| ObjectiveThis study intends to develop a new technology for the detection of blood group antibody,which uses the in vitro Red blood cell(RBC)membrane antigen extraction technology to extract RBC membrane antigen.The extracted RBC membrane antigen is coupled to the microsphere to prepare blood group antibody microarray.The blood group antibody microarray used for screening and identifying the blood group antibody in human serum,which overcomes some defects in existing detection methods such as the low sensitivity,non-standardization of detection,the waste of resources caused by short storage period of the reagent and the hemolytic transfusion reaction caused by antibody leakage,to achieve accurate detection of blood group antibody,then to ensure the blood transfusion safety and blood transfusion efficacy.Methods1.The RBC membrane was extracted by the RBC membrane antigen method,which coupled with the appropriate magnetic microspheres.Then optimized the amount of RBC membranes and the reaction conditions to obtain the maximum signal-to-noise(S/N)ratio.After the microarray preparation and reaction conditions were optimized,the sensitivity,specificity,stability and repeatability of the microarray were verified:1)We measured the fluorescent signal intensity across 8 different concentrations of anti-s and anti-B antibodies in a 3-fold dilution series spanning ? 3 orders of magnitude using our microarray and the microcolumn gel method.The lowest limit of detection(LOD)and dynamic range of the two detection methods were then calculated and compared.2)BSA was used as non-target antigen to verify the detection specificity of the microarray.3)The RBC membrane was prepared into a microarray before and after storage for two years,and the signal values before and after storage were detected to verify the stability of the RBC membrane antigen;4)the same batch of samples were tested in intra-experiment and inter-experiment,and compared the coefficient of variation(CV).2.We used the blood group antibody microarray to detect 15 commonly used blood group antibodies(anti-C,E,c,e,Jka,Jkb,M,N,S,s,Fya,K,k,Lea,P1),and compared the results with the microcolumn gel method.The agglutination inhibition experiment was used to verify the inconsistent results of the two detection methods.Then genotyped the corresponding antigen which may have a cross-reactivity,and finally the similarity antigens were compared by Blast software to analyze the possible causes of cross-reaction.3.We used our blood group antibody microarray,microcolumn gel method and glass-bead microcolumn method to detect 43 patients' specimens,and compared the consistency of the results;In 43 cases 20 samples were selected,which were used for the repetitive detection of blood group antibody microarray,and the consistency of the two test results was compared.Results1.The RBC membrane antigens were then conjugated to beads where the optimal signal-to-noise ratio employs 45?g of RBCs membrane antigens per 1.25×105 beads and an blood group antibody-array incubation of 1 h at 21?.The sensitivity and dynamic range of our antibody array was?700-fold higher than the microcolumn gel method that is routinely used in the clinic,and our antibody array only recognized the specific antigen-antibody reaction,and did not cross-react with other non-target antigens.The reproducibility results showed that the intra-and inter-CV values were 3.32%and 4.34%respectively,both within 5%,indicated that our antibody array has good repeatability.The stability results showed that there was no difference in the detection signal values of the RBC membrane antigen before and after storage for two years,P=0.9487,indicated that the RBC membrane antigen could be preserved for at least 2 years.2.We used blood group antibody microarray to detect 15 antibody reagents and compared the results with the microcolumn gel method.9 of 15 antibodies(anti-Lea,K,E,P1,N,Fya,M,s,k)were consistent with the two methods and 6 of 15 antibodies(anti-C,c,e,JKa,JKb,S)were inconsistent.The six inconsistent antibodies were detected by agglutination inhibition test,the results of agglutination inhibition experiments consistent with our antibody microarray.The genotyping results showed that there was no antigen matching the detection antibody on the red blood cells,but the homology with the corresponding antigen protein sequence was above 98.8%.3.Blood group antibody microarray,microcolumn gel method and glass-bead mcrocolumn method were used to detect RBC allogeneic unexpected antibodies in 43 patients.35/43(81.4%)of results were consistent through three methods;37/43(86.1%)of results were consistent between microcolumn gel method and our microarray;41/43(90.7%)of results were consistent between glass-bead microcolumn method and our microarray;35/43(81.4%)of results were consistent between glass-bead microcolumn method and the microcolumn gel method.Among the 21 negative samples,the three detection methods were consistently negative,however,22 cases of positive samples showed inconsistent results when tested.Our microarray detected 22 cases,microcolumn gel method detected 16 cases,glass-bead microcolumn method detected 18 cases.Conclusions1.We have successfully built a blood group antibody microarray,which has the advantages of high sensitivity,high specificity,high throughput,good repeatability and long shelf life.2.At present,some conventionally used blood group antibody reagents have a certain cross-reaction,and the cross-reaction can only be detected in a highly sensitive method,so our blood group antibody microarray has great advantages in evaluating the quality of the blood group antibody reagent.3.The blood group antibody microarray can be used for the detection of RBC unexpected antibodies,and can detect weak antibodies that are currently missed by conventional detection methods(microcolumn gel method).So it can effectively avoid the occurrence of hemolytic transfusion reactions caused by the missed antibody,in order to ensure safe and effective blood transfusion. |
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