Human CD Antigens Research Articles

Development of Stable CHO-K1 Cell Lines Overexpressing Full-Length Human CD20 Antigen.

CD20 is a differentiation-related antigen exclusively on the membrane of B lymphocytes. CD20 amplification is observed in numerous immune-related disorders, making it an ideal target for immunotherapy of hematological malignancies and autoimmune diseases. MAb-based therapies targeting CD20 have a principal role in the treatment regimens. Fc gamma receptors mediate CD20 internalization in hematopoietic cells; therefore, this study aimed to establish non-hematopoietic stable cell lines overexpressing full-length human CD20 antigen as an in vitro model for CD20-related studies. CD20 gene was cloned into the transfer vector. The lentivirus system was transfected to packaging HEK 293T cells, and the supernatants were harvested. CHO-K1 cells were transduced using recombinant viruses, and a stable cell pool was developed by the antibiotic selection. CD20 expression was confirmed at the mRNA and protein levels. Simultaneous expression of GFP protein facilitated the detection of CD20-expressing cells. Immunophenotyping analysis of stable clones demonstrated expression of CD20 antigen. In addition, the mean fluorescence intensity was significantly higher in the CD20-CHO-K1 clones than the wild-type CHO-K1 cells. This study is the first report on using second-generation lentiviral vectors for the establishment of a non-hematopoietic cell-based system, which stably expresses full-length human CD20 antigen. Results of stable CHO cell lines with different levels of CD20 antigen are well suited to be used for CD20-based investigations, including binding and functional assays.

Construction and evaluation of wild and mutant ofatumumab scFvs against the human CD20 antigen

Several monoclonal antibodies targeting the CD20 have been produced and Ofatumumab is a case in point. Although whole antibodies target cancer cells effectively, their applications are restricted in some ways. Single-chain fragment variable antibodies, rather than employing the entire structure of antibodies, have proven a practical approach for creating completely functional antigen-binding fragments. In current research, the DNA coding sequence of and of the wild and mutant forms of ofatumumab were joined with a flexible linker separately. Using the E. coli BL21 (DE3) expression system, the -linker- genes were cloned into the pET-28 a (+), and the associated recombinant proteins were produced. Purified and refolded scFvs (scFv-C and scFv-) represented a concentration of around 0.7 mg/ml from 1 L of initial E. coli culture with a molecular weight of about 27 kDa. Affinity measurement disclosed anti-CD20 scFv- possesses a higher affinity constant compared to anti-CD20 scFv- The recombinant scFvs exclusively attach to Raji cells but not to Jurkat cells, according to a cell-ELISA analysis. The MTT test signified anti-CD20 scFvs could affect cell viability in Raji cells but had no impact on Jurkat cells and also, Raji cells viability was affected more significantly by anti-CD20 scFv-

CELL SURFACE MOLECULAR MARKERS FOR IDENTIFICATION OF CANCER STEM CELL POPULATIONS (SYSTEMATIC REVIEW)

The present-day concepts of cancer stem cells (CSC) could be considered as the innovative breakthrough in understanding the nature of cancer that opens new vistas in clinical approach. The review summarizes the available data and the trends in the search of molecular markers that may be advantageous for CSC identification and isolation. The combinations of markers useful for studying CSC phenotype in glioblastoma, neuroblastoma, head and neck squamous cell carcinoma, melanoma, hepatocellular carcinoma, small-cell lung carcinoma, gastric cancer, colorectal cancer, pancreatic cancer, thyroid cancer, breast cancer, cervical cancer, ovarian cancer, renal cell carcinoma, prostate cancer, bladder cancer, and osteosarcoma are reviewed with the emphasis on the genes coding for marker molecules, their chromosomal localization as well as molecular weights of corresponding proteins. The data on the molecular and functional features of 35 CSC markers promising for CSC identification including 26 human CD antigens are briefly analyzed. Furthermore, two cytoplasmic proteins aldehyde dehydrogenase-1 and nestin that may be also recommended for CSC detection are characterized. The involvement of epithelial-to-mesenchymal transition in generation of CSC population is also assessed. The development of targeted therapies for CSC eradication that should be safe for normal stem cells of the corresponding organs and tissues is outlined.

