Objective To evaluate the correlation between alterations in DNase1 and DNase1L3 enzyme activities and impairment of NET degradation in patients with sporadic SLE, and to investigate the underlying mechanism. Methods 46 sporadic SLE patients and 30 age- and sex-matched healthy individuals were recruited. Serum levels of DNase1, DNase1L3 and corresponding autoantibodies were detected by ELISA. DNase1 and DNase1L3 were isolated by immunoprecipitation; NETs and enzyme degradation activities were detected using a modified immunofluorescence. DNase1L3 secretion by PBMCs was analyzed by ELISPOT, Western blotting and reverse transcription PCR. Results Levels of H3-dsDNA and Ela-dsDNA complexes were significantly elevated in SLE patients. LDGs in SLE population was significantly higher than in the control group, and LDGs was positively correlated with H3-dsDNA and Ela-dsDNA NETs complexes. The ability of SLE patients to degrade NET in vitro was significantly lower than that of the control group. Degradation experiments of DNase1 and DNase1L3 in different proportions showed that the decrease in DNase1L3 activity was the primary contributor to the elevated NET residue level. The concentration of DNase1L3 autoantibodies in SLE patients was significantly elevated compared to the control group. In addition, the capacity of PBMCs to secrete DNase1L3 was significantly lower in the SLE patients compared to the control group. Conclusion Decreased secretion of DNase1L3 and the presence of relevant autoantibodies notably impede NET degradation in patients with SLE, offering new directions for the monitoring and treatment of SLE patients.
Humans ; Autoantibodies ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Extracellular Traps ; Lupus Erythematosus, Systemic

Here, we present a 40-year-old female with multiple pruritic occasionally painful vesicles, papules, and plaques in a circinate pattern on seborrheic areas, progressing to erosions and scales. Clinical findings led to the diagnosis of pemphigus foliaceus (PF). Initial treatment with prednisone and clobetasol ointment, however, did not fully suppress blister formation and healing of erosions. Skin punch biopsy revealed a subcorneal split and intracorneal neutrophilic infiltrates, while enzymelinked immunoassay (ELISA) revealed elevated anti-desmoglein 1 (Dsgl), consistent with PF. Doxycycline was then added to the previous regimen, resulting in remission. We discuss the role of doxycycline as a cost-effective adjunctive treatment in patients with refractory PF.
Pemphigus ; Clobetasol ; Enzyme-Linked Immunosorbent Assay

Introduction: Epidermolysis Bullosa Pruriginosa (EBP) is a rare subtype of the inherited Dystrophic ~ Epidermolysis Bullosa spectrum of diseases and results from a gene mutation in COL7AL Though predominantly an autosomal dominant disease, autosomal recessive and even sporadic have been reported. Case Summary: Case Summary:We report a case of a 12-year-old Filipino male presenting with a chronic history of numerous scratching-induced blisters predominantly distributed on the extensor aspect of his arms and legs without concomitant oral lesions, nail dystrophy, or hair findings, and without a family history of similar lesions. Histopathologic assessment, Direct Immunofluorescence (DIF), and Indirect Immunofiuorescence (IIF) showed a subepidermal split with scant inflammatory infiltrates, no immunofluorescence, and absent userrated linear immunofluorescence at the dermal-side of the Salt Split Skin slide, respectively, which were all consistent with EBP. Enzyme-Linked Immunosorbent Assay (ELISA) for Anti-Collagen VII antibodies was slightly elevated, which may suggest an alternative diagnosis of Epidermolysis Bullosa Acquisita (EBA). This slight elevation may be due to the mutated Collagen Vil protein becoming antigenic and therefore provoking an immune response. To conclusively distinguish EBP from EBA, a COL7AI gene mutation analysis was recommended. With a diagnosis of EBP cannot totally rule out EBA, the patient was initially managed with dapsone monotherapy, counseled regarding behavioral modification to reduce scratching and trauma, advised wound care and close monitoring for the development of oropharyngeal lesions, and recommended for COL7A1 genetic mutation analysis. Conclusion This report demonstrates a case of EBP with elevated Anti-Collagen VII antibodies. The diistinction between EBP and EBA is important because this changes the management: EBP is largely supportive, while EBA may benefit from immunosuppressive therapy.
Epidermolysis Bullosa Pruriginosa ; Enzyme-Linked Immunosorbent Assay ; Epidermolysis Bullosa Acquisita

