A novel ELISA-based approach for quantitative analysis of biomolecules in formalin-fixed paraffin-embedded tissues

Abstract

Aims: Enzyme-linked immunosorbent assay (ELISA) is a widely used method for protein quantification in biological specimens. However, its application to formalin-fixed paraffin-embedded (FFPE) tissue is limited due to inconsistent results and a lack of standardization. We aimed to develop and apply a novel formula to standardize ELISA results derived from FFPE tissue samples and to assess its feasibility using inflammatory skin disease biopsies. Methods: FFPE skin tissue blocks from individuals diagnosed with psoriasis (n=6), cutaneous lupus erythematosus (CLE, n=6), lichen sclerosis (LS, n=6), and healthy controls (n=6) were included. Tissue homogenates were prepared and tumor necrosis factor-α (TNF- α) levels were measured using a commercial ELISA kit. A new calculation formula was developed, normalizing absorbance values to protein concentration and biopsy size. Results: Application of the new formula enabled more consistent quantification of the TNF-α levels across heterogenous sized FFPE specimens. TNF- α levels were significantly elevated in all patients group compared to the controls (p<0.05), TNF-α concentration in CLE samples was significantly higher than in psoriasis and LS samples. Conclusion: This study introduces a novel, standardized formula that allows reproducible ELISA quantification in FFPE specimens. It may serve as a practical tool in retrospective and archival tissue-based research.

Keywords

• ELISA
• FFPE tissue
• TNF-alpha
• skin biopsy
• inflammation
• cytokine

Formalinle fikslenmiş parafin gömülü dokularda biyomoleküllerin kantitatif analizi için yeni formül

Abstract

Amaçlar: Enzim bağlantılı immünosorbent test (ELISA), biyolojik örneklerde protein miktarının ölçülmesinde yaygın olarak kullanılan bir yöntemdir. Ancak, formalinle fikse edilmiş parafin bloklanmış (FFPE) dokulara uygulanması, tutarsız sonuçlar ve standardizasyon eksikliği nedeniyle sınırlıdır. Bu çalışmada, FFPE doku örneklerinden elde edilen ELISA sonuçlarını standardize etmek amacıyla yeni bir formül geliştirmeyi ve inflamatuar deri hastalığı biyopsileri üzerinde uygulanabilirliğini değerlendirmeyi amaçladık. Yöntemler: Psoriasis (n = 6), kutanöz lupus eritematozus (CLE, n = 6), liken sklerozus (LS, n = 6) tanısı konmuş bireyler ile sağlıklı kontrollerden (n = 6) elde edilen FFPE deri doku blokları çalışmaya dahil edildi. Doku homojenatları hazırlandı ve tümör nekroz faktörü-α (TNF-α) düzeyleri ticari bir ELISA kiti kullanılarak ölçüldü. Absorbans değerlerini protein konsantrasyonu ve biyopsi boyutuna göre normalize eden yeni bir hesaplama formülü geliştirildi. Bulgular: Yeni formülün uygulanması, farklı boyutlardaki FFPE örneklerinde TNF-α düzeylerinin daha tutarlı şekilde ölçülmesini sağladı. TNF-α düzeyleri tüm hasta gruplarında kontrol grubuna kıyasla anlamlı derecede yüksekti (p < 0.05). Ayrıca, CLE örneklerindeki TNF-α konsantrasyonu, psoriasis ve LS örneklerinden anlamlı olarak daha yüksekti. Sonuçlar: Bu çalışma, FFPE örneklerinde tekrarlanabilir ELISA kantifikasyonu sağlayan yeni ve standardize bir formül sunmaktadır. Söz konusu formül, retrospektif ve arşiv doku temelli araştırmalarda pratik bir araç olarak kullanılabilir.

Keywords

• ELISA
• FFPE doku
• TNF-alfa
• Deri biyopsisi
• İnflamasyon
• Sitokin

References

1. Tabatabaei MS, Ahmed M. Enzyme-linked immunosorbent assay (ELISA). Methods Mol Biol. 2022;2508:115-134. doi:10.1007/978-1-0716-2376-3_10
2. Lin AV. Direct ELISA. Methods Mol Biol. 2015;1318:61-67. doi:10.1007/ 978-1-4939-2742-5_6
3. Lakshmipriya T, Gopinath SCB, Hashim U, Murugaiyah V. Multi-analyte validation in heterogeneous solution by ELISA. Int J Biol Macromol. 2017;105(Pt 1):796-800. doi:10.1016/j.ijbiomac.2017.07.115
4. Weber D, Davies MJ, Grune T. Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions. Redox Biol. 2015;5: 367-380. doi:10.1016/j.redox.2015.06.005
5. Boehncke W-H, Schön MP. Psoriasis. Lancet. 2015;386(9997):983-994. doi:10.1016/S0140-6736(14)61909-7
6. Rio CD, Millan E, Garcia V, Appendino G, DeMesa J, Munoz E. The endocannabinoid system of the skin. A potential approach for the treatment of skin disorders. Biochem Pharmacol. 2018;157:122-133. doi: 10.1016/j.bcp.2018.08.022
7. Kirtschig G. Lichen sclerosus-presentation, diagnosis and management. Dtsch Arztebl Int. 2016;113(19):337-343. doi:10.3238/arztebl.2016.0337
8. Geller L, Antonov NK, Lauren CT, Morel KD, Garzon MC. Pityriasis lichenoides in childhood: review of clinical presentation and treatment options. Pediatr Dermatol. 2015;32(5):579-592. doi:10.1111/pde.12581

