Research progress in the rapid preparation of monoclonal antibodies of mouse hybridoma / 生物工程学报

Mouse hybridoma monoclonal antibody is the most commonly used antibody in immunology because of its stable source, easy preparation in later stage and high yield. The traditional time-consuming and laborious hybridoma preparation technology could not meet the growing market demand. In this paper, we describe the rapid preparation techniques involved in antigen design and screening, B cell enrichment and screening, transgenic myeloma cells, fusion technology improvement, positive hybridoma cell screening and rapid detection of monoclonal antibody performance, to provide a reference for rapid preparation of mouse hybridoma monoclonal antibody.

PD-S15 fusion protein specifically target PD-1 and rapidly expands NK/T cells / 中国肿瘤生物治疗杂志

@#Objective: To investigate the function of anti-PD-1 (scFv)/IL-15/IL-15Rα-sushi fusion protein (PD-S15) to specifically bind to PD-1 in vitro and to explore its effect on NK/T cell proliferation. Methods: The human anti-PD-1 (scFv) gene sequence and human IL-15/IL-15Rα-sushi fusion gene sequence were synthesized chemically. The recombinant expression plasmid pUC57-PD-S15 was constructed by enzyme digestion and ligation of the two target genes, and then transiently transfected into HEK293T cells by lipofectamineTM 2000. The supernatants of cell culture medium were acquired, and the expression of PD-S15 fusion protein in cell culture supernatants was detected by Wb assay. PBMCs and TILs were cultured in mediums with different proportion of PD-S15/X-VIVOTM15, respectively. Then, the capacity of PD-S15 fusion protein to bind to PD-1 in vitro and its effect on the proliferation of PBMCs and the proportion of CD3+CD8+, CD3+CD4+ and CD3-CD56+ subsets were detected by flow cytometry. The effect of PD-S15 fusion protein on the proliferation of TILs was detected by cytometry. Results: The successful construction of pUC57-PD-S15 eukaryotic expression plasmid was confirmed by double enzyme digestion and sequencing, and then successfully transfected into HEK293T cells. The relative molecular weight of the target protein was approximately 55 000, and was in line with expectations. PD-S15 fusion protein could specifically combine with PD-1 in vitro (P<0.05) and stimulate NK/T cell proliferation (P<0.05). Compared with classical TILs culture method, the efficiency of activation and amplification of T cells in vitro by PD-S15 culturemethodwasbetter (P<0.01). Conclusion: PD-S15 fusion protein can specifically target PD-1 and rapidly expand NK/T cells in vitro, which lays a foundation for the selective expansion of CD8+PD-1+ antigen-specific T lymphocytes from tumor tissues and even peripheral blood.

Polymeric Nanoparticles Containing Both Antigen and Vitamin D₃ Induce Antigen-Specific Immune Suppression

The active form of vitamin D3, 1,25-dihydroxyvitamin D₃ (aVD₃), is known to exert beneficial effects in the treatment of autoimmune diseases because of its immunosuppressive effects. However, clinical application of aVD₃ remains limited because of the potential side effects, particularly hypercalcemia. Encapsulation of aVD₃ within biodegradable nanoparticles (NPs) would enhance the delivery of aVD₃ to antigen presenting cells, while preventing the potential systemic side effects of aVD₃. In the present study, polymeric NPs containing ovalbumin (OVA) and aVD₃ (NP[OVA+aVD₃]) were prepared via the water-in-oil-in-water double emulsion solvent evaporation method, after which their immunomodulatory effects were examined. Bone marrow-derived immature dendritic cells (DCs) treated with NP(OVA+aVD₃) did not mature into immunogenic DCs but were converted into tolerogenic DCs, which express low levels of co-stimulatory molecules and MHC class II molecules, produce lower levels of pro-inflammatory cytokines while increasing the production of IL-10 and TGF-β, and induce the generation of Tregs. Intravenous injection with NP(OVA+aVD₃) markedly suppressed the generation of OVA-specific CTLs in mice. Furthermore, OVA-specific immune tolerance was induced in mice orally administered with NP(OVA+aVD₃). These results show that biodegradable NPs encapsulating both antigen and aVD₃ can effectively induce antigen-specific immune suppression.