A simple, fast, and visual method for detecting antibodies against peste des petits ruminants virus (PPRV) using colloidal gold strips was developed. In this study, the pET-32a-N was transformed into Escherichia coli Rosetta (DE3) for expression. Hybridoma cell lines were generated by fusing SP2/0 myeloma cells with splenocytes from immunized mice with the expressed and purified N protein of PPRV. The PPRV N protein was labeled with colloidal gold particles as the gold-labeled antigen. The N protein served as the gold standard antigen and as the test (T) line-coated antigen, while the monoclonal antibody served as the quality control (C) line-coated antibody to assemble the colloidal gold immunochromatographic test strips for detecting antibodies against the N protein of PPRV. Hybridoma cell line designated as 1F1 was able to stably secrete the monoclonal antibody against the N protein of PPRV. The titer of 1F1 monoclonal antibody in ascites was 1:128 000 determined by indirect enzyme-linked immunosorbent assays (ELISA), and the immunoglobulin subtype of the monoclonal antibody was IgG1, with kappa chain. The obtained monoclonal antibody was able to specifically recognize the N protein of PPRV, as shown by Western blotting and indirect immunofluorescent assay (IFA). The developed colloidal gold test strip method was able to detect PPRV antibodies specifically, and there was no difference between different batches of the test strips. Testing of a total of 122 clinical sera showed that the compliance rate of the test strip with ELISA test was 97.6%.The test strip assay developed in this study has good specificity, reproducibility, and sensitivity, and it can be used for the rapid detection of PPRV antibodies.
Animals ; Mice ; Peste-des-Petits-Ruminants/prevention & control* ; Antibodies, Monoclonal ; Reproducibility of Results ; Peste-des-petits-ruminants virus ; Antibodies, Viral ; Enzyme-Linked Immunosorbent Assay ; Goats

Objective To prepare and identify rabbit anti-cyclin dependent kinase 6 (CDK6) antibody. Methods The recombinant pET21a (+)/CDK6 was successfully constructed, then the recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells and was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) for protein expression, which was detected by SDS-PAGE and Western blot analysis. The expressed protein was purified by nickel-chelating nitrilotriacetic acid (Ni-NTA) agarose and then analyzed by SDS-PAGE. Japanese white rabbits were immunized with purified CDK6 protein for many times every two weeks. The blood was collected at 0, 2, 4 and 6 weeks after immunization, and serum was separated from blood. The titer was detected by indirect ELISA. Western blot analysis, immunofluorescence assay and immunohistochemistry were employed to determine the specificity. Results High purity CDK6 protein and high specificity of rabbit anti-CDK6 antibody were successfully prepared. The titer of CDK6 rabbit serum antibody reached 1:30 000 after immunization, which could specifically recognize the CDK6 protein expressed in cervical cancer cell line and cervical cancer tissues. Conclusion The high titer and specificity of rabbit anti-CDK6 antibody is successfully prepared.
Animals ; Female ; Humans ; Rabbits ; Antibodies ; Antibody Specificity ; Blotting, Western ; Cyclin-Dependent Kinase 6 ; Enzyme-Linked Immunosorbent Assay ; Uterine Cervical Neoplasms

Objective To prepare rabbit polyclonal antibody against mouse IQ and ubiquitin-like domain-containing protein (IQUB) and detect its expression in the mouse testis. Methods Full-length coding sequence of IQUB was inserted into the pET-30a(+) vector to construct pET-30a-IQUB recombinant prokaryotic plasmid. Transformation of pET-30a-IQUB plasmid into E. coli BL21 was performed, and protein expression was induced with isopropyl-beta-D-thiogalactoside (IPTG). The protein was purified through histidine-tagged fusion protein purification column, then denatured by treatment of urea with gradient concentration. New Zealand rabbits were immunized with the denatured protein to produce IQUB polyclonal antibody. Antibody titer was detected by ELISA, and Western blot analysis and immunofluorescence assay were employed to validate the effectiveness and specificity of IQUB antibody. Results pET-30a-IQUB recombinant plasmid was constructed, and protein expression of IQUB was induced successfully with IPTG. The titer of IQUB polyclonal antibody reached 1:1 000 000. The antibody specifically recognized the endogenous IQUB protein of testis in the wild-type adult mouse. IQUB was expressed in spermatogenic cells of different stages. It was localized in the acrosome and flagellum of mature sperms. Conclusion The highly specific rabbit anti-mouse IQUB polyclonal antibody is successfully prepared, which can be used for Western blot and immunofluorescence histochemistry.
Male ; Rabbits ; Animals ; Mice ; Ubiquitins ; Escherichia coli/genetics* ; Isopropyl Thiogalactoside ; Antibodies ; Enzyme-Linked Immunosorbent Assay

