Study On The Assessment And Evaluation Of Immunogenicity Of Animal Tissue-derived Biomaterials

[Objective]Alpha-gal antigen is the main target responsible for hyper-acute immune rejection induced by animal tissues and organs.Alpha-gal antigen remained in animal tissues-derived biomaterials after the deantigen treatment.The purpose of this study was to establish detection and evaluation methods for immunogenicity of alpha-gal in animal tissues-derived biomaterials.[Methods]1?Establishment of standardized methods for Gal antigen detection of animal tissues-derived biomaterialsWe established enzyme-linked immune inhibited method by using Gal antigen specificity antibodies(M86),and using synthetic Gal-BSA with Gal antigen negative biomaterial cracking supernatant fluid as standard curve samples,using the Gal antigen positive biomaterial(from pig liver)and Gal antigen negative biomaterial(from human placental tissue)to monitor sensitivity and specificity of experimental system.We used established standardized method to quantitative detect Gal antigen residues of acellular matrix biomaterial(such as bovine derived biological bone repair materials,bovine pericardial tissue derived repair patch and acellular pig conjunctiva).The homogeneity and stability of Gal antigen positive biomaterial prepared from pig liver was also investigated.Gal antigen content of Gal antigen positive biomaterial was detected by collaborative calibration.2?Preparation,immunological characteristics and application of GGTA1 knockout miceGGTA1 gene knockout mice were prepared by bacterial chromosome homologous recombination technique.The main organs and tissues of GGTA1 KO mice were taken and mRNA was extracted.RT-PCR was used to identify the phenotype change of GGTA1 KO mice.Gal antigen content in main organs and tissues of GGTA1 KO mice was determined by a standardized method.GGTA1 KO mice were immunologically stimulated with rabbit red blood cells(foreign antigen)for investigating the change in the expression levels of total antibodies and anti-gal antibodies in mice.After GGTA1 KO mice subcutaneously implanted of animal tissue derived biomaterials(such as bovine derived biological bone repair materials,bovine pericardial tissue derived repair patch and cell-free pig conjunctiva),immunogenicity of these biomaterials was evaluated including total antibody expression level,anti Gal antibody expression level,cytokine expression level,lymphocyte activation level or subtype analysis and local material histopathologic evaluation.In these studies,the feasibility of evaluating the immunogenicity of animal tissue-derived biomaterial using GGTA1 KO mice was investigated.3?Preparation and immunological characteristics of iGb3S knockout miceiGb3S knockout mice were prepared by bacterial chromosome homologous recombination technique.The main organs and tissues of iGb3S KO mice were taken and mRNA was extracted.And the phenotype changes of iGb3S KO mice were identified by RT-PCR.Gal antigen content in main organs and tissues of iGb3S KO mice was detected by a standardized method.iGb3S KO mice were stimulated with rabbit red blood cells(foreign antigen)for investigating the change in the expression levels of total antibodies,subtypes and anti-gal antibodies in mice.4?Preparation and immunological characteristics of GGTA1/iGb3S knockout miceGGTA1 KO mice were mated with iGb3S KO mice to screen homozygous GGTA1/iGb3S double knockout mice.Gal antigen content in main organs and tissues of homozygous GGTA1/iGb3S DKO mice was determined by a standardized method.GGTA1/iGb3S DKO mice and GGTA1 KO mice were immunologically stimulated by rabbit red blood cells(foreign antigen).The changes in the expression levels of anti-gal antibodies,cytokine expression levels and lymphocyte subtypes were compared to investigate the difference in the immunological characteristics of the two Gal-deficient mice,and to evaluate the application prospects of GGTA1/iGb3S DKO mice.