ELISA SİSTEMİNDE PROTEİN-İNORGANİK HİBRİT KONJUGATIN KULLANILABİLİRLİĞİ

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

USABILITY OF PROTEIN-INORGANIC HYBRID CONJUGATE IN ELISA SYSTEM

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.

Keywords:

Hybrid conjugate, Protein-inorganic hybrid structure, ELISA,

PDF

___

1.Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry 8, 871–874 (1971).
2.Wei T, Du D, Zhu M.-J, Lin Y, Dai Z. An Improved Ultrasensitive Enzyme-Linked Immunosorbent Assay Using Hydrangea-Like Antibody-Enzyme-Inorganic Three-in-One Nanocomposites. ACS Appl. Mater. Interfaces 8, 6329–6335 (2016).
3.Ram J, Nakane P, Rawlinson D, Pierce G. Enzyme labelled antibodies for ultrastructural studies. Fed Proc 25, (1966).
4.Shah K, Maghsoudlou P. Enzyme-linked immunosorbent assay (ELISA): the basics. Br. J. Hosp. Med. Lond. Engl. 2005 77, C98-101 (2016).
5.Ball JM, Henry NL, Montelaro RC, Newman MJA. versatile synthetic peptide-based ELISA for identifying antibody epitopes. J. Immunol. Methods 171, 37–44 (1994).
6.Stearns NA, Zhou S, Petri M, Binder SR, Pisetsky DS. The Use of Poly-L-Lysine as a Capture Agent to Enhance the Detection of Antinuclear Antibodies by ELISA. PLOS ONE 11, e0161818 (2016).
7.Tong S, Ren B, Zheng Z, Shen H, BaoG , Tiny Grains Give Huge Gains: Nanocrystal-Based Signal Amplification for Biomolecule Detection - ACS Nano (ACS Publications). Available at: https://pubs.acs.org/doi/10.1021/nn400733t. (Accessed: 11th May 2019)
8.Jeanson A, Cloes JM, Bouchet M, Rentier B. Comparison of conjugation procedures for the preparation of monoclonal antibody-enzyme conjugates. J. Immunol. Methods 111, 261–270 (1988).
9.Speroni F, Elviri L, Careri M, Mangia A. Magnetic particles functionalized with PAMAM-dendrimers and antibodies: a new system for an ELISA method able to detect Ara h3/4 peanut allergen in foods. Anal. Bioanal. Chem. 397, 3035–3042 (2010).
10.Lin H. et al. Modified Enzyme-Linked Immunosorbent Assay Strategy Using Graphene Oxide Sheets and Gold Nanoparticles Functionalized with Different Antibody Types - Analytical Chemistry (ACS Publications). Available at: https://pubs.acs.org/doi/10.1021/ac401075u. (Accessed: 11th May 2019)
11.Qu Z. et al. Ultrasensitive ELISA Using Enzyme-Loaded Nanospherical Brushes as Labels. Anal. Chem. 86, 9367–9371 (2014).
12.Ge J, Lei J, Zare RN. Protein-inorganic hybrid nanoflowers. Nat. Nanotechnol. 7, 428–432 (2012).
13.Altinkaynak C, Kocazorbaz E, Özdemir N, Zihnioglu F. Egg white hybrid nanoflower (EW-hNF) with biomimetic polyphenol oxidase reactivity: Synthesis, characterization and potential use in decolorization of synthetic dyes. Int. J. Biol. Macromol. 109, 205–211 (2018).
14.Shende P, Kasture P, Gaud RS. Nanoflowers: the future trend of nanotechnology for multi-applications. Artif. Cells Nanomedicine Biotechnol. 46, 413–422 (2018).
15.Lee SW, Cheon SA, Kim MI, Park TJ. Organic-inorganic hybrid nanoflowers: types, characteristics, and future prospects. J. Nanobiotechnology 13, 54 (2015).
16.Altinkaynak C, Tavlasoglu S, Özdemir N, Ocsoy I. A new generation approach in enzyme immobilization: Organic-inorganic hybrid nanoflowers with enhanced catalytic activity and stability. Enzyme Microb. Technol. 93–94, 105–112 (2016).
17.Altinkaynak C. et al. Preparation of lactoperoxidase incorporated hybrid nanoflower and its excellent activity and stability. Int. J. Biol. Macromol. 84, 402–409 (2016).
18.Somturk B. et al. Synthesis of urease hybrid nanoflowers and their enhanced catalytic properties. Enzyme Microb. Technol. 86, 134–142 (2016).
19.Lin Z. et al. Facile synthesis of enzyme-inorganic hybrid nanoflowers and its application as a colorimetric platform for visual detection of hydrogen peroxide and phenol. ACS Appl. Mater. Interfaces 6, 10775–10782 (2014).
20.Sun J. et al. Multi-enzyme co-embedded organic-inorganic hybrid nanoflowers: synthesis and application as a colorimetric sensor. Nanoscale 6, 255–262 (2014).
21.Wu Z. et al. Enantioselective transesterification of (R,S)-2-pentanol catalyzed by a new flower-like nanobioreactor. RSC Adv. 4, 33998–34002 (2014).
22.Zhang Y, Ge J, Liu Z. Enhanced Activity of Immobilized or Chemically Modified Enzymes. ACS Catal. 5, 4503–4513 (2015).
23.Yin Y. et al. An enzyme–inorganic hybrid nanoflower based immobilized enzyme reactor with enhanced enzymatic activity. J. Mater. Chem. B 3, 2295–2300 (2015).
24.Gulmez C, Altinkaynak C, Özdemir N, Atakisi O. Proteinase K hybrid nanoflowers (P-hNFs) as a novel nanobiocatalytic detergent additive. Int. J. Biol. Macromol. 119, 803–810 (2018).
25.Zhu L. et al. Rapid Detection of Phenol Using a Membrane Containing Laccase Nanoflowers. Chem. – Asian J. 8, 2358–2360 (2013).
26.Zeng J, Xia Y. Hybrid nanomaterials: Not just a pretty flower. Nat. Nanotechnol. 7, 415–416 (2012).
27.Lei Z. et al. Recent advances in biomolecule immobilization based on self-assembly: organic–inorganic hybrid nanoflowers and metal–organic frameworks as novel substrates. J. Mater. Chem. B 6, 1581–1594 (2018).
28.Wang R, Zhang Y, Lu D, Ge J, Liu Z, Zare RN. Functional protein–organic/inorganic hybrid nanomaterials. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology - Wiley Online Library. Available at: https://onlinelibrary.wiley.com/doi/full/10.1002/wnan.1210. (Accessed: 21st May 2019)