In Silico Analysis for Determination and Validation of Human CD20 Antigen 3D Structure

CD20 has been known as an attractive therapeutic target for refractory diseases such as B-cell chronic lymphocytic leukemia, rheumatoid arthritis and multiple sclerosis. Determining the 3D structure of the CD20 antigen could help to achieve a better deduction of its functions and its interactions with ligands. In this regard, we have launched an in silico protein modeling strategy to unveil the probable 3D structure of CD20 molecule. Various protein modeling approaches including homology modeling, Fold recognition and ab initio method were employed to build a qualified mode Protein BLAST tool from NCBI database was used to find a suitable template and the selected template was fed as input structure of the modeling software. Thereafter, the quality of the obtained models was evaluated invoking the model quality assessment software. CD20 Topology prediction shows that 4 trans membrane helixes. The best model predicted by LOMETS was selected for analyses. Refinement of 3D structure as well as determination of its B-cell epitopes, clefts and ligand binding sites was carried out on the structure. In conclusion, CD20 antigen 3D prediction led to design and production of a new monoclonal antibody.

Development and Characterization of a New Antipeptide Monoclonal Antibody Directed to Human CD20 Antigen.

The rapid expansion of immunotherapeutic approaches for treatment of various diseases, including cancers, has been greatly facilitated by the invention of new generation of antibodies. Clinical studies have indicated that anti-CD20 mAb-based therapies represent an effective treatment for various diseases with overexpression of CD20 on their cell surface, such as non-Hodgkin's lymphoma, hemolytic anemia, as well as autoimmune diseases like rheumatoid arthritis. Technically, due to a short extra membrane domain, the recombinant CD20 protein is a difficult antigen to raise immune responses. In search for new monoclonal antibodies, the authors used an antigenic polypeptide, which yielded numbers of new binders that may lead to production of anti-CD20 antibodies, with improved diagnostic or clinical attributes. Mice were immunized with extra membrane loop of human CD20 (exCD20) polypeptide. The exCD20 antigen showed a desired immune response and was able to develop a monoclonal antibody, 3B4C10, which reacted well with peptide antigen as well as native antigen on the surface of Raji B-cell line. The antibody 3B4C10 with a balanced K(on) and K(off) may be applicable in the construction of affinity columns or beads for isolation and purification of CD20-positive cells and cancer stem cells.

Effect of Dexamethasone, LPS, or INFγ on the production of TNFα, IL-12, IL-6, IL-10, CXCL8, CXCL10, and CLC3, by bovine macrophages infected in vitro with Mycobacterium avium paratuberculosis.

Effect of Dexamethasone, LPS, or INFγ on the production of TNFα, IL-12, IL-6, IL-10, CXCL8, CXCL10, and CLC3, by bovine macrophages infected in vitro with Mycobacterium avium paratuberculosis.

Immunological evaluation of predicted linear B‐cell epitopes of human CD20 antigen

The importance of B-lymphocyte-restricted differentiation antigen Bp35 (CD20) as a target for immunotherapeutic depletion of B cells is irrefutable. Several anti-human CD20 (anti-hCD20) monoclonal antibodies are expressed at different stages of development. However, resistance to anti-CD20 therapy has made the search for new alternatives imperative. Identification of B-cell epitopes within hCD20 using in silico tools can provide new opportunities to develop monoclonal antibodies with different binding sites. Furthermore, identification of the relationship between amino acid sequences of predicted B-cell epitopes and immune responses facilitates the determination of immunogenic regions of proteins by using their primary structure. Experimental evaluation of predicted linear B-cell epitopes as candidate peptides and bioinformatics allows us to explore this relationship. In this study, we selected three candidate epitopes within the extra membrane loop of hCD20 with the aid of five immunoinformatics predictor web servers and evaluated mouse humoral response to keyhole-limpet-hemocyaninconjugated peptides, and P4 and P5 peptides (the extracellular loop of hCD20 without and with a disulfide bond, respectively). Injection of the peptides yielded results that confirmed the prediction and selection of candidates. ELISA and flow cytometry corroborated the in silico selections. The B-cell epitopes P1, P2, and P3 were effective for immunization of mice.

