Objective To establish an initial diagnostic method for β-thalassemia (BT) using a double antibody sandwich ELISA approach. Methods The hybridoma producing monoclonal antibodies against anti-HbF were screened using a trailing count method. The mAbs were evaluated through ELISA,modified immunocytochem-istry,and Western blot analysis. A double antibody-sandwich ELISA assay was established by labeling the pairs of mAbs with ALP using the glutaral method,and this detection system was used to analyze 40 serum samples. Results The results demonstrate the successful generation of nine hybridoma cell lines capable of secreting highly active anti-HbF monoclonal antibodies (mAbs). Specifically,four mAbs (3F7,4G1,6C1,and 9H7) exhibited exclusive reactivity towards HbF without any cross-reactivity with hemoglobin variants (HbA and HbA2). These four HbF-specific mAbs displayed exceptional specificity and sensitivity,with a maximum titer of 1:256000 and the highest affinity constant (Ka) recorded at 2.36×108 L/mol. Among these mAbs,optimal antibody pairing was achieved using capture antibody 3F7 in conjunction with ALP-4G1 for the development of a sandwich ELISA detec-tion method. By employing this approach,fetal and healthy human blood samples were successfully quantified for HbF levels with an impressive detection sensitivity reaching up to 80％. Conclusion This sandwich ELISA dem-onstrated precise quantification of HbF levels,making it suiTab.for both research and diagnostic purposes in the field of β-thalassemia.

Objective To establish an initial diagnostic method for β-thalassemia (BT) using a double antibody sandwich ELISA approach. Methods The hybridoma producing monoclonal antibodies against anti-HbF were screened using a trailing count method. The mAbs were evaluated through ELISA,modified immunocytochem-istry,and Western blot analysis. A double antibody-sandwich ELISA assay was established by labeling the pairs of mAbs with ALP using the glutaral method,and this detection system was used to analyze 40 serum samples. Results The results demonstrate the successful generation of nine hybridoma cell lines capable of secreting highly active anti-HbF monoclonal antibodies (mAbs). Specifically,four mAbs (3F7,4G1,6C1,and 9H7) exhibited exclusive reactivity towards HbF without any cross-reactivity with hemoglobin variants (HbA and HbA2). These four HbF-specific mAbs displayed exceptional specificity and sensitivity,with a maximum titer of 1:256000 and the highest affinity constant (Ka) recorded at 2.36×108 L/mol. Among these mAbs,optimal antibody pairing was achieved using capture antibody 3F7 in conjunction with ALP-4G1 for the development of a sandwich ELISA detec-tion method. By employing this approach,fetal and healthy human blood samples were successfully quantified for HbF levels with an impressive detection sensitivity reaching up to 80％. Conclusion This sandwich ELISA dem-onstrated precise quantification of HbF levels,making it suiTab.for both research and diagnostic purposes in the field of β-thalassemia.

OBJECTIVE To prepare nanoparticle-based targeting preparation loaded with triptolide(TP),and evaluate its quality,targeting ability and cytotoxic effects.METHODS Polymer nanoparticles carrying TP-targeted folic acid(FA)receptor(TP@PLGA-PEG-FA)were fabricated using poly(lactic-co-glycolic acid)/polyethylene glycol/FA(PLGA-PEG-FA)as the carrier by emulsion and volatilization technique.The morphology and distribution were observed,and their particle size,Zeta potential,polydispersity index(PDI),drug loading capacity and encapsulation efficiency were measured.Their stability,blood compatibility,in vitro drug release,uptake by RAW264.7 cells(localization with fluorescent dye Cy3.5),and in vitro cytotoxicity were evaluated.RESULTS TP@PLGA-PEG-FA exhibited spherical shape and uniform distribution,with particle size of(122.60±0.02)nm,Zeta potential of(-17.6±0.6)mV,and PDI of 0.26±0.02;drug loading capacity and encapsulation efficiency of TP were measured to be(7.78±0.05)%and(68.62±0.03)%,respectively.The hemolysis rates of 100,200,300,400 μg/mL TP@PLGA-PEG-FA were 0.77%,0.92%,1.34%and 1.63%,respectively.There were no significant changes in particle size,PDI and Zeta potential when TP@PLGA-PEG-FA were placed in 4℃water for 14 days and in DMEM culture medium containing 10%fetal bovine serum at 37℃for 12 h.The cumulative release rate of TP@PLGA-PEG-FA was(84.83±0.29)%in phosphate buffer at pH5.5 for 72 h,which was significantly higher than the cumulative release rates in phosphate buffer solutions at pH7.4 and 6.5 for 72 h[(42.37±0.35)%and(63.83±0.29)%,respectively](P＜0.05).Activated RAW264.7 cells took up significantly more Cy3.5@PLGA-PEG-FA than they took up Cy3.5@PLGA-PEG-FA+free FA and Cy3.5@PLGA-PEG.When the mass concentration of TP was≥15.63 ng/mL,the survival rates of activated cells in the TP@PLGA-PEG-FA groups were significantly lower than those of the same mass concentration of free TP groups(P＜0.05).CONCLUSIONS The prepared TP@PLGA-PEG-FA has high stability,good blood compatibility,active targeting and cytotoxicity to inflammatory cells.