Extraction And Purification Of Pulmonary Surfactant-associated Protein A And Preparation And Pilot Applied Study Of Monoclonal Antibody Against Human SP-A

Objective 1. To extract and purify high-purity and high-activity pulmonary surfactant-associated protein A (SP-A) from human bronchoalveolar lavage fluid (BALF) . 2. To prepare monoclonal antibody against human SP-A by using purified SP-A for the immunization antigen injected into the peritoneal cavities of BALB/C mice. 3. To set up the methods of detection of SP-A by a two-site sandwich ELISA used purified SP-A mAb and to prepare for the preparation of the kit which can detect the content of human SP-A rapidly.Methods 1. Two healthy body pulmonary were contributed by the sib of dead. Human SP-A was isolated and purified by the following steps: BALF was obtained by lavaged the body pulmonary four times with 0.9% sodium chloride. BALF was centrifuged at 10000Xg for 40 minutes after the removal of cells and cell debris by centrifugation at 500Xg for 15 minutes. The pellet was suspended in 6 M urea. This suspension contained recovery SP-A was layered on a maltosyl-agarose column and eluted using a gradient of EDTA. The obtained SP-A was further purified by gel - filtration on superpose-6 column. The final product was identified by SDS-PAGE and western blotting analysis.2. The SP-A isolated from BALF was fully emulsified in Freund' s adjuvant and injected into the peritoneal cavities of BALB/C mice every week. The same process was carried out three time. Then purity SP-A antigen was injected into the Spleen at the fourth time. BALB/C mice were injected purity SP-A antigen through tail vein three days before cell fusion. Mice was boosted and its eyepit blood was collected to detect the antibody titer. The immune spleen cells isolated from the mice were fused with SP2/0 cell line using50% polyethyleneglycol (PEG) 3500 and distributed to wells of 96 -well microplates. Hybrid cells of spleen and myeloma cells were filtrated by the selectivity HAT medium. After two weeks the selectivity HAT medium was replaced by HT medium. The positive clones were detected by indirect ELISA technique and cloned three times in limiting dilution to monoalonality. By above process we obtained hybrid cell line which could product SP-A mAb stably. The secrete activity of the cell lines was determined by frequently freeze and recovery. The hybrid cell lines was injected to the peritoneal cavities of BALB/C mice to product ascites. The ascites was collected after 7~10d. The SP-A mAb was purified from the ascites by salt out methods. The immune activity of SP-A mAb was detect by indirect ELISA. The SP-A mAb was further identified by western bolting analysis. The sub - type of the SP-A mAb was detected by the mice monoclonal antibody sub-typing kit of sigma.3. The affinity constant of SP-A mAb were detected by indirect ELISA. Two-site sandwich ELISA was carried out to detect the content of the SP-A by using purified SP-A mAb which have higher affinity constant. The second antibody was labeled with HRP. The best antibody combination was filtrated .Result 1. We obtained 40 mg high-purity SP-A finally from the BALF of two body pulmonary by the affinity chromatography with maltosyl-agarose column. There are five clear band on the SDS-PAGE gel with apparent molecular weights (Mr) of 36 70 and >118 kDa. All the visible bands were identified to be special SP-A by western blotting analysis.2. The antibody titer of serum before injection through tail vein was 1/128000 and was 1/256000 at the time of fusion. The fusion efficiency was 80%. The antibody positive secrete efficiency was 85%. We obtained 11 hybrid cell line which could product SP-A mAb stably finally (1B6D9E1C2, 1C1E7E7D2, 1B6D7D3C6,1B6B4F7G9,1A5F6H2D5, 1D5G9C10H3, 1C1E9D3G5, 2F2F5E9D4, 2F11D3C2A8, 2B6C4F6E7, 2B6C4E2H1). The sub-type of five antibodies(1B6D9E1C2, 1C1E7E7D2, 1B6D7D3C6,1B6B4F7G9, 1A5F6H2D5) with higher antibody titer were detect to be IgG1 or IgG2b respectively. All of the hybrid cell lines were determined to have stable secrete antibody activity by freeze and recovery frequently.3. We obtained 58 ml ascites from 15 mice by injection of the hybrid cells into the per...