks coincidence rates of was 100%.
The optimal condition of the colloid gold dipsticks was selected,and the sensitivity,specificity,repeatability,reproducibility,recovery rate and stability were detected.The results showed that the colloid gold dipsticks were preseved for five months at 4℃,and still kept high specificity,sensitivity,repeatability and reproducibility.
采用胶体金免疫层析法,制备相思子毒素胶体金试纸,最低检测限在0.1μg/mL～0.5μg/mL之间,与蓖麻毒素、蓖麻凝集素和相思子凝集素不发生交叉反应。模拟样本水样的检测限为0.1μg/mL,土样浊液为0.5μg/mL,且重复性好,37℃破坏试验能保存11天半,相当于2-8℃保存17个月左右。用本实验组装的试纸进行检测,结果19份为阳性,符合率为95%。12份不同浓度的相思子毒素经过处理以后,分别用平板凝集试验(PAT)和试纸条进行检测。结果与PAT符合率为100%。
In this study,Immunoglobulin G(IgG)antibodies against abrins were prepared and purifed,the optimal factors of label were selected,and the colloid gold dipsticks for abrins developed through immunochromatomatographic colloid gold method.
The hybridoma cell strain of abrins 4G1 and 3C2 were expand-raised enable the cell to be 5.0×10~6,the Balb/C mouse were immunized to produce the monoclonal antibody ascites.The antibodies were purifed with caprylic acid-ammonium sulfate, then further purified with HiTrap Protein GHP affinity column.
The titer of the obtaind antibodies were above 1.0×10~(5.5),the affinity constants were among2.43×10~6～5.3×10~7,antibodies IgG contents were among 0.242 mg/mL～1.323 mg/mL and purity was 91.59%.The Multi-clone antibodies were purified.SDS-PAGE showed the same size of fragment as the anticipated it,abrins antibodies titer was 1:25600,content of post-purified antibodies' protein was 11.32 mg/mL,concentration of IgG was 10.35mg/mL,and the purity was 91.59%.
The colloid gold particles were prepared using the citric acid reducing process. The mean diameters of colloid gold particles were determined 15nm,25nm,30nm and 40nm by electron microscope and ultraviolet scanning,the panicle sizes were consistent,and well-distributed.Choosing 4G1,as the golden sign antibodies and taking Multi-clone,3C2 as the coating antibodies,using the colloid gold immunity chromatographic assay prepared colloid gold dipsticks,and its sensitivity,specificity, repeat ability and stability were tested,the optimal coating density was 30μg/mL abrin 4G1,and the optimal coating pH value was 8.8,the optimal blocking protein was BSA,the optimal labelling time was 25 min,and the the optimal temperature was 25℃.
The colloid gold dipsticks were prepared.The lowest testing limit of the dispicks was among 0.1μg/mL-0.5μg/mL.No cross reaction was abserved among ricin,ricinus agglutinin and abrus agglutinin,detection limit of water samples was 0.1μg/mL,the detection limit of soil samples was 0.5μg/mL,and the its repeat ability was satisfactory.12 abrins samples diluted with PBS were detected with plate agglutination test and the strip respectively,the results showed that 12 the coincidence positive of rate was 95%,The plate agglutination test(PAT)and the dipsticks coincidence rates of was 100%.
The optimal condition of the colloid gold dipsticks was selected,and the sensitivity,specificity,repeatability,reproducibility,recovery rate and stability were detected.The results showed that the colloid gold dipsticks were preseved for five months at 4℃,and still kept high specificity,sensitivity,repeatability and reproducibility.
引文
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[3]Evensen,G.Mathiesen,A.and Sundan,A.Direct molecular cloning and expression of two distinct abrin A-chains.J.Biol.Chem,1991,266(11):6848-6852
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[7]Tahirov TH,Lu TH,Liaw YC,etal.Crystal structure of abrin2a at 2.14A[J].J Mol Biol,1995,250:354-367.
[8]Tahirov TH,Lu TH,Liaw YC,et al.Crystal structure of abrin2a at 2.1A[J].J Mol Biol,1995,250:346-354.
[9]李丽琴,郑晓军,陈乐贵.相思子毒素多克隆抗体研究.防化研究,2001,4:19-21.
[10]Narayanan S,Surolia A.Ribosome-inactivating protein and apoptosis:abrin causes cell death via mitochondrial pathway in Jurkat cells[J].Biochem J.2004, Jan 1,377(Pt 1):233-40.
[11]Goto LS,Beltramini LM.Abrus pulchellus type-2 KIP,pulchellin:heterologous expression and refolding of the sugar-binding B chain[J].Protein Expr Purif.2003,Sep 31(1):12-8
[12]Hung V,Kuttan G.Immunopotentiating activity of abrin,a lectin from Abrus precatorius Linn[J].Indian J Exp Biol.2002,Aug,40(8):910-3.
[13]Wood,K.A,Lord,J.M,Wawrzynczak.Preproabrin:genomic cloning,characterisation and the expression of the A-chain in Escherichia coli.Eur.J.Biochem,1991,198(3):723-732
[14]Hung,C.H,Lee,M.C,Lee,T.C and Lin,J.Y.Primary structure ofthree distinct isoabrins determined by cDNA sequencing.Conservation andsignificance.J.Mol.Biol,1993,229(1),263-267
[15]Ohba Hung CH,Lee MC,Chen J K,et al.Cloning and expression of three abrin A-chains and their mutants derived by site specific mutagenesis in Escherichia coil[J].EurJBiochem,1994,219:83-87.
[16]Ohba H,Moriwakis.Plant-derived abrin-a induces apoptosis in cultu- reed leukemic cell lines by different mechanisms[J].Toxicol Appl Pharmacol,2004Mar 1,195(2):182-93.
[17]Wang,L.-C,Kang,L,Hu,T-M.etal.Abrin-a A chain expressed as soluble form in Escherichia coil from a PCR-synthesized geneis catalytically and functionally active Biochimie,2004,86(4-5):327-333
[18]Silva AL,Goto LS.Pulchellin,a highly toxic type 2 ribosome- inac- tivating protein from Abrus pulchellus.Cloning heterologous expression of A-chain and structural studies[J].FEBS J,2005,272(5):1201-10.
[19]DickersKJ,Bradberry,SM.Abrinpoisoning[J].ToxicolRev,2003,22(3):137-42
[20]Feldherr Carl M.Feldherr Ph.D.The nuclear Annuli as pathways for nucleocytoplasmic Exchanges.The Journal of Cell Biology,11(14):65-72
[21]孙秀兰,赵晓联,汤坚,等.单分散胶体金的制备工艺的优化.免疫学杂志,2004,20(2):151-154
[22]XL,Zhao XL,Tang J,et al.The optimization of the preparation croft of the colloidal gold.Immunol Mag,2004,20(2):151-154
[23]杨文玉,崔亚丽,代丽君.单分散纳米级胶体金的合成.陕西师范大学学报(自然科学版),2003,31(2):71-73
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