Monoclonal anti-A/B

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A new approach to comparing anti-CD20 antibodies: importance of the lipid rafts in their lytic efficiency

A new approach to comparing anti-CD20 antibodies: importance of the lipid rafts in their lytic efficiency

B-cell-ablative therapies have been proposed for B-cell non-Hodgkin’s lymphoma (NHL), representing a major advance in its treatment. One of these therapies involves anti-CD20 monoclonal Abs (mAbs), which are directed against the CD20-expressing members of the B-cell lineage, from late pre-B-cells through mature B-cells. Rituximab (RTX) was the first mouse–human chimeric mAb to be approved by the Food and Drug Administration (FDA) for the treatment of relapsed, or refractory, low-grade NHL. 7

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B cell targeted therapies

B cell targeted therapies

Thus, the new concept is not only to target cytokines but also to target the cellular elements, such as B cells, that cause or perpetuate RA. How, then, may B cells be targeted? There are several ways to target B cells. First, one may target cytokines that promote B cell function and survival, such as IL-6 and B lymphocyte stimulator (BLyS). This may be done in several ways. One can produce an antibody to BLyS or use a soluble receptor such as transmembrane activator and calcium- modulator and cyclophilin ligand interactor-immunoglobulin (TACI-Ig) to block positive signaling through BLyS receptors. Second, B cells may be depleted by monoclonal antibodies that inhibit CD19, CD20, CD21, or CD22 B cell surface antigen. Finally, the co-stimulatory molecule may be targeted, preventing B cells from contributing to the inflammatory process through processing autoantigen and presenting it to T cells [2], as well as by producing cytokines and autoantibody. Treatment with anti-CD20 antibody destroys mature B cells in central lymphoid organs, the synovium, and the peripheral blood.
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B cell subpopulations in humans and their differential susceptibility to depletionwith anti CD20 monoclonal antibodies

B cell subpopulations in humans and their differential susceptibility to depletionwith anti CD20 monoclonal antibodies

Vaccination studies in patients treated with rituximab can provide indirect data on B-cell subpopulations that may be resistant to depletion with anti-CD20 mAbs. However, published data are diffi cult to interpret because of the small number of patients, eff ects of concomitant therapy and the background disease itself on the humoral response to vaccines and, in particular, because studies included patients at various stages of B-cell depletion or repopulation at the time of vaccination. Most studies have looked at responses to infl uenza vaccines and showed absent or decreased humoral responses to vacci- nation in patients previously treated with rituximab when compared with normal controls or patients not treated with rituximab [58-64]. Some studies described a positive relationship between the antibody responses to vaccination and number of circulating B cells at the time of vaccination [64] or the time from last rituximab treat- ment [60,62]. Interestingly, when circulating infl uenza- specifi c B cells were studied 6  days after vaccination, specifi c IgM-B cells were decreased in patients treated with rituximab 6 months previously when compared with controls but IgA B cells and IgG B cells were similar [61]. In a study in lymphoma patients, responses to recall antigens in the infl uenza vaccine were also seen but not to the new antigen [65]. Th ese studies suggest that memory B cells are more resistant to depletion than naïve B cells and can survive treatment with rituximab and be recruited in a secondary immune response.
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Production and analysis of human monoclonal IgG anti-DNA antibodies

Production and analysis of human monoclonal IgG anti-DNA antibodies

The British Isles Lupus Assessment Group (BILAG) designed disease activity criteria based on the principle of the physician's intention to treat (Hay et al, 1993a)(see Appendix). This index assesses the activity of the disease in eight major organs or systems avoiding the drawbacks of a global scoring system. The BILAG index also makes an allowance for change in activity over time . The organ/systems are general features, locomotor system, nervous system, renal involvement, dermatological involvement, pleuro- pericardial involvement, vasculitis, and haematological involvement. Immunological tests do not contribute to the score. From this assessment scores are generated ranging from A, most active, to E, no activity in that particular organ system. Although not designed for this purpose the BILAG index can be adapted to provide a global score using the following scoring system, A=9 points, B=3, C=1, D=0, E=0. The BILAG index has been validated against other global scores (Gladman et al, 1992b). A and B scores are considered to represent active disease in individual systems and a total score of greater than 6 indicates active disease as a whole. The apparent complexity of BILAG reflects the
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Application of Subtype Specific Monoclonal Antibodies for Rapid Detection and Identification of Influenza A and B Viruses

