표면 플라즈몬 공명 분광법과 효소 면역 측정법에 의한 항 pB1 항체와 apoliprotein B-100의 결합능 비교 분석

Abstract

The incidences of obesity and its associated disorders are recognized as rapidly increasing serious health problem of global population, threatening the lives of millions of people. New approaches and finding the appropriate therapy to combat obesity remain high priority and is the prime target in the field of life sciences. Although, extensive research on obesity has revealed a number of potential molecular targets, only a few safe, therapeutically effective products have yet been developed for this disease. The most realistic option to address the above issue is to cause interference in the lipid homeostasis of the body by impeding the function of low density lipoproteins (LDL) in a natural condition through immunization and development of an antibody that will bind specifically to apolipoprotein B (apoB) of LDL. However, in order to pursue this strategy as a potential therapeutic target, thorough and meticulous studies are needed to evaluate the binding of this antibody to apoB either alone or as a part of LDL and very low density lipoprotein (VLDL). Incidentally, apoB mimetic peptide pB1 was designed and developed. In the present study the binding of the antibody induced from mimetic peptide pB1 (anti-B4T) to apoB was analyzed. In order to be sanguine whether any sort of binding exists between anti-B4T Ab and apoB, WB was performed. Interestingly, WB data revealed that a definite binding exist between anti-B4T Ab and apoB but reproducibility was not up to the level of satisfaction. Therefore, to analyze the binding affinity of anti-B4T to apoB, ELISA was performed. Apparently, ELISA result suggests that anti-B4T Ab has a prominent binding to apoB which was validated by using a positive control F₂C₄, an antibody being induced from apoB itself. As per our expectation the binding intensity of apoB and F₂C₄ was around 100 times more than anti-B4T and apoB. Eventually, SPR was performed to evaluate the kinetics parameters. The KD values for the binding of F₂C₄ and apoB, pB4 and B4T are 1.21 X 10^(-11), 5.68 X 10^(-9), and 1.77 X 10^(-3) respectively. The KD representing the binding of anti-B4T Ab and apoB, pB4 and B4T are 3.11 X 10^(-7), 5.4 X 10^(-8), and 9.31 X 10^(-10) respectively. Notably, ELISA and WB data revealed a striking resemblance. However, in some cases the binding analysis data between ELISA and SPR are contradictory, especially for F₂C₄ and anti-B4T binding of LDL, probably due to hydrophobicity and complex molecular architechture of LDL that is not compatible for SPR analysis. It is noteworthy that WB, ELISA and SPR are considered all together for accurate and reliable characterization of binding. Binding analysis by these techniques agreed that there is a moderate but definite molecular recognition and interaction of apoB by anti-pB1 antibody. Further intrigue studies with this antibody will open the door for the possibilities of promising therapeutic approach in controlling and inhibition of obesity using anti-pB1 Ab.