Objective To produce rabbit polyclonal antibody against mouse testis expressed 38 (TEX38). Methods Full-length open reading frame sequence of TEX38 was amplified and inserted into the pET-30a-(+) vector to construct pET-30a-TEX38 prokaryotic plasmid. The recombinant plasmid was transformed into E.coli BL21, and expression was induced with isopropyl β-D-thiogalactopyranoside (IPTG). New Zealand white rabbits were immunized with TEX38 protein after purification and denaturation, then TEX38 polyclonal antibodies were collected from rabbit serum samples. ELISA was performed to detect the antibody titer. Western blot and immunofluorescence staining were performed to determine the specificity of TEX38 polyclonal antibodies. Results The pET-30a-TEX38 recombinant plasmid was constructed, and TEX38 prokaryotic protein was expressed and purified successfully. After immunization, the titer of TEX38 antibody reached 1:1 000 000. Western blot analysis and immunofluorescence staining showed that TEX38 was localized in the mouse spermatogenic cells and sperms with a good specificity. Conclusion The rabbit polyclonal antibody against mouse TEX38 is successfully produced, and the expression of TEX38 in mouse spermatogenic cells and sperms is validated.
Male ; Rabbits ; Animals ; Mice ; Testis ; Antibodies ; Enzyme-Linked Immunosorbent Assay ; Immunization ; Spermatozoa ; Escherichia coli

Objective To prepare rabbit polyclonal antibody specifically against human lactate dehydrogenase C4 (LDHC4). Methods Site-directed mutation was performed by PCR to generate the mutated LDHC gene, and the mutated gene was ligated into the pET-28a vector to form the pET-28a-LDHC recombinant expression vector. The recombinant vector was introduced into E. coli BL21 (DE3), and LDHC4 protein was obtained by induced expression. The recombinant protein was used as an antigen to immunize New Zealand rabbits, and the antiserum was obtained after three boosted immunizations. The titer of the antiserum against LDHC4 were detected by ELISA. Western blot was used to detect the specificity of the antiserum, and immunohistochemistry was used to detect the expression of LDHC4 in human triple-negative breast cancer tissue. Results A specific rabbit anti-human LDHC4 polyclonal antibody was obtained with an antibody titer of 1:51 200. The antibody can be used for Western blot and immunohistochemistry. Conclusion The specific rabbit anti-human LDHC4 polyclonal antibody is successfully prepared.
Humans ; Rabbits ; Animals ; Escherichia coli/genetics* ; Antibodies ; Enzyme-Linked Immunosorbent Assay ; L-Lactate Dehydrogenase/metabolism* ; Blotting, Western ; Antibody Specificity

OBJECTIVE: To observe the levels of soluble programmed cell death protein 1 (sPD-1) and soluble programmed cell death ligand 1 (sPD-L1) in peripheral blood of lymphoma patients, and reveal their clinical significances. METHODS: The peripheral blood specimens and clinical data of 64 newly diagnosed lymphoma patients and 30 healthy volunteers were collected. The levels of sPD-1 and sPD-L1 were detected by enzyme-linked immunosorbent assay (ELISA), and their correlations with clinical characteristics of the patients including pathological type, stage, lactate dehydrogenase (LDH) level, T cell subsets were analyzed. RESULTS: The levels of both sPD-1 and sPD-L1 in peripheral blood of lymphoma patients were higher than those of normal controls (P <0.05). There were no significant differences in sPD-1 and sPD-L1 levels in peripheral blood between Hodgkin lymphoma and non-Hodgkin lymphoma patients. Different pathological subtypes of lymphoma had different levels of sPD-1. The level of sPD-1 in patients with T-cell lymphoma was higher than that in patients with B-cell lymphoma (P =0.001). The levels of both sPD-1 and sPD-L1 in patients with Ann Arbor stage III and IV were higher than those in patients with stage I and II (P <0.05). The level of sPD-L1 in patients with abnormally increased LDH was higher than that in patients with normal LDH (P =0.001), but there was no significant difference in sPD-1 level. T cell subset analysis showed that the level of sPD-L1 was negatively correlated to CD4⁺ T cell content (r =-0.265). CONCLUSION The levels of sPD-1 and sPD-L1 in peripheral blood of lymphoma patients are related to the pathological type, Ann Arbor stage, LDH content and T cell subsets, and will be potential biomarkers in predicting the prognosis of lymphoma.
Humans ; Clinical Relevance ; Prognosis ; T-Lymphocyte Subsets/metabolism* ; Lymphoma, T-Cell, Peripheral ; Enzyme-Linked Immunosorbent Assay ; B7-H1 Antigen/metabolism*

c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni²⁺-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.
Animals ; Rabbits ; Escherichia coli/metabolism* ; Enzyme-Linked Immunosorbent Assay ; Moths/genetics* ; Blotting, Western ; Larva/genetics* ; Isoantibodies/metabolism* ; Antibody Specificity