Services & Instrumentation

Our Platform

The Recombinant Antibody Production Core (RAPC) provides custom recombinant monoclonal antibodies to academic researchers in Texas using a rapid, high-throughput, and cost-effective platform. The core serves as more than a fee-for-service core, they become your collaborators and consultants on selecting and targeting your antigen while also aiding in assay development to vet your antibodies with respect to antigen targeting, antibody specificity, and antibody affinity.

The McBride Lab developed a propriety method for isolating single-antigen-specific plasma cells following inoculation into mice. Cells are sorted, the immunoglobulin genes are amplified and sequenced, and the sequences are analyzed. Curated antibody-encoding genes are cloned into expression vectors for recombinant antibody production and purification. The antibodies can then be tested in collaboration with the end-user, using a number of assays based on user priorities. This process results in sequence-defined, recombinant antibodies in as few as 30-90 days. Many of the core's current customers are collaborators on longer-term projects.

Services

Basic Services

The core's unique process performs screening and sequence analysis up-front, not post-antibody production, and in contrast to hybridoma technology, this process does not require high antibody titers. The RAPC's recombinant antibody production methods overcome many of the obstacles of classical methods, especially lackluster antibody reproducibility and poor antibody characterization. The techniques used in the core are especially well-suited for generating antibodies that recognize challenging targets, such as antigens with single amino acid polymorphisms or post-translational modifications. Enhancements such as fluorescent or affinity tags can be easily engineered into your antibodies. In the future, the core's central technology will be optimized for the identification of candidate antibodies for human therapeutic development.

• Recombinant antibody project consultation (feasibility, antigen design)
    Standard recombinant antibodies
    Antibodies recognizing post-translational modification
    Antibodies recognizing specific antigen conformations

• Recombinant antibody production, purification, and characterization
    Mouse immunization
    Immunoglobulin sequencing
Immunoglobulin gene cloning
    Antibody validation assays based on client needs

• Recombinant antibody engineering
Fluorescent, epitope, or affinity tags
Single chain antibodies

Optimization for different species

• Conversion of existing hybridoma cell lines to recombinant antibody production stocks

Additional Services

Development of new services and applications is driven by client needs. We are open to new research collaborations that will broaden our scope. Examples of ongoing and completed projects include:

• Antibody profiling of human tumors (e.g. melanoma, sarcoma, non-small cell lung cancer, ovarian cancer)
    Profiling the immunoglobulin repertoire of tumor-associated B cells
    Recapitulating antibodies expressed by tumor-associated B cells
    Defining B cell clonal expansion, evolution, and selection
    Comparing circulating B cell populations and tumor-associated B cell populations

• Developing human antibodies to cell surface targets
    Production of cell-surface antigens for immunization
    Potential for creating humanized mice to produce “human” antibodies
    Developing candidate antibodies for therapeutic development

Instrumentation

Our process uses state-of-the art equipment to generate sequence-defined, recombinant antibodies in as little as one month. We are able to rapidly isolate individual B cells using a BD FACS ARIA Fusion and ready the single cells for RT PCR using our epMotion liquid handler. PCR is accomplished rapidly using an Applied Biosystems Veriti 384-well thermocycler coupled with an Agilent Zag DNA Analyzer for parallel capillary electrophoresis for amplicon analysis. Selected, purified DNA samples are sequenced and selected, matched heavy and light chain genes are cloned into expression vectors before being co-electroporated into modified CHO cells using a BTX HT-100 high-throughput electroporation system. Antibody-expressing cells are grown in a Kuhner CO2 shaker-incubator and the expressed antibodies are harvested and purified with protein-A binding in automated 96-well format. Select users may also arrange to use specific pieces of core equipment for their own research, when not in use by core personnel.

Recombinant Antibody Production Projects

Production of Recombinant Antibodies - General Overview

Our overall process includes several platforms that we used for isolation of single B cells, the recovery of immunoglobulin sequences by immunoglobulin profiling and sequence analysis, followed by cloning and expressing the immunoglobulin genes and purifying and testing the resulting recombinant antibodies.

Currently, we can perform immunoglobulin recovery on B cells isolated from patient tumor samples, peripheral blood mononuclear cells (PBMCs), and mouse spleens. Immunoglobulin profiling uses 5’ single-cell RNA-seq or B-cell receptor-seq. Sequences are analyzed using one or more methods, and the selected immunoglobulin-encoding sequences are cloned into expression vectors. The cloned genes are expressed in cells to recombinantly produce antibodies. The antibodies are then purified and tested in one or more assays.

Production of Recombinant Antibodies Against Viral Uracil-DNA Glycosylase (vUNG) Overview

In this example, we developed antibodies against gammaherpesvirus uracil DNA glycosylase that recognize the viral protein (vUNG) but not the related mouse protein. In our approach, we identify those cells that recognize the antigen of interest early in the process through a combination of antigen binding and fluorecence-activated cell sorting.

Capture of Antigen-specific Plasma Cells (vUNG)

In this example, plasma cells expressing antibodies specifically recognizing a recombinant gammaherpes virus antigen (vUNG) inoculated into mice were separated from those displaying non-specific binding.

Defining the Immunoglobulin Repertoire (vUNG)

Expressing and Testing the Recombinant Antibodies (vUNG)

Finally, the immunoglobulin genes encoding the antibody heavy and light chains are cloned into expression vectors and transfected into cells that will secrete the antibodies.

The secreted antibodies are purified using a high-throughput, automated protein-A purification system. Antibody integrity is checked by SDS-PAGE, and the antibodies are characterized in a downstream assay, in this case an ELISA.

The ELISA data graphed below illustrate the specificity of the recombinant anti-vUNG antibodies against the target v-UNG protein (left) compared to the related, non-target murine UNG (right).

Recombinant Antibodies Recognizing Post-Translationally Modified Protein (AID phospho-serine 38)

In this example project, we generated antibodies that recognized a post-translationally modified protien (phosphorylated activation-induced deaminase) but not the unmodified protein. The workflow diagram illustrates our process from inoculation of mice to testing the recombinantly expressed antibodies by immunoblotting.