抗人胃癌细胞AGS单克隆抗体的制备和初步鉴定
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摘要
本研究用人胃癌细胞AGS免疫Balb/c小鼠,采用杂交瘤技术制备了抗人胃癌细胞的单克隆抗体(McAb),初步鉴定了杂交瘤和McAb的生物学特性,包括:杂交瘤的染色体、McAb的效价和亚类以及McAb的特异性。
一人胃癌细胞AGS(抗原)的培养
常规方法体外培养人胃癌细胞AGS。通过绘制生长曲线,研究了AGS细胞的增殖规律,结果表明:AGS细胞生长增殖的潜伏期为1-2 d,对数生长期为2-5d,停滞期为5-8 d。以DMSO为冷冻保护剂,对AGS细胞进行冷冻复苏。以该方法冻存的细胞解冻后的贴壁率为96.31%。这说明该方法对AGS细胞进行冷冻是切实可行的。
二抗人胃癌细胞AGS单克隆抗体杂交瘤细胞株的制备、筛选和克隆
1.制备
用AGS细胞免疫5只雌性Balb/c小鼠,间隔3周免疫1次。双方阵法确定了间接ELISA法的最佳抗原包被浓度为2×10~5/ml,阳性血清最适工作稀释度为1:10~4。间接ELISA法检测抗体效价达到1:10~4的3d后,通过PEG4000使免疫B淋巴细胞和小鼠骨髓瘤细胞SP2/0融合。融合3d后出现杂交瘤细胞,7-10 d后观察到杂交瘤细胞克隆生长。经HAT培养基选择培养后,细胞的融合率为51.04%。
2.筛选
间接ELISA法的结果显示:只有1B4孔的上清液重复3次检测的P/N均大于2.1,呈阳性。
3.克隆
用显微操作法挑出杂交瘤1B4的单个细胞并接种于96孔细胞培养板,将杂交瘤培养上清液按1:10~4稀释,间接ELISA法第一次检测后,有9个孔呈阳性。第二次检测后,仅有1B4孔杂交瘤(命名为1B4)分泌的抗体仍呈强阳性。
三抗人胃癌细胞AGS单克隆抗体杂交瘤细胞株和McAb的初步鉴定
1.杂交瘤细胞染色体
杂交瘤细胞染色体的平均数为92条,符合融合细胞染色体的特点。
2.McAb的效价
间接ELISA法检测到杂交瘤1B4上清液中McAb的效价可达1:10~4。
3.McAb的亚类
Ig类及亚类的鉴定结果说明该McAb属于IgG1亚类。
4.McAb的特异性
免疫化学法和间接ELISA法的结果均提示:McAb 1B4与宫颈癌细胞株Hela、肝癌细胞株HepG和正常人胎肺细胞均呈阴性反应,即不存在明显的交叉反应;但它与胃癌细胞株AGS呈明显阳性反应,说明该McAb可能对人胃癌有一定的特异性。
Bal/c mice were immunized with human gastric cancer cell line AGS. The research prepared the monoclonal antibody (McAb) against human gastric cancer carcinoma with the lymphocytes hybridoma technique and identified the elementary properties including: hybridoma chromosomes, titers of the McAb, subclass of the McAb and specific reaction.
1. Culture of human gastric cancer cell line AGS
AGS cells were cultured in vitro with the general methods. Proliferation-promoting rule of AGS cells was studied by protracting the growth curve. AGS cells were cryo-preserved with DMSO as cryoprotectants in manual way. After freeze-thaw cell viability was 96.31% in AGS cells. The results indicates that the cryo-preservation with DMSO as cryoprotectants in manual way is an effective way for cryo-preserving AGS cells.
2. Hybridoma preparation, selection and clone
(1) Preparation: 5 female Balb/c mice were immunized once every three weeks with human gastric cancer cell line AGS. Concentration optimization of coated antigen and antibodies in indirect ELISA were 2×10~5/ml and 1:10~4, respectively. SP2/0 cells and spleen cells fused with the help of PEG4000 after 3d when the titers of antibodies was 1:10~4. Hybridoma clones cultured in HAT were observed one week later. Fusion rate was 51.04% .
(2) Selection: Hybridoma were selected by indirect ELISA. The result suggests that P/N of the supernatant of 1B4 were more than 2.1 in three indirect ELISA, that is to say, hybridoma 1B4 secretes antibodies.
(3) Clone: Every cell was picked out of hybridoma 1B4 with micromanipulation and cultured in 96 culture plate. Antibodies only were found in hybridoma 1B4. The antibodies were called McAb 1B4.
3. Identification of the elementary properties of hybridoma cell 1B4 and McAb 1B4
(1) The average of the hybridoma cell 1B4 is 92 which accords with the characteristic of fusion cell.
(2) The titers of antibodies in supernatant of hybridoma cell 1B4(McAb 1B4) is 1:10~4.
(3) It was revealed that McAb 1B4 belonged to IgGl subclass.
