Mouse melanoma (B16F10) and breast (4T1) cancer cell lines were purchased from the Pasture Institute. The cells were cultured in RPMI-1640 medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) with a humidity of 70% and 5.0% CO2 as previously mentioned (
Rabbits and mice were used in the current study, which were purchased from the Pasture Institute. A total of 6 female Balb/c, inbred, 2-month-old mice were used to prepare spleen cells. For this purpose, the mice were euthanized by intraperitoneal injection of 250 mg/kg body weight pentobarbital (Sigma, 3636 under sterile conditions and their spleens removed. Subsequently, the spleen cells were extracted, counted and their viability checked by using trypan blue staining. To prepare cells, spleens were removed from scarified mice and transferred to a Petri Dish containing Isotonic saline. Spleens were then minced using a Scalpel blade and the mixture was passed through a four-layered gauze to remove large debris. Isotonic saline containing cells were then washed twice with isotonic saline and centrifuged at 600 x g for 2 min at room temperature. Subsequently, cells were further suspended in isotonic saline. To stain cells with Τrypan blue (Merck, 50 µl prepared cells was mixed with 50 µl Trypan blue stain at room temperature. Following 15 min, one drop of mixed cells with trypan blue was applied to a Neubauer slide (HBG, Germany) and counted using light microscope (magnification, x400).
The animals were maintained in an appropriate animal research facility and mice kept in groups of six per cage (rabbits one/cage) and fed with clean food and water. The rabbits were housed at 20-25˚C with a humidity of 70-80% with a 12 h light/dark cycle. The animal research protocols of the present study were approved by Isfahan University of Medical Sciences Ethics Committee with approval number IR.mui.REC.1394.824.
Volumes of 1 ml containing 2 mg of the different parasite antigens and sonicated breast and melanoma cancer cells were emulsified in the same volume (1 ml for each antigen) of Freund's adjuvant (Sigma-Aldrich; Merck KGaA) at room temperature and each antigen injected subcutaneously into a male 4-month-old, white New Zealand rabbit (n=5 in total). Injections were repeated fortnightly. In all rabbits, complete Freund's adjuvant was used for the first injections and incomplete Freund's adjuvant for the boosters. Following the third booster, 1 ml blood samples from the ear vein of each rabbit were collected. To prepare sera, the blood samples were centrifuged at 3,000 x g for 5 min at room temperature. The sera were then checked for the presence of specific antibodies using home-made ELISA tests as described in our previous study (
For ELISA, 96 wells plates were coated with the antigens (
Mouse cancer cells were harvested from culture medium and normal lymphocytes obtained from normal mouse spleens. All the cells were washed with PBS and incubated for 1hour at 37˚C with different antisera (1:100) which raised in rabbits against different mentioned antigens, namely anti-
The experiments were repeated three times and data are presented as the mean ± standard deviation. A linear mixed model test was used for statistical analysis. In comparison of means, P<0.05 was considered to indicate significance.
The reaction of antiserum against
The reaction of antisera against
The reaction of anti-
Results of the present work indicated that anti-
Reaction of anti-
The effect elicited by cross-reaction of antibodies raised against
In previous years there has been considerable focus on enhancing antibody activity through conjugation with cytotoxic drugs (
One of the main problems in the treatment of cancer is that most of the drugs used to target cancer cells are also cytotoxic to normal tissues. Attempts have been made to overcome this problem by coupling anticancer drugs to antibodies which have some degree of specificity for cancer antigens. In this regard there are numerous patents regarding use of antibodies for drug delivery. For instance, in a patented method by Sahin
Through further study it may be possible to conjugate humanized purified anti-
In conclusion, in the present study it was indicated that anti-
The present study was prepared from thesis by a PhD student (Mrs Mahshid Shakibapour) and an MSc student (Miss Fereshteh Mohamadi) from Isfahan University of Medical Sciences.
The present work was supported by a grant (grant no. 3953009) from Isfahan University of Medical Sciences (Isfahan, Iran).
The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.
FM and MS performed the experiments. SS performed the experiments and wrote the first draft of the manuscript. AA consulted immunohistochemical procedures. ST assisted with experiments. HD supervised experiments and prepared final version of the manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Reaction of antisera against mouse melanoma cancer cells, normal rabbit serum and
Reaction of antisera against breast cancer cells, normal rabbit serum and
Reaction of normal rabbit serum and antisera against
Results of flow cytometry analysis (M2 cell percentage of total cells) indicating the level of reaction of different antisera with the surface of mouse melanoma cancer cells (B16F10) and mouse normal spleen cells.
Anti-melanoma (B16F10) antiserum | Anti- |
Normal rabbit serum | Background fluorescence | |
---|---|---|---|---|
B16F10 | 10.28±0.49 | 11.14±1.48 |
1.91±0.31 | 1.11 |
Spleen cell | - | 9.32±0.21 | 7.79±0.79 | 3.18 |
aP<0.05 B16F10 cells vs. spleen cells.
Results of flow cytometry analysis (M2 cell percentage of total cells) indicating the level of reaction of different antisera with the surface of mouse breast cancer cells (4T1) and mouse normal spleen cells.
Anti-4T1 antiserum | Anti- |
Normal rabbit serum | Background fluorescence | |
---|---|---|---|---|
4T1 | 62.14±7.97 | 81.77±22.2 |
2.06±0.69 | 0.17 |
Spleen cells | - | 7.79±0.79 | 9.32±0.21 | 3.18 |
aP<0.05 vs. spleen cells.
Results of flow cytometry analysis (M2 cell percentage of total cells) indicating reaction of antisera against Trichomonas vaginalis and hydatid cyst crude protoscolices antigen with the surface of breast cancer cells, melanoma cancer cells or normal mouse spleen lymphocytes.
Anti- |
Anti-hydatid cyst protoscolices antiserum | Background florescence intensity | |
---|---|---|---|
Melanoma cancer cells | 3.74±0.74 | 2.11±0.35 | 1.11±0.14 |
Breast cancer cells | 5.75±1.23 | 3.64±0.82 | 0.17±0.09 |
Normal mouse spleen lymphocytes | 11.23±2.39 | 9.43±1.97 | 3.18±0.68 |