Development of a Sensitive Chemiluminescence Immunoassay for the Quantification of Folic Acid in Human Serum.

Xiang Chen,Xuan Huang,Ting Zhang,Hadji Ahamada,Qiyang Zhou,Chunxin Wang,Zheng Yu,Zhonghu Bai

doi:10.1155/2019/5402903

Abstract

Folic acid (FA) is an important vitamin for human growth, especially for pregnant women. FA deficiency is associated with megaloblastic anemia, neural tube defects, cardiovascular diseases, irritability, diarrhea, and psychiatric disorders. Normally, FA molecules bind to folate-binding protein (FBP) in the serum as complex. Before quantify the FA concentration, a releasing procedure should be conducted. Alkaline condition and tris(2-carboxyethyl)phosphine (TCEP) are used to release binding FA to freeing state. In this work, a chemiluminescence immunoassay (CLIA) for human serum FA was established by competition model. Streptavidin (SA) was labeled to magnetic beads by an 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDAC/NHS) method. Activated biotin molecules were labeled to FBP molecules purified from milk. FA was labeled to horseradish peroxidase (HRP) by EDAC to activate the FA molecules. The pretreated samples or standards were added into the reaction tube with biotin-FBP and FA-horseradish peroxidase (HRP), FA in the sample compete with FA-HRP for binding to biotin-FBP, the signal is inversely proportional to the FA concentration. The method established shows good thermostability and performance. The limitation of detection (LOD) is 0.44 ng/mL. The intra-assay coefficient of variation (CV) is 3.6%–7.1%, the interassay CV is 4.2%–7.5%, and the recovery rate is 92.1%–103.5%. Cross reactivity (CR) was remarkably low with aminopterin, folinic acid, and methotrexate. The method shows good correlation with the FA CLIA product from Beckman Coulter; the equation is y = 0.9618x−0.1434 while the R2 value is 0.9224. The established method is sensitive, rapid, and accurate which can fully satisfy for the clinical requirement.

Highlights

Folate is the generic term for all B-vitamins that exhibit vitamin activity similar to that of folic acid. e various compounds of this class are designated as “folates”
TM Link Sulfo-NHS-LC-Biotinylation Kit and HABA solution are obtained from ermo Fisher; EAH-Sepharose 4B and Akta purifier are purchased from GE healthcare; folate-binding protein (FBP) is obtained from Scripps; horseradish peroxidase (HRP) is purchased from BBI solutions; Streptavidin is purchased from NeuroPeptide from China; Microscope are purchased from Olympus; Auto microplate chemiluminescent analyzer is supplied by Baiming biotechnology from China; and Auto magnetic beads chemiluminescent analyzer is supplied by Zecheng biotechnology from China
Characterization of FBP and Biotin-FBP Conjugates. e purified FBP was coated on the surface of the microwells

Summary

Introduction

Folate is the generic term for all B-vitamins that exhibit vitamin activity similar to that of folic acid. e various compounds of this class are designated as “folates”. Folic acid is the most oxidised and most stable form of folate. It is an organic compound necessary for the maintenance of normal body processes, synthesis of proteins and nucleic acids, metabolism of amino acids [1, 2], and division of cells. Highperformance liquid chromatography spectrometry is accurate for FA detection and can be used for the simultaneous detection of several vitamins [10]. This method is quite expensive and time consuming

Methods

Results

Conclusion