Majid, the pre-bleed specifically gives you a negative control for the mouse.  It will tell you if there is non-specific reactions.  This bleed should not contain antibodies against your antigen.  Bleeds after your immunization contains antibodies against your antigen.  Immunizations are done at 3 week intervals. We collect post-immunization bleeds at 2 weeks after the immunization. When you screen serum samples from a specific mouse you should see an increase in the antibody titre after each immunization and this plateau at approximately 35 days after the first immunization.  We normally determine the titre using an ELISA. Hope this helps.  Just mail me if you require more info.  We do a lot of monoclonal antibody production in my lab.  I fuse the spleen cells of the immunized mouse with SP2 myeloma and grow the hybrids in 10 x 96 well plates.  I screen the plates after 2 weeks and clone positives immediately.  For low immunogenicity antigens I normally do secondary in vitro immunization prior to fusion - this increase my success rate of getting positive clones.

Take a look at the following manuscript

Konduri V1, Decker WK, Halpert MM, Gilbert B, Safdar A. Modeling dendritic cell vaccination for influenza prophylaxis: potential applications for niche populations.J Infect Dis. 2013 Jun 1;207(11):1764-72.

Abstract

BACKGROUND:

Cancer patients can exhibit negligible responses to prophylactic vaccinations, including influenza vaccination. To help address this issue, we developed in vitro and in vivo models of dendritic cell (DC) immunotherapy for the prevention of influenza virus infection.

METHODS:

Human cord blood (CB)-derived or mouse splenocyte-derived DCs were loaded with purified recombinant hemagglutinin (rHA). T-cell responses to HA-loaded CB-derived DCs were determined by ELISpot. Protective efficacy was determined by vaccination of BALB/c mice with a single injection of 10(6) autologous DCs. DC migration to peripheral lymphoid organs was verified by carboxyfluorescein succinimidyl ester staining, and HA-specific antibody titers were determined by enzyme-linked immunosorbent assay. Mice were then challenged intranasally with BALB/c-adapted A/New Caledonia influenza virus derived from four consecutive lung pool passages. Antigen-presenting cell (APC) dysfunction was modeled using the MAFIA transgenic system, in which the Csf1r promoter conditionally drives AP20178-inducible Fas.

RESULTS:

CB-derived human DCs were able to generate de novo T-cell responses against rHA, as determined by a system of rigorous controls. Mice vaccinated intraperitoneally developed HA titers detectable at serum dilutions of >1:1000. HA seroconverters survived virus challenge, whereas unvaccinated controls and vaccinated nonseroconverters lost weight and died. Furthermore, use of a model of APC-specific immunosuppression revealed that DC vaccination could generate HA-specific antibody titers under conditions in which protein vaccination could not.

CONCLUSIONS:

The model demonstrates that DC immunotherapy for the prevention of influenza is feasible, and studies are underway to determine whether populations of immunosuppressed individuals might ultimately benefit from the procedure.

Majid, the pre-bleed specifically gives you a negative control for the mouse.  It will tell you if there is non-specific reactions.  This bleed should not contain antibodies against your antigen.  Bleeds after your immunization contains antibodies against your antigen.  Immunizations are done at 3 week intervals. We collect post-immunization bleeds at 2 weeks after the immunization. When you screen serum samples from a specific mouse you should see an increase in the antibody titre after each immunization and this plateau at approximately 35 days after the first immunization.  We normally determine the titre using an ELISA. Hope this helps.  Just mail me if you require more info.  We do a lot of monoclonal antibody production in my lab.  I fuse the spleen cells of the immunized mouse with SP2 myeloma and grow the hybrids in 10 x 96 well plates.  I screen the plates after 2 weeks and clone positives immediately.  For low immunogenicity antigens I normally do secondary in vitro immunization prior to fusion - this increase my success rate of getting positive clones.

The description of Edmund Pool said it all. The procedure described was my experience in Central Laboratory at Working in England.  Like Edmund said, the pre-bleeding will show  if there are confounding factors and that will guide the design of quality control for the experiment.