Investigation on antigen-specific T cell responses in type 2 diabetes mellitus patients / 中华内分泌代谢杂志

Objective To investigate the antigen-specific T cell functionality in type 2 diabetes mellitus patients. Methods Peripheral blood from 38 type 2 diabetes mellitus patients and 47 health controls (control group) have been collected. The proportions of CD4+and CD8+T cell as well as the ratio of CD4+/CD8+were monitored by flow cytometry. Meanwhile, antigen- nonspecific and specific Th1 responses were compared between two groups through detecting interferon (IFN)-γ, interleukin 2 (IL-2), and tumor necrosis factor (TNF)-α producing cells upon propylene glycol monomethyl ether acetate (PMA)/ionomycine and epstein-barr virus ( EBV) peptides stimulation, respectively followed by an intracellular cytokine staining. Results Compared to control group, the proportion of CD4+T cell and the ratio of CD4+/CD8+were significantly increased in type 2 diabetes mellitus group (P＜0.05) whereas CD8+T cells exhibited no significant difference between two groups. Antigen-nonspecific Th1 responses in type 2 diabetes mellitus patients were significantly decreased, demonstrated by lower percentages of IFN-γ, IL-2, and TNF-α producing CD4+T cells when compared to control group , while CD8+T cells in type 2 diabetes mellitus patients exhibited similar cytokine production patterns. However, when stimulated by EBV specific peptides, the percentages of IFN-γ, IL-2, and TNF-α producing CD8+T cells were significantly higher in type 2 diabetes mellitus patients than those in control group (P＜0.05). HbA1Cwas positively correlated with the percentage of EBV-specific TNF-α producing CD8+T cells (P＜0.05). Conclusion In type 2 diabetes mellitus, the secretion capacity of CD4+and CD8+T cell was significantly decreased and the antigen-specific responses represent the presence of an abnormal activated status, which indicates that chronic hyperglycemia may damage T cells function and aggravate chronic inflammation.

Expression and purification of rabies virus glycoprotein and analysis of its specific binding capacity to memory B cells / 生物工程学报

We aimed to express and purify three rabies virus glycoproteins with different tags and sizes. After analyzing their binding function, we wish to obtain a rabies virus glycoprotein with higher affinity and ability to specifically bind memory B cells. Experiments were carried out to express full length, as well as the ectodomain RVG by gene engineering method. Combined with the antibody of CD19 and CD27, the candidate protein labeling with fluorescence was used to analyze its binding function. Flow cytometry was used to detect the anti-rabies virus specific memory B cells in PBMCs, and confirm the binding ability between the candidate proteins and anti-rabies virus-specific memory B cells. We successfully constructed three expression vectors pGEX-5X-1-RVG, pET28a-RVG and pET30a-G. Three glycoproteins GST-RVG, His-RVG and His-G were obtained by optimized expression and purification conditions. The antigen specificity of purified GST-RVG, His-RVG and His-G were identified by Western blotting and ELISA. The affinity of these three purified glycoproteins to anti-rabies virus antibody were detected by competitive ELISA. Anti-rabies virus specific memory B cells in positive PBMCs gained from people who had ever been injected with the vaccine can be detected by flow cytometry. Thus, we got a recombinant rabies virus glycoprotein that had high-affinity and could sort antigen specific memory B cells.

Bordetella bronchiseptica antigen enhances the production of Mycoplasma hyopneumoniae antigen-specific immunoglobulin G in mice

We previously demonstrated that Bordetella (B.) bronchiseptica antigen (Ag) showed high immunostimulatory effects on mouse bone marrow cells (BMs) while Mycoplasma (M.) hyopneumoniae Ag showed low effects. The focus of this study was to determine if B. bronchiseptica Ag can enhance the M. hyopneumoniae Ag-specific immune response and whether the host's immune system can recognize both Ags. MTT assay results revealed that each or both Ags did not significantly change BM metabolic activity. Flow cytometry analysis using carboxyfluorescein succinimidyl ester showed that B. bronchiseptica Ag can promote the division of BMs. In cytokine and nitric oxide (NO) assays, B. bronchiseptica Ag boosted production of tumor necrosis factor-alpha in M. hyopneumoniae Ag-treated BMs, and combined treatment with both Ags elevated the level of NO in BMs compared to that from treatment of M. hyopneumoniae Ag alone. Immunoglobulin (Ig)G enzyme-linked immunosorbent assay using the sera of Ag-injected mice clearly indicated that B. bronchiseptica Ag can increase the production of M. hyopneumoniae Ag-specific IgG. This study provided information valuable in the development of M. hyopneumoniae vaccines and showed that B. bronchiseptica Ag can be used both as a vaccine adjuvant and as a vaccine Ag.

