Significance of Bilirubin as Anti-Oxidant in Gastrointestinal Surgery

本文报道了一种新的抗胆红素单克隆抗体的制备和鉴定。采用改进的MBS（metaboleimidobenzoyl N-hydroxysuccinimide ester）法，将半抗原非结合的白蛋白IX α（UCB）共价偶联到牛血清白蛋白（BSA）上，保留了分子内氢键和三维结构。经体细胞融合和筛选试验，获得了与UCB反应而与胆红素二葡萄糖醛酸苷和胆红素单葡萄糖醛酸苷不反应的抗胆红素单克隆抗体，用于测定人血清或尿液中极低浓度的胆红素。我们利用了目前可获得的抗体（5 M2）和另一种抗胆红素单斜抗体（24 G7），其与UCB以及结合胆红素反应，用于酶-免疫吸附试验（ELISA）。用ELISA法测定了许多病人血清中的胆红素浓度，并与传统的重氮法进行了比较。当5 M2用于ELIZA时，两种方法测定的胆红素浓度在任何样品中均无差异。然而，当在ELISA中使用24 G7时，在总尿量减少的患者的情况下，存在显著的差异。这表明存在一种降解的胆红素，即物质“X”，当发生任何类型的应激时，其在血清中增加，并将在尿液中排出。此外，观察到健康患者的尿液中存在物质“X”。

A new anti-bilirubin monoclinal antibody has been prepared and characterized. Using modified MBS (metamaleimidobenzoyl N-hydroxysuccinimide ester) method, haptenic unconjugated bilirubin-IXalpha (UCB) covalently coupled to bovine serum albumin (BSA) retaining its intramolecular hydrogen bonds and three-dimensional structure. After somatic cell fusion and screeing assay, we obtained anti-bilirubin monoclonal antibodies which reacted with UCB but did not react with bilirubin diglucuronide or monoglucuronide.To determine extremely low concentrations of bilirubin in human serum or urine, we utilized an antibody presently available (5M2) and another anti-bilirubin monoclinal antibody (24G7) which reacted with UCB as well as with conjugated bilirubin for enzyme-linked immunosorbent assay (ELISA). Bilirubin concentration in serm specimens obtained from many patients, determined using ELISA was compared with that determined by the conventional diazo method. When 5M2 was used in ELIZA, there was no discrepancy between bilirubin concentration determined by the two method in any sample. However, when 24G7 was used in ELISA, in the case of patients whose total urine volume was decreased, there was a significant discrepacy. This suggests the existence of a degraded bilirubin, substance "X", that is increased in the serum when any kind of stress occurs which will be excreted in the urine, In addition existence of substance "X" in the urine of healthy patients was observed.