Khosravi Javad, Krishna Radha G, Bodani Umesh, Diamandi Anastasia, Khaja Najmuddin, Kalra Bhanu, Kumar Ajay
Diagnostic Systems Laboratories (Canada) Inc., Toronto, Ontario, Canada M5G 1L7.
Clin Biochem. 2007 Jan;40(1-2):86-93. doi: 10.1016/j.clinbiochem.2006.07.004. Epub 2006 Aug 10.
Development of an ELISA for phosphorylated isoform of IGFBP-1. Serine phosphorylation is an important regulator of IGFBP-1 bioactivity, but specific immunoassays for its measurement are currently lacking.
Assay design was based on a novel approach of first capturing the phosphorylated and non-phosphorylated IGFBP-1 by an anti-IGFBP-1 antibody and then selectively detecting the phosphorylated form by an anti-phosphoserine antibody. Method development involved pair-wise evaluation of the candidate antibodies and determinations of analytical performance and specificity. Specificity was monitored by reactivity with dephosphorylated IGFBP-1, with antibodies against other phosphorylated residues that are not expressed, and by comparative analysis of sample containing different IGFBP-1 phosphorylation profile.
Analytical evaluation demonstrated acceptable performance; detection limit 0.3 microg/L, dynamic range 1.56-100 microg/L; intra- and inter-assay CVs 2.1-8.6%; mean recovery (+/-SD) 97.8+/-9.2%, and mean recovery of sample dilution 93.4+/-6.0%. The phosphorylated and total IGFBP-1 medians in non-pregnant adult serum, which mostly contain the highly phosphorylated isoform, were 11.9 and 18.6 microg/L, respectively, and the sample values were tightly correlated (r=0.99). As expected, the corresponding medians in 1st trimester (17.4 and 63.0 microg/L) and 2nd trimester (30.9 and 75.8) samples with altered IGFBP-1 phosphorylation were significantly different (p<0.001). Similarly, a fraction (1.29%) of total IGFBP-1 (13.3 mg/L) in amniotic fluids was found to be phosphorylated (0.172 mg/L). There was no reactivity with dephosphorylated IGFBP-1.
The present ELISA is highly specific for the phosphorylated isoform of IGFBP-1 and its availability should help expedite further investigations of IGFBP-1 phosphorylation.
开发一种用于检测胰岛素样生长因子结合蛋白-1（IGFBP-1）磷酸化异构体的酶联免疫吸附测定（ELISA）法。丝氨酸磷酸化是IGFBP-1生物活性的重要调节因子，但目前缺乏用于检测其磷酸化水平的特异性免疫测定方法。
该检测方法基于一种新的策略，即先用抗IGFBP-1抗体捕获磷酸化和非磷酸化的IGFBP-1，然后用抗磷酸丝氨酸抗体选择性地检测磷酸化形式。方法开发包括对候选抗体进行两两评估，并测定分析性能和特异性。通过与去磷酸化的IGFBP-1反应、与针对未表达的其他磷酸化残基的抗体反应，以及对具有不同IGFBP-1磷酸化谱的样本进行比较分析来监测特异性。
分析评估显示该方法性能良好；检测限为0.3μg/L，动态范围为1.56 - 100μg/L；批内和批间变异系数分别为2.1% - 8.6%；平均回收率（±标准差）为97.8±9.2%，样本稀释后的平均回收率为93.4±6.0%。非妊娠成年血清中大多含有高度磷酸化的异构体，其中磷酸化IGFBP-1和总IGFBP-1的中位数分别为11.9μg/L和18.6μg/L，样本值紧密相关（r = 0.99）。正如预期的那样，在妊娠早期（17.4μg/L和63.0μg/L）和妊娠中期（30.9μg/L和75.8μg/L）样本中，IGFBP-1磷酸化发生改变，相应的中位数有显著差异（p < 0.001）。同样，在羊水样本中，总IGFBP-1（13.3mg/L）的一小部分（1.29%）被发现是磷酸化的（0.172mg/L）。与去磷酸化的IGFBP-1没有反应。
本ELISA法对IGFBP-1磷酸化异构体具有高度特异性，其应用应有助于加快对IGFBP-1磷酸化的进一步研究。
