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Protocols

Immunization

Immunization is necessary to present an antigen in a suitable form to induce the most vigorous humoural immune response to an animal. This essential step will result in the production of cells secreting antibody against your chosen antigen

Antigen concentrations

Mice - 50-100 µg per injection except for the final boost when 100-200 µg should be used.

Rats - 50-100 µg per injection except for the final boost when 100-200 µg should be used.

Immunization Schedule

Day one
Day Ten to Fifteen
Day Twenty to Thirty
Day Thirty or Forty

N.B. Healthy and well-cared-for animals are essential to obtain good antibody responses.

It is absolutely essential that the correct training according to the regulations of the relevant country be completed satisfactorily for any procedures involving animals. In additional to training, injecting and bleeding animals safely and painlessly requires skill and patience.

Appendix A

Intraperitoneal injection

The concentration of the antigen should be between 50-100 μg per mouse.  The volume to be injected should be no more than 300 μl per mouse.

The method below is for two mice:

Appendix B

Subcutaneous injection

The concentration of the antigen should be between 50-100 μg per mouse.  The volume to be injected should be no more than 300 μl.

The method below is for 2 mice:

Appendix C

The concentration of the antigen should be between 50-100 μg per mouse and diluted in sterile PBS only. NO adjuvant should be used.

The volume to be injected should be no more than 300 μl per mouse.

Intravenous injection

N.B. Be careful of the length of time the mouse is left under the lamp. If the temperature is too hot for your hand, then it is too hot for the mouse.

Appendix D

Adjuvants

The two main adjuvants that we use are Freund’s adjuvant (complete or incomplete) (BD Difco 263810 and BD Difco 263910) and Titremax gold (Sigma T2684).

Both are extremely good at producing an immune response.

Freund’s Adjuvant
Complete Freund’s Adjuvant (CFA)

CFA is a mineral oil containing a suspension of whole or pulverized heat-killed mycobacteria. When emulsified together with a solution of the antigen of interest to form a water-in-oil emulsion, CFA is effective in potentiating both cellular and humoral immune responses to the injected antigen. Its adjuvant activity is a result of the sustained release of antigens from the oily emulsion and stimulation of a local innate immune response resulting in enhanced adaptive immunity. An essential component of this response is an intense inflammatory reaction at the site of antigen deposition, resulting from an influx of leukocytes and their interaction with the antigens. 

Incomplete Freund’s Adjuvant (IFA)

For most applications, CFA is usually only necessary for the initial immunization, while Incomplete Freund's Adjuvant (IFA), which lacks mycobacteria, is the adjuvant of choice for subsequent immunizations.

Titremax gold

TitreMax Gold is a new and improved water-in-oil adjuvant. TitreMax Gold Adjuvant contains three essential ingredients: a block copolymer (CRL-8300), squalene (a metabolizable oil), and a unique microparticulate stabilizer. It has been especially formulated with squalene to produce stable water-in-oil emulsions. It is considerably easier to emulsify than Freund's adjuvant. The resulting emulsion is less viscous, making it easy to inject through small needles. TitreMax Gold appears to be less toxic and just as effective as other adjuvants.