Screening for Positive Hybridoma Cells by ELISA
The hybridoma method is a mainstay for monoclonal antibody (mAb) production (including the hapten-associated small molecule antibody), during the whole workflow, the accurate and specific screening of positive hybridomas usually determines the success. Commonly, positive antibody-secreting subclones are screened and selected by a solid-phase enzyme-linked immunosorbent assay (ELISA). As a specialist in customized antibody development, Creative Biolabs offers a broad range of hybridoma screening services for the production of mAbs to promote basic scientific research and clinical applications.
Hybridoma and Positive Hybridoma Cells Screening
MAbs are useful biological tools for various analytical applications, including clinical chemistry, food analysis, environmental monitoring, and human therapeutics. Hybridoma technology is still the most common method for the generation of mAbs. An efficient hybridoma screening procedure is a crucial step that should be fast, reliable, and easy to accomplish. Especially, it should detect positive hybridoma cells with a minimum of both false positives and false negatives.
Fig.1 Anti-hapten antibody production and application for immunoassay. (Tian, 2018)
Classification of ELISA Screening
There are two kinds of ELISA methods feasible for hybridoma screening, indirect ELISA and direct ELISA. In the hapten-specific antibody discovery, indirect ELISA is the de facto standard screening method.
- Indirect ELISA
- Direct ELISA
The basic principle of indirect ELISA is the covalent conjugation of antigen and antibody, and the free components are washed away. Usually, the first step is to immobilize a hapten-protein conjugate on the microplate surface; the second step is to add diluted hybridoma culture supernatant containing anti-hapten mAbs; the third step is to add enzyme-labeled secondary (anti-mouse IgG) antibody which can combine with anti-hapten mAbs. Then, the color reaction of the substrate determines whether or not there is a corresponding immune response, and the depth of the color reaction is proportional to the amount of the antibody or antigen.
Direct ELISA begins with the absorption of anti-hapten mAbs onto a plate. A hapten-enzyme conjugate labeled with detectable enzyme is then used as a tracer. Direct ELISA is rarely used for routine hybridoma screenings, because its sensitivity is generally lower compared to indirect ELISA, and individual high-affinity mAbs might be classified as a false negative.
Fig.2 Direct ELISA and indirect ELISA.
Available Services for Hybridoma
Creative Biolabs has extensive experience in developing hybridoma-based antibodies to a wide range of hapten targets. The hybridoma-based antibody shows highly specific and natural affinity maturation. We provide one-stop hybridoma-based antibody production services, including positive hybridoma screening by ELISA.
- Standard Hybridoma Services
- Featured Post-Hybridoma Services
ELISA screening of hybridomas has the characteristics of high sensitivity and strong adaptability. If you are interested in our hybridoma associated services, please do not hesitate to contact us to get more suggestions and scientific support.
- Tian, W.; et al. Antibody production and application for immunoassay development of environmental hormones: a review. Chemical and Biological Technologies in Agriculture. 2018, 5(1): 1-12.