This is the final step in hybridoma method and the purpose of this step is to collect the end product that was obtained by fusion method and also known as biological cloning.
In order to obtain hybridoma clones limiting dilution cloning is used and the basis of this method is poisson distribution. It provides the superiority than other methods because of its simplicity and the equipments that used are not so costly after all.
To start this step firstly the cells that were put into the 96 well plates are made to be grown into fresh small groups. After this, sorting for the antigen attachment were done or it can be further cloned by mouse ascites method. The use of “limiting dilution” guarantees that each of the wells is accompanied solely by a single clone. There are some complications too that the resulting antibodies might show toxicity. In order to prevent such phenomenon mouse ascites expansion method can be applied. What Ishaque and Al-Rubeai quoted in 1998 that “Also, it is the experience of many that a brief period of growth by the mouse ascites method produces cell lines that at later in vitro and in vivo stages show enhanced hardiness and optimal antibody production.”