[Results]1?A standardized method for Gal antigen detection of animal tissues-derived biomaterial was established(YY/T 1561-2017).Gal antigen positive biomaterial with good homogeneity and stability was developed as the national standard substance(no.380001-201701).The Gal antigen in bovine derived biological bone repair materials,bovine pericardial tissue repair patches and acellular porcine conjunctiva was determined by a standardized method:(3.61±0.33)×1012/mg(dry weight),(1.13±0.44)×1014/mg(dry weight)and(1.85±0.09)×1013/mg(dry weight),respectively.The corresponding Gal antigen clearance rate(relative to raw materials)was 55.6%,82.4%and 99.8%,respectively.The reference value of Gal antigen positive biomaterial was:(1.733±0.437)×1014/mg.2?GGTA1 KO mice was successfully constructed,the population was established,and the species was preserved.It was confirmed that GGTA1 gene was the main gene regulating the synthesis of Gal antigen,causing the reduction of Gal antigen expression about 97%?99 GGTA1 KO mice showed a significant increase in the specific anti-Gal antibody level after the stimulation of rabbit red blood cells(foreign antigen),proving that GGTA1 KO mice are sensitive animal models to evaluate the specific immunological response of foreign antigen based on Gal antigen.Application results showed that GGTA1 KO model mice after subcutaneous implanted of antigen removed animal tissues-derived biomaterial and raw material,mainly caused the increase of anti-Gal antibodies and/or partial cytokine expression level in raw material implanted group.The level of raw material implanted group is significantly higher than that of the antigen removed group,which means GGTA1 KO mice can sensitively reflect the remaining Gal antigen immunogenicity risk of animal tissues-derived biomaterial.3?iGb3S KO mice were successfully constructed,the population was established,and the species was preserved.It has been confirmed internationally for the first time that iGb3S gene is involved in the synthesis of Gal antigen in mice,causing a reduction of Gal antigen expression about 5%-21%.After iGb3S KO mice were stimulated by rabbit red blood cells(foreign antigen),the levels of total antibodies and anti-gal antibodies were not significantly increased,indicating that there was no change in immunological reaction of foreign antigen stimulation.4?Gal antigens in the main organs of GGTA1/iGb3S DKO mice was under detection limit,indicating that GGTA1 and iGb3S are the two regulatory genes of Gal antigen synthesis.100%Gal antigen can be eliminated by simultaneous knockout of the two genes.The comparative study of GGTA1/iGb3S DKO mice and GGTA1 KO mice showed that the expression level of anti-Gal antibody was significantly increased after two times of rabbit red blood cells stimulation,while the expression level of lymphocyte subtypes and cytokines was not significantly changed.GGTA1/iGb3S DKO mice were not found to be more sensitive to the stimulation of rabbit red blood cells than GGTA1 KO mice.GGTA1/iGb3S DKO mice and GGTA1 KO mice both are sensitive models for evaluating Gal antigen residue heterogeneous risk of immune rejection.[Conclusions]1?In this study,a standardized method for Gal antigen detection of animal tissues-derived biomaterial was established,and Gal antigen positive biomaterial and Gal antigen negative biomaterial were successfully developed.2?GGTA1 KO mice were successfully constructed in this study,and it was confirmed that GGTA1 gene was the main gene involved in the regulation of Gal antigen synthesis in mice,and GGTA1 KO mice showed strong sensitivity to the stimulation of foreign antigens such as rabbit red blood cells.It was confirmed that GGTA1 KO model mice can be used to evaluate the immunogenicity risk of animal tissues-derived biomaterials,in which anti-Gal antibodies and cytokines can be used as specific evaluation indicators.3?This study successfully constructed iGb3S KO mice,and confirmed that iGb3S gene is a secondary gene involved in the regulation of Gal antigen synthesis in mice,and iGb3S KO mice are not sensitive to the stimulation of foreign antigens such as rabbit red blood cells.4?The GGTA1 and iGb3S DKO mice of were successfully obtained,which confirmed that the simultaneous knockout of GGTA1 and iGb3S could cause complete deletion of Gal antigen expression in the main organs of mice.GGTA1/iGb3S DKO mice showed a strong sensitivity to the stimulation of foreign antigens such as rabbit red blood cells,which is a suitable animal model to evaluate the immunogenicity risk of animal tissues-derived biomaterials.However,GGTA1/iGb3S DKO mice are not found to be more sensitive to the stimulation of rabbit red blood cells than GGTA1 KO mice.