Positron Emission Tomography of 64Cu-DOTA-Rituximab in a Transgenic Mouse Model Expressing Human CD20 for Clinical Translation to Image NHL

This study aims to evaluate (64)Cu-DOTA-rituximab (PETRIT) in a preclinical transgenic mouse model expressing human CD20 for potential clinical translation. (64)Cu was chelated to DOTA-rituximab. Multiple radiolabeling, quality assurance, and imaging experiments were performed. The human CD20 antigen was expressed in B cells of transgenic mice (CD20TM). The mice groups studied were: (a) control (nude mice, n = 3) that received 7.4MBq/dose, (b) with pre-dose (CD20TM, n = 6) received 2mg/kg pre-dose of cold rituximab prior to PETRIT of 7.4MBq/dose, and (c) without pre-dose (CD20TM, n = 6) PETRIT alone received 7.4MBq/dose. Small animal PET was used to image mice at various time points (0, 1, 2, 4, 24, 48, and 72h). The OLINDA/EXM software was used to determine the human equivalent dose for individual organs. PETRIT was obtained with a specific activity of 545 ± 38.91MBq/nmole, radiochemical purity >95%, and immunoreactivity >75%. At 24h, spleenic uptake of PETRIT%ID/g (mean ± STD) with and without pre-dose was 1.76 ± 0.43% and 16.5 ± 0.45%, respectively (P value = 0.01). Liver uptake with and without pre-dose was 0.41 ± 0.51% and 0.52 ± 0.17% (P value = 0.86), respectively. The human equivalents of highest dose organs with and without pre-dose are osteogenic cells at 30.8 ± 0.4μSv/MBq and the spleen at 99 ± 4μSv/MBq, respectively. PET imaging with PETRIT in huCD20 transgenic mice provided human dosimetry data for eventual applications in non-Hodgkins lymphoma patients.

Development of a quantitative, cell-line based assay to measure ADCC activity mediated by therapeutic antibodies

Antibody-dependent cellular cytotoxicity (ADCC) contributes to clinical efficacy of a broad range of antibody therapeutics. However, reproducible quantitation of ADCC activity on a cellular level remains highly challenging, as ADCC assays rely on primary effector cells associated with laborious cell purification procedures, resulting in highly donor-dependent results. Here, we report the development of an in vitro ADCC method based on an engineered human natural killer cell line as effectors. While eliminating the limitations of primary cells, this assay exhibits all the hallmarks of traditional ADCC assay systems. We have used this assay to measure the ADCC activity of a humanized IgG1 antibody directed against the human CD20 antigen. Our data show that this assay is capable to measure small changes in ADCC and can therefore be used to test therapeutic antibodies against cell-surface targets for their depleting activity.

