| Backgrounds:In the first decades of the discovery of complements(C),complements was thought to be a simple lytic cascade reaction aimed to kill bacteria infecting the host organism.With the continuous development of immunology and molecular biology,the understanding of complements has been gradually deepened.Nowadays,the view that complements is a complex innate immune surveillance system that plays a key role in homeostasis,inflammation and defense against pathogens in the host is widely accepted.The researches over the years have shown that the complement system is involved in all aspects of the functioning of the body’s immune system.It should occupy an important place in contemporary immunological research and in the clinical laboratory assessment of the immune function of the organism.However,the current protocols for testing the complement system in clinical laboratories do not adequately reflect the clinical value of complement system testing,and clinical laboratories lack low-cost,high-volume assays for testing the immunological function of complement.In this study,based on the immunological principles related to the activation of the complement system,we propose two aspects of the immunological function of the complement test,the total complement activity(AC),which reflects the own components of the complement system,and the complement activators activated complements activity(ACA),which reflects complement activators(mainly circulating immune complexes).And based on the CH50 hemolysis assay and the phenomenon of dissociated erythrocyte agglutination based on erythrocyte fragmentation,a sensitive and accurate assay suitable for high-volume testing of the immunological function of the complement system in clinical laboratories was gradually established.This method allows for the combined analysis of AC and ACA.In this study,New Zealand rabbits were used as experimental subjects to explore the relationship between complement immunological function and MCH gene polymorphisms.Methods:1.Quantitative complement is activated and consumed with complement activators in the serum to be tested,and the remaining complement is activated and consumed by adding sensitized human O-erythrocytes,and the ACA is quantified by the degree of hemolysis.The interference factors of the method were investigated and the test conditions were optimized.The resolution,stability and value of the method for clinical application were validated.2.The number of fragments produced by complement lysis of erythrocytes was counted by flow cytometry to quantify the AC of complement.Erythrocyte agglutination was measured by flow cytometry and used as a basis for quantifying the amount of hemolysin.The erythrocyte fragmentation index EFI indicates the test result with the following formula:EFI=M*M2/(M1+M2),where M is the number of fragments of erythrocytes going to the background,M1 is the number of unagglutinated erythrocytes,M2 is the number of agglutinated erythrocyte groups,and M2/(M1+M2)is the agglutination coefficient which indicates the degree of erythrocyte agglutination.Mildly varying the concentrations of hemolysin and erythrocytes for optimization of the test conditions.The resolution,stability and value of the method for clinical application were validated.3.The effect of corresponding erythrocyte fragments on erythrocyte agglutination was explored with three types of erythrocytes,A,B and O,and the level of effect of erythrocyte fragments on erythrocyte agglutination was quantified by auto hematology analyzer.4.Using sensitized human O-erythrocytes as an indicator system,the effect of erythrocyte fragments on erythrocyte agglutination from lysis of sensitized erythrocytes by complement and residual complement after consuming quantitative complement by complement activators was examined by auto hematology analyzer.The mean hemoglobin concentration of erythrocytes(MCHC)of the reaction system was used to quantify the AC and ACA of the serum to be examined.The interference factors of the method were investigated and the test conditions were optimized.The resolution,stability and value of the method for clinical application were validated.5.The method of the detection of complement activators activated complements activity using human O-erythrocytes as the indicator system and a flow cytometry-based assay for the detection of total complement activity were used to detect changes in complement immunological function after inoculation of New Zealand rabbits with the corresponding antigen.PCR,PCR-SSCP and gene sequencing were used to detect the polymorphism of MHC gene in New Zealand rabbit,and to analyze the relationship between complementary immunological function and MCH gene polymorphism in New Zealand rabbit.Results:1.The degree of hemolysis assay OD was inversely correlated with ACA,r was-0.986,p was 0.002.The main interfering factors of the experiment were sera from different individuals,the mixed sera were used to eliminate the interference and optimize the experimental results by ratio.the hemolysis degree(HD)was used to indicate the detection result,HD=(detection value OD/negative value OD)*100,which is inversely proportional to ACA.The inter-batch results showed good stability with a CV of 6.5%.HD differences between males and females were significant(P=0.015)while the normal distribution for both genders was conformed.The HD recommended reference range for men is 56-88 while for women is 51-86.Serum HD of healthy subjects and autoimmune disease patients showed a significant difference(P=0.001).Autoimmune disease patients have lower HD which was a result of having stronger ACA.2.There was a linear relationship between haemolysin and erythrocyte agglutination(r=0.998,P<0.001).The erythrocyte fragmentation index(EFI)was used to indicate the assay results,a good linear relationship existed between EFI and TCA(r=0.991,P<0.001).The results were not affected by slight fluctuations in the concentrations haemolysin or erythrocytes.The inter-batch CV=8.6%of the test results showed good stability.There was a significant difference in the EFI between nephrotic syndrome patients and healthy individuals,P<0.001,and EFI was reduced in nephrotic syndrome patients compared to healthy individuals.3.After the corresponding addition of 1:8 diluted isotype erythrocyte fragments for the three ABO agglutinated erythrocytes,the erythrocyte counts(0.34X1012,0.51X1012,0.78X1012;0.40X1012,0.46X1012,0.62 X1012)increased in the agglutination-inhibited and dissociated agglutination groups for each type compared to their agglutination controls(0.20X1012,0.25X1012,0.49X1012).AC and AC A can be detected by auto hematology analyzer,with AC being inversely proportional to MCHC and ACA being positively proportional to MCHC.The assay results were not affected by mild fluctuations in hemolysin concentration.The inter-batch CV of the assay results for AC,ACA,and ACA/AC were 5%,9%,and 11.7%,respectively,with good stability.There was a significant difference with P was 0.018 in ACA of serum from patients with acute phase reaction and healthy individuals and there were significant differences with P were 0.014,0.002,0.041 in AC,ACA,ACA/AC of serum from patients with nephrotic syndrome and healthy individuals.4.The first and fourth exons of rabbit RLA-DRB1 mRNA were prone to deletion,with deletion frequencies of 29.2%and 12.5%,respectively.ACA of CA in serum before and after antigen inoculation in rabbits was significantly different,P was 0.011.Compared with the difference in ACA after antigen inoculation in rabbits with other genotypes(-0.030 ±0.044),the difference(-0.010±0.014)was lower in rabbits with the RLA-DQA3 genotype,and the rabbit RLA-DQA3 gene had a suppressive effect on the increase in ACA after antigen inoculation.Conclusion:1.The ACA assay while utilizing human O-erythrocytes as an indicator system is sensitive and accurate,and has potential in clinical applications.2.The flow cytometry-based assay for TCA was sensitive and accurate and had potential value for clinical application.3.Homotypic erythrocyte fragments have an inhibitory and dissociative effect on the agglutination of the corresponding erythrocytes.The method for detecting the immunological function of the complement system based on the phenomenon of dissociating erythrocyte agglutination is sensitive,accurate,has potential clinical application and has the advantage of being easily scalable.The combined analysis of its assay results could highlight the role of the complement system’s own components and complement activators in influencing complement immunological function.4.The exon of rabbit RLA-DRB1 mRNA showed deletion-prone and the second exon of rabbit RLA-DQA showed abundant polymorphism.The rabbit RLA-DQA3 gene has the effect of suppressing the increase of ACA after antigen inoculation. |
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