Application of Subtype Specific Monoclonal Antibodies for Rapid Detection and Identification of Influenza A and B Viruses

We established a rapid method for the identification of influenza A and B virus strains: the peroxidase- antiperoxidase (PAP) staining method with two subtype-specific murine monoclonal antibodies, C179 (H1 and H2 specific) and F49 (H3 specific), and an anti-influenza B virus rabbit polyclonal serum. The types and subtypes of 160 strains were examined, and 158 strains were identified to be the same by the hemagglutination- inhibition (HI) test and the PAP method. In contrast to the results by the HI test, two strains were revealed to be a mixture of two subtypes (H1 and H3) by the PAP method, which was confirmed by plaque cloning. We further analyzed clinical specimens by the PAP method by directly inoculating specimens into Madin-Darby canine kidney cells in microplates. After 40 h of incubation, the types and subtypes of viruses in 52 of 152 specimens were clearly identified. Since the reactivities of the two monoclonal antibodies are not influenced by the antigenic drift of influenza virus, the newly developed method should be applicable not only for rapid diagnosis but also for the epidemiological study of influenza.
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Ofatumumab: a novel monoclonal anti-CD20 antibody

Ofatumumab: a novel monoclonal anti-CD20 antibody

developed a total of 8 grade 3 AEs judged to be related to therapy. Thirteen patients experienced a total of 20 infections; 18 infections were grade 1 or 2 and included 11 cases of upper respiratory tract infection. Two grade 3 infections (urinary tract infection, neutropenic sepsis) were reported as SAEs but were judged unrelated to ofatumumab. There was no relation- ship between dose and toxicity, and the MTD was not reached. Sixteen of 38 evaluable patients (43%) responded (Table 2); 5 patients achieved a CR, and 2 attained an unconfirmed CR (CRu). The OR rate varied between 20% and 63% across cohorts, and there was no dose-response relationship. Nine of 14 evaluable patients who previously received rituximab responded (64%), including 3 of 4 rituximab-refractory patients. Six of 10 patients in cohort 4 (1000 mg dose), 6 of 10 patients responded, including 4 of 5 patients who had failed prior rituximab therapy. Median time to progression (TTP) was 8.8 months for all subjects and 32.6 months for responding patients, and median duration of response was 29.9 months. Thus, ofatumumab demonstrated significant activity in FL patients who had relapsed after prior rituximab therapy. Ofatumumab caused immediate, profound B-cell depletion lasting 6 to 10 months after end of therapy, and 65% of evaluable patients converted from Bcl-2 positive to Bcl-2 negative peripheral blood. Clinical response did not correlate with C max or AUC, but t 1/2 and clearance correlated with clinical response at week 26 but not week 19.
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Endogenous interferon specifically regulates Newcastle disease virus-induced cytokine gene expression in mouse macrophages.

Endogenous interferon specifically regulates Newcastle disease virus-induced cytokine gene expression in mouse macrophages.

This hypothesis was further supported by the finding that addition of identical amounts of mouse rIFN-a4 in the presence of monoclonal anti-IFN-,B also exerted a suppressive effect on su[r]

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Neutralization of IL-8 decreases tumor PMN-MDSCs and reduces mesenchymalization of claudin-low triple-negative breast cancer

Neutralization of IL-8 decreases tumor PMN-MDSCs and reduces mesenchymalization of claudin-low triple-negative breast cancer

