Two Balb/c mice (6-8 weeks old) were immunized with purified human ferritin ⁽¹⁷⁾. Briefly, each mouse was immunized 5 times with 50 µg of ferritin every 2 weeks. The first immunization was performed using complete Freund's adjuvant. Incomplete Freund's adjuvant was used for subsequent immunizations. One week after the last immunization, blood was collected by a vertical incision of the tail vein followed by determination of antibody titers by ELISA. Finally, three days before the cell fusion, 20 µg of ferritin (without any adjuvant) were injected intravenously ⁽¹⁸⁾.

Mouse serum titrations and screening of hybridoma supernatants were performed by Enzyme-linked immunosorbent assay (ELISA). The wells of ELISA plate (Nunc, Roskilde, Denmark) were coated with 50 µl of ferritin (10 µg/ml) dissolved in Phosphate Buffered Saline (PBS) and then incubated at 37°C for 1 hr followed by overnight incubation at 4°C. Then the plates were washed 3 times with PBS containing 0.05% Tween 20 (PBS-T) for 3 min. followed by blocking with 2.5% bovine serum albumin (BSA) at 37°C for 1 hr. Wells were then washed 3 times and mouse sera (in two-fold serial dilutions starting from 1:500) were added and incubated for 1 hr and wells were again washed with PBS-T. Rabbit anti mouse Ig conjugated to horseradish peroxides (1:1000) (Avicenna Research Institute, Tehran, Iran) were added to the wells and incubated for 1 hr at 37°C. After washing, 50 µl of tetramethylbenzidine (TMB) (Sigma-Aldrich, Missouri, USA) substrate was added to each well and the plates were incubated at room temperature in the dark. After 15 min, the reaction stopped by adding 15 µl of stop solution (20% H₂SO₄) to each well. The Optical Density (OD) of the reactions was measured at 450 nm by an ELISA reader (BioTek, Winooski, VT, USA). The mouse with higher titer of antibody was selected for fusion. To screen the antibody production of hybridoma cells, the same method was done on the cell supernatants.

Mouse myeloma Sp2/0 cells, used as fusion partners, were cultured and propagated in RPMI-1640 culture medium (Gibco, Gran Island, NY, USA) and 10% Fetal Bovine Serum (FBS) (GIBCO Invitrogen, USA). Spleen cells from the immunized mouse were mixed with the Sp2/0 cells at a ratio of 1:5 (1 Sp2/0 and 5 spleen cells). The mixture was washed twice with pre-warmed RPMI-1640 (37°C). Then, pre-warmed 50% polyethylene glycol (PEG) 1500 (Sigma-Aldrich, Germany) was used for fusion. Selective HAT medium (Sigma-Aldrich, Germany) was then used for selection of hybridoma cells. The reactivity of culture supernatants was then tested by ELISA ⁽¹⁸⁾. Finally, positive hybridomas were cloned by limiting dilution process ⁽¹⁹⁾.

Anti-ferritin mAb 2F9-C9 was purified from culture supernatants by affinity chromatography using a Hi-Trap protein G column (GE Healthcare, Uppsala, Sweden). Briefly, culture supernatants were filtered through 0.45 µm filters and pH was adjusted to 7.5. The elution was performed using Glycine-HCl (0.1 M, pH = 2.7). The eluted antibody was dialyzed against PBS at pH = 7.5 and the reactivity of the purified antibody was determined by ELISA method as mentioned above.

Goat anti mouse IgG₁, IgG2a, IgG2b, IgG3, IgA and IgM (Sigma-Aldrich, Missouri, USA) at 1/1000 dilution were coated in the wells of ELISA plate (Nunc). Supernatant of the growing hybridoma 2F9-C9 was added to each well. The isotype of 2F9-C9 mAb was then determined according to the ELISA method described above.

