Studies On Preparation Of Monoclonal Antibody Based Immunoaffinity Resin And Application In Human Plasma High Abundant Proteins Removal

About thousands of proteins presented in the human plasma with the concentration varied from pg/mL to mg/mL.In the exploration of plasma proteome,this highly dynamic concentration of plasma proteins deterred the detection of low abundant proteins.To obtain the information of low abundant proteins,plasma sample should be pre-separated.The pre-separation include two approaches:removal of high abundant proteins or enrichment of low abundant proteins.High abundant proteins removal methods include density centrifugation,chemicals precipitation,molecular weight cut,ligand(Cibaron blue,antibody,peptide etc.) affinity chromatography and so on.The method of enriching low abundant proteins include lectin binding glycoproteins,metal surface binding phosphor-proteins, hydrophobic surface or beads binding low molecular weight proteins or peptides. Poly colonal antibody based affinity resin was mostly evaluated and applied in high abundant proteins removal by abroad researchers.The shortcomings of commercial high abundant proteins removal system were incompatible buffer system with each other,nonspecific binding and un-homogenous activity loss.Up to now,there is still no domestic institute or company engaging in study or produce such immunoaffinity resin for plasma proteome research.The covalently immobilization of antibody usually adapt CNBr activated Sepharose. Unfortunately,this method were not provide a site-directed immobilization,that is to say,the antibody binding domain maybe blocked by Fab amino groups binding to media other than the Fc amino groups.The binding activity of immobilized antibody depended on the reactive ratio of Fc amimo group to Fab amino group.Hydrazide-aldehyde immobilization provided a site-directed immobilization method, but in practice,hydrazide groups-aldehyde group reaction depends on the glycon proportion in antibodies and usually occurred self-reaction in experiments.Protein G/A-mAb cross link required bifunctional cross linker such as DMP(Dimethyl pimelinediimidate chloride) and reacted in mild work buffers.The nonspecific binding of immunoaffinity resin existed in the interaction between sample proteins and container surface,support media and samples proteins themselves.Thus,chromatography system,immoaffinity resin and sample treatment affected the results.The profile of nonspecific binding proteins should be supplied after high abundant proteins removal for differential comparison.Monoclonal antibody technology was practical and feasible to quality control.The unique binding site of monocolonal antibody can provide a high reproducibility in nonspecific binding.Objective:1) Select the best efficient antibody immobilization method.2) Screening the mAbs for high binding capacity and robust behavior in bound-elution.3) Identifying the nonspecific bound proteins.Methods:1) antibody immobilization method were referred to the instruction of CNBr-activated Sepharose 4 Fast Flow(GE Healthcare,USA),CarboLink^TM Coupling Gel(PIERCE Cat 20391) and POROS(?) 20XL Media Immobilizing Antibody for Perfusion Immunoassays.2)14 strains of mAb against albumin and 12 strains mAb against IgG were immobilized at the ratio of 1mg antibody to 0.6mL gel (50%slurry).600μL gel were packed into SpinX tube and load 15μL human raw plasma then incubated for 20 minutes at room temperature,then centrifuging 1 min at 3000g for collecting flow through sample,adding 600μL 0.1M PBS washing 3 three times,mix all flow through samples for SDS-PAGE analysis.100μL 0.1M Gly-HCl pH2.5 was applied as elution buffer to dissociate the bound proteins for SDS-PAGE ananylsis.3) loading 0.2mg,0.5mg,1mg and 5mg albumin or IgG for testing immobilized mAb binding capacity.4) Bound proteins were separated by 2-DE and cut the protein spot from the gel then degraded by trypsin,peptides were harvested then loaded into MALDI-TOF/TOF for identification.Results:1)Protein G/A-mAb cross link method immobilized the largest amount of available mAbs than other two method.2) Two mAbs(HPSIIB002,HPSIIB007) against albumin and three mAbs(HPSIS010,HPSIS020,HPSIA025 and HPSIIB011) against IgG demonstrated the reliable behavior of bound-elution.3)1mg mAb against albumin and 0.333mg mAb against IgGs can remove most of albumin/IgG in 10μL raw plasma sample.The treated plasma sample showed a high resolution 2-DE results. In ten cycles of removal run,the total albumin/IgGs binding capacity of immunoaffinity resin decreased little while the fragments capacity decreased proved by Western-blot.4) The nonspecific binding proteins including Vitamin D binding protein,Fibrinogen,Transthyretin,Keratin 10,Transferrin,C3,Proapolipoprotein, CD5 antigen-like protein and Alphal-Antitrypsin were identified by 2-DE MALDI-TOF/TOF approach.Conclusion:1) we obtained 2 mAb and 4 mAbs for producing albumin/IgG removal immunoaffinity resin.2) we determined Protein G/A-mAb cross link method as a best performance of immobilization mAbs.3) Immnoaffinity resin showed reliable performance in 10 cycles run of bound-elution for high abundant proteins removal.4) Nonspecific binding proteins were identified by MALDI-TOF/TOF method.Thus,we developed a high abundant proteins removal immunoaffinity resin and convenient to perform without chromatography instruments.The albumin/IgGs removal immunoaffinity resin are applied in the proteome research ordered by the Department of Chinese Triditional Medicine,Southern Medical University.This research route can be also applied in other high abundant proteins removal such as the sample from saliva,cerebrospinal fluid and urine.