Preparation And Experimental And Clinical Study Of Monoclonal Antibody Against Advanced Oxidation Protein Products

Advanced oxidation protein products (AOPPs) are dityrosine-containing protein crossing products isolated from the plasma of hemodialysis (HD) patients, which was firstly reported by Witko-Sarsat in 1996. AOPPs represent a generic name for final products of oxidative modification of proteins caused by oxidative stress in HD patients. AOPPs rise as novel uremic toxins with the progression of chronic renal failure (CRF) and oxidative stress and accumulate in HD patients'plasma and tissues. It HSA been clarified that AOPPs are a novel class of pro-inflammatory mediators with monocytes being their elective cellular target. AOPPs are able to trigger the oxidative burst of monocytes and the release of inflammatory cytokines, and take part in the process of systemic micro-inflammation of CRF patients, which is a crucial pathogenic link to long-term uremic complications, such as dysregulation of immune system, accelerated atherosclerosis and dialysis-related amyloid arthropathy. Plasma AOPPs are mainly composed of high molecular weight-AOPPs (HMW-AOPPs) and low molecular weight-AOPPs (LMW-AOPPs). HMW-AOPPs, aggregates of oxidized albumin, which play the main role in HD long-term complications, are our focal point. However, the formation of AOPPs is irreversible and their combination with proteins in vivo greatly hinders the removal through any hemodialysis modality, which results in the limitation of therapy.Up to now, AOPPs were measured by spectrophotometry on a microplate reader and were calibrated with chloramine-T solutions. But this method lacks sensitivity and specificity. Otherwise it will give us much false positive information. Compared with spectrophotometry, immunolocal method is specific, sensitive and HSA less false positive rate. Therefore, if a test of immunology can be developed, it may be a good succedaneum or supplement for spectrophotometry in the diagnosis of AOPPs relative oxidation stress.AOPPs serve as a pro-inflammatory mediator which participates in many pathologic and physiologic reactions. It HSA vital significance to observe AOPPs deposition situation in tissues and cells'plasma using the AOPPs antibody through the immunohistochemical methods and western blotting, ELISA and so on. AOPPs aggravate dysregulation of immune system, atherosclerosis and dialysis-related amyloid arthropathy through the deposition in the blood vessel. Therefore, it is possible to use its corresponding antibody coupled dialyzer to block AOPPs'function and provide one new treatment.According to the clues above, in order to study the AOPPs prevention therapeutic methods and AOPPs detection, we prepare its monoclonal antibody by using purified AOPPs as antigen and establishe a double sandwich enzyme immunoassay for quantitative detection of AOPPs.Methods1. To achieve purified human serum proteins (HSA), a gel filtration was performed on a GE Healthcare Hitrap 26/60 sephacryl S-300 high Resolution adapted to a FPLC system. AOPPs-HSA was prepared in vitro by incubation of 200 mg/ml purified HSA and 200mmol/l of hypochlorous acid (HOCl) for 30min. To achieve separations of AOPPs-HSA bound to HSA, a gel filtration was performed as HSA. Nondenaturing and denaturing electrophoresis was performed according to Laemmli's procedure to identify HSA molecular weight and purity. The molecular weigh of AOPPs-HSA can be determined from known protein standard. In order to identify activity of purified AOPPs-HSA, we investigate the effect of AOPPs on the secretion of tumor necrosis factor a (TNF-a) in monocytes.2.The monoclonal antibodies against AOPPs were generated by fusion spleen cells from immunized BALB/c mouse with purified preparations of AOPPs antigen and SP2 /0 mouse myeloma cells. The culture of hybridoma lines were first screened by indirect ELISA, then mouse ascites were prepared. Immunoglobulin isotype was determined using a commercially available isotyping kit.The specificities of McAbs were characterized by indirect ELISA and Western Blotting.3. The purified monoclonal antibody specific to antigen AOPPs were labeled with HRP by periodinate sodium. Optimal paired McAbs were selected, which 1H1 McAb was employed as coated McAb and 1-C5 McAb as detecting McAb after HRP labeling. Following the optimization of working concentrations of coated and detecting mAbs, we successfully established ELISA for detecting AOPPs. Evaluation tests of methodology were done, including recovery test, repeatability test and linear range investigation.4. AOPPs of thirty-six control, eighteen undialyzed patients with advanced chronic renal failure and fifity-six HD patients were detected in plasma by sandwich enzyme immunoassay and spectrophotometry respectively. The influence of high speed gentrifugalism to AOPPs examination was