The multifaceted roles of IL-8 in cancer make it a promising therapeutic target. Blockade of IL-8 signal- ing could modulate tumor phenotype, reduce recruitment of immune-suppressive cells to the tumor site, and inhibit cancer stem-like cells, thereby attacking multiple avenues that contribute to tumor progression and treatment resistance with a single approach. While there have been reports employing this tactic with IL-8 receptor blockade both in the preclinical and clinical settings (15–18), to our knowledge this study is the first to utilize an IL-8–specific antibody to target IL-8 signaling in a preclinical model of claudin-low TNBC. Here, the high-affinity, fully human, clinical-stage monoclonal anti–IL-8 antibody, termed HuMax-IL8 (19, 20), was explored both in vitro and in vivo with xenografts of claudin-low breast cancer cells. These studies demonstrated the ability of an anti–IL-8 monoclonal antibody to (a) reduce mesenchymal features in can- cer cells; (b) reduce the frequency of polymorphonuclear MDSCs (PMN-MDSCs) found at the tumor site, an effect that was enhanced when HuMax-IL8 was combined with docetaxel treatment in MDA-MB-231 xenografts; and (c) enhance the susceptibility of claudin-low TNBC cells to lysis mediated by immune effec- tor NK and antigen-specific T cells in vitro. Altogether, these data provide the rationale for combination therapies involving the use of HuMax-IL8 antibody with chemotherapy or immune-based therapies in the claudin-low breast cancer group and, potentially, in other tumors driven by an IL-8 autocrine loop.
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A unique cell surface antigen identifying lymphoid malignancies of B cell origin

A unique cell surface antigen identifying lymphoid malignancies of B cell origin

A monoclonal antibody (anti-B1) specific for a unique B cell surface differentiation antigen was used to characterize the malignant cells from patients with leukemias or lymphomas. All tumor cells from patients with lymphomas or chronic lymphocytic leukemias, bearing either monoclonal kappa lambda light chain, expressed the B1 antigen. In contrast, tumor cells from T cell leukemias and lymphomas or acute myeloblastic leukemia were

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Control of human B cell tumor growth in severe combined immunodeficiency mice by monoclonal anti B cell antibodies

Control of human B cell tumor growth in severe combined immunodeficiency mice by monoclonal anti B cell antibodies

Severe combined immunodeficiency (scid) mice develop EBV (+)B cell tumors after infusion of EBV(+)B cells or of B cells and EBV. In this study, scid mice were infused with B cell lines derived from three patients who developed a B lymphocyte proliferative disorder after bone marrow or organ transplantation. Intraperitoneal injection of 5 x 10(6) B cells induced tumor growth in all mice, leading to death within 60 d. Human B cells were identified in spleen and bone marrow by means of immunofluorescence or EBV genome amplification, and human IgM was detected in serum. Infusion of murine monoclonal antibodies specific for human B cell membrane antigens CD21, CD24, and CD23 was effective in 80% of animals, against two of the three cell lines preventing tumor development or inducing remission according to the time of treatment. The effect was antibody dose dependent and was optimal with four intravenous infusions of at least 0.1 mg 4 d apart. Human IgM in serum and human B cells in spleen and bone marrow became undetectable when peritoneal tumors regressed completely. Infusions of IgG1 isotype-matched anti-CD4
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Biologic activity and safety of belimumab, a neutralizing anti B lymphocyte stimulator (BLyS) monoclonal antibody: a phase I trial in patients with systemic lupus erythematosus

Biologic activity and safety of belimumab, a neutralizing anti B lymphocyte stimulator (BLyS) monoclonal antibody: a phase I trial in patients with systemic lupus erythematosus

The frequency of circulating plasma cells correlates with SLE disease activity and with the titer of anti-dsDNA autoantibod- ies [4]. Therefore, B-cell and plasma cell depletion may be an appropriate therapeutic approach in the treatment of SLE. B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) ligand superfamily of cytokines that is expressed and secreted by monocytes, macrophages, den- dritic cells, and granulocyte colony-stimulating factor activated neutrophils [5,6]. BLyS exists in both membrane-bound and soluble forms. The biologically active, soluble form of BLyS is enzymatically cleaved from the cell membrane and can bind to any of three receptors: TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor) [7]; BCMA (B-cell Maturation Antigen) [8]; and BAFF-R (B-cell lymphocyte activating factor receptor)/BLyS receptor 3 [9,10], localized primarily on B lymphocytes. BLyS contributes to B-cell proliferation and differentiation, and it is important in immunoglobulin class switching [5,11]. Constitutive over- expression of BLyS in transgenic mice results in the develop- ment of an autoimmune-like disease that is characterized by hypergammaglobulinemia, autoantibody production, and glomerulonephritis [8,12,13]. In murine lupus, treatment with a BLyS antagonist significantly reduces the occurrence of pro- teinuria and prolongs survival [8,14]. Moreover, elevated BLyS blood levels have been found in some patients with SLE [15,16], and observational studies demonstrated that BLyS concentrations change over time in the majority of SLE patients [17,18]. Increases in BLyS levels correlated with increased disease activity and were predictive of future dis- ease activity, suggesting that BLyS may be a biomarker for SLE [17].
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Inhibition of IL-13 by Antisense Oligonucleotide Changes Immunoglobulin Isotype Profile in Cultured B-Lymphocytes

