Study On The Quantitative Immunoassay For Biomarkers Of Bone Metabolism

Osteoporosis affects about 200 million people worldwide,ranking seventh among the common diseases and multiple diseases.According to the WHO survey,Osteoporosis will be the second largest medical problem after cardiovascular disease.At present,bone mineral density（BMD）is the main diagnostic standard in the diagnosis and prognosis monitoring of osteoporosis.BMD can only statically reflect the bone shape,but the change of bone mass cannot be detected.In view of the lack of accurate and specific biomarkers in the dynamic monitoring of bone metabolism,biomarkers of bone metabolism have been a research hotspot in the past decade.Recently identified markers of bone metabolism with clear significance include:Bone-GLA-protein（BGP）,parathyroid hormone（PTH）,total collagen amino end extension peptide（P1NP）,25 hydroxyvitamin D（25-OH VD）.These markers can accurately evaluate the efficiency of bone conversion at the level of intact bone tissue,so as to realize quantitative auxiliary diagnosis of osteoporosis in clinical practice.However,how to realize the clinical detection of these biomarkers and the clinical value of the biomarkers and diseases is still a difficulty,mainly due to the difficulty in obtaining antibodies for bone metabolism markers and the immaturity of detection methodology.In China,due to the above-mentioned technical difficulties,only Roche Diagnostics from Switzerland can produce and supply the reagents for bone metabolism detection at home and abroad.This greatly restricts the promotion of markers of bone metabolism with clear value in China,which is a great economic burden for the majority of patients and cannot benefit the majority of patients.In this study,a pair of P1NP monoclonal antibodies were obtained by peptide immunization,and the P1NPα1 chain was successfully expressed by Escherichia coli expression system to purify the natural P1NP protein from the pleural effusion of tumor patients.Vitamin D antibody with high specificity and affinity was successfully immunized with VD₃.Relying on the chemiluminescence immunoassay technology platform,the chemiluminescence immunoassay reagent for bone metabolism marker was developed,and the clinical samples were tested,and the correlation with Roche results was very good.A large number of samples from patients with different diagnoses were also collected to test for BGP and 25 hydroxyvitamin D.Through the data analysis,it can have a certain guiding significance for clinical diagnosis and treatment.Bone metabolism markers were measured for patients with lung transplantation before and after surgery.The study found that compared with the traditional bone density detection,bone metabolism markers can better monitor the dynamic changes of bone mass in a certain stage,which is of great significance for the diagnosis and treatment of osteoporosis in patients with lung transplantation after surgery.The main research contents of this paper are as follows:（1）Establish a method for clinical detection of key biological raw materials for bone metabolism markersThe P1NP expression system of Escherichia coli was constructed to express theα1 chain of P1NP protein for subsequent antibody screening.The natural P1NP protein was successfully isolated and purified from human pleural effusion for the screening of antibodies and the preparation of calibration products.Three differentα1 chain short peptides were designed to immunize mice,and the P1NP monoclonal antibodies were screened for further development of immune products.A similar approach was used to develop vitamin D antibody.Mice were immunized with VD₃ antigen,and the monoclonal antibodies of vitamin D were screened and purified for the development of subsequent reagents.（2）Development of the automatic chemiluminescence reagent for bone metabolism markerBGP,PTH,P1NP and 25-OH VD chemiluminescence detection methods were established respectively.The precision of BGP reagent was less than 10%.The recovery was in the range of100%±10%.The reduction level of luminescent value was within 10%after 7 days accelerated test.Method comparison between BGP reagent and Roche reagent showed that the correlation coefficient R² was 0.98,which showed a very good correlation with Roche results.The hook effect is not found when the content of BGP in the sample is as high as 4000ng·m L^-1.The linear factor R of P1NP reagent was greater than 0.99.The limit of detection was3.11ng·m L^-1.The precision of the three batches of P1NP reagents was lower than 10%.When the concentration of P1NP in the sample was greater than 4000 ng·m L^-1,no hook effect was found.Method comparison with Roche P1NP reagent showed that the correlation coefficient R² was 0.98,which showed a good consistency.The precision of 25 hydroxyvitamin D reagent between batches and batches is less than10%,and the recovery was in the range of 100%±10%.The results of accelerated stability test show that after 7 days of acceleration,the luminous value is reduced within 10%.The developed reagents were compared with Roche and Diasorin respectively.The results showed that compared with Roche,the correlation coefficient R² was 0.9.Compared with Dia Sorin,the correlation coefficient R² was 0.93,both of them are very good.Roche,Dia Sorin and developed method in this study were compared with the gold standard in the liquid phase mass spectrometry,Roche and liquid mass spectrum ratio on the slope was 1.1,the correlation coefficient R² was 0.80,Dia Sorin and liquid mass spectrum ratio on the slope was 0.8,the correlation coefficient R²was 0.79,the establishing method and liquid phase mass spectrum ratio on the slope was 0.79,the correlation coefficient R²of 0.90.Based on the comprehensive results,the detection method established in this study is even more than that of two imported manufacturers.The limit of detection PTH reagent was 0.2 pg·m L^-1.The linear factor r is greater than0.99.The precision of three samples with different concentrations was less than 10%.Through the method comparison with Roche’s PTH reagent,the slope was 0.99,and the correlation coefficient R²was 0.96,which is very consistent with Roche’s results.（3）The study of relationship between bone metabolic markers and clinical diseasesThe relationship between BGP and 25 hydroxyvitamin D and various clinical diseases was studied.Women with high BGP levels were more likely to be obese（24%）and to have cancer（51%）.Men with high BGP levels were more likely to have cancer（56%）and cardiovascular disease（32%）.The study found that 94.98%of the patients tested for25-hydroxyvitamin D had concentrations below 30 ng·m L^-1(reference value:30 ng·m L^-1).Of those with low vitamin D levels,50%were obese;About 20%are cancer patients;14%were fracture patients.According to the distribution statistics of 25 hydroxyvitamin D content in different populations,the results showed that the content of 25 hydroxyvitamin D in human body may be closely related to cancer,diabetes,cardiovascular and cerebrovascular diseases and osteoarthritis.This study also established for the first time a male reference range for P1NP.The relationship between bone metabolism markers and lung transplantation was first studied.The bone metabolism detection reagent developed in this project was used to regularly track and detect the changes in bone metabolism markers before and after lung transplantation patients.The results showed that in the monitoring of bone mass in a short time,bone metabolism markers can better indicate the change of bone mass,while the traditional bone mineral density detection is lagging behind the bone metabolism markers,which is of great significance for clinical diagnosis and treatment.