(4) The results of immunocytochemical method and indirect ELISA all suggested that McAb 1B4 was negative to Hela cell line, HepG cell line and lung cells from human embryo, but positive to AGS cell line. It shows that McAb 1B4 may be specific to human gastric cancer.
一人胃癌细胞AGS(抗原)的培养
常规方法体外培养人胃癌细胞AGS。通过绘制生长曲线,研究了AGS细胞的增殖规律,结果表明:AGS细胞生长增殖的潜伏期为1-2 d,对数生长期为2-5d,停滞期为5-8 d。以DMSO为冷冻保护剂,对AGS细胞进行冷冻复苏。以该方法冻存的细胞解冻后的贴壁率为96.31%。这说明该方法对AGS细胞进行冷冻是切实可行的。
二抗人胃癌细胞AGS单克隆抗体杂交瘤细胞株的制备、筛选和克隆
1.制备
用AGS细胞免疫5只雌性Balb/c小鼠,间隔3周免疫1次。双方阵法确定了间接ELISA法的最佳抗原包被浓度为2×10~5/ml,阳性血清最适工作稀释度为1:10~4。间接ELISA法检测抗体效价达到1:10~4的3d后,通过PEG4000使免疫B淋巴细胞和小鼠骨髓瘤细胞SP2/0融合。融合3d后出现杂交瘤细胞,7-10 d后观察到杂交瘤细胞克隆生长。经HAT培养基选择培养后,细胞的融合率为51.04%。
2.筛选
间接ELISA法的结果显示:只有1B4孔的上清液重复3次检测的P/N均大于2.1,呈阳性。
3.克隆
用显微操作法挑出杂交瘤1B4的单个细胞并接种于96孔细胞培养板,将杂交瘤培养上清液按1:10~4稀释,间接ELISA法第一次检测后,有9个孔呈阳性。第二次检测后,仅有1B4孔杂交瘤(命名为1B4)分泌的抗体仍呈强阳性。
三抗人胃癌细胞AGS单克隆抗体杂交瘤细胞株和McAb的初步鉴定
1.杂交瘤细胞染色体
杂交瘤细胞染色体的平均数为92条,符合融合细胞染色体的特点。
2.McAb的效价
间接ELISA法检测到杂交瘤1B4上清液中McAb的效价可达1:10~4。
3.McAb的亚类
Ig类及亚类的鉴定结果说明该McAb属于IgG1亚类。
4.McAb的特异性
免疫化学法和间接ELISA法的结果均提示:McAb 1B4与宫颈癌细胞株Hela、肝癌细胞株HepG和正常人胎肺细胞均呈阴性反应,即不存在明显的交叉反应;但它与胃癌细胞株AGS呈明显阳性反应,说明该McAb可能对人胃癌有一定的特异性。
Bal/c mice were immunized with human gastric cancer cell line AGS. The research prepared the monoclonal antibody (McAb) against human gastric cancer carcinoma with the lymphocytes hybridoma technique and identified the elementary properties including: hybridoma chromosomes, titers of the McAb, subclass of the McAb and specific reaction.
1. Culture of human gastric cancer cell line AGS
AGS cells were cultured in vitro with the general methods. Proliferation-promoting rule of AGS cells was studied by protracting the growth curve. AGS cells were cryo-preserved with DMSO as cryoprotectants in manual way. After freeze-thaw cell viability was 96.31% in AGS cells. The results indicates that the cryo-preservation with DMSO as cryoprotectants in manual way is an effective way for cryo-preserving AGS cells.
2. Hybridoma preparation, selection and clone
(1) Preparation: 5 female Balb/c mice were immunized once every three weeks with human gastric cancer cell line AGS. Concentration optimization of coated antigen and antibodies in indirect ELISA were 2×10~5/ml and 1:10~4, respectively. SP2/0 cells and spleen cells fused with the help of PEG4000 after 3d when the titers of antibodies was 1:10~4. Hybridoma clones cultured in HAT were observed one week later. Fusion rate was 51.04% .
(2) Selection: Hybridoma were selected by indirect ELISA. The result suggests that P/N of the supernatant of 1B4 were more than 2.1 in three indirect ELISA, that is to say, hybridoma 1B4 secretes antibodies.
(3) Clone: Every cell was picked out of hybridoma 1B4 with micromanipulation and cultured in 96 culture plate. Antibodies only were found in hybridoma 1B4. The antibodies were called McAb 1B4.
3. Identification of the elementary properties of hybridoma cell 1B4 and McAb 1B4
(1) The average of the hybridoma cell 1B4 is 92 which accords with the characteristic of fusion cell.
(2) The titers of antibodies in supernatant of hybridoma cell 1B4(McAb 1B4) is 1:10~4.
(3) It was revealed that McAb 1B4 belonged to IgGl subclass.
(4) The results of immunocytochemical method and indirect ELISA all suggested that McAb 1B4 was negative to Hela cell line, HepG cell line and lung cells from human embryo, but positive to AGS cell line. It shows that McAb 1B4 may be specific to human gastric cancer.
引文
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