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6. Rapid primary screening process:
ELISA can be done. Here, adsorbtion of antigen to the bottom of 96-well plates, incubation for growth of hybridomas occur. The desired antibody in the sample remains bound to antigen and is detected by an immune-conjugate. Immune-conjugate is consists of two components, one antibody is distinct for an epitope that remains in the constant domain in the first antibody. It acts as anti-antibody. Second one is alkaline phosphatase enzyme. Immune-conjugate is retained in the well during the immobilization at first incubation of antibody. After washing colorless substrate of enzyme (ex. p-nitrophenyl phosphate) is converted to a colored product (ex. p-nitrophenol) by the enzyme (ex. alkaline phosphatase). After incubation and the termination of enzyme function, ELISA reader quantify the optical density of product.(Kulkarni, 2002)

Adapted from: ELISA Guide – Creative Diagnostics (no date). Available at: https://www.creative-diagnostics.com/ELISA-guide.htm (Accessed: 4 November 2018).

Figure: Screening process by ELISA (Enzyme-linked immunosorbent assay)

7. Cloning:
After screening, cloning can be done by three different techniques (cloning by technique of limiting dilutions, cloning using semi-solid agars and cloning and selection using the fluorescence-activated cell sorter). The limiting dilution method allows the enumeration of cells in the culture, dilution and fractional adsorption into new wells in which each well is consist of only one cell. Then, cell regeneration process is repeated to assure presence of monoclonal property. Another method is soft agar method that allows the proliferation of enormous malignant cells in a low agar content semi-solid medium. The dispersion of culture into single cells and the presence of spaced colonies due to such cell concentration ensure the presence of monoclonal antibody. Both techniques are used by combining these methods.(Hurrell, 2018)

Adapted from: Cloning Method. EuroMAbNet (no date). Available at: https://www.euromabnet.com/protocols/cloning.php (Accessed: 4 November 2018).

Figure: Cloning process by technique of limiting dilutions

8. Cryopreservation:
It is essential against the loss of beneficial lines. Hybridoma should be froze down as soon as possible to reduce the loss of chromosome.(Hurrell, 2018)

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