Inhibition of IL-13 by Antisense Oligonucleotide Changes Immunoglobulin Isotype Profile in Cultured B-Lymphocytes

restricted to T lymphocytes [11, 14, 18]. It has been suggested that following the ligation of CD-40 and the addition of cytokine, human B-lymphocytes increase the IL-13 m-RNA [18]. Therefore, we cultured purified human tonsillar B-cells in the presence of anti CD-40 monoclonal antibody as a T- B cell interaction substitute plus IL-4 to promote IL-13 secretion. It has been reported that human B- cells produce IL-13 that involves in Ig class switching from IgM to IgE on RNA transcription at unrearranged  heavy chain locus [5]. We studied IL-13 inhibition and the consequences on immunoglobulin levels. Previous reports indicated the effects of anti IL-13 and anti IL-13 receptor: 1 and 2 monoclonal antibodies in neutralizing IL-13 in B-cell culture and further down regulation effects on IgE production [19-21]. Although these antibodies partially decreased IgE concentration, no investigation was performed on the other Ig classes. Although many reports indicate cytokine specific antisense oligos and their effects on Ig production, there are no reports about the role of human IL-13 antisense on Ig levels [22]. Accordingly, we applied an antisense ODN, which was already designed for human IL-13 inhibition in our laboratory.
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Establishment of anti mesothelioma monoclonal antibodies

Establishment of anti mesothelioma monoclonal antibodies

Diagnostic or therapeutic mAbs are now typically gen- erated by immunizing mice with synthetic peptides or target antigens that are purified to some extent [23, 24]. Except for nude or SCID mice, mice reject inoculated live malignant human tumor cells. During the first or sec- ond challenge, tumor cells may be primarily killed by NK and CD8 cytotoxic T cells or ingested by macrophages. However, during the course of repeated immunizations, mouse B cells are generated that produce antibodies against the tumor cells. These antibodies probably make a major contribution to the rejection of the tumor cells. This hypothesis served as the rationale for our experi- ments that were aimed at establishing anti-mesothelioma mAbs. We report here the generation of anti-human mesothelioma mAbs for diagnosis and treatment. This was accomplished by immunizing a mouse with live mes- othelioma cell lines. The hybridomas were selected for their ability to produce antibodies that bound to meso- thelioma cell lines not used for immunization. Some of these newly established mAbs reacted specifically with mesothelioma cells or inhibited their proliferation.
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Proteomic Analysis of Phagocytosis in the Enteric Protozoan Parasite Entamoeba histolytica

Proteomic Analysis of Phagocytosis in the Enteric Protozoan Parasite Entamoeba histolytica

We confirmed the phagosome localization of representative phagosome proteins Hgl, Igl, CP1, DPAP, Rab7A, and Rap2 by immunofluorescence assay as previously described (30) (Fig. 1). Trophozoites were incubated with carboxylate-modi- fied beads for 24 h or red blood cells for 5 to 30 min. Mono- clonal antibodies against representative surface membrane proteins Igl (9) and Hgl (3F4) (22) reacted with the membrane of phagosomes containing beads or red blood cells (results of Igl in a red blood cell-ingesting ameba and Hgl in a bead- ingesting ameba not shown). Anti-myc monoclonal antibody 11MO reacted with phagosomes containing red blood cells (B) and beads (data not shown), as well as small vesicles, in the transformant expressing myc-tagged Rab7A (30) and myc- tagged Rap2. Polyclonal rabbit antisera raised against the rep- resentative luminal digestive proteins CP1 (a gift from Sharon L. Reed) and DPAP also nicely reacted with the luminal part of the bead (A) or red blood cell-containing phagosomes (data not shown). Protein profiles of phagosomes obtained using
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B cell epitopes on infliximab identified by oligopeptide microarray with unprocessed patient sera

