Assessment of molecular recognition element for the quantification of human epidermal growth factor using surface plasmon resonance|
Rosti, Ira Amira; Ramanan, Ramakrishnan Nagasundara; Ling, Tau Chuan & Ariff, Arbakariya B.
Background: A method for the selection of suitable molecular recognition element (MRE) for the quantification of human epidermal growth factor (hEGF) using surface plasmon resonance (SPR) is presented. Two types of hEGF antibody, monoclonal and polyclonal, were immobilized on the surface of chip and validated for its characteristics and performance in the quantification of hEGF. Validation of this analytical procedure was to demonstrate the stability and suitability of antibody for the quantification of target protein.
Results: Specificity, accuracy and precision for all samples were within acceptable limit for both antibodies. The affinity and kinetic constant of antibodies-hEGF binding were evaluated using a 1:1 Langmuir interaction model. The model fitted well to all binding responses simultaneously. Polyclonal antibody (pAb) has better affinity (KD = 7.39e-10 M) than monoclonal antibody (mAb) (KD = 9.54e-9 M). Further evaluation of kinetic constant demonstrated that pAb has faster reaction rate during sample injection, slower dissociation rate during buffer injection and higher level of saturation state than mAb. Besides, pAb has longer shelf life and greater number of cycle run.
Conclusions: Thus, pAb was more suitable to be used as a stable MRE for further quantification works from the consideration of kinetic, binding rate and shelf life assessment.
antibody; BIAcore; biosensors; epidermal growth factor; validation