An analysis of clinical and imaging characteristics of atopic myelitis / 中华内科杂志

Objective To study the clinical and imaging characteristics of Chinese atopic myelitis (AM) patients.Methods Three diagnosed AM patients were retrospectively analyzed for the clinical data,serum IgE level,antigen specific IgE,cerebrospinal fluid,spinal MRI and therapeutic efficacy profiles.Results All the three patients were male and presented as subacute AM with the onset at 25,47 and 49 years old respectively.Two patients were allergic to pollen and other drugs,while another patient suffered from allergic rhinitis.Elevated serum total IgE and mite antigen specific IgE were found in all cases.Paraesthesia in limb extremities and positive Lhermitte sign were the main clinical features,while no optic,motor,urinary and defecation disturbance were found.Oligoclonal banding of cerebrospinal fluid and serum aquaporin 4 (AQP4) antibody were both negative in all cases.Spinal MRI showed lesions were hypointense on T1 and hyperintense on T2 at the posterior column of T2-3 segment with abnormal enhancement in case 1,hypointense on T1 and hyperintense on T2 at C2/3 segment with mild swelling in case 2 and hypointense on T1 and hyperintense on T2 at C3-5 segments with swelling and abnormal enhancement in case 3.Vitamin B were used in one patient,while the other two patients improved after the treatment with high-dose corticosteroids.Conclusions Subacute myelitis predominantly presents as paraesthesia in limb extremities with elevated serum total IgE and mite antigen specific IgE,while severe motor disorders are rare.Swelling and abnormal enhancement lesions at the posterior column of cervical cord are the common imaging features.Treatment with corticosteroids is recommended to be sustained for 3-6 months.

Evaluation of adjuvant effects of fucoidan for improving vaccine efficacy

Fucoidan is a sulfated polysaccharide derived from brown seaweed, including Fucus vesiculosus. This compound is known to have immunostimulatory effects on various types of immune cells including macrophages and dendritic cells. A recent study described the application of fucoidan as a vaccine adjuvant. Vaccination is regarded as the most efficient prophylactic method for preventing harmful or epidemic diseases. To increase vaccine efficacy, effective adjuvants are needed. In the present study, we determined whether fucoidan can function as an adjuvant using vaccine antigens. Flow cytometric analysis revealed that fucoidan increases the expression of the activation markers major histocompatibility complex class II, cluster of differentiation (CD)25, and CD69 in spleen cells. In combination with Bordetella bronchiseptica antigen, fucoidan increased the viability and tumor necrosis factor-alpha production of spleen cells. Furthermore, fucoidan increased the in vivo production of antigen-specific antibodies in mice inoculated with Mycoplasma hyopneumoniae antigen. Overall, this study has provided valuable information about the use of fucoidan as a vaccine adjuvant.

Antigen Specific IgG4 in Patients with Gastrointestinal Complaints.

Aims: To find the antigen specific IgG4 in patients with gastrointestinal complaints and in control group, to demonstrate suitability of detection of IgG4 by using antigen panel. Place and Duration of Study: Laboratory Management and Consultancy, Riga, Latvia, October 2012 - February 2013. Methodology: The study included 147 patients (46 men, 101 women, aged from 1 to 76) with different complaints regarding gastrointestinal habitus. Antigen specific IgG4 analysis by using regional adopted antigen panel was executed. Results: Almost all patients with gastrointestinal complaints -141 out of 147-have at least 2nd level antibodies, while in control group 15 out of 24 have 2nd level of antibodies. Conclusion: IgG4 antigen specific antibody tests by panels could be used as a screening tool for food intolerance detection.

Proliferation of antigen specific T cells in vitro and analysis of phenotype and function / 中华微生物学和免疫学杂志

Objective To explore a new peptide-based approach independent of HLA to generate antigen-specific CD+ CD8+T cells. Methods Peripheral blood mononuclear cells(PBMC) were stimula- ted for 6 h with IE-1 peptide pool. Then the activated IFN-γsecreting ceils were tested by immunomagnetic selection. And the selected cells were cultured with radio-inactivated PBMC in medium with 100 IU/ml IL-2 for 4 weeks. Results The generated T cell lines consisted of IE-1 specific CD4+ T (6.88%) and CD8+ T cells 92.99%, which demonstrated antigen-specific killing and cytokine secretion. Conclusion T ceils can be proliferated with this new procedure, and maintain its phenotype and antigen-specific function.

Two Cases of Atopic Myelitis

Myelitis is caused by various infectious organisms or an autoimmune mechanism. We report two cases of myelitis with increased serum total IgE and mite antigen specific IgE. The patients had paresthesia and Lhermitte's signs without weakness nor bladder dysfunctions. A spine MRI revealed a T2-high signal lesion with focal enhancement in the upper cervical cord. The patients showed favorable responses to steroid treatment. Atopic myelitis should be considered as a form of myelitis.