I am sorry for my late contribution, however, I would suggest that you try the protocol which I have used in the production of monoclonal antibodies against camel Igs. Have  a look at my list of publication. Good luck..

hi dear 

thanks so much for your response.

dear professor poolوMy questions sent to your email address ([email protected])as you told

thanks

Hi Majid

I did not receive the mail.

Regards

Dear professor pool

Thank you for your tips and let me ask you a question

My PhD thesis is the producing monoclonal antibodies for human cardiac Troponin I and i want to design a rapid test for it.

My major problem is that:

1-       i do not know whether my mice are immunized or not? Because I'm not sure about my Elisa method

               Can i have your Elisa protocol?

2-      If you have some points (key point) about it, tell me please.

3-      What is your methods for injection and immunization of mice?

And i think there are things about it that whatever try, i do not understand (I try for around one year and i can't confirm my mice were immunize exactly or not)

Maybe my antigen is a weak immunogen because it has 90% homology to human troponin.

Is it 10%  of the difference is not enough?

 [email protected]  is your emails?

my email is: [email protected]

Thanks so much

Hi Majid

Sent the following reply to your e.mail address. Hope you saw it. I attach my reply again below:

Question 1- i do not know whether my mice are immunized or not? Because I'm

not sure about my Elisa method

Can i have your Elisa protocol?

Answer to Question 1:

a. I only use Nunc Maxisorb plates for ELISAs. These plates give us good reproducibility.

b. Disolve your antigen (Troponin) to give 1-10 ug/ml in Phosphate buffered saline (PBS). Coat wells at 100 ul/well overnight at 4 C.

c. Decant coating solution. Wash the plate 4 times with PBS containing 0.1 % Tween 20.

d. Block the plate with 200ul/well PBS containing 2 % albumin for 2 hours at room temperature. You can also use skimmed milk for blocking.

e. Tap dry. Apply 100 ul/well diluted mouse antibody. Antibody is diluted in PBS/0.1% Tween 20. We normally assay a whole range of dilutions for both the pre- and post-immunization antisera. We do 10 fold dilutions starting at 1/100. All dilutions are assayed in triplicate. We also include a no antibody control on all plates. Incubate the plate at room temperature for 1 hour.

f. Wash the plate 4 times with PBS containing 0.1 % Tween 20.

g. Add 100 ul/well of anti-mouse antibody conjugated to peroxidase diluted in PBS containing 0.1 % Tween 20(most commercial antibody conjugates must be diluted 1 in 5000). Incubate 1 hour at room temperature.

g. Wash the plate 4 times with PBS containing 0.1 % Tween 20.

h. Add 100 ul per well of TMB substrate. Monitor colour development (Colour goes blue).

i. Stop the reaction with 50 ul/well acid.

j. Read plate at 450nm.

Question 2 was answered above.

Question 3- What is your methods for injection and immunization of mice?

Answer question 3: We immunize mice with Freunds complete adjuvant for the first immunization. We take a pre-bleed before we start immunization. Then we mix 100 ul of a 1mg/ml antigen solution with 100 ul Complete Freunds adjuvant. Mix with a fine needle until you get a stable emulsion. This looks like thick cream. Inject 50 ul of this per mouse. There will be enought for 3 mice.

After 3 weeks we take a second blood sample

We follow this with an immunization using incomplete Freunds adjuvant. We mix 100 ul of a 1mg/ml antigen solution with 100 ul incomplete Freunds adjuvant. Mix with a fine needle until you get a stable emulsion. Inject 50 ul of this per mouse.

We repeat bleeds and immunizations at 3 week intervals with incomplete Freunds untill we get good antibody titres.

Question 4: Maybe my antigen is a weak immunogen because it has 90% homology to human

troponin.

Is it 10% of the difference is not enough?

Answer: A 10 % difference should be fine.

If you have finances for travel and reagents you can come to my lab to learn the techniques.