B cell epitopes on infliximab identified by oligopeptide microarray with unprocessed patient sera

Several studies have shown that anti-drug-antibodies (ADA) are induced in a substantial number of patients who are treated with therapeutic monoclonal antibodies. It is assumed that the formation of ADA in such patients is responsible for several side effects reported for thera- peutic antibodies [20, 21]. Whether or not ADA develop in a patient is dependent on various factors such as the therapeutic antibody itself, application route, dosage, fre- quency of application, and the immunological history of patients with or without co-medication [1, 2, 4, 5, 15, 22]. All of these factors are classical parameters in vaccine development—not surprisingly, since ADA induction can be considered as an undesired immunization event. A key parameter in every immunization/vaccination effort is the immunogenicity of the antigen administered. Immunogenicity is governed by the physical status of the antigen (aggregate/monomer), its valency (mono- or multivalent) and the presence of B and T cell epitopes. In addition, cross-recognition caused by pre-existing immu- nity against a similar antigen may also play a role.
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A profile of immune response to herpesvirus is associated with radiographic joint damage in rheumatoid arthritis

A profile of immune response to herpesvirus is associated with radiographic joint damage in rheumatoid arthritis

Our approach to quantify the broad responsiveness of ex vivo cytokine production was previously described [17]. Functional peripheral blood mononuclear cells (PBMCs) were procured by Ficoll density gradient centrifugation. Within one to two hours, 4 × 10 5 PBMCs were cultured in 200 μ L of medium (RPMI-1640 + 10% fetal bovine serum + 1× penicillin, streptomycin, and glutamine) in the presence of one of a panel of six stimuli, or in med- ium alone, in a 96-well culture plate. The rationale for the use of each stimulus was detailed in our previous publication [17]. The stimulation panel included immo- bilized anti-CD3 and anti-CD28 monoclonal antibodies (anti-CD3/anti-CD28; Dynabeads Human T-Activator, Invitrogen, Grand Island, NY, USA); bacterial CpG oli- gonucleotides (CpG); combined lysates of purified CMV and EBV, containing both viral peptides and DNA (CMV/EBV; Advanced Biotechnologies, Columbia, MD, USA); PMA with ionomycin (PMA/ionomycin; Sigma, St. Louis, MO, USA); phytohemagglutinin (PHA; Sigma); and staphylococcus enterotoxins A and B (SEA and SEB; Toxin Technology, Sarasota, FL, USA). The final concentrations of each stimulant were as follows: anti-CD3/anti-CD28, 0.5 × 10 6 beads per culture well (1:1 ratio of beads to PBMCs per manufacturer’s instructions); PHA, 5 μg/mL; Staphylococcus enterotoxin A, 10 ng/mL, with Staphylococcus enterotoxin B, 10 ng/ mL; CMV, 1 μg/mL, with EBV, 1 μg/mL; CpG, 10 μg/ mL; PMA, 1 μg/mL, with ionomycin, 700 ng/mL. The PBMCs were incubated at 37°C in 5% CO 2 for 48 hours;
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Minute Virus of Mice NS1 Interacts with the SMN Protein, and They Colocalize in Novel Nuclear Bodies Induced by Parvovirus Infection

Minute Virus of Mice NS1 Interacts with the SMN Protein, and They Colocalize in Novel Nuclear Bodies Induced by Parvovirus Infection

