Complete freund s adjuvant

Five 6- to 7-week-old female ICR mice (Clea, Tokyo, Japan) were immunized intraperitoneally (i.p.) with 100 μg of purified rBcMSA1-GST or rBcSA1-GST in an equal volume of Freund’s complete adjuvant (Difco Laboratories, USA) for the primary immunization, respectively. Two booster immunizations were given at 14 day intervals by i.p. using 100 μg of the same protein emulsified in Freund’s incomplete adjuvant. Ten days after the last booster shot, the whole blood was collected and serum was harvested and stored at -30 °C.

Peptides were synthesized using reagents and amino acid derivatives produced by Merck (Germany) and Fluka (Switzerland). Immunological studies were carried out with the keyhole limpet hemocyanin (KLH) solution in PBS at a concentration of 5 mg/ml (Sigma–Aldrich, USA), 25% aqueous solution of glutaric dialdehyde (Sigma, USA), Freund’s complete adjuvant, Freund’s incomplete adjuvant (Difco Laboratories, USA), goat antimouse immunoglobulin antibodies conjugated to horseradish peroxidase (Imtek, Russia), and 96-well Maxisorp plates (Nunc, Denmark). Peptides were chosen according to the sequence of the human p75 (TNR16_HUMAN) UniProtKB), and their synthesis was carried out on Wang resin, using the Fmoc/But-scheme. All peptides contained additional C-terminal glycine residue. TBTU/ DIEA method was applied for elongation of peptide chain. Cleavage of peptides from the resin was carried out in a mixture of TFA (94%), triisopropylsilane (1%), 1,2-ethane dithiol (2.5%), and water (2.5%) for 2 h. The peptides were purified by reverse phase HPLC in Phenomenex Jupiter 10 μ C18 300A 250×10 mm in an acetonitrile gradient from 10 to 70% in 0.1% TFA solution. Synthetic peptides were characterized by the data of amino acid analysis, analytical HPLC, and mass spectrometry.

The 8-week-old BALB/cByJ female mice were first immunized with 50 μg of the purified rNP (in 250 μl of PBS) emulsified with equal volume of Freund’s complete adjuvant (Difco Laboratories, BD, Franklin Lakes, NJ) by intraperitoneal injection. Mice were then injected on a weekly basis for two more weeks using 50 μg of rNP emulsified with Freund’s incomplete adjuvant (Difco Laboratories). Mice were injected a fourth time without adding the adjuvant, then sacrificed 3 days after the last injection to harvest splenocytes. The splenocytes were fused with Spll/0-ag/14 myeloma as described previously [38 (link)]. Hybridoma cells secreting anti-rNP antibodies in cultural media were screened by indirect ELISA using the crude leaf sap of N. benthamiana infected with TZSV-13YV639 as the antigen. Subsequently, the selected antibody-secreting hybridoma cells were cloned by limiting dilution method. Antibodies were further produced in ascitic fluids by intraperitoneal injection of 10⁶ hybridoma cells into Pristane-primed BALB/cByJ female mice.

All experiments were related to the day post-immunization (DPI). At day 0 DPI, the rats were subjected to anesthesia with an intraperitoneal injection of a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine (Vetoquinol, Biowet). Next, 100 µl of the immunization mixture was injected subcutaneously at the lower left and right back of the animal (100 µl at each site). The mixture consisted of Freund’s Complete Adjuvant (DIFCO Laboratories) with 50% guinea pig spinal cord homogenate in phosphate-buffered saline (PBS) (Merck Millipore), 1:1, supplemented with Mycobacterium tuberculosis (DIFCO Laboratories), 1 mg/ml of the mixture.
The bodyweight of the animals was measured daily or every second day and clinical symptoms were evaluated starting on 10 DPI, using a 5-grade scale where 0—no symptoms, 1—limp tail, 2—hind limb paresis, 3—incontinence, paraplegia, 4—quadriplegia, 5—death. The number of days with clinical symptoms was counted.
The mean score for the evaluated clinical symptoms was counted. The body mass ratio (mass at the day of euthanasia/mass at day 0) was calculated. The percentage of days with clinical symptoms observed from 10 DPI was counted. The mean score, the body mass ratio, and the percentage of days with clinical symptoms observed were presented graphically.

Mice were injected subcutaneously at two sites on the femoral region with 200 μl of a mixture of MOG _35–55 peptide emulsified 1:1 with Freund’s complete adjuvant (Difco Laboratories, Detroit, MI, USA). MOG treated mice were also boosted with pertussis toxin (List Laboratories, Campbell, CA, USA; 200 ng) I.P. on both the day of injection and also 48 h later. Control mice were injected with equal volumes of CFA and given saline instead of pertussis toxin. Tissues were harvested at days 10, 20, 30 and 60 post injection after the animals had been fasted for 4 h before being anesthetized with Avertin (2,2,2-tribromoethanol, 32 mg; Aldrich, Milwaukee, WI, USA).