I recommend dilutions of the post immunization sera from 1:25, 1:50, 1:100, 1:250, 1:500, 1:1000 and 1:2000. In parallel you should use the same dilutions utilizing prä-immune sera or sera of a not immunized mouse. Using an ELISA you should see an reaction up to a 1:100 dilution (minimum). If you have a good immunogene the test should be positive up to 1:2000. If you have access to a flow cytometer I would use your Immunogen-positive and –negative cells and test the recommended dilutions with this approach. Be sure to have immunized at least three times before the fusion!

However, you could also test the amount of IgG in the sera of immunized and non-immunized sera in parallel and detect it with an HRP-coupled goat anti-mouse IgG antibody. IgG in not-immunized sera should definitively be lower that of immunized sera.

If you need further assistance I could offer you my expertise in the fiel of hybridoma making. Just contact me ([email protected]) and I will look at your data.

Thanks, Johannes. But my doubt is how to calculate the antibody titer. I read this: "A blood serum sample containing antibody is diluted serially (1:2, 1:4, 1:8, 1:16... and so on). Using an appropriate detection method (colorimetric, chromatographic etc.), each dilution is tested for the presence of detectable levels of antibody. The assigned titer value is indicative of the last dilution in which the antibody was detected. For example, if antibody was detected in each of the tubes listed above, but not in a 1:32 dilution, the titer is said to be 16. If it is detected in the 1:2 and 1:4 dilutions, but no others, the titer is said to be 4. Therefore the titer is the degree to which the antibody-serum solution can be diluted and still contain detectable amounts of antibody." Is it correct indeed?

Other thing is I am not using cell lysates. I am using cells in 96 well plate, because my protein is expressed at membrane cell. I need the protein in nactive conformation. Did you get it? It is the whole cell ELISA. My negative control is cells without my protein of interest.

I recommend dilutions of the post immunization sera from 1:25, 1:50, 1:100, 1:250, 1:500, 1:1000 and 1:2000. In parallel you should use the same dilutions utilizing prä-immune sera or sera of a not immunized mouse. Using an ELISA you should see an reaction up to a 1:100 dilution (minimum). If you have a good immunogene the test should be positive up to 1:2000. If you have access to a flow cytometer I would use your Immunogen-positive and –negative cells and test the recommended dilutions with this approach. Be sure to have immunized at least three times before the fusion!

However, you could also test the amount of IgG in the sera of immunized and non-immunized sera in parallel and detect it with an HRP-coupled goat anti-mouse IgG antibody. IgG in not-immunized sera should definitively be lower that of immunized sera.

If you need further assistance I could offer you my expertise in the fiel of hybridoma making. Just contact me ([email protected]) and I will look at your data.

Hi , Johannes. I am using a Hek293T cells (adherent cells). But I prepare the plate with PDL. Poly-D-lysine is a nonspecific attachment factor for cells useful in promoting cell adhesion to solid substrates. You can use it in your experiment (see the attached).

Thanks Bernhard. I will contact you by email soon. I really need some help in hybridoma.

Hi, have you tried testing your serum by IFA. I used to do that with whole cells to get monoclonal antibodies against membrane antigens, in paralel by ELISA.. By IFA you can see exactly what is going on. The control should be non imune serum (mice) and another control with just the second antibody. Hope it helped.

Hi Priscila,

If ELISA is easier than IFA, I guess IFA should be the most appropriate way to do it, due to you can recognize a pattern and discard cross reactivity with other cell proteins. In addition to that a negative control assay should be carrying out using a pre immune serum and cell non expressing the antigen. The assay is a little bit more complicated and you need a fluorescence microscope. Anyway, in a near future you will need to test the supernatants too, and in this case you will need ELISA. If your ELISA works I suggest you just choose one criteria and apply always the same. Serial dilutions will be fine.

If you need any extra help fell free to contact me.

Usually you should see an increase in end titer with the repeated booster. With the higher end titer, the chances are more for a positive hybridoma. lalitha kabilan

Plot ODs (or whatever output values you have for your ELISAs) for your dilution series (subtract background, ideally from preimmune serum controls) and find the half-maximum. That is your titer. Half-maximal binding is the most reliable and least subjective measure of titer. BTW, I have produced many hybridomas, including some very commercially successful ones without ever checking titer in the animals (e.g. see below). There are so many other factors contributing to success that knowing the titer is of little concern, so long as you have followed a reasonable immunization protocol. You only need one good positive clone to be successful!

http://www.nature.com/nature/journal/v316/n6031/pdf/316820a0.pdf

http://www.nature.com/ng/journal/v1/n5/pdf/ng0892-321.pdf

Thanks, Norbert. But I can't get this one: http://www.nature.com/nature/journal/v316/n6031/pdf/316820a0.pdf

Could you send the pdf to my, please?

Priscila, it works for me. If you have access to Nature (as you must, if the second PDF file was accessible to you) it should work. Try again. If you still can't get it, let me know how I can get it to you. Unfortunately, I can't post it as part of my contributions on ResearchGate because of copyright issues (ridiculous for such an old paper).

Norbert, unfortunatly I can't get the paper. If is possible, could you sent the paper to me, please? My email is [email protected]

Thanks

I am haven't read all the answers..there are many useful suggestions especially those to do with titration. I am just wondering why you are using whole cells for your elisa to assay a response to a single protein. Isn't it easier at this point to just do an ELISA with the mouse sera and the purified protein? I thought this way, you will eliminate quite a bit of background reaction and it will lead you to a quicker way to screen your hybridomas....Then you could use the cell with or without the protein for your elisa's after you cultuvate your monoclonals to a decent level where you can use hybridoma supernatants. Mouse sera at the stage of screening is very precious (quantitywise) and I would suggest you test it against the purified protein only. My general rule of thumb is to have positive titers of 1/1000 and above, with the background staying negative. Good luck!