The Development of Polyclonal Antibody Against Glucose-6-Phosphate Dehydrogenase (G6PD) for G6PD Deficiency Newborn Screening in Indonesia
Abstract
Background/Aim: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an enzymatic disorder affecting carbohydrate metabolism, is a common cause of haemolytic anaemia and jaundice in newborns. Despite the proven benefits of G6PD screening, Indonesia faces significant challenges in implementing nationwide programs. Standard screening via dried blood spot (DBS) samples has limitations, prompting the need for improved diagnostic methods. This study aimed to develop a more sensitive and accessible screening method by creating polyclonal antibodies against G6PD.
Methods: G6PD protein was isolated from newborn cord blood and confirmed using western blot analysis. The rabbits were immunised with purified G6PD antigen. The polyclonal antibody with the highest titre was purified by SAS method.
Results: Western blotting confirmed the presence of G6PD at approximately 56 kDa in the isolated protein fraction. Immunised rabbits showed a significant increase in antibody titre compared with the control group. This research successfully isolated G6PD and produced polyclonal antibodies against it. However, further work is required to fully characterise the antibody and evaluate its suitability for G6PD deficiency screening. This antibody development holds promise for creating a dipstick kit for G6PD deficiency screening, thus overcoming the limitations of the DBS method. Colloidal gold-based immunochromatographic strips could be a suitable platform for such a kit due to their ease of use and rapid results.
Conclusion: Presented findings constitute a solid foundation for introducing a novel immunochemical reagent and developing a rapidly scalable protocol for producing G6PD-specific antibodies.
References
Lee HY, Ithnin A, Azma RZ, Othman A, Salvador A, Cheah FC. Glucose-6-phosphate dehydrogenase deficiency and neonatal hyperbilirubinemia: insights on pathophysiology, diagnosis, and gene variants in disease heterogeneity. Front Pediatr. 2022 May 24;10:875877. doi: 10.3389/fped.2022.875877.
Roper D, Layton M, Rees D, Lambert C, Vulliamy T, De la Salle B, D'Souza C; British Society for Haematology. Laboratory diagnosis of G6PD deficiency. A British Society for Haematology Guideline. Br J Haematol. 2020 Apr;189(1):24-38. doi: 10.1111/bjh.16366.
Malik SG, Oktavianthi S, Wahlqvist ML, Asih PBS, Harahap A, Satyagraha AW, Syafruddin D. Non-nutritional anemia: Malaria, thalassemia, G6PD deficiency and tuberculosis in Indonesia. Asia Pac J Clin Nutr. 2020;29(Suppl 1):S32-S40. doi: 10.6133/apjcn.202012_29(S1).04.
Lukitasari L. [Analysis of change patterns in glucose-6-phosphate dehydrogenase (g6pd) enzyme activity, enos levels, and f2-isoprostane in preeclampsia patients in Surabaya]. Universitas Arilangga. Indonesian.
Pfeffer DA, Ley B, Howes RE, Adu P, Alam MS, Bansil P, et al. Quantification of glucose-6-phosphate dehydrogenase activity by spectrophotometry: A systematic review and meta-analysis. PLoS Med. 2020 May 14;17(5):e1003084. doi: 10.1371/journal.pmed.1003084.
George RS, Moat SJ. Effect of dried blood spot quality on newborn screening analyte concentrations and recommendations for minimum acceptance criteria for sample analysis. Clin Chem. 2016 Mar;62(3):466-75. doi: 10.1373/clinchem.2015.247668.
Zailani MAH, Raja Sabudin RZA, Ithnin A, Alauddin H, Sulaiman SA, Ismail E, et al. Population screening for glucose-6-phosphate dehydrogenase deficiency using quantitative point-of-care tests: a systematic review. Front Genet. 2023 Jun 14;14:1098828. doi: 10.3389/fgene.2023.1098828.
