Muhamad Arif Budiman, Mohamad Sadikin, Ani Retno Prijanti


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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Iyer R, Tomar SK. Folate: a functional food constituent. J Food Science. 2009;74:114–122.

Ball GFM. Vitamins in foods: analysis, bioavailability, and stability. Taylor & Francis Group. Boca Raton, FL. 2006.

Pfeiffer CM, Fazili Z, Zhang M. Folate analytical methodology. In: Bailey LB, editor. Folate in health and disease. 2nd ed. Boca Raton (FL): CRC Press, Taylor & Francis Group; 2010. p. 517–74.

Ibrahim W, Tousson E, El-Masry T, Arafa N, Akela M. The effect of folic acid as an antioxidant on the hypothalamic monoamines in experimentally induced hypothyroid rat. Toxicol Ind Health. 2012;28:253-61.

Depkes [internet]. Tabel angka kecukupan gizi bagi orang Indonesia [cited 21 april 2017]. Available from:

Wen SW, Guo Y, Rodger M, White RR, Yang Q, Smith NG, Perkins LS, Walker MC. Folic acid Supplementation in pregnancy and the risk of pre-eclampsia — A cohort study. PLoS One. 2016;11(2): e0149818.

Subandrate, Gunarti DR, Sadikin M. Properties of folate binding protein purified from bovine’s milk. Hayati J of Biosciences. 2012;19(3):105-109.

Crowther JR. The ELISA guide book: methods in molecular biology. New Jersey: Humana Press Inc Totawa; 2001.

Lequin, R. M. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clinical chemistry. 2005;51(12):2415-2418.

Sadikin M, Jusman SWA, Priyanti AR. Pengembangan perangkat pereaksi untuk pengukuran kadar asam folat dalam serum: isolasi dan purifikasi protein ikat folat (PIF) dari susu sapi. Laporan penelitian FKUI. 2000:1-8.

Salter DN, Scott KJ, Slade H, Andrews P. The preparation and properties of folate-binding protein from bovine’s milk. Biochem J. 1981;193:469-476.

Nixon PF, Jones M, Winzor DJ. Quantitative description of the interaction between folate and the folate-binding protein from bovine’s milk. Biochem J. 2004;382:215-21.

Balbol LN, Jagerstad M. Folate-binding protein in milk: a review of biochemistry, physiology, and analytical methods. Critical Reviews in Food Science and Nutrition. 2011;52: 411-412.

Christensen U, Holm J, Hansen SI. Stop-flow kinetic studies of interaction of bovine folate binding protein (FBP) and folate. Biosci Rep. 2006;26:291-9.

Mardiana. Penggunaan protein ikat folat (PIF) dari susu sapi untuk mengukur kadar folat serum [Thesis]. Jakarta: Universitas Indonesia; 2005.

World Health Organization [Internet]. Serum and red blood cell folate concentrations for assessing folate status in populations [cited 2 July 2016] Available from:

Salter DN, Ford JE, Scott KJ, dan Anderson P. Isolation of folate binding protein from bovines milk by the use of affinity chromatography. Fe Eur Biochem Soc let. 1972;20:302-6.

Puthusseri B, Divya P, Lokesh V, Kumar G, Savanur MA, Neelwarne B. Novel folate binding protein in Arabidopsis expressed during salicylic acid-induced folate accumulation. J Agric Food Chem. 2018;66(2):505-11.

Mahmood T, Yang P-C. Western blot: technique, theory, and troubleshooting. N Am J Med Sci. 2012;4(9):429-34.

Nygren L, Sternesjo A, Bjorck L. Determination of folate-binding proteins from milk by optical biosensor analysis. International Dairy J. 2003;13:283–90.

Riyanto. Validasi & verifikasi metode uji. Yogyakarta: Depublish; 2014:32-33.

Thomsson O, Ström-Holst B, Sjunnesson Y, Bergqvist AS. Validation of an enzyme-linked immunosorbent assay developed for measuring cortisol concentration in human saliva and serum for its applicability to analyze cortisol in pig saliva. Acta Veterinaria Scandinavica. 2014;56(1):55.

Andreasson U, Liaudet AP, Van wallwijk vandoorn LJC, lennow K, Chiasserini D, Engelborghs S, et al. A practical guide to immunoassay method validation. Front Neurol. 2015;6:93-101.

O’Riodan N, Kane M, Joshi L, Hisckey RM. Structural and functional characteristics of bovine milk protein glycosylation. Glycobiology J. 2014;24: 220-36.

Wingfield PT. Protein Precipitation Using Ammonium Sulphate. Curr Protoc Protein Sci. 2001;appendix 3:appendix-3F.

Tavares GM, Croguennec T, Lê S, Lerideau O, Hamon P, Carvalho AF, dan Bouhallab S. Binding of folic acid induces specific self-aggregation of lactoferrin: thermodynamic characterization. Langmuir. 2015;31: 12481–8.

Treloar T, Grieve PA, Nixon PF. One-step affinity purification of folate-binding protein, a minor whey protein. Australian J Dairy Technology. 2000;55(2):96.

Jansen EHJM, Beekhof P, Cremers JWJM, Schenk E. Long-term (in)stability of folate and vitamin B-12 in human serum. Clin Chem Lab Med. 2012;50:1761-3.


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