Preparation And Clinical Trials Of Anti-human GPBB Antibody

Objective: Glycogen phosphorylase(GP, E.C.2.4.1.1) is a key enzyme of glycogen catabolism. GP is composed of two identical subunits. The molecular weight of monomer is 94KD. GP has three isozymes: brain isoform(GPBB), skeletal muscle isoform(GPMM)and liver isoform(GPLL). GPBB has 87% and 80% homology with GPMM and GPLL, respectively, this suggests that GPBB has different antigen epitope with other two isozymes. Recently, the studies show that GPBB may be a high sensitive marker for ischemia myocardium injury. GPBB is very important for the earlier diagnosis of ischemia myocardium injury and AMI. However, it is difficult that GPBB is isolated from human heart. For this reason, the present study predicted the antigen epitope of GPBB using computer analysis software to find the specific fragment of GPBB. The compound peptide antigens with eight-branch structure were used for preparation anti-GPBB polyclonal and monoclonal antibodies. The ELISA of serum GPBB was established.Methods:1.Prediction of antigen epitope and synthesis of compound peptide antigen of GPBB: First, GOLDKEY analysis software for nucleic acid and protein was used topredict the mono-parameter of Hopp&Woods hydrophilicity, Welling antigenicity, secondary structure , accessibility of GPBB respectively. Second, the differences in amino acid sequence among GPBB, GPMM and GPLL were detected. At last, according to the above results, the three compound peptides(GPBBP1,GPBBP2,GPBBP3) with eight branch structure were synthesized with ABI MODEL 431A. 2.Preparation and identification of anti-GPBB antibody: The rabbits were routinely immuned to prepare the polyclonal antibodies(anti-GPBBP1, anti-GPBBP2, anti-GPBBP3). Balb/c mouses were immuned with GPBBP3. The spleen cells from the mouse immuned were used for cell fusion with myeloma cells. The positive cell strain was screened with limiting dilution. The hybridoma cell strain 1A8 which can excretion antibody was established. The titer of antibodies was detected with ELISA. The antibody specificity was evaluated with Western blot and ELISA. The sandwich ELISA for the detection of homogenate of tissue and cell strain was established. 3.The establishment and clinical application of ELISA for level of serum GPBB: The semi-quantity method for serum GPBB was established with anti-GPBBP3 as the coated antibody and HRP marked anti-GPBBP2 as the detection antibody. Three patients with AMI, 200 normal persons and 649 persons of general check-up were subjected to detect the level of serum GPBB.Results:1.The selected specific antigen epitopes wereGPBBP1(N4-N15,PLTDSEKRKQIS), GPBBP2(N590-N601,RIKRDPAKAFVP), GPBBP3(N786-N804,AQVDQLYR NPKEWTKKVIR), respectively. The compound peptide antigens with eight-branch structure were synthesized.2.The titer of anti-GPBBP1, anti-GPBBP2 and anti-GPBBP3 polyclonal antibodies were 1:25600, 1:102400, 1:204800,respectively. The titer of monoclonal antibody 1A8 was 1:10000. All of three antibodies could react with native GPBB from heart. The detection system for the serum GPBB was specific.3.Results from sandwich ELISA suggested that all of 200 normal persons were negative. 3 AMI patients were strong positive. Among the 649 persons of general check-up, 626 persons were negative(96.46%). 23 persons in which were positive or strong positive(3.54%), in which 9 persons had the instability myocardial ischemia. Conclusion: 1.The prediction of antigen epitope is accurate. High titer antibodies of anti-GPBB were obtained with the compound peptide immuning animals. The antibody had high specificity.2.Serum GPBB level in AMI and myocardial ischemia patients was markedly increased and could be used for clinical diagnosis reference.