Abstract: Objective This study aimed to investigate the effects of treating red blood cells (RBCs) with four different proteinaases on the detection of anti-E and anti-c antibodies, and to compare the sensitivity among three detection methods. The goal is to provide a scientific basis for selecting the optimal enzyme treatment detection protocol in clinical practice. Methods Five RBC samples with different Rh phenotypes (3 cases of CcEe, 1 case of Ccee, and 1 case of CCEe) were selected. RBCs were treated using a two-step enzyme method with chymotrypsin, trypsin, pronase, and papain. Prior to enzyme treatment, antibody titers for anti-E and anti-c were determined by the Microcolumn Gel Test (MGT), the polybreneethylene Glycol-enhanced Microcolumn Gel Test (PEG-MGT), and the Polybrene method. Following enzyme treatment, the MGT method was used uniformly for antibody detection. Cells with CcEe and CCEe phenotypes were tested for anti-E; cells with CcEe and Ccee phenotypes were tested for anti-c. Analyses were performed on: differences among the three pre-treatment methods; differences among the four enzyme treatment groups; and different titers between post-enzyme treatment and pre-enzyme treatment. Results Anti-E results: Anti-E: Before enzyme treatment, a significant difference in detected titers was found between the PEG-MGT and MGT method (P<0.05). After treatment with the four enzymes, a significant difference in titer was observed between chymotrypsin and pronase (P<0.05). When comparing the titers of MGT after enzyme treatment with those before treatment, significant differences were observed between the pronase and MGT groups, as well as between the papain and MGT groups (P<0.05). Anti-c results: Before enzyme treatment, a significant difference in detected titers was found between the Polybrene and MGT methods (P<0.05). After treatment with the four enzymes, a significant difference in titer was observed between chymotrypsin and papain (P<0.05). When comparing post-enzyme treatment MGT titers with pre-enzyme treatment titers, significant differences were found between the MGT method and the trypsin, pronase, and papain groups (P<0.05). Conclusion Before enzyme treatment, the optimal method for detecting anti-E is PEG-MGT, while the optimal method for detecting anti-c is the Polybrene method. After enzyme treatment, the pronase-MGT combination is the best solution for detecting anti-E, whereas the papain-MGT combination serves as the optimal method for detecting anti-c. Enzyme treatment techniques and PEG can significantly enhance the detection sensitivity of specific antibodies, serving as an important supplement to conventional detection methods.

Key words: Protease, Enzyme-treated red blood cells, Antibody titer, Microcolumn gel test, Polybreneethylene glycol-enhanced microcolumn gel test, Polybrene