Detection of Helicobactor pylori by polymerase chain reaction: A comparison in sample preparation.

Gastric biopsy samples obtained from 14 patients with upper abdominal pain, clinically diagnosed as acid peptic disease, were analysed for the presence of Helicobacter pylori (H. pylori) by Polymerase Chain Reaction (PCR) using partially (template A) and completely purified DNA (template B). Antigen specific primer was used to analyse the sample by PCR method. The presence of H. pylori in the samples was confirmed by running a positive control. The presence of H. pylori was also detected by urease method using standard protocol. Among the 14 samples studied, 8 showed the presence of H. pylori with both templates A and B. Among these 8 samples only 3 showed positive for the presence of H. pylori with urease method. The present work discusses the results obtained in the detection of H. pylori in template A and B by PCR method.

Selective Expansion of TCR V beta 3+CD4+ T Cells in Collagen-induced Arthritis in DBA/1 Mice

BACKGROUND: Collagen-induced arthritis (CIA) in mice is animal model of autoimmune disease known as rheumatic arthritis in human. We investigated CII-specific CD4+ T cell receptor usage in CIA mice. METHODS: In CIA model, draining lymph node (dLN) CD4+ T cells and splenocytes at 3rd, 5th, 8th week, we investigated CII-specific T cell proliferation, production of IL-17, IFN-gamma, TNF-alpha, IL-4 and IL-10. And we also performed anti-CII IgG Ab measurements in serum level, TCRVbeta usage and T cell clonality with RT-PCR-SSCP analysis. Also, we performed proliferative response against CII when CII-specific T cell subset is deleted. RESULTS: CIA mice showed more increase in the serum level of anti-CII IgG than normal mice after induction of arthritis. And the level of anti-CII IgG2a in CIA mice was increased after 3rd week after primary immunization, while anti-CII IgG1 was decreased. Draining LN CD4+T cells have proliferated against CII stimulation at 3rd week after 1st immunization. CD4+T cells derived from dLN of CIA mice produced proinflammatory cytokine IFN-gamma, IL-17 etc. Draining LN CD4 T cells of CIA presented higher proportion of CD4+Vbeta +subsets compared to those of normal mice at 3rd week after 1st immunization, and they were increased in proportion by CII stimulation. Draining LN CD4+ T cells without TCRVbeta +/Vbeta 8.1/8.2+/Vbeta 10b+cells were not responsive against CII stimulation. But, CII-reactive response of TCRVbeta 3-/Vbeta 8.1/8.2-/Vbeta 10b- T cells was recovered when Vbeta 3+ T cells were added in culture. CONCLUSION: Our results indicate that CD4+Vbeta 3+ T cells cells are selectively expanded in dLN of CIA mice, and their recovery upon CII re-stimulation in vitro, as well as the production Th1-type cytokines, may play pivotal role in CIA pathogenesis.

Induction of Cellular Immune Response by Dendritic Cells Pulsed with Glioma Apoptotic Bodies

OBJECTIVE: The aim of this study is to verify the hypothesis that human dendritic cells(DCs) can process antigens from glioma cell apoptotic bodies and induce antigen-specific effector T cells. METHODS: DCs generated in the presence of granulocyte macrophage-colony stimulating factor(GM-CSF) and interleukin-4(IL-4) from peripheral blood mononuclear cells(PBMCs) of healthy donors with human leucocyte antigen(HLA) A*0201 were cultured for 7 days. Glioma apoptotic bodies(GABs) from T98G glioblastoma cells following 18 hour-actinomycin D treatment were co-incubated with DCs for 3 days. CD8 T cells isolated from peripheral blood of same donors were cultured in media containing IL-2 and were stimulated by GAB-pulsed DCs three times at a weekly interval. The interferon-gamma(IFN-gamma), a cytokine related to cytotoxicity, concentrations of cell culture supernates were measured by enzyme immunoassay technique. RESULTS: Induced DCs had DC's own phenotypic characteristics such as highly expressed major histocompatibility complex(MHC) class II, CD1a and CD86 molecules. They also had high endocytotic activity. Preteatment of T98G glioma cells with actinomycin D resulted in 53% of cells undergoing apoptosis. IFN-gamma production of effector T cells stimulated by GAB-pulsed DCs was significantly higher than that of T cells stimulated by non-pulsed DCs. CONCLUSION: Naive CD8 T cells can be activated by human GAB-pulsed DCs to become antigen-specific effector T cells. Using GABs as a antigen source may be a novel approach in future DC-based immunotherapeutic trials for malignant glioma.

