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

Yıl 2025, Cilt: 6 Sayı: 5, 447 - 451, 24.10.2025

Serkan Türeli , Onur Alp Kuloğlu , Elif Karakoç , Sevilay Karahan , Özay Gököz Alp Usubütün , Pergin Atilla

Öz

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.

Anahtar Kelimeler

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

Etik Beyan

The study is approved by Hacettepe University Non-interventional Clinical Researches Ethics Board (register number 2021/18-24). Informed consent was obtained from all patients participating in the study.The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki.

Destekleyen Kurum

Hacettepe University Scientific Research Projects Coordination Unit (THD-2022-19666)

Proje Numarası

THD-2022-19666

Teşekkür

Thanks to Hacettepe University Scientific Research Projects Coordination Unit for funding this project.

Formalinle fikslenmiş parafin gömülü dokularda biyomoleküllerin kantitatif analizi için yeni formül

Öz

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.

Anahtar Kelimeler

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

Proje Numarası

THD-2022-19666

Kaynakça

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