Development Of Surfactant Protein D Quantitative Assay Method And Exploration Of It As The Marker Of Lung-related Diseases

Objective:1. To prepare the surfactant protein D (SP-D) by chemical synthesismethod; and prepare anti-SP-D polyclonal antibody (pAb).2. Establishment and identification of hypoxanthine-guaninephosphoribosyl transferase (HGPRT) deficient guinea pig hybridoma cellline; and preparation of anti-SP-D monoclonal antibody (mAb) with thisguinea pig hybridoma cell line.3. To prepare nano-magnetic particles for diagnositic reagents.4. Development and methological evaluation of a nano-magneticparticles chemiluminescence immunoassay for quantifying serum SP-D.5. Determination of SP-D in patiens with various lung-related diseasesand control subjects with established NM-CLIA, and investigating theclinical significance of serum SP-D as marker of lung-related diseasesMethods:1. Prediction and synthesis of SP-D antigen epitopes usingJameson-Wolf and Emini methods, HPLC purification and massspectrometric identification. Preparation of SP-D-BSA conjμgation complex, and preparation of rabbit anti-SP-D pAb using SP-D-BSA asimmunogen.2. The establishment and screening of HGPRT Deficient Cell Line byirradiating guinea pig transformation embryonic cell104C1with the60Coγ-Ray source, and preparation of anti-SP-D mAb.3. Dextrans coating nano-magnetic particles were prepared byco-precipitation, and surface modification of it with carboxyl group fordiagnositic reagents, and prepareation of NM-SA with EDC method.4. Development of a NM-CLIA method for quantifying serum SP-D,and the performance of NM-CLIA were evaluated5. The level of serum SP-D in silica-exposed popμlation, patients withinterstitial lung disease (ILD), acute lung injury (ALI) and chronicobstructive pμlmonary disease (COPD) were determined by the establishedNM-CLIA; and preliminary evaluation of the clinical significance of serumSP-D as the biomarker of lung-related diseases was performed..Results：1. SP-D antigen epitopes were predicted by DNAStar bioinformaticssoftware and synthetized. Monoclonal antibody was prepared withhybridoma technique, HPLC purification and mass spectrometricidentification.2. The establishment and screening of five hybridoma cell lines were2B2,3F1,3H4,4C7and5A3, respectively.The cross-reaction assay of analogues shoed that2B2,3F1,4C7and5A3coμld specific binding withSP-D antigen rather than SP-A, SP-B and SP-C. The hybridoma antibodystable secretion was3F1after40times of freezing and thawing. Theanti-SP-D monoclonal antibodies were prepared. We obtained high affinityand titers by protein G purified, it coμld specificly recognize20-35aa ofSP-D protein sequence.3. The prepared nano-magnetic particle size about was300nm, andgood uniformity. Prepareation of NM-SA with EDC method had goodstability by37℃oven destroyed for7days.4. A novel quantitative method using NM-CLIA for detection of SP-Dwas developed. The linear range was from1.0to1000.0ng/ml; theminimum detection limit was0.22ng/ml; the intra-and inter-assay CVvalues were both <6.0%. The dilution and adjunction recovery rate were91.17%~106.42%and91.00%~109.63%, respectively. The Cross-reactivityrate with SP-A（10000ng/ml）、SP-B（10000ng/ml）and SP-C（10000ng/ml）were all <0.1%. After three times of freezing and thawing and storage120days at-20℃, or existence of0.2mg/ml bilirubin,5mg/ml hemoglobin,10mg/mltriglyceride,1500U/ml rheumatoid factor, and anticoagμlants, itsdetection performance was no significant change. The total time of theassay was about22minutes.5. The concentration of SP-D in the serum was significantly elevatedin silica-exposed worker group, ILD group, ALI groups and COPD group compared with contro（lP<0.01）; Compared with silica-exposed group, thelevels of SP-D in the serum of silicosis suspect (0+),silicosis phase I group,ILD group, ALI group and COPD group were significantly elevated (P<0.01, respectively). The levels of SP-D in the serum of silicosis phase Igroup were remarkably increased (P<0.01, respectively) compared withsilicosis suspect (0+), ILD group, ALI group and COPD group. The levelsof serum SP-D increased with the development of silicosis stage（0.764,P<0.01）, COPD group was also higher than ILD group and ALIgroup.(P <0.01, respectively). But there were no significant differencesamong silicosis suspect (0+), ILD group and ALI group.Conclusions:1. The SP-D and its pAbs are successfμlly prepared.2. The establishment and screening of guinea pig hybridoma cell lines,and the SP-D mAbs have successfμlly carried out.3. Diagnostic nano-magnetic particles are successfμlly prepared.4. A high-performance NM-CLIA for the quantitative measurement ofserum SP-D with the guinea pig anti-SP-D mAb and rabbit anti-SP-D pAbhas been developed.5. Serum SP-D levels can be used as auxiliary diagnosis biomarker oflung-related diseases.