The purpose of this study was to prepare high affinity monoclonal antibodies(mAbs)a-gainst bovine viral diarrhea virus(BVDV)and establish a double antibody sandwich ELISA detec-tion method.BVDV was purified by differential ultracentrifugation and used to immunize BALB/c mice.Hybridoma cells were prepared by fusing spleen cells from the immunized mice with SP2/0 cells.Positive cells were screened by indirect ELISA.A double-antibody sandwich ELISA method for detecting BVDV was developed using monoclonal antibody 4D11 as the capture antibody and HRP-labeled monoclonal antibody 3F3 as the detection antibody.The results of the ELISA and the determination of the variable region gene sequence of monoclonal antibodies indicated that the two monoclonal antibodies recognize different antigenic epitopes.Specificity tests showed that two monoclonal antibodies specifically recognize BVDV and did not cross-react with other bovine viru-ses associated with diarrhea.Indirect immunofluorescence assay and Western blot assay demonstra-ted that both mAbs exhibited strong reactivity with BVDV.The double antibody sandwich ELISA detection method established in this study had good specificity.The sensitivity test revealed that the method could detect a minimum virus amount of 3.1 × 104 TCID50.The reproducibility test showed that the inter-batch coefficient of variation(Cv)was between 2.47％and 7.44％,and the intra-batch Cv was between 1.71％and 9.89％,indicating good reproducibility.The establishment of this method provides an effective technical tool for the rapid diagnosis and prevention and con-trol of BVDV.

The purpose of this study was to prepare high affinity monoclonal antibodies(mAbs)a-gainst bovine viral diarrhea virus(BVDV)and establish a double antibody sandwich ELISA detec-tion method.BVDV was purified by differential ultracentrifugation and used to immunize BALB/c mice.Hybridoma cells were prepared by fusing spleen cells from the immunized mice with SP2/0 cells.Positive cells were screened by indirect ELISA.A double-antibody sandwich ELISA method for detecting BVDV was developed using monoclonal antibody 4D11 as the capture antibody and HRP-labeled monoclonal antibody 3F3 as the detection antibody.The results of the ELISA and the determination of the variable region gene sequence of monoclonal antibodies indicated that the two monoclonal antibodies recognize different antigenic epitopes.Specificity tests showed that two monoclonal antibodies specifically recognize BVDV and did not cross-react with other bovine viru-ses associated with diarrhea.Indirect immunofluorescence assay and Western blot assay demonstra-ted that both mAbs exhibited strong reactivity with BVDV.The double antibody sandwich ELISA detection method established in this study had good specificity.The sensitivity test revealed that the method could detect a minimum virus amount of 3.1 × 104 TCID50.The reproducibility test showed that the inter-batch coefficient of variation(Cv)was between 2.47％and 7.44％,and the intra-batch Cv was between 1.71％and 9.89％,indicating good reproducibility.The establishment of this method provides an effective technical tool for the rapid diagnosis and prevention and con-trol of BVDV.

Objective    To construct a prognostic model of esophageal squamous cell carcinoma (ESCC) based on immune checkpoint-related genes and explore the potential relationship between these genes and the tumor microenvironment (TME). Methods     The transcriptome sequencing data and clinical information of immune checkpoint genes of samples from GSE53625 in GEO database were collected. The difference of gene expression between ESCC and normal paracancerous tissues was evaluated, and the drug sensitivity of differentially expressed genes in ESCC was analyzed. We then constructed a risk model based on survival-related genes and explored the prognostic characteristics, enriched pathway, immune checkpoints, immune score, immune cell infiltration, and potentially sensitive drugs of different risk groups. Results    A total of 358 samples from 179 patients were enrolled, including 179 ESCC samples and 179 corresponding paracancerous tissues. There were 33 males and 146 females, including 80 patients≤60 years and 99 patients>60 years. 39 immune checkpoint genes were differentially expressed in ESCC, including 14 low expression genes and 25 high expression genes. Drug sensitivity analysis of 8 highly expressed genes (TNFRSF8, CTLA4, TNFRSF4, CD276, TNFSF4, IDO1, CD80, TNFRSF18) showed that many compounds were sensitive to these immunotherapy targets. A risk model based on three prognostic genes (NRP1, ICOSLG, HHLA2) was constructed by the least absolute shrinkage and selection operator analysis. It was found that the overall survival time of the high-risk group was significantly lower than that of the low-risk group (P<0.001). Similar results were obtained in different ESCC subtypes. The risk score based on the immune checkpoint gene was identified as an independent prognostic factor for ESCC. Different risk groups had unique enriched pathways, immune cell infiltration, TME, and sensitive drugs. Conclusion     A prognostic model based on immune checkpoint gene is established, which can accurately stratify ESCC and provide potential sensitive drugs for ESCC with different risks, thus providing a possibility for personalized treatment of ESCC.

Objective:To investigate the clinical and molecular features of patients with CD7 +relapsed or refractory acute myeloid leukemia(r/rAML)and the prognosis of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods:172 r/rAML patients who underwent allo-HSCT in department of hematology, Aerospace Center Hospital between January 1st 2017 and December 31st 2020 were retrospectively analyzed The patients were were divided into CD7 + group( n=75) and CD7 - group( n=97) according to the expression CD7 in the initial immunophenotype. Mann-Whitney U and Chi-square test were used to compare the clinical data, molecular and cytogenetic characteristics of the two groups of patients. Kaplan-Meier method was used to analyze the median progression-free survival (PFS) and median overall survival (OS) of the two groups of patients, and Cox regression screenthe prognostic factors of the patients. Results:The median follow-up time was 19 months. The recurrence rates were 23.71% and 50.67%, respectively in CD7 - and CD7 + group (χ 2=13.428 P<0.001). In relapsed patients, 86.96 percentage of CD7 - group did not express CD7 while 86.84 percentage of CD7 + group expressed CD7. The median PFS was 25 and 5 months in CD7 - and CD7 + group (χ 2=8.695, P=0.003), and the medianOS was 34 and 15 months in CD7 - and CD7 + group (χ 2=2.579, P=0.108). Univariate analysis showed that the CD7 +group, had the lower rates of morphological remission (χ 2=10.014, P=0.002), molecular remission (χ 2=22.809, P<0.001), and more male patients (χ 2=5.281, P=0.022). The incidence of CEBPA double-site mutation was higher (23.4% vs 8.2%, χ 2=8.180, P=0.004) and the rearrangement of RUNX1::RUNX1T1 was lower(4.0% vs18.6%, χ 2=8.362, P=0.004)in CD7 +group than in CD7 -group. Multivariate analysis showed that pre-transplant tumor load was the only prognostic factor for PFS (HR, 1.600; 95% CI, 1.203 to 2.127; P=0.001) and OS (HR, 1.737; 95% CI, 1.273 to 2.369; P<0.001) in r/r AML patients. Conclusion:CD7 expression is a risk factor for poor prognosis in r/r AML patients, and CD7 expression is stable after relapse. Positive CD7 can be used as a target for immune targeted therapy.