Hybridoma Selection

Our Smart Selection Approach

Hybridomas are immortal somatic cell hybrids that secrete antibodies. Hybridoma generation is a critical step in making monoclonal antibodies. A major barrier to monoclonal antibody discovery is hybridoma selection. Hundreds to thousands of hybridoma cells have to be manually screened in the hope of identifying antibody-secreting clones.

BiCell Scientific Inc has developed a “smart” selection approach to identify antibody-secreting hybridoma cells and streamlined the process for monoclonal antibody production. Our "smart" selection approach is included into monoclonal antibody production service in Custom Antibodies.

Flow chart of “smart” hybridoma selection

Step 1:

Mouse is immunized with antigen

Step 2:

Splenocytes from immunized mouse are isolated

Step 3:

Splenocytes are fused with myeloma cells (our electrofusion protocol can generate 1 hybridoma clone out of 104 splenocytes)

Step 4:

Hybridoma cells are treated by a proprietary crosslinking protocol to immobilize antibodies on the cell surface

Step 5:

Fluorophore labeled antigen molecules allow identifying positive hybridoma cells by direct binding to the hybridoma cell surface

Step 6:

Fluorescence based cell sorting

Step 7:

Dilution cloning and clonal selection


Electrofusion efficiently fuses myeloma cells with splenocytes. Circle indicates fusion event. Myeloma cells: large nuclei; splenocytes: small nuclei.


Antigen labeling of hybridoma cells. Hybridoma cells are transiently crosslinked to immobilize antibodies on the cell surface. FITC-labeled antigen binds to and decorates the hybridoma cell that secretes the correct antibody.


Hybridoma cell plating and screening. Antibody secreting hybridoma cells are cultured and their supernatants screened in 96-well plates. Over 50% of hybridoma cell clones show positive binding to antigen. Procedure: plate was coated with peptide antigen at 1ug/ml in PBS, followed by incubation with 50ul supernatant for 1hr and secondary antibody (HRP) for 1hr. HRP substrate (blue color) develops within 5 min. No acid was added. Image was taken with iPhone.

Our Workflow Chart

Steps Timeline (weeks) Deliverables/Internal testing milestone QC standard
Phase I: Antigen preparation

(peptide antigen,13-19 aa length, will be designed against target protein and its region of interest, synthesized and conjugated to KLH)

2 weeks / COA





Phase II: Animal immunization

(3 Sprague Dawley rats or 5 Swiss Webster mice) will be immunized with antigen; conventional protocol – three injections)



8-10 weeks 100 ul anti-serum ELISA after 3rd injection
Phase III: Cell fusion and screening

(fusion of mouse myeloma cells with splenocytes from one best responder animal to generate hybridoma and perform antigen based hybridoma selection, enriching, and plating)



4-6 weeks Supernatant from 96 wells ELISA from supernatant
Phase IV: Expansion and antibody purification

(customer choosing one clone; expanding clone and purifying antibodies with affinity chromatography)


6-8 weeks One hybridoma cell line (to customer); purified monoclonal antibody from the selected hybridoma cell line (1mg)

(BiCell Scientific will bank all remaining clones; customer can request additional clones by paying a service fee for hybridoma recovery)

ELISA from supernatant

"I am really impressed with your approach. We tried multiple times previously to create monoclonal and polyclonal antibodies to claudin-2 and MLCK1. We have had limited success generating polyclonals and no success generating monoclonals. You have generated outstanding monoclonals to both. I look forward to continuing to work with you."

Jerrold R. Turner, M.D., Ph.D.

Brigham and Women’s Hospital | Harvard Medical School

"The polyclonal antibody you generated for KIAA0408 is stunning! KIAA0408 is a novel cilium molecule that has never been studied. So, clearly there will be a lot of demand for it as we have discovered a very interesting finding and the story will be published in a high impact journal. I am strongly inclined to generate monoclonal antibody for this protein too and we should think about patenting it."

Univ.-Prof. Jay Gopalakrishnan PhD

Heinrich-Heine-Universität | Universitätsklinikum Düsseldorf

"Your ARL13B antibody works beautifully!!! We’re so happy to have a cilia-specific antibody made in rat! I can send you high resolution images to be posted on your website."

Julie Craft Van De Weghe, PhD

School of Medicine | University of Washington

"The assay is a homophilic interaction mediated cell adhesion on purified protein (in this case, immobilized purified Pcdhga9 to Pcdhga9 expressed on cell surface). Compared to control, cell adhesion is reduced in the presence of Pcdhga9 monoclonal antibody supernatants!"

Divyesh Joshi, PhD

School of Medicine | Yale University

"The rabbit hybridoma supernatants of anti-APOBEC3 project are tested positive by ELISA, and we are very happy about it! We previously tried a company, Abclone. Their Project "A" has immune response that is <10,000 titer in antiserum, which would explain why there is no positive mAb after fusion. Their project "B" didn't have any immune response in rabbit."

Harshita B Gupta, PhD.

School of Medicine | UT Health San Antonio

Contact us for questions or custom requests!