Malakaneh M, Rasaee M J, Rahbarizadeh F, Madani R, Forozandeh M M, Khabiri K, Alimohammadian M H
Department of Biochemistry, Faculty of Medicine, Birjand University of Medical Sciences, Khorasan, Iran.
Hybridoma. 2001 Apr;20(2):117-21. doi: 10.1089/02724570152057616.
An active ester derivative of neopterin was prepared using 4-(N-maleimidomethyl) cyclohexan 4-carboxilic acid N-hydroxy succinimide ester (MCH-NHS), conjugated to bovine serum albumin (BSA) and injected for antibody production (for both monoclonal and polyclonal antibodies). High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. Neopterin was conjugated to the enzyme penicillinase by a one-step glutaraldehyde method, which was used as tracer. A novel enzyme immunoassay was developed using this conjugate to screen and characterize the monoclonal antibody (MAb) produced in these experiments. After limiting dilutions, it was found that antibody produced by one clone with a Ka value of 7.6 x 10-7 mol/L was specific for a number of structurally related molecules. This clone was found to be of IgG class and IgG2a subclass. The standard curvewas constructed with a sensitivity of 10 pg/well (100 pg/mL) covering up to 1 ng/mL.
使用4-(N-马来酰亚胺甲基)环己烷-1-羧酸N-羟基琥珀酰亚胺酯（MCH-NHS）制备了新蝶呤的活性酯衍生物，将其与牛血清白蛋白（BSA）偶联并注射用于产生抗体（用于单克隆抗体和多克隆抗体）。去除产生高滴度抗体的脾细胞，并与源自Sp2/0的骨髓瘤细胞融合。通过一步戊二醛法将新蝶呤与青霉素酶偶联，用作示踪剂。使用该偶联物开发了一种新型酶免疫测定法，以筛选和鉴定在这些实验中产生的单克隆抗体（MAb）。经过有限稀释后，发现由一个克隆产生的抗体，其Ka值为7.6×10⁻⁷mol/L，对许多结构相关分子具有特异性。发现该克隆属于IgG类和IgG2a亚类。构建的标准曲线灵敏度为10 pg/孔（100 pg/mL），覆盖范围高达1 ng/mL。
