PTPRC Recombinant Monoclonal Antibody

Molecular Structure and Target Protein

PTPRC recombinant monoclonal antibodies target CD45, also known as protein tyrosine phosphatase receptor type C (PTPRC), a transmembrane protein encoded by the PTPRC gene located on chromosome 1q31-32. CD45 consists of approximately 1,258 amino acids with a molecular weight ranging from 180 to 220 kDa due to extensive glycosylation and post-translational modifications, including phosphorylation and disulfide bond formation . As a transmembrane protein, CD45 is primarily localized to the plasma membrane of hematopoietic cells, where it plays essential roles in cell signaling, activation, and differentiation .

The PTPRC protein undergoes alternative splicing to generate multiple isoforms, including CD45RA (ABC and BC isoforms) and CD45RB, which are expressed differentially across immune cell populations. The CD45RA isoform is a protein of 205-220 kDa that represents a specific variant of the human leukocyte common antigen . These isoforms arise from differential splicing of three exons (A, B, and C), generating at least five distinct variants: ABC, AB, BC, B, and O . The distribution of these isoforms varies significantly across immune cell types and developmental stages, providing valuable markers for distinguishing cell subpopulations.

Biological Function and Significance

CD45 functions as a critical regulator of immune cell activation and signaling, particularly in T and B lymphocytes. It acts as a key tyrosine phosphatase that modulates signaling pathways downstream of antigen receptors, cytokine receptors, and co-stimulatory molecules . By dephosphorylating specific tyrosine residues on signaling molecules, CD45 helps maintain the balance of intracellular signaling cascades critical for immune cell responses .

In T-cell biology, CD45 is essential for T-cell activation through the antigen receptor and serves as a positive regulator of T-cell coactivation upon binding to DPP4 . Upon T-cell activation, CD45 recruits and dephosphorylates proteins such as SKAP1 and FYN, influencing downstream signaling events . Additionally, it dephosphorylates LYN, thereby modulating LYN activity and further influencing cellular signaling pathways .

CD45 expression is found on all nucleated hematopoietic cells, including T cells, B cells, natural killer cells, monocytes, macrophages, and granulocytes . The differential expression of CD45 isoforms is physiologically significant, with CD45RA expressed on 40-50% of peripheral CD4 T-cells, 50% of peripheral CD8 T-cells, B-cells, and leukemic B-cell lines . T-cells expressing CD45RA are considered naïve or virgin T-cells, while those expressing CD45RO represent memory T-cells, allowing for functional distinction between immune cell subsets .

Production Methodology

The production of PTPRC recombinant monoclonal antibodies represents a sophisticated biotechnological process that ensures high specificity and consistency. The initial step involves introducing PTPRC antibody genes into plasmid vectors, followed by the transfection of these engineered plasmids into suitable host cells for expression using exogenous protein expression technology . Following successful expression, the antibodies undergo purification through affinity chromatography to isolate the target protein with high purity .

Advanced methodologies such as phage display technology have further enhanced the development of PTPRC antibodies. This approach allows for efficient screening of human single-chain variable fragments (scFvs) against specific antigens, enabling the production of fully human IgG1 monoclonal antibodies with superior specificity and reduced immunogenicity . The process typically involves:

1. Identification of potential antigenic peptides through bioinformatic analysis

2. Synthesis of selected peptides for antibody screening

3. Screening of human scFv libraries through phage display

4. Selection of high-affinity fragments for full antibody production

5. Expression and purification of complete monoclonal antibodies

6. Validation through various analytical techniques

These antibodies undergo rigorous validation for specific applications, including ELISA, flow cytometry, and immunohistochemistry, ensuring their reliability for research purposes .

Types and Classification of PTPRC Antibodies

PTPRC recombinant monoclonal antibodies are available in various formats and classifications, each designed for specific applications and targeting different epitopes of the CD45 protein. The following table presents a comprehensive overview of different PTPRC antibody types currently available:

These antibodies vary in their specificity toward different CD45 isoforms, making them valuable tools for distinguishing between cell populations. For instance, antibodies targeting CD45RA can effectively identify naïve T-cells and B-cells, while those against CD45RB target a different subset of immune cells . The selection of the appropriate antibody depends on the specific research question, target cell population, and intended application.

Technical Benefits Over Traditional Antibodies

PTPRC recombinant monoclonal antibodies offer numerous advantages over traditional monoclonal antibodies produced through hybridoma technology. The recombinant approach provides:

1. Increased sensitivity: Recombinant antibodies typically demonstrate enhanced binding affinities, with some PTPRC antibodies exhibiting nanomolar binding constants as confirmed by surface plasmon resonance (SPR) studies . This increased sensitivity enables the detection of lower antigen concentrations, enhancing research outcomes.

2. Confirmed specificity: Through genetic engineering, recombinant antibodies can be designed to target specific epitopes with exceptional precision. This results in minimized cross-reactivity and more reliable experimental data. For example, recombinant anti-PTPRC antibodies have demonstrated the ability to selectively bind to tumor T lymphocytes expressing specific TCR segments without binding to other lymphocytes or blood cell components .

3. High repeatability: The genetically defined nature of recombinant antibodies ensures consistent performance across experiments, enhancing reproducibility in research settings.

4. Animal-free production: Many recombinant antibody production systems eliminate or significantly reduce the need for animal immunization, addressing ethical concerns associated with traditional antibody production methods .

5. Customizable features: Recombinant technology allows for the incorporation of specific tags, conjugates, or modifications to enhance functionality. For instance, PTPRC antibodies can be conjugated with fluorochromes like PE-Cy5 for flow cytometry applications or biotin for detection systems .

