USABILITY OF PROTEIN-INORGANIC HYBRID CONJUGATE IN ELISA SYSTEM

Yıl 2020, , 226 - 238, 30.07.2020

Murat Ekremoğlu , Cevahir Altınkaynak

https://doi.org/10.18036/estubtdc.604509

Cited By: 1

Öz

In the Enzyme-linked immunosorbent assay method (ELISA), it
is important to increase the immobilization efficiency of the functional
molecules on the microplate and increase the signal-to-noise ratio in order to
detect the target molecules with high sensitivity and selectivity. For this
purpose, antibody functionalized materials can generate remarkable signal
amplification with high enzyme capacity by using conventional immobilization
methods. Recently, hybrid structures containing protein/enzyme and Cu₃(PO₄)₂,
which is a different enzyme immobilization method, have higher catalytic
activity compared to the free form of protein-containing molecules as they have
hierarchical microstructures and form large active surface areas.

In this study; Using protein-inorganic hybrid structure
synthesis method, hybrid functionalized conjugate systems with enzyme, antibody
and Cu₃(PO₄)₂ were synthesized all together
and characterization of the resulting structures was performed by SEM, EDX, XRD
and FTIR analysis. When the findings obtained from the researches were
evaluated, the hybrid nano structure showed 183.5 EU/mg peroxidase activity and
free HRP enzyme showed 59.01 EU/mg activity. The performance of the ELISA
system was measured using hybrid conjugate constructs prepared using various
TNF-alpha specific antibodies at a concentration of 5-1000 mg mL^-1. The performance of hybrid
conjugate structure containing multiple organic molecules in ELISA system was
higher than other structures. This method is a highly practical method that can
replace enzyme-labeled antibody method in ELISA.

Anahtar Kelimeler

Hybrid conjugate, Protein-inorganic hybrid structure, ELISA

ELISA SİSTEMİNDE PROTEİN-İNORGANİK HİBRİT KONJUGATIN KULLANILABİLİRLİĞİ

Öz

Enzim bağlı immünosorbent analizinde (ELISA); hedef
molekülleri yüksek hassasiyet ve seçicilikle tespit etmek için fonksiyonel
moleküllerin mikro plaka üzerindeki immobilizasyon verimlerinin arttırılması ve
sinyal-gürültü oranının yükseltilmesi önemlidir.  Bu amaçla konvansiyonel immobilizasyon
yöntemleri kullanılarak antikorla fonksiyonelleştirilmiş malzemeler yüksek
enzim kapasitesi ile dikkate değer sinyal artışına neden olabilir) Son yıllarda
farklı bir enzim immobilizasyon yöntemi olan protein/enzim ve Cu₃(P0₄)₂
içeren hibrit yapılar hiyerarşik mikro yapılara sahip olmaları ve geniş aktif
yüzey alanları oluşturmaları neticesinde içeriğindeki protein yapılı moleküllerin
serbest formuna göre daha yüksek katalitik aktivite gösterebilmektedir.

Bu çalışma kapsamında; protein-inorganik hibrit yapı sentez metodu
kullanılarak enzimi, antikoru ve Cu₃(P0₄)₂’ın
hepsi bir arada olacak şekilde antikorla fonksiyonelleştirilmiş hibrit konjugat
sistemleri sentezlenmiş, oluşan yapıların karakterizasyonları SEM, EDX, XRD ve FTIR
analizleri ile gerçekleştirilmiştir. Araştırmalar sonucu elde edilen bulgular
değerlendirildiğinde, hibrit nano yapı 183,5 EU/mg peroksidaz aktivitesi
gösterirken serbest HRP enzimi 59,01 EU/mg aktivite göstermiştir. TNF-alfa’ya
spesifik çeşitli antikorlar kullanılarak hazırlanan hibrit konjugat yapılar 5-1000 µg mL^-1
aralığındaki konsantrasyonlarda kullanılarak ELISA sistemindeki
performansı ölçülmüştür. Çoklu organik molekül içeren hibrit konjugat
yapının ELISA sistemindeki performansı diğer yapılara göre daha yüksek
bulunmuştur. Bu metot; ELISA’da antikorların enzimle işaretlenmesi yöntemi yerine
geçebilecek pratik olarak uygulanabilirliği kanıtlanmış bir uygulamadır.

Anahtar Kelimeler

Hibrit konjugat, protein-inorganik hibrit yapı, ELISA

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