IMMUNOLOGY

This book aims to cover the basic concepts and practical applications of immunology, introducing this complex field to students in biomedical sciences. Immunology has 14 chapters, each with self-assessment questions, and three appendices. The book is logically structured, starting with explaining the immune response before taking the reader through the components of the immune system and development of lymphocytes. The basal immunology is finished by covering the innate and adaptive immune response and regulations thereof, and after five chapters on immunology and infection, hypersensitivity, tolerance and autoimmune disease, transplantation and tumour immunology, and immunodeficiencies, practical aspects of immunology and immunotherapy are discussed. The appendices include tables of human CD antigens, selected cytokines and their functions and a thorough glossary. Each chapter starts with a list of learning objectives with a short summary of what the chapter covers; the figures are simple and easy to understand. In addition, boxes with nice-to-know information are scattered around the book, providing the reader with fun facts and useful definitions that make the complicated subject bearable to read and easier to grasp. The language is simplified compared with other textbooks within this field, which makes this book more reader-friendly, and as the authors have included a list of suggested further reading after each chapter, the reader can easily find the more advanced literature if necessary. At the first, quick glance, Immunology might appear boring and less attractive to read. The figures are in grey tones, with the exception of an immunohistochemistry figure, as are the few boxes and tables, and the text follows a strict outline. On the other hand, the clean structure to text, figures and boxes renders the book very tidy and easy to read, with no confusing elements. The index guides the reader to the right sections, the glossary provides good definitions and explanations, and the list of human CD antigens is in-depth and up to date. As the book includes no separate chapter on cytokines, the table with selected cytokines and their functions is very useful, also covering the source of the cytokines and the receptors they act upon. The book does cover the basics of immunology, and serves well as an introductory book to the field. Autoimmune diseases are well described, comprising symptoms and immunological explanations for them where such are known, treatment strategies and future therapies. The chapter on immunology in practice gives a useful overview of the most commonly used techniques. Having a rather small format, it is easy to carry to lectures and the pages have enough space for own notes. As the field of immunology is rapidly evolving and expanding, it is difficult to write books that will always be up-to-date, but as a work of references for basic immunological concepts, this book is perfect.

In Vivo Biodistribution and Lifetime Analysis of Cy5.5-Conjugated Rituximab in Mice Bearing Lymphoid Tumor Xenograft Using Time-Domain Near-Infrared Optical Imaging

Rituximab is a chimeric monoclonal antibody directed against human CD20 antigen, which is expressed on B-cell lymphocytes and on the majority of B-cell lymphoid malignancies. Herein we report the conjugate of rituximab with the near-infrared (NIR) fluorophore Cy5.5 (RI-Cy5.5) as a tool for in vitro, in vivo, and ex vivo NIR time-domain (TD) optical imaging. In vitro, RI-Cy5.5 retained biologic activity and led to elevated cell-associated fluorescence on tumor cells. In vivo, TD optical imaging analysis of RI-Cy5.5 injected into lymphoma-bearing mice revealed a slow tumor uptake and a specific long-lasting persistence of the probe within the tumor. Biodistribution studies after intraperitoneal and endovenous administration were undertaken to evaluate differences in the tumor uptake. RI-Cy5.5 concentration in the organs after intraperitoneal injection was not as high as after endovenous injection. Ex vivo analysis of biologic tissues and organs by both TD optical imaging and immunohistochemistry confirmed the probe distribution, as demonstrated by imaging experiment in vivo, showing that RI-Cy5.5 selectively accumulated in the tumor tissue and major excretion organs. In summary, the study indicates that NIR TD optical imaging is a powerful tool for rituximab-targeting investigation, furthering understanding of its administration outcome in lymphoma treatment.

GA101, a Novel Humanized Type II CD20 Antibody with Glycoengineered Fc and Enhanced Cell Death Induction, Exhibits Superior Anti-Tumor Efficacy and Superior Tissue B Cell Depletion In Vivo.