9. Akkaya DAD. Liken sklerozuslu kadın olgularda serum prolaktin düzeylerinin araştırılması. 2007.
10. Kuhn A. Kutanöz lupus eritematozus tedavisinde S2k kılavuzu. 2017.
11. Nancy Weigle M. Psoriasis. Am Fam Physician. 2013;87(9):626-633.
12. Little AJ, Vesely MD. Cutaneous lupus erythematosus: current and future pathogenesis-directed therapies. Yale J Biol Med. 2020;93(1):81-95.
13. Armstrong AW, Read C. Pathophysiology, clinical presentation, and treatment of psoriasis: a review. JAMA. 2020;323(19):1945-1960. doi:10. 1001/jama.2020.4006
14. Lu D, Wang H, Wang M, Pei X, Du X, Zhang X. The efficacy of microwave therapy combined with focused ultrasound or white spot ointment combined with infrared light in the treatment of female vulvar sclerotic lichen. Medicine (Baltimore). 2025;104(12):e41716. doi:10.1097/MD.0000000000041716
15. Nicholson EM, Greenlee JJ, Hamir AN. PrPSc detection in formalin-fixed paraffin-embedded tissue by ELISA. BMC Res Notes. 2011;4(1):432. doi:10.1186/1756-0500-4-432
16. Knight AR, Taylor EL, Lukaszewski R, et al. A high-sensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells. Free Radic Biol Med. 2018;120:246-54. doi:10.1016/j.freeradbiomed. 2018.03.026
17. Chaubey KK, Gangwar N, Pawaiya RS, et al. Evaluation of newly developed 'six recombinant secretary proteins based 'cocktail ELISA' and 'whole cell lysate' based 'indigenous ELISA' and tissue microscopy' with 'gold standard' histo-pathology for the diagnosis of Johne's disease in slaughtered goats and buffaloes. Comp Immunol Microbiol Infect Dis. 2019;66:101338. doi:10.1016/j.cimid.2019.101338
18. Sadeghipour A, Babaheidarian P. Making formalin-fixed, paraffin embedded blocks. Methods Mol Biol. 2019;1897:253-268. doi:10.1007/978-1-4939-8935-5_22
19. Coscia F, Doll S, Bech JM, et al. A streamlined mass spectrometry-based proteomics workflow for large-scale FFPE tissue analysis. J Pathol. 2020; 251(1):100-112. doi:10.1002/path.5420
20. Pennock ND, Jindal S, Horton W, et al. RNA-seq from archival FFPE breast cancer samples: molecular pathway fidelity and novel discovery. BMC Med Genomics. 2019;12(1):195. doi:10.1186/s12920-019-0643-z
21. Taylor CR, Levenson RM. Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment II. Histopathology. 2006;49(4):411-424. doi:10.1111/j.1365-2559.2006.02513.x
22. Sato K, Takaishi M, Tokuoka S, Sano S. Involvement of TNF-alpha converting enzyme in the development of psoriasis-like lesions in a mouse model. PLoS One. 2014;9(11):e112408. doi:10.1371/journal.pone. 0112408
23. Yost J, Gudjonsson JE. The role of TNF inhibitors in psoriasis therapy: new implications for associated comorbidities. F1000 Med Rep. 2009;1: 30. doi:10.3410/M1-30
24. Farrell AM, Dean D, Millard PR, Charnock FM, Wojnarowska F. Cytokine alterations in lichen sclerosus: an immunohistochemical study. Br J Dermatol. 2006;155(5):931-940. doi:10.1111/j.1365-2133.2006. 07414.x
25. Tran DA, Tan X, Macri CJ, Goldstein AT, Fu SW. Lichen sclerosus: an autoimmunopathogenic and genomic enigma with emerging genetic and immune targets. Int J Biol Sci. 2019;15(7):1429-1439. doi:10.7150/ijbs.34613
26. Aringer M, Smolen JS. The role of tumor necrosis factor-alpha in systemic lupus erythematosus. Arthritis Res Ther. 2008;10(1):202. doi:10. 1186/ar2341
27. Wang R, Zhang X, Wang S. Differential genotypes of TNF-alpha and IL-10 for immunological diagnosis in discoid lupus erythematosus and oral lichen planus: a narrative review. Front Immunol. 2022;13:967281. doi:10.3389/fimmu.2022.967281