To produce monoclonal antibody (mAb) specifically against human thrombomodulin (hTM), an immune-tolerizing procedure was employed to generate monoclonal antibodies specific to hTM. Female BALB/c mice were first immunized with CHO cells following at 10 min, 24 h, 48 h by intraperitoneal injection of different doses of cyclophosphamide (CP) 2 times at an interval of 2 weeks, thereby tolerizing the mice to common epitopes shared between CHO and CHO-TM5 cells. Subsequently the selected mice with the lowest titer of serum polyclonal antibody by cellular enzyme-linked immunoabsorbent assay (CELISA) were immunized with CHO-TM5 cells, which have stable high level expression of hTM, to produce antibodies specific to hTM 3 times at an interval of 2 weeks. On the third day after the third immunization, mouse with the highest titer of serum polyclonal antibody was sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96 well plates for screening with CELISA. To improve probability to obtain specific mAb, CELISA was applied twice. The first CELISA was done with polyethylene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but having CHO cells monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5 +CHO- hybridoma cells. BALB/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded mAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. Detection of CELISA showed that 100 mg/kg dose of CP could tolerize the mouse to common epitopes shared between CHO and CHO-TM5 cells. And CELISA also discovered that all hybridomas positive for CHO-TM5 cells were negative for CHO cells. Five lines of positive hybridoma cells had been obtained altogether and 2F7 was selected randomly for next investigation. The Ig subclass of the mAb 2F7 was IgG1 and the titer of ascitic mAb was 1?10-6. Furthermore, the content of ascitic mAb was 19.56 g/L and chromosome numbers is 98. Flow cytometry, CELISA and Western blotting assays demonstrated that mAb 2F7 could specifically recognize hTM expressed on CHO-TM5 and human umbilical vascular endothelial cells (HUVEC). Meanwhile, the tissue specificity of mAb 2F7 was also identified by immunohistochemical ABC staining. On the other hand, Western blotting assays indicated that mAb 2F7 could recognize the antigen protein with 105 ku molecular mass under reduction condition. Moreover, the dissociation constant of mAb 2F7, 1.22? 10-9 mol/L, indicated the affinity higher than some others. The results suggest that the immunotolerizing protocol provides a convenient general method for producing antibodies specific to desired protein isoforms. mAb 2F7 can specifically recognize the natural hTM expressed mainly on vascular endothelial cells, which will potentially useful for investigating the functions and clinic values of hTM.

OBJECTIVES: Hereditary spherocytosis (HS) is the most common hereditary defect of the red cell membrane, mainly characterized by anemia, jaundice, and splenomegaly. Due to the atypical clinical manifestations and negative family history of some patients, as well as the low sensitivity and specificity of traditional laboratory examinations, it is easy for it to escape diagnosis or be misdiagnosed. At present, it has been confirmed that the mutation of ANK1, SPTB, SPTA1, SLC4A1 and EPB42 genes can cause the deletion of their corresponding coding proteins, and thus lead to the defect of erythrocyte membrane. This study aims to analyze the feasibility and clinical application value of HS gene diagnosis. METHODS: Data of 26 patients from Hunan, China with HS admitted to the Department of Hematology, Second Xiangya Hospital of Central South University from January 2018 to September 2021 were retrospectively collected, and their clinical manifestations and results of laboratory examinations were analyzed. Next-generation sequencing (NGS) combined with Sanger sequencing were applied. The mutation of HS pathogenic gene and the variation of uridine diphosphate-glucuronosyl transferase 1 family polypeptide A1 (UGT1A1), a key enzyme in the regulation of bilirubin metabolism, were detected. The results of pathogenic gene variations were interpreted pathogenic gene variations in accordance with the Standards and guidelines for the interpretation of sequence variants published by the American College of Medical Genetics and Genomics (ACMG). The clinical characteristics of patients with different gene variants were analyzed, and the clinical diagnosis and genetic diagnosis were compared. RESULTS: Among the 26 patients with HS, there were 23 cases of anemia, 25 cases of jaundice, 24 cases of splenomegaly, and 14 cases of cholelithiasis. There were 16 cases with family history and 10 cases without family history. The results of HS mutation test were positive in 25 cases and negative in 1 case. A total of 18 heterozygous mutations of HS pathogenic genes were detected in 19 families, among which 14 were pathogenic, 1 was likely pathogenic and 3 were of unknown significance. SPTB mutations (12) and ANK1 mutations (4) were the most common. The main variation types were nonsense mutation (9). There were no significant differences in peripheral blood cell parameters and hemolysis indicators between the SPTB mutant group and the ANK1 mutant group (all P>0.05). The rate of splenectomy in ANK1 mutation group was higher than that in SPTB mutation group, and the difference was statistically significant (χ²=6.970, P=0.014). There were no significant differences in peripheral blood cell parameters and hemolysis indicators among different mutation types (nonsense mutation, frameshift mutation, splice site mutation and missense mutation) (all P>0.05). Among the 18 clinically confirmedpatients, there were 17 cases whose diagnosis is consistent with the genetic diagnosis. Eight patients were clinically suspected, and all of them were confirmed by detection of HS gene mutation. Twenty-four patients with HS underwent UGT1A1 mutation detection, among which 5 patients carried UGT1A1 mutation resulting in a decrease in enzyme activity, and 19 patients had normal enzyme activity. The level of total bilirubin (TBIL) in the group with reduced enzyme activity was higher than that in the group with normal enzyme activity, and the difference was statistically significant (U=22, P=0.038). CONCLUSIONS Most patients with HS have anemia, jaundice and splenomegaly, often accompanied by cholelithiasis. SPTB and ANK1 mutations are the most common mutations in HS pathogenic genes among patients in Hunan, China, and there was no significant correlation between genotype and clinical phenotype. Genetic diagnosis is highly consistent with clinical diagnosis. The decrease of UGT1A1 enzyme activity can lead to the aggravation of jaundice in HS patients. Clinical combined gene diagnosis is beneficial for the rapid and precision diagnosis of HS. The detection of UGT1A1 enzyme activity related gene variation plays an important role in evaluation of HS jaundice.
Humans ; Codon, Nonsense ; Hemolysis ; Retrospective Studies ; Splenomegaly ; Bilirubin