The immune reaction in mucosal and systemic immune system was induced after intranasal immunization with bivalent Shigella vaccines / 中国免疫学杂志

Objective:To observe the effect on different mucosal sites and system immune sites after intranasal immunization with bivalent Shigella vaccines Methods:BALB/c mice were divided into three groups at random , 10 mice per group Mice were intranasally immunized respectively with FSM 2117or FS 5416 (4?10 7CFU) three doses with an interval of two weeks The NALT, NP, spleen, PP, MLN, lymphocytes were isolated on the seventh day after the last immunization to assay the change of the cell phenotype with FACS The nasal ?lung?intestine?genital tract lavage fluid and serum were taken to assay the specific IgA or IgG against F2a or Sonni LPS with ELISA Results:The specific IgA and IgG in different mucosal sites and serum increased significantly after intranasal immunization with two Shigella vaccines compared with the control (P

The Relation of Food-Specific IgE to Recurrent Wheezing Attack

PURPOSE: There were some reports that IgE-mediated food hypersensitivity is associated with subsequent development of atopic disease in wheezy infants. So we carried out this study to show whether the food specific IgE antibodies are useful to predict recurrent wheezing attack in wheezy infants. METHODS: A total of 190 children younger than 4-year-old were enrolled in this study. They were divided into 3 groups according to the number of experienced wheezing attacks as 1st, 2nd and more than 3rd attack group. There were 30 age-matched controls who had not wheezing nor family members of allergy. We measured peripheral blood eosinophil count, total serum IgE level, RAST f1(egg white), f2(milk), D1(Dp) and D2(Df) from the all participants. RESULTS: The proportion of patients having more than 250/mm3 cosinophils in each group increased as increasing wheezing attacks(P0.05). There was no significant difference in RAST f1 levels among the four groups. In contrast, the proportion of positive RAST f1 increased significantly as the number of wheezing attacks increased(P0.05). CONCLUSION: Egg white specific IgE antibody appear to be a risk factor for recurrent wheezing in infants and young child.

S Antigen Specific Rat Helper T Cell Line Induced Experimental Autoimmune Uveoretinitis / 대한면역학회지

No abstract available.

Studies on Diagnostic Methods to Detect Drug Allergies to Kampo Medicines. (I): Preliminary Analysis of Antigens from Crude Drugs and Antigen-Specific Antibodies using Shosaiko-to Extracts / 日本東洋医学雑誌

Recently, there has been an increase in reports of allergic reactions to Kampo medicines. In order to elucidate the mechanisms of these so-called Kampo allergies, suitable methods are necessary to detect allergens in crude drugs and allergenspecific antibodies in sensitized individuals. In this study, methodology and sensitivity were studied using enzyme-linked immunosorbent assay and Western blot analysis. The antigens used were haptenized baicalin and high-molecular-weight components of Ginseng Radix, both derived from Shosaiko-to extract.<br>Attempts were also made to further identify antigens from Shosaiko-to and detect antigenspecific antibodies in immunized rabbits.

Relationship between concentrations of antigen-specific IgG, nitric oxcide in CSF and intracranial hypertension and epilepsy in patients with neurocysticercosis / 临床神经病学杂志

Objective To evaluate the relationship between concentrations of nitric oxide (NO) and antigen-specific immunoglobulin G (IgG) in CSF and intracranial hypertension and epilepsy in patients with neurocysticercosis.Methods The NO concentration and the IgG level were measured in 40 cases of neurocysticercosisk, and the results were compared according to the patients whether had intracranial pressure or epilepsy. 23 patients with varicosis of great saphenous vein required subarachnoid anesthesia were served as control group.Results Compared with control group, the NO concentration in CSF significantly increased in patient group (P0.05) in the patients with epilepsy as compared with the cases without epilepsy.Conclusions The CSF NO concentrations are higher in the patients with neurocysticercosis who had intracranial hypertension or epilepsy. The IgG levels are also increased in the patients who had intracranial hypertension.

ANTIGEN SPECIFIC ACTIVITIES OF THE TRANSFER FACTOR AGAINST HERPES SIMPLEX VIRUS TYPE I / 中国免疫学杂志

TF was prepared from spleen cellsof both HSV-1-immunized mice(TF_(HSV)-1)and control group animals(TFn)4 weeks after immunization.The leukocyte adherence inhibitionindex(LAII)demonstrating the spe-cific activity of TF_(HSV)-1 in vitrowas found to be significantly higherthan that of control(P