Shetty PP, Belle VS, Biswas M. Glucose 6 phosphate dehydrogenase deficiency quantitative test in dried blood spot- a potential marker for adult unknown G6PD deficiency. Trop Doct. 2023 Jan;53(1):100-3. doi: 10.1177/00494755221125615.
Luzzatto L, Arese P. Favism and glucose-6-phosphate dehydrogenase deficiency. N Engl J Med. 2018 Jan 4;378(1):60-71. doi: 10.1056/nejmra1708111.
Javadi M, Deravi S, Zarei S, Mahdavi N, Ranjbaran M. Prevalence of G6PD deficiency in Iranian neonates with jaundice: a systematic review and meta-analysis. J Matern Fetal Neonatal Med. 2022 Dec;35(25):5813-20. doi: 10.1080/14767058.2021.1895738.
Halim SA, Bahar R, Abdullah WZ, Zon EM, Yusoff SM. Performance comparison between conventional fluorescent spot test and quantitative assay in detecting g6pd deficiency in neonates. Oman Med J. 2023 Jul 31;38(4):e524. doi: 10.5001/omj.2023.86.
Greco C, Arnolda G, Boo NY, Iskander IF, Okolo AA, Rohsiswatmo R, et al. Neonatal jaundice in low- and middle-income countries: lessons and future directions from the 2015 don Ostrow Trieste yellow retreat. Neonatology. 2016;110(3):172-80. doi: 10.1159/000445708.
De Jesús VR, Borrajo GJ, Mei J, Adams W, Chunko K, Das S, et al. Dried blood spot specimen collection for newborn screening suggested citation. CLSI 2021;41(5).
Moat SJ, Bonham JR, Cavanagh C, Birch M, Griffith C, Shakespeare L, et al. Consistency in the assessment of dried blood spot specimen size and quality in U.K. Newborn Screening Laboratories. Int J Neonatal Screen. 2024 Sep 5;10(3):60. doi: 10.3390/ijns10030060.
CareStartTM G6PD RDT – Access Bio [Internet]. [Cited: 11-Feb-2024]. Available from: https://accessbio.net/products/rapid-diagnostic-tests/carestart-g6pd-rdt.
BinaxNOW G6PD | Abbott Point of Care [Internet]. [Cited: 11-Feb-2024]. Available from: https://www.globalpointofcare.abbott/us/en/product-details/binaxnow-g6pd.html.
Bulut ME, Kına N, Büyükyanbolu E, Özer VY, Aktaş E, Bayraktar B. A highly-sensitive rapid test for the diagnosis of streptococcal pharyngitis: BD veritor ™ system. Int J Pediatr Otorhinolaryngol. 2020 Jun;133:109980. doi: 10.1016/j.ijporl.2020.109980.
Porte L, Legarraga P, Vollrath V, Aguilera X, Munita JM, Araos R, et al. Evaluation of a novel antigen-based rapid detection test for the diagnosis of SARS-CoV-2 in respiratory samples. Int J Infect Dis. 2020 Oct;99:328-33. doi: 10.1016/j.ijid.2020.05.098.
Yamasaki E, Sakamoto R, Matsumoto T, Morimatsu F, Kurazono T, Hiroi T, et al. Development of an immunochromatographic test strip for detection of cholera toxin. Biomed Res Int. 2013;2013:679038. doi: 10.1155/2013/679038.
Posthuma-Trumpie GA, Korf J, van Amerongen A. Lateral flow (immuno) assay: its strengths, weaknesses, opportunities and threats. A literature survey. Anal Bioanal Chem. 2009;393(2):569–82. doi:10.1007/s00216-008-2287-2
Wojnarski B, Lon C, Sea D, Sok S, Sriwichai S, Chann S, et al. Evaluation of the CareStart™ glucose-6-phosphate dehydrogenase (G6PD) rapid diagnostic test in the field settings and assessment of perceived risk from primaquine at the community level in Cambodia. PLoS One. 2020 Jan 31;15(1):e0228207. doi: 10.1371/journal.pone.0228207.
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).