GA101 is a novel monoclonal antibody of IgG1 type which binds with high affinity and selectivity to the extracellular domain of the human CD20 antigen on B cells. In contrast to rituximab which is a chimeric antibody and recognizes a type I epitope, GA101 is humanized and recognizes a type II epitope which is also localized in the extracellular loop of CD20. The recognition of the type II epitope together with a modification of the elbow hinge region results in enhanced direct non-caspase dependent cell death induction, and concomitant reduction in CDC upon binding to CD20. In addition, using GlycoMab technology, the Fc-region of GA101 was glycoengineered to contain bisected, afucosylated carbohydrates. As a result GA101 has increased affinity for the low and high affinity FcγRIIIa receptor expressed on natural killer cells, macrophages and monocytes. Consequently, GA101 mediated a 5–50 fold enhanced induction of effector cell mediated ADCC. In B-cell depletion assays with whole blood from healthy donors, an assay combining all mechanisms of action, GA101 was significantly more potent and efficacious in depleting B cells than rituximab. In preclinical NHL testing these properties translated into superior anti-tumoral efficacy of GA101 in direct comparison to rituximab against a number of aggressive NHL xenograft models. In cynomolgus monkeys the induction of B cell depletion mediated by GA101 and subsequent B cell recovery were investigated. GA101 induced complete, rapid and long-lasting B cell depletion both in peripheral blood and in lymphoid tissue e.g. spleen and lymph nodes. The efficacy of GA101 (10 and 30 mg/kg) at depleting B cells in different lymphoid tissues of cynomolgus monkeys was compared with that of rituximab (10 mg/kg) following 2 i.v. doses administered on days 0 and 7. Notably, GA101 showed statistically superior depletion of total B cells from lymph nodes compared to Rituximab from day 9 to 35 onwards with B cell numbers decreased by over 95%. These results demonstrated that GA101 was more efficacious at depleting B cells from lymph nodes and spleen of cynomolgus monkeys compared to rituximab. Compared to existing antibodies, GA101 constitutes the first type II CD20 antibody engineered for increased ADCC with significantly enhanced efficacy in a variety of preclinical models. Based on these data it is assumed that the combination of the recognition of a type II epitope together with improved ADCC potency might translate into superior efficacy in the clinical treatment of CD20 positive malignant diseases.

Cross-reactivity of human leukocyte differentiation antigen monoclonal antibodies on carp and rainbow trout cells

Three hundred and seventy-seven monoclonal antibodies (mabs) directed against human CD antigens and non-classified human leukocyte surface antigens were assayed for their reactivity with common carp ( Cyprinus carpio L.) and rainbow trout ( Oncorhynchus mykiss) peripheral blood leukocytes (PBL) and thymocytes within the animal homologue section of the 8th International Workshop on Human Leukocyte Differentiation Antigens (HLDA8). Four of the mabs clearly reacted with rainbow trout PBL and two with carp PBL. Positive mabs were investigated further by two-colour flow cytometry with established mabs directed against carp and rainbow trout leukocyte subpopulations. None of these mabs were suitable for Western blotting and immunoprecipitation. Three mabs were found to stain cells in fixed cryostate sections of the lymphatic organs thymus, pronephros and spleen. In this study, for the first time an anti-CD14 mab was found to cross-react with fish cells. This mab could be a valuable tool complementing the limited toolbox of population-specific mabs in fish. The low number of cross-reactive mabs analyzed in this workshop is another indication for the great phylogenetic difference between mammals and osteichthyes.

Reactivity of monoclonal antibodies to human CD antigens with cells from mink

Three hundred and seventy six monoclonal antibodies (mAbs) raised against human leukocyte surface antigens were analyzed by flow cytometry for cross reactivities against mink leukocytes. We found 53 mAbs (14%) to cross react. This study defined cross reactions to the following human markers: CD1a, CD9 (4 mAbs), CD10, CD11a (2 mAbs), CD14 (3 mAbs), CD18 (5 mAbs), CD20 (atypical reaction), CD21, CD25 (atypical reaction), CD29 (3 mAbs), CD32, CD41, CD42a, CD44 (4 mAbs), CD45, CD45RO, CD47 (2 mAbs), CD49d (3 mAbs), CD61 (2 mAbs), CD62P, CD66abcd, CD71, CD75s, CD79b (2 mAbs), CD86, CD88, CD104 (atypical reaction), CD172a, CD236R (glycophorin C, (atypical reaction)), Xg(a) carbohydrate antigen, Rhesus antigen and two unspecified PAN-reactive mAbs. In order to characterize the molecular mass of the corresponding cross reacting mink markers, the mAbs were used to immunoprecipitate the surface antigens. Fourteen mAbs out of the 53 mAbs reactive with mink leukocytes gave reproducible IP findings. The masses of the precipitated antigens were generally in good agreement with those of the homologous human markers. We also performed immunohistochemical staining analyses on formalin fixed, paraffin embedded mink tissue from lymph node and spleen, and found 7 out of 22 mAbs to give a positive signal. Generally, the immunohistological analyses resulted in expected staining patterns.