observed in spectrophotometry and ELISA. Correlated data were analyzed by linear association test.Results1. Elution patterns obtained from HSA fractionation by size-exclusion chromatography were visualized as three peaks. Under both nondenaturing and denaturing conditions, fraction 3 was detected as a single band with molecular weight of around 67 kDa, which was purified HSA. AOPPs-HSA were prepared by exposure of purified HSA to HOCl. Elution patterns obtained from exposure products fractionation by size-exclusion chromatography were visualized as 6 peaks. Protein aggregation was clearly seen by the presence of a smear on top of the gels in PAGE and SDS-PAGE of fraction 1-4, presumably as a result of albumin cross-linking. But under denaturing condition, the smear was less intense than that under nondenaturing condition. Under nondenaturing and denaturing conditions, a single band of around 67 kDa in all the 6 fractions was detected, presumably as a result of non cross-linking albumin. The molecular weight of purified AOPPs-HSA by molecular sieve was 670KDa.2. AOPPs concentration in purified AOPPs-HSA was 20.38μmol/l examined by spectrophotometer, much higher than that in HSA. Purified AOPPs-HSA induced TNF-a secretion in monocytes, while HSA didn't. Time effect showed that TNF-a secretion increased to the highest level after 12 hours of AOPPs-HSA stimulation.3. Four hybridoma cell lines were successfully achieved by hybridoma technique, using purified AOPPs-HSA fraction 1 to immune BALB /c mice. Of the four strains, three could stably secret McAbs with a high specificity to AOPPs, named as 1-C6, 1-H1 and 3-A10, one secreted McAb with a high specificity to AOPPs and HSA, named as 1-C5. All the strains had low affinity to AOPPs-BSA, BSA and CD26. 1-C5, 1-C6 and 1-H1 were identified as the immunoglobulin IgG1 isotype, 3-A10 as IgG2a.4. A sandwich enzyme immunoassay for quantitative detection of AOPPs contents was developed using 1-H1 as coating antibody and horseradish peroxidase (HRP) labelled 1-C5 as labelled antibody. The optimum dilutions for coating antibody and labeled antibody were 55μg/ml and 1:500 respectively. The lowest limitation of detection was 12ng/ ml and the linearity of the standard curve was preferable from 20 to 300 ng/ ml. The coefficients of variation for intra-assay and inter-assay precision were 4.8% and 7.17 % respectively. The mean sample recovery rate was 98%.5. AOPPs of thirty-six healthy controls, eighteen undialyzed patients with advanced chronic renal failure and fifity-six patients on hemodialysis were detected in plasma by sandwich enzyme immunoassay and spectrophotometry respectively.In vivo, plasma level of AOPPs was the highest in HD patients. Undialyzed patients with advanced chronic renal failure had higher level than healthy controls. 16 out of 18 serum samples (88.9 %) from undialyzed patients with advanced chronic renal failure were shown to be positive by spectrophotometry while 10 out of 18 serum samples (55.6 %) were shown to be positive by ELISA. In addition , 51 out of 56 serum samples (91.1 %) from HD patients were shown to be positive by spectrophotometry while 42 out of 56 (75 %) were shown to be positive by ELISA. High-speed centrifugation decreased apparently AOPPs level when using spectrophotometry,while had no influence on AOPPs level when using ELISA. AOPPs level detected by double-sandwished ELISA correlated positively with that by spectrophotometry(r=0.543, P<0.001, n=110). AOPPs level correlated positively with the level of creatinine (r=0.664, P<0.001) and BUN (r=0.606, P<0.001) and inversely with Hb (r= -0.535, P<0.001).Conclusion1. After the successful purification of human serun albumin and AOPPs-HSA, we collected purified AOPPs protein and successfully used it as an antigen to immunize BALB/c mice. Four hybridoma cell lines were achieved successfully with hybridoma technique, three of which could stably secret specific McAb against AOPPs, while one against AOPPs and HSA.2. A sandwich enzyme immunoassay for quantitative detection of AOPPs contents was developed by using 1-H1 as coating antibody and horseradish peroxidase (HRP) labelled 1-C5 as labelled antibody. The method can establish a rapid assay for the measurement of AOPPs antigen and obviates the disadvantages associated with spectrophotometer measurement.3. In vivo, plasma level of AOPPs was the highest in HD patients. Undialyzed patients with advanced chronic renal failure had higher levels than healthy controls. The level of AOPPs correlated positively with the level of creatinine and with BUN and inversely with Hb.4. All the methods developed in this study, such as antibody preparation and identity, and Sandwich ELISA, can not only be used as effective tools to monitor plasma AOPPs, but also facilitate the understanding of the disease related with AOPPs and antibody preventation therapy.