Quality and Consistency Factors

A major advantage of recombinant monoclonal antibodies is their exceptional batch-to-batch consistency, which addresses one of the significant limitations of traditional hybridoma-derived antibodies. This consistency stems from several factors:

1. Defined genetic sequence: Recombinant antibodies are produced from a known genetic sequence, eliminating the variability introduced by hybridoma drift or inconsistent animal immune responses.

2. Controlled expression systems: Production in well-characterized expression systems allows for standardized culture conditions and protein expression.

3. Standardized purification protocols: Consistent purification methods ensure uniform antibody quality across different manufacturing batches.

4. Long-term sustainable supply: The genetic information encoding the antibody can be stored indefinitely, ensuring continuous availability of identical antibodies for longitudinal studies.

5. Reduced lot-to-lot variation: Studies have demonstrated that recombinant PTPRC antibodies maintain consistent binding properties and specificity across different production lots, enhancing experimental reproducibility .

For researchers, these quality factors translate into more reliable experimental results, reduced need for antibody validation across batches, and improved data comparability across different research studies or time points.

Laboratory Techniques and Protocols

PTPRC recombinant monoclonal antibodies serve as versatile tools across various laboratory techniques. Their applications include:

1. Flow Cytometry (FC): PTPRC antibodies are extensively used in flow cytometry for immunophenotyping of hematopoietic cells. Different isoform-specific antibodies can distinguish between naïve and memory T-cells, making them valuable for studying immune cell development and activation states . Recommended dilutions for flow cytometry typically range from 1:20 to 1:500, depending on the specific antibody and experimental conditions .

2. Western Blotting (WB): These antibodies can detect CD45 protein in cell or tissue lysates, enabling quantitative analysis of protein expression. The high specificity of recombinant antibodies reduces background signal and improves data quality .

3. Immunohistochemistry (IHC) and Immunocytochemistry (ICC): PTPRC antibodies are used to visualize CD45 expression in tissue sections or fixed cells, aiding in the identification of immune cell infiltration in tissues and the characterization of hematopoietic malignancies .

4. Enzyme-Linked Immunosorbent Assay (ELISA): Certain PTPRC recombinant antibodies are validated for ELISA applications, providing quantitative measurement of CD45 levels in biological samples .

5. Immunoprecipitation (IP): Some clones, such as UCH-L1, are suitable for immunoprecipitation studies, allowing researchers to isolate CD45 and its binding partners for further analysis .

Protocol considerations for optimal results with PTPRC recombinant monoclonal antibodies include:

• Proper sample preparation to preserve epitope integrity

• Appropriate blocking steps to minimize non-specific binding

• Careful titration to determine optimal antibody concentration

• Selection of suitable detection systems based on the application

• Inclusion of appropriate positive and negative controls

Clinical Research Implications

Beyond basic research applications, PTPRC recombinant monoclonal antibodies hold significant value in clinical research contexts:

1. Diagnostic Applications: CD45RA antibodies are useful in differentiating T-cell lymphomas (typically CD45RO positive) from B-cell lymphomas (typically CD45RA positive), aiding in the accurate classification of hematological malignancies . This distinction is critical for proper diagnosis and treatment selection.

2. Immune Monitoring: These antibodies facilitate the monitoring of immune cell populations in various disease states, including immunodeficiencies, autoimmune disorders, and infectious diseases. For instance, tracking naïve versus memory T-cell ratios provides insights into immune status and response to therapies.

3. Therapeutic Target Identification: Research using PTPRC antibodies has contributed to the identification of potential therapeutic targets in immune-related disorders. Studies have demonstrated that targeting specific V segments of the TCR beta chain with human monoclonal antibodies represents a potential therapeutic option for patients with mature T-cell neoplasms .

4. Biomarker Development: CD45 isoform expression patterns serve as potential biomarkers for disease progression or response to therapy in various conditions, with PTPRC antibodies enabling their reliable detection and quantification.

5. Cell Therapy Research: In the evolving field of cell-based therapies, these antibodies aid in the characterization and quality control of therapeutic cell products, ensuring their safety and efficacy.

The clinical research applications of PTPRC recombinant monoclonal antibodies continue to expand as our understanding of immune cell biology and its role in disease pathogenesis deepens.

Advancements in Immunological Understanding

Recent research utilizing PTPRC recombinant monoclonal antibodies has significantly enhanced our understanding of immune cell biology and pathology:

Therapeutic Potential and Future Directions

The development of PTPRC recombinant monoclonal antibodies has opened new avenues for potential therapeutic applications:

1. Targeted Therapy for T-cell Neoplasms: The development of fully human IgG1 monoclonal antibodies targeting specific V segments of the TCR beta chain represents a promising therapeutic direction for patients with mature T-cell neoplasms . This approach offers the potential for highly specific treatments that target malignant T-cell clones while sparing healthy immune cells.

2. Immunomodulatory Applications: Given CD45's role in regulating immune cell activation, antibodies that modulate its function could potentially be used to treat autoimmune disorders or enhance immune responses in certain contexts.

3. Diagnostic Applications in Lymphoma Classification: CD45RA antibodies have demonstrated utility in differentiating T-cell lymphomas from B-cell lymphomas, potentially improving diagnostic accuracy and treatment selection .

Future research directions in this field include:

1. Development of antibody-drug conjugates targeting specific CD45 isoforms for targeted therapy of hematological malignancies

2. Engineering bispecific antibodies that simultaneously target CD45 and other immune checkpoints to enhance anti-tumor immune responses

3. Further exploration of CD45's role in various immune disorders to identify new therapeutic targets

4. Refinement of recombinant antibody production methods to improve yield, reduce costs, and enhance accessibility for research and clinical applications

5. Investigation of CD45 as a potential target for immunotherapy approaches in solid tumors with significant immune cell infiltration