Cross-reactivity of mAbs to human CD antigens with cells from cattle

A panel of 377 commercially available mAbs were submitted to the animal homologue section of the 8th International Workshop on Human Leukocyte Differentiation Antigens (HLDA8, Adelaide, Australia) for cross-reactivity studies on different animal species. In this study we describe the results of testing the mAbs on cattle cells by flow cytometry and Western blot. Eight commercial suppliers participated, providing mAbs to a total of 144 CD antigens plus controls. Fifty-two mAbs were identified as potentially staining cattle cells in the first round screen. In the second phase, 38 mAbs were confirmed as staining cattle cells. This included some that may recognise polymorphic determinants and others with atypical distribution patterns compared to humans. mAb to human CD9, CD11a, CD14, CD18, CD21, CD23, CD29, CD44, CD45R, CD47, CD49d and CD172a cross-reacted with bovine cells and mAb to CD22, CD88, CD119 and CD163 stained CD antigens that have not previously been identified in cattle.

Cross-reactivity of mAbs to human CD antigens with sheep leukocytes

A panel of 377 commercially available mAbs was submitted to the animal homologue section of the Eighth International Workshop on Human Leukocyte Differentiation Antigens (HLDA8, Adelaide, Australia) for cross-reactivity studies in a range of vertebrate species. Eight commercial suppliers participated by providing isotype controls and mAbs specific for a total of 144 CD antigens. In this study, we describe the results of flow cytometric testing of the reactivity of these mAbs with leukocyte populations isolated from blood, bronchoalveolar lavage, and ileal Peyer's patches of sheep. A total of 52 mAbs were identified as potentially reacting with sheep blood leukocytes in the first round of screening with blood leukocytes. In the second phase, reactivity of selected mAbs was further analyzed by repeating the screening with blood leukocytes at an independent facility. Screening of selected mAbs for reactivity with myeloid antigens was completed with alveolar macrophages and screening for reactivity with B cell antigens was completed with ileal Peyer's patch B cells. This screening identified mAbs that consistently reacted with both putative myeloid (CD10, CD22, CD23, CD27, CD29, CD32, CD49d, CD81, CD86, CD88, CD163, CD165) and B cell (CD10, CD22, CD23, CD27, CD29, CD32, CD49d, CD81, CD86, CD88, CD165) activation or differentiation antigens. Further studies will be required to determine if each mAb cross-reacts with an orthologous leukocyte antigen.

Canine CD20 gene

The human CD20 antigen, a 35 kDa cell surface nonglycosylated hydrophobic phoshpoprotein is expressed consistently on almost all human B-cells, and its monoclonal antibody is used for the therapy on human B-cell lymphoma. In the present study, canine CD20 gene was cloned and sequenced, and the expression of CD20 mRNA was investigated in canine peripheral blood mononuclear cells (PBMCs), and lymph nodes from healthy dogs, and canine lymphoma cells. Using canine cDNA as a template, full-length of canine CD20 gene was sequenced by 5′-RACE and 3′-RACE methods. The full-length of the cDNA sequence of canine CD20 was 1239 bp encoding 297 amino acids. The amino acid sequences of canine CD20 showed 73 and 68% sequence similarities with those of human and mouse, respectively. Canine CD20 was predicted to contain domains of amino acid sequences consisting of two extracellular domains (EM), four transmembrane domains (TM), and three intracellular domains (IC) as in human CD20. Canine CD20 mRNA was detected in PBMCs and lymph node from healthy dogs, and B-cells of canine lymphoma, but not in T-cell lymphoma cells and non-T and non-B-cell lymphoma cells by RT-PCR analysis. From these results, canine CD20 might be targeted for monoclonal antibody therapy against B-cell lymphoma of dogs.

