Development of inositol 1,4,5-trisphosphate assay kit

在肌醇磷脂介导的信号转导系统中，信号是通过磷脂酰肌醇4，5-二磷酸（PIP_2）分解为肌醇1，4，5-三磷酸（IP_3）和甘油二酯（DG）而产生的。因此，决定反应强度的最重要因素之一似乎是PIP_2（产生脂质的信号）、IP_3和DG（第二信使）。本研究试图建立一种新的方法，利用抗PIP_2抗体或抗IP_3抗体，定量检测细胞内的PIP_2和IP_3。制备了抗PIP_2和抗IP_3单克隆抗体。抗PIP_2抗体对PIP_2有高度特异性，与PIP或PI无交叉反应。另一方面，由于抗IP_3抗体与1，3，4，5-IP_3或1，3，4，5-IP_4的交叉反应程度低10倍，因此，我们建立了一种薄层免疫染色检测微量IP_2的方法。从细胞中提取PIP_2（1 × 10^6）并上样至TLC板后，展开板。用抗体检测PIP_2。在此方法中，测定了痕量（20 pmol 1 - 1 nmol）的PIP_2，表明灵敏度比通常测定无机磷酸盐的方法高1,000倍。

In inositol phospholipid-mediated signal transduction system, signals are caused through the breakdown of phosphatidyl inositol 4,5-bisphosphate (PIP_2) to inositol 1,4,5-trisphosphate (IP_3) and diacylglycerol (DG). Therefore, one of the most important factor to determine the strength of responses seems to be PIP_2 ( a signal producing lipid), IP_3 and DG (2nd messenger). But these materials are contained too low to measure in cells.We tried to develop a new method to quantify PIP_2 and IP_3 using anti-PIP_2 antibody or anti-IP_3 antibody. Monoclonal anti-PIP_2 antibody and anti-IP_3 antibody were developed. anti-PIP_2 antibody showed a high specificity for PIP_2 and did not cross react with PIP ot PI. On the other hand, anti-IP_3 antibody was found to be fairly low specificity for IP_3, since the antibody cross reacted with 1,3,4-IP_3 or 1,3,4,5-IP_4 with a 10-fold lesser extent.Therefore, we extablished a method to measure a trace amount of PIP_2 by TLC immunostaining. After PIP_2 was extracted from cells (1 x 10^6) and applied on TLC plate, the plate was developed. PIP_2 was detected with the antibody. In this method, a trace amount (20 pmo1 - 1 nmol) of PIP_2 was determined, suggesting 1,000 times higher sensitivity than usual method for measuring inorganic phosphate.