Optimization and intensification of synthesis protein-protein and protein-hapten conjugates for highly sensitive immunoassays

O. Yu. Galkin, N. P. Metalnikova, Yu. P. Gorgo

Abstract


It is known a lot of methods of conjugation of antibodies with enzymes that can produce conjugates with high antigen-binding and enzymatic activity. A variety of functional groups of enzyme and antibody molecules is used for immunoenzymatic conjugates synthesis (for example, amino, sulfhydryl, and aldehyde groups). Enhancement of diagnostic kits can be realized by improving of immunoenzymatic conjugates, and methods for its synthesis and purification. Intensification and optimization of the protein conjugates synthesis is possible by increasing the influence of macrokinetic component of the conjugation reaction. Hardware and tool implementation of this principle is the replacement of the adsorbent which is used in conjugation with an inert filter material and circulation the reaction mixture through filter material. The optimal conditions for the synthesis of the conjugates have been determinate. It has been proved the possibility of the protein conjugates synthesis for a significantly smaller (10 min) period of time compared to traditional methods. Temperature is significantly less effect on the reaction rate of conjugation in the case of the optimized method, when macrokinetic factors influence on its course more intensively. It has been shown that proteins and antibody conjugates with biotin and peroxidase conjugates prepared by an optimized method are more active, and they can be used to develop highly sensitive immunoassays.


Keywords


protein conjugates; antibodies; enzymes; biotin; macrokinetic factors immunoassay

References


Galkin O.Yu. Approaches to the synthesis of conjugates for enzyme immunoassay test-systems and evaluation of their use for diagnostics of infectious diseases // Український журнал клінічної та лабораторної медицини. – 2010. – Т.5, №4 – С. 54-60.

Saegerman C., De Waele L., Gilson D. et al. Evaluation of three serum i-ELISAs using monoclonal antibodies and protein G as peroxidase conjugate for the diagnosis of bovine brucellosis // Veterinary Microbiology. – 2004. – Vol. 100, 1-2. – P. 91-105.

Lombardi Vincent C., Schooley David A. A method for selective conjugation of an analyte to enzymes without unwanted enzyme-enzyme cross-linking // Analytical Biochemistry. – 2004. – Vol. 331, 1. – P.40 45.

Hermanson G.T. Bioconjugate techniques. – San Diego: Academic Press, 2000. – 760 p.

Goding J. Monoclonal antibodies. Principles and practice. – San Diego: Academic press, 1996. – 492 p.

Tijssen P. Practice and theory of enzyme immunoassays // Lab. Techiques in Biochem. and Molecular Biology. – 1985. – 15. – 674 p.

Habeeb A.F.S.A. Determination of free amino groups in protein by trinitrobenzene sulfonic acid // Anal. Biochem. – 1966. – Vol. 14. – P. 328-336

Яблонский Г.С., Быков В.И., Горбань А.Н. Кинетические модели каталитических реакций. – Новосибирск: Наука, 1983. – 254 с.

Берлин А.А. Макрокинетика // Соросовский образовательный журнал. – 1998. – №3. – С. 48-54.

Николаенко И.В., Галкин А.Ю., Раевская Г.Е. и др. Получение моноклональных антител к Fс фрагменту IgG человека и применение иммуноферментных конъюгатов на их основе // Клиническая лабораторная диагностика. – 2005. – №11. – С. 8-11.

Галкін О.Ю., Савченко А.А., Нікітіна К.І. та ін. Одержання та дослідження властивостей нових моноклональних антитіл до IgЕ людини // Український біохімічний журнал. – Т. 85, № 5. – 2013. – С.81-85.


Full Text: PDF (Українська)

Refbacks

  • There are currently no refbacks.
Archive
2014 2 36
2015 2 19
2016 1 2
2017 1 2
2018 1 2
2019 1  

User

Information

Journal Content

Browse

Language