Flow cytometry identification and characterization of mononuclear cell subsets in the neotropical primate Saimiri sciureus (squirrel monkey)

The neotropical primate squirrel monkey is used in many areas of biomedical research including neuroendocrinology, immunology and infectious diseases. However, research has been hampered by the lack of immunological tools for this primate. A series of 67 commercially available monoclonal antibodies to human CD antigens or cytokines were tested on Saimiri mononuclear cells and the specificity was assessed by double staining using flow cytometry. Monoclonal antibodies defining the main mononuclear cells subsets (monocytes, B, T, including CD4 and CD8 T cells) as well as activation markers have been identified. The conditions to specifically identify the various cell subsets using two color flow cytometry and establish their relative proportions have been set-up. We also have established normal values of the main circulating mononuclear cell subsets for adult Saimiri sciureus monkeys from the breeding unit of Institut Pasteur in French Guiana. The distribution between spleen, blood and lymph nodes has been compared. These tools allow documenting the phenotype of most Saimiri mononuclear cell subsets and assessing their activation level. This opens new perspectives for vaccinology and immunopathology research in this experimental non-human primate host, in particular for malaria research.

Rapid method for monitoring galactosylation levels during recombinant antibody production by electrospray mass spectrometry with selective-ion monitoring

This report describes a simple and rapid method to determine the relative amounts of glycoforms differing in terminal galactose on a recombinant antibody produced in Chinese hamster ovary (CHO) cells. The method uses a single quadrupole mass spectrometer coupled to an HPLC system to quantify the glycoform amounts found on a recombinant antibody that binds to the human CD20 antigen. Samples from the recombinant antibody process are reduced and injected directly into the HPLC system where the heavy and light chain antibody fragments, as well as host-cell protein contaminants, are separated chromatographically. Mass-selective detection is performed in the selected-ion monitoring (SIM) mode to monitor the most abundant (38 +) ions corresponding to the glycoforms found on the heavy chain of the recombinant antibody. Results obtained using the assay demonstrate good sensitivity, linearity and reproducibility. Comparison to a method using capillary electrophoresis (CE) of the labeled free oligosaccharides demonstrates similar quantitation of the glycoforms in the recombinant antibody. The LC–MS method provides a simple and rapid means for accurately quantifying antibody glycoforms directly from cell culture and other process samples.

Cross-reactivity with bovine cells of monoclonal antibodies submitted to the 6th International Workshop on Human Leukocyte Differentiation Antigens

Twelve subpanels of monoclonal antibodies (MAb) included within the 6th International Workshop on Human Leukocyte Differentiation Antigens (6th HLDA) were assayed for reactivity with bovine peripheral blood leukocytes. Sixty-nine of the 807 MAb (8.6%) stained bovine cells. These MAb represented 30 different human CD groups. Nine of the MAb to different human CD antigens (CD19, CD23, CD39, CD47, CD86, CD117, CD120b, CDw149, CD165) potentially recognized antigens on cattle cells that had not previously been identified. These were investigated further by two-colour immunofluorescence to compare the cellular expression of the antigen on cattle cells with that reported for the different CD antigens in humans. Four of the MAb that belonged to CD23, CD39, CD47, and CDw149 stained bovine cells in a manner that indicated an almost identical cellular distribution of the antigen to that reported in humans. This implied that these MAb reacted with the homologous cattle molecules. Further work would be necessary to confirm specificity of CD19, CD86, CD117, CD120b and CD165 MAb. Other cross-reacting MAb either recognized antigens already defined in cattle or antigens not yet clustered in humans. The study has identified valuable new reagents for studies of cattle and confirmed that most common cross-reactive MAb are to epitopes on integrins.