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
- Dilute the antigen in 150 μl-200 µl of sterile PBS for each animal.
- Mix the antigen with 150 μl-200 µl of the adjuvant (see Appendix D).
- Inject two BALB/c female mice intraperitoneally, if you work in Europe, or subcutaneously if you work within the UK (see Appendix A).
Day Ten to Fifteen
- Repeat the injection.
Day Twenty to Thirty
- The injection this time should contain antigen in sterile PBS only (no adjuvant). This injection can be administered intraperitoneally or subcutaneously, whether you work in Europe or the UK.
Day Thirty or Forty
- Collect a tail bleed from the immunised mice.
- Allow the blood to clot.
- Make serial dilutions of the resulting serum in PBS.
- Test the mouse serum samples in an ELISA against the antigen and/or another staining technique. The results should be compared with those obtained using similar dilution of normal mouse serum. This step will enable you to check if the animals have recognized the antigen and identify which mouse has the best immune response.
- Leave at least 3 weeks between the third injection and the final antigen boost before performing the fusion.
- Administer the final boost intravenously (see Appendix C) or intraperitoneally three or four days before the fusion day.
- This final boost can contain between 100-200 μg of antigen diluted in 150 μl of sterile PBS.
- Three to four days after the final boost you should sacrifice the mouse, remove the spleen aseptically and place it in a sterile container containing 5 ml medium.
- Proceed with the fusion technique.
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:
- The antigen should be diluted in approximately in 300 μl of PBS and mixed with 300μl of adjuvant in an eppendorf tube.
- Vortex the eppendorf to resuspend the adjuvant and to form an emulsion between the aqueous antigen and oil-based adjuvant. The solution should be uniformly white at this stage with no separation into two layers.
- Draw the solution into 1.0 ml syringe.
- Place a 23 or 25 gauge needle on the syringe.
- Displace any air bubbles present in the syringe barrel or in the needle by carefully depressing the plunger until the emulsion reaches the needle.
- Hold the mouse securely using one hand and using the other hand carefully inject the antigen-adjuvant emulsion into the peritoneal cavity.
- N.B. All adjuvants are potentially harmful. Be careful during antigen preparation and injection.
- Return the animal to its cage and match for five minutes to ensure that all is well.
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:
- The antigen should be diluted in approximately 300 μl of PBS and mixed with 300μl of adjuvant in an eppendorf tube.
- Vortex the eppendorf to resuspend the adjuvant and to form an emulsion between the aqueous antigen and oil-based adjuvant. The solution should be uniformly white at this stage with no separation into two layers.
- Draw the solution into 1.0 ml syringe.
- Place a 23 or 25 gauge needle onto the syringe.
- Displace any air in the syringe barrel or in the needle by carefully depressing the plunger until the emulsion reaches the needle.
- Hold the mouse securely using one hand and using the other hand carefully inject the antigen-adjuvant emulsion between the skin and the peritoneal cavity, do not go through into the peritoneal cavity.
- B.B. All adjuvants are potentially harmful. Be careful during antigen preparation and injection.
- Return the animal to its cage and observe for five minutes to ensure that all is well.
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
- Isolate the mouse in a small cage or container.
- Heat the mouse with an infrared lamp. This will increase the blood supply to the tail, enlarging the veins and making them easier to locate and inject. The veins of the tail should be easily visible.
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.
- Swab a portion of the tail about 1.5 cm below the tail base with alcohol.
- Displace any air in the syringe barrel or in the needle by carefully depressing the plunger until the emulsion reaches the needle. N.B. This step is essential to prevent a fatal air embolism occurring in the mouse.
- Place a 26 or 27 gauge needle onto a 1.0 ml syringe.
- Hold the tail firmly with one hand and guide the needle into one of the veins.
- Ensure that the needle is inside the vein, slowly deliver the injection.
- Pause a few seconds.
- Remove the needle and return the mouse to its cage.
- Observe the mouse for five minutes to ensure that all is well.
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.