抗三肽囊素单克隆抗体的制备

为研制抗三肽囊素(bursin,KHG-NH₂)的单克隆抗体,本试验采用EDC法将三肽囊素分子赖氨酰(K)上的氨基与载体蛋白(BSA或OVA)定向偶联,分别制备三肽囊素免疫抗原(BSA-KHG-NH₂)和检测抗原(OVA-KHG-NH₂)。以BSA-KHG-NH₂免疫BALB/c小鼠,应用杂交瘤技术,筛选到18株能稳定分泌抗三肽囊素单克隆抗体的杂交瘤细胞株。这18株单克隆抗体与三肽囊素人工抗原的结合均能被三肽囊素、GKHG-NH₂四肽阻断,但不能被KHGK四肽阻断,表明18株单抗与三肽囊素分子的结合具有高度特异性。以本研究制备的单抗2D2建立间接竞争ELISA方法,检测三肽囊素含量,检测下限为4 ng/mL,线性范围为4~500 ng/mL,加标检测回收率为85.0%~92.4%。

Preparation of Monoclonal Antibodies against Bursin

In order to produce monoclonal antibodies (McAbs) against bursin, the artificial antigens of bursin (BSA-KHG-NH₂and OVA-KHG-NH₂) were prepared by EDC method, in which the amino group on -Lys of bursin molecules was conjugated to bovine serum albumin (BSA) or chicken ovalbumin (OVA), respectively. BALB/c mice were immunized four times by BSA-KHG-NH₂, and eighteen hybridoms producing anti-bursin monoclonal antibodies were obtained by limited dilution. The binding of artificial antigen OVA-KHG-NH₂ to the McAbs were competitively inhibited by free bursin(KHG-NH₂) and GKHG-NH₂peptide,respectively, but couldn't be inhibited by free KHGK peptide. These results indicated that the 18 McAbs specifically bonded to bursin molecules. An IC-ELISA method to detect bursin was developed using McAb 2D2, in which the detection limits was about 4 ng/mL and the linear range of the inhibition curve was about 4 to 500 ng/mL, the recovery rate of bursin in the samples ranged from 85.0% to 92.4%.