On-line Microdialysis Sampling Combined With Flowinjection Chemiluminescence System For Measurement Of Antibody Affinity

With the continuous development of biology and medicine,the technology of monoclonal antibody(McAb)has been widely used in the fields of immunotherapy,immunoassay and immunological purification.In the course of preparation and screening of McAb,one of the most important work is to evaluate the affinity of McAb secreted by different hybridoma cell lines.Therefore,it is of great significance to develop rapid,simple and accurate methods for the measurement of antibody affinity.Microdialysis(MD)is a sampling technique with the advantages of in situ,in vivo sampling and online,real-time detection.Small-molecular substances in the external medium diffuse passively through the dialysis membrane into the probe lumen due to the concentration gradient,while macromolecules such as proteins and cells can be excluded,so as to realize the sampling of small-molecular substances.Flow injection(FI)-chemiluminescence(CL)assay is a promising analytical technique combining FI injection technique with CL detection,showing a series of advantages such as simple manipulation,high sensitivity,rapid analysis speed and low cost.As a trace analysis technology,the principle of FI-CL is mainly on the basis of the linearity between the concentration of analytes and chemical luminescence intensity.In this study,a novel label-free method based on on-line MD sampling combined with FI-CL detection was designed for measuring antibody affinity to hapten in a homogeneous system,by using ?-agonist McAb as models.Herein,a novel methodological platform was established to evaluate the affinity of McAb against small molecular hapten.Part 1 Measurement of the affinity of terbutaline McAb utilizing on-line MD sampling combined with FI-CL.In this work,a novel label-free approach based on on-line MD sampling combined with FI-CL detection was designed for measuring terbutaline McAb affinity to hapten in a homogeneous system.The unbound hapten(terbutaline)was sampled on-line by the MD probe and injected into the FI-CL system for quantification,while McAb and McAb-terbutaline conjugation were excluded.The CL intensity was used to quantify the amount of terbutaline in the dialysate based on its enhancement on the emission intensity of the CL system of luminol-K3[Fe(CN)6] in basic medium.The results were evaluated by MD recovery to obtained concentrations of the unbound hapten.Then they were treated with Scatchard analysis and Klotz analysis to calculate the affinity constant.The affinity constants obtained using Scatchard analysis and Klotz analysis were 4.9 × 106 M-1 and 4.9 × 106 M-1,respectively,indicating the terbutaline McAb was an antibody with medium affinity and showing good agreement with those obtained from thiocyanate elution test.This developed method showed many merits such as simple manipulation,low-cost and short assay time,and provided a new pathway for measuring McAb affinity.Part 2 Measurement of the ractopamine McAb affinity utilizing on-line MD sampling combined with FI-CL.A novel label-free method based on MD on-line sampling combined with FI-CL realtime detection was developed to directly measure the affinity between ractopamine and its McAb.The unbound ractopamine was sampled on-line by the MD probe and injected into the FI-CL system for quantification,while McAb and immunological complex were excluded.The amount of hapten in the dialysate was quantified by the CL intensity based on the fact that ractopamine could enhance significantly the CL emission of luminolKMnO4 system in basic medium.Afterward the detected concentration of unbound ractopamine in the dialysate was calibrated with MD probe recovery to obtain that in the immunoreaction solution.The affinity constants of ractopamine McAb calculated by Scatchard analysis and Klotz analysis were both 1.0 × 106 M-1,showing negligible difference and indicating the ractopamine McAb was an antibody with medium affinity.The results were in good agreement with those obtained from thiocyanate elution test.Therefore,we believe that the designed protocol is a facile and reliable approach for evaluating antibody affinity,and can be applied extensively in immunochemistry.