sid proteins accumulate within SAABs. The smaller APAR bodies identified at 15 h postinfection and the larger SAABs identified at 30 h postinfection contained VP1, VP1-containing intermediates, and fully assembled MVM capsids (Fig. 8). The capsids are likely due to de novo synthesis since capsids are not detected at earlier time points (data not shown). However, there were differences in the overall cellular distribution of assembled capsids compared to those of VP1 and VP1-con- FIG. 8. Viral capsid components (VP1) and the assembled MVM capsid accumulate within SAABs. Double-label experiments were performed on blocked infected A9 cells 15 and 30 h postrelease. (A) NS1 and VP1 colocalize at the 15- and 30-h time points. NS1 (primary antibody: anti-NS1 polyclonal; secondary antibody: anti-rabbit FITC conjugate) and VP1 (primary antibody: anti-VP1 polyclonal peptide antibody; secondary antibody: anti-mouse TRITC conjugate) are indicated. (B) NS1 and the assembled MVM capsid colocalize at the 15- and 30-h time points. NS1 (primary antibody: anti-NS1 polyclonal; secondary antibody: anti-rabbit FITC conjugate) and the capsid (primary antibody: anticapsid monoclonal antibody [30]; secondary antibody: anti-mouse TRITC conjugate) are indicated. Insets show nuclear regions of interest magnified by an additional factor of 2. Bar ⬇ 30 ␮m.
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Role of the Metabolic Minor Components in the Regulation of Intermolecular Interaction

Role of the Metabolic Minor Components in the Regulation of Intermolecular Interaction

After examining in silico the potential of natural metabolites and antigenic determinants, we passed to the series of model experiments. Influence of pyruvate and lactate on the antigen-antibody complexes AB0 system was carried out considering changes of degree and speed of onset of agglutination. We found that the second blood group was the most sensible to the effects of pyruvate and lactate—the time of agglutination onset increased by 16% and 40% respectively. Antigen determinant A of the fourth blood group is more sensible to the influence of external stimules in contrast to the glycoprotein B. The degree of agglutination of the antigens of A (II)-B (IV) blood groups remained stable comparing with the control and amounted 4+. In general, we note that lactate in- fluences more than pyruvate on the A and B determinants: formation of complexes antigen-antibody was longer (Figure 2(a)). Red blood cells A (II) and AB (IV) groups which have glycoprotein A on the surface, slower en- gage with the monoclonal antibodies than B (III) and AB (IV) blood groups on the membrane which have anti- genic determinant agglutinogen B.
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Anti T cell immunoglobulin and mucin domain 2 monoclonal antibody exacerbates collagen induced arthritis by stimulating B cells

Anti T cell immunoglobulin and mucin domain 2 monoclonal antibody exacerbates collagen induced arthritis by stimulating B cells

CD4 T cells were purified from the spleen of DBA/1 mice by passing it through a nylon wool column (Wako Pure Chemical Industries, Osaka, Japan) and by using an auto-MACS columns with CD4 T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) accord- ing to the manufacturer ’ s instructions. Purified CD4 T cells were stimulated with immobilized anti-CD3 mAb (5 μ g/ml) in the presence or absence of anti-CD28 mAb (5 μ g/ml). B cells were also purified by using the auto-MACS column with B cell isolation kit. Purified B cells were stimulated with anti-IgM antibody (Ab) (5 μg/ml), anti-CD40 mAb (5 μg/ml) and/or recombi- nant mouse IL-4 (20 ng/ml) for 48 hours. The anti-CD3 (145-2C11), anti-CD40 (HM40-3), and recombinant mouse IL-4 were purchased from eBioscience (San Diego, CA, USA). Goat anti-mouse IgM F(ab’)2 Ab was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Anti-CD28 (PV-1) mAb was kindly provided by Dr. R. Abe (Tokyo University of Science, Chiba, Japan) and Dr. C. June (University of Pennsylvania, Philadelphia, PA, USA).
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Blood Group Discrepancies at a Regional Blood Center

Blood Group Discrepancies at a Regional Blood Center

This cross-sectional study was conducted in the immunohematology laboratory of Yazd Blood Transfusion Center from March 2010 to March 2017. Demographic data of donors and previous history of blood donation were obtained from integrated software of Yazd Blood Transfusion Center. All blood donor samples received during the study period were analyzed. The exclusion criteria were deferred donors. All donor samples were tested for ABO typing using the tube method. Monoclonal antisera: anti-A, anti-B (Iranian Blood Research and Fractionation, Tehran, Iran) and in-house cells (group A1, B, and O reagent red blood cells) were used for forward and reverse grouping, respectively. The tests were performed according to standard operational procedures (SOPs) of the Iranian Blood Transfusion Organization (IBTO).
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