Custom Monoclonal Antibody Production

We provide both rat and mouse monoclonal antibodies. In particular, our proprietary mouse immunization approach allows us to provide mouse monoclonal antibodies within 70 days.

To generate monoclonal antibodies that fit your specific purposes, we tailor our protocols in every major step of antibody production, including antigen preparation [peptide synthesis or protein expression in E.coli, yeast, insect or mammalian cells], animal immunization and hybridoma screening.

o Antigen preparation

• We express and purify your recombinant proteins with an appropriate E. coli. Yeast, insect or mammalian cell expression system according to the specific purposes of the final antibodies.
• Our proprietary chimeric protein expression method can produce a recombinant protein immunogen in a soluble form in bacterial cells that retains the specificity and immunogenicity of the original protein, and generate antibodies that bind to the protein surface. This approach highly increases the possibility of getting IHC-positive monoclonal antibodies, and antibodies that recognize the native protein and good for ELISA, IP, IHC, Immuno-fluorescence and Western Blotting. In rare cases, codon optimization at a few critical positions is necessary to have a satisfactory expression level of the recombinant protein.
• Alternatively, we perform peptide synthesis and conjugation.

o Immunization of rats or mice

• We immunize animals with customized protocols that involve proper adjuvants, inoculation routes, dosage and timing. We can develop antibodies targeting impure native antigens or rare antigens of a minimum amount by adjusting the immunization strategies.
• For antibodies intended for immunohistochemical [or immunocytochemical] staining, in addition to the natural immunogens, e.g. recombinant protein, we may alternatively use denatured antigens in animal immunization and hybridoma screening. Our theory is that antigens to be stained in IHC are denatured [completely or partially], altered [by cross-linking] or precipitated by paraffin embedding and various fixations. We may use fixative-treated immunogens in animal immunization. To enhance the chance of getting the expected monoclonal antibodies that recognize the target antigens in their native forms, we believe it is also important to use the native proteins [enriched are fine] to boost [immunize] the animals one to two times, so that the epitopes shared by the recombinant and native proteins will be the dominant immunogens in antibody production.
• Our fast track mouse immunization protocol allows us to provide monoclonal antibodies within 70 days.

o Development of hybridoma

• We fuse splenocytes with proper myeloma cells using optimized PEG mediated fusion protocols.
o We screen hybridoma clones with customized protocols according to the final applications of the requested antibodies, including but not limited to immunoprecipitation, immunoblotting, various ELISA and particularly, IHC methods.
• IHC screening of monoclonal antibodies.To have antibodies for IHC is tricky. We perform hybridoma screening with IHC [as well as ICC] using paraffin-embedded slides (but not cryosections) or tissue microarrays at extra charges. Customer may provide the slides or Tissue Arrays; otherwise, we may procure the slides or arrays at customers’ expense. In IHC, it is not easy to know the specificity of the antibodies, since a perfect control is usually not available, except in the cases in which there is a gene-knock out tissue to be used as a control [or a specific transgenic fusion protein can serve as a reference staining]. For this reason, an independent assay, such as ELISA, Western Blotting or IP is required to confirm the specificity of the antibody’s binding to the target antigen. Then the antibody will be tested in IHC assays and the staining must be specific, i.e. positive not to all tissues or cells and the staining is stronger than the control isotype-matched negative control antibodies. For this reason, to get IHC-positive antibodies, we first use ELISA, Western Blotting [for denatured epitopes] and/or Immuno-precipitation [for native epitopes] to select the positive binders. In practice, we found that if we obtain many ELISA/Western/IP positive clones, the chance for us to have an IHC positive clone is very high. It is important to conduct ELISA, Western Blotting and/or Immuno-precipitation to validate the specificity of the IHC-positive antibodies, although this strategy is widely-forgotten.
o Omni-Hybridoma Platform. We have established an Omni-Hybridoma Platform, in which newly fused hybridoma cells are subcloned, grown and selected using an agarose-based semi-solid medium containing growth factors, B-cell stimulators and medium supplements optimized for the growth of single hybridoma cells. This revolutionary platform ensures that a large number of hybridoma clones can be selected after each cell fusion. In traditional hybridoma plating methods, multiple rounds of limiting dilutions are required to obtain monoclonal hybridoma clones. A serious problem is that some hybridoma clones overgrow others prior to cloning. Often, these faster growing cell lines do not synthesize antibodies, resulting in a failure to obtain the best antibody-producing hybridoma clones. Also, it is extremely labor-intensive and time-consuming to single-cell clone a hybridoma clone by performing multiple rounds of limited dilutions, thus making it difficult to isolate a large number of hybridoma clones from each cell fusion. The Omni-Hybridoma Platform is designed to select and clone hybridoma cell clones immediately after fusion. This eliminates the possibility of overgrowth of potentially valuable slow-growing clones by fast-growing clones. In this platform, hybridoma colonies are clonal from the start; the number of clones to be screened for the secretion of a specific antibody is minimized because all daughter cells are found in the same colony. Most importantly, since HAT selection and cloning of hybridomas are performed simultaneously in a single step, large numbers of hybridomas can be selected and tested. In fact, more than 1,500 hybridoma clones can be single-cells cloned in ten 15cm dishes after a single fusion, thus significantly increasing the number of candidate hybridoma clones that are specific for the target immunogen.
o We deliver your positive hybridoma clones in culture or in cryopreservation as well as the derived monoclonal antibodies.

o Ascites production or in vitro antibody manufacturing

• Inoculation of hybridoma cells in to relevant animals to produce ascites. We normally use immuno-sufficient mice to produce ascites. Immuno-deficient mice are available for ascites production for non-murine hybridoma cell lines or murine cell lines of distinct genetic backgrounds. Optimized methods are used to generate tumors and accumulate ascitic fluid. Using nude or SCID mice in ascites production will eliminate the contamination from endogenous mouse IgG.
• Cell culture in protein-free medium. We use stationary flasks or 5-50liter bioreactors to manufacture monoclonal antibodies. We have an antibody fermentation capacity of over 300L.