Construction Of Magnetic Bead-based Protein Microarray For ABO/RH(D) Blood Grouping

During blood donation and transfusion, ABO and RhD typing is compulsorily requiredin routine work. The main blood typing methods in clinical includes plate method, test tube,microporous plate method, the microcolumn gel method and automated hematologyanalyzer and so on. These methods are based on the agglutination of red blood cells so thatthey must be affected by hemolysis, moreover, they have lower detection flux. Blood typingusing gene chip can avoid the disadvantages of serological methods to some extent, but theoperation is complicated, time-consuming, and it needs to rely on expensiveinstruments.This study combined immunomagnetic beads and protein chip and did bloodtyping according to the principle of response between antigen and antibody. At the sametime, the intervention of complement C1q as common tracer realized the synchronousdetection of antigen and antibody. This study will lay a good foundatoin for a high-flux,automated, convenient and fast blood typing method.Objectives:To construct a magnetic bead-based protein microarray for ABO and Rh(D) bloodgrouping.Methods:1. Magnetic bead-coupled protein was spotted on the surfaces of aldehyde slide,Polymer Slide-D and glass slide with oxidated agarose-gel. The redundant proteins wereremoved after overnight immobilization. Then the signal of each spot was detected forcomparing the immobilization rate on the three different slides. Immobilizing antibody onthe surface of the three slides, followed by an incubation with secondary antibody labeledby magnetic bead. The activity of the immobilized proteins was analyzed according to theresults. The most appropriate slide for the following experiments can be selected in this way.Protein microarrays were established in different conditions, such as agarose concentration,temperature, immobilization concentration and time. Finally, the optimal experiment conditions were confirmed by comparing the detection results of these microarrays.2. Construct the ABO/Rh(D) typing protein microarray in the optimal experimentconditions. The antibody of anti-A and anti-B, red blood cells(RBCs) of group A and B, andRBCs of group O and Rh(D)-positive were diluted with dilution buffer, then these sampleswere detected with protein microarray and tube method respectively. The sensitivity of theblood grouping microarray was evaluated by analyzing the results. A random sample withthe microarray for four times was tested, and the coefficient of variation(CV) within andbetween the groups was calculated. Finally,30donated blood samples were identified withthe microarray and tube method respectively for assessing veracity and specificity of thechip.Results:1.With comparison of the signal intensity, the immobilization rate of agarose-gelsubstrate were the highest(P<0.05). The test results revealed the proteins on the surface ofaldehyde slide had nearly no activity. On the contrary, the proteins on agarose-gel substrateshowed the highest activity, followed by these on the Polymer Slide-D. In addition, theparallel spots on gel substrate are more homogeneous with lower standard deviation value.Therefore we choosed glass slide with oxidated agarose-gel for follow-on tests. The optimalconditions were confirmed through a series of experiments. The substrate was fabricatedwith1%agarose gel, then spotted with the protein probes(100μg/ml in printing buffer) andincubated in a humid chamber at24℃for8h. Next, the microarray was blocked withBSA(1mg/ml) and stored at4℃for use. At last, the slide was placed upside down andwashed with PBST(0.05%) for the last step of test.2. The forward test titer was up to1:8192and the reverse test was1:4096by detectingdifferent dilution rate of samples with the arrays. By contrast, that is1:256and1:16withtube method. As for the reproducibility test, the values of CV within and between thegroups were distributed in14%to22%. Results of the30clinical specimens detected by thearrays and tube methods were the same. Each positive spot on the arrays was strong andthere was no cross-reaction.Conclusions:1. The glass slide with oxidated agarose-gel was the best of the three differentsubstrates because of higher immobilization rate and activity of protein.2. We constructed a magnetic bead-based protein microarray for ABO and Rh(D) blood typing successfully. And sensitivity, veracity and specificity of the chip was verifiedto be comparable to tube method.