Human serum folate can be measured using folate binding protein linked to enzyme-labeled protein ligand binding assay (ELPLBA) as well as ELISA

Muhamad Arif Budiman, M. Sadikin, A. Prijanti
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Abstract

Background: Folate is an important substance used for purine and pyrimidine nucleotide synthesis. One measurement of folate that already establishes is using ELISA (Enzyme-linked immunosorbent assay) method. Folate binding protein is a protein that can bind folate, therefore it considered can be used as a tool that can replace antibody dependent ELISA method. Objectives: The aim of this research was to create a method for folate measurement in serum called Enzyme-labeled protein ligand binding assay (ELPLBA) by replacing antibody as used in ELISA method with folate binding protein (FBP) that purified from the whey of milk. Methods: The method is tested using 20 serum samples and compared to ELISA. Folate binding protein was purified from bovine’s milk using ammonium sulfate up to 90% saturated, DEAE-cellulose anion exchange chromatography and affinity chromatography. SDS-PAGE and western blot were used to establish the protein band of FBP that has molecular weight of ~25-35 kDa. ELPLBA was arranged with stationary phase using aminohexyl-agarose, and folic acid linked on it using carbodiimide. Results: The result show there was no significant difference of folate concentration between ELPLBA (14.804 ± 2.795) and ELISA method (13.859 ± 3.638), p = 0.363. Conclusion:  ELPLBA method show similarity for determination of folate in serum which was the same as standard folate measurement (ELISA).
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叶酸结合蛋白与酶标记蛋白配体结合试验(ELPLBA)和ELISA可用于测定人血清叶酸
背景:叶酸是嘌呤和嘧啶核苷酸合成的重要物质。已经建立的叶酸的一种测量方法是使用ELISA(酶联免疫吸附试验)方法。叶酸结合蛋白(Folate binding protein)是一种能够结合叶酸的蛋白,因此被认为可以作为一种替代抗体依赖ELISA法的检测工具。目的:用从乳清中纯化的叶酸结合蛋白(FBP)代替ELISA法中使用的抗体,建立测定血清中叶酸的酶标蛋白配体结合法(ELPLBA)。方法:采用20份血清标本对该方法进行检验,并与ELISA法进行比较。采用饱和硫酸铵达90%,deae -纤维素阴离子交换层析和亲和层析纯化牛乳中的叶酸结合蛋白。利用SDS-PAGE和western blot技术建立分子量为~25-35 kDa的FBP蛋白条带。用氨基己基琼脂糖将ELPLBA与固定相结合,用碳二亚胺将叶酸连接在ELPLBA上。结果:ELPLBA法测定叶酸浓度(14.804±2.795)与ELISA法测定叶酸浓度(13.859±3.638)差异无统计学意义(p = 0.363)。结论:ELPLBA法测定血清中叶酸的方法与标准叶酸测定法(ELISA)具有相似性。
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