Forgive me if I am misinterpreting your question. I assume your cells are growing in serum or some other protein-containing medium. It seems like it might be doable if you rinse a population of cells free of protein by several centrifugations in serum free medium and then re-culture them in serum-free medium. Samples of the medium could be taken at different times for your protein assay. You could determine protein (antibody) production in time as a function of cell number. Cells that do not produce any antibody could be used as a control.

Thank you very much for your response Sir Don Kaiser .

The medium used in this experiment is RPMI 1640 supplemented with 10% FBS. serum-free medium is not available in my lab right now. and due to financial limitation, I can not perform direct quantification of the antibody concentration by ELISA or HPLC. For that, I am asking about anyway to explain this result from total protein concentration.

Tel now, I explained my result by incorporating the interference of the residual FBS and the host cell proteins (HCPs). for example, while the cells consumed most of the FBS during the early growth phases, the total protein concentration continues to be approximately at the same level. this could indicates that cells produced IgG antibodies in addition to the HCPs.

sorry for too long explanation.... please give me your opinion about my explanation.

Thank you very much

A simple precipitation with ammonium sulphate followed by a dialysis could allow you to palliate the absence of HPLC and Ig specific ELISA test, followed by a colorimetric assay, BCA for example. In any case it is impossible to evaluate the production of antibodies by the method you are using. The culture supernatant contains 100 times more proteins (serum, BSA, cellular proteins, metabolic residues, etc.) than a hybridoma can produce in antibodies. The ammonium sulphate precipitation is very reliable but only if you use a serum-depleted and BSA-free culture medium. Another alternative is filtration / concentration on Vivaspin, 300k for IgMs (very reliable), 100k for IgGs, followed by colorimetric assay of the concentrate.

Thank you very much Jean-Jacques Pin

Based on my graph, can we say at least that there is an evidence of antibody production by my cells?? Jean-Jacques Pin

Dear Karim I am sorry to tell you that it is impossible to evaluate the amount of antibodies by your assay method on a crude extract of culture supernatant. The amount of antibody produced is much too low compared to the amount of total protein contained in this culture supernatant. The simplest method is a capture ELISA, using an antibody directed against the heavy chain as a sensor and then an antibody coupled to the HRPO directed against the light chain as a developer. This makes it possible to precisely quantify the secreted Ig, to be certain that the antibody produced is dimeric, and in addition this test is not very expensive to develop.

I will try to find a solution through your recommendations.

Thank you very much Dear Jean-Jacques Pin .