To determine the reactivity of 2F9-C9 with free ferritin and ferritin in human sera, 2F9-C9 mAb (50 µl/well, 5 µg/ml) was coated in a 96 well plate and then, different dilutions of ferritin from 0 to 250 ng/ml as well as human sera (1:5 dilution) were added (50 µl/well). HRP-conjugated anti-ferritin polyclonal antibody (Avicenna Research Institute, Tehran, Iran) was then added. After washing, TMB was added as substrate and the test continued as described in ELISA section. For comparison, 1:5 diluted human sera were also tested by a commercial ferritin kit (RADIM, Florence, Calenzano, Italy).

Hela (Human cervical carcinoma) cells were harvested by 0.5% trypsin and 0.1% EDTA (Gibco) and permeabilized by permeabilizing solution (Becton Dickinson, USA). Cells were then blocked with 5% sheep serum for 10 min, and then cells were incubated with 100 µl of 2F9-C9 mAb (10 µg/ml) for 1.5 hr at 4°C. Anti-HIV Env (Avicenna Research Institute, Tehran, Iran) with IgG1/K isotype was used as an isotype control ⁽²⁰⁾. After 3 times of washing with cold PBS, FITC-conjugated sheep anti-mouse Ig (Avicenna Research Institute, Tehran, Iran) (1:50) was added to cells and incubated for 45 min in the dark at 4°C. Cells were washed and sorted by flow cytometry. The data was analyzed using Flowmax software (Partec, Nuremberg, Germany).

Lysates of the cell lines Raji (human bur-kitt's lymphoma), PC3 (human prostate cancer), K-562 (human myelogenous leukemia), human and mouse liver lysates (lysis buffer: 150 mM NaCl, 1 mM EDTA, 50 mM Tris HCl pH = 7.4, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS), supplemented PI (phosphatase inhibitor) (Roche, Basel, Switzerland) and 1% PIC (protease inhibitor cocktail) (Sigma-Aldrich, Missouri, USA) were prepared. The protein concentrations of the lysates were measured by BCA protein assay kit (Thermo Scientific, Rockford, IL. USA). Twenty µg of cell lysates and 200 ng purified ferritin were run on a 15% SDS–PAGE gel.

After electrophoresis, resolved proteins were transferred onto PVDF membranes (Millipore Corporation, Billerica, Mass chusetts, USA). The membranes were blocked with 5% non-fat milk in PBS-T overnight at 4°C. After gentle washing with PBS-T, 2F9-C9 mAb (5 µg/ml) was added to the membrane and incubated for 1.5 hr at room temperature. The membrane was washed extensively with PBS-T and incubated with HRP-conjugated Rabbit anti-mouse Ig (Avicenna Research Institute, Tehran, Iran) (1:2500) for 1 hr at room temperature followed by washing and developing with ECL Chemiluminescence detection system (GE Healthcare).

For validation of protein band specificity detected in Western blot, reactivity of anti-ferritin mAb was blocked with a saturating concentration of ferritin (30:1 ferritin to antibody molar ratio). In this regard, ferritin was added to 2F9-C9 mAb for 1 hr at 37°C and then the mixture was added to PVDF membrane. Unblocked anti-ferritin mAb was added to another PVDF as a positive control. The incubations, washings and development of bands were performed as the above ⁽²¹⁾.

The affinity constant (K_aff) of 2F9-C9 mAb was determined by ELISA ^{(21, 22)}. Briefly, different concentrations of ferritin (5000, 2500, 1250, 625, 312.5, 156, 78 and 39 ng/ml) were coated in 96 well ELISA plates. Serial dilutions of 2F9-C9 mAb (5000, 1250, 312.5, 78, and 19.5 ng/ml) were added to each coated well. Sigmoid curves were plotted using the OD values obtained for different concentrations of mAb. The affinity constant was measured as described elsewhere ⁽²³⁾.

After immunization of mice with ferritin, the titers of anti-ferritin antibodies in the mice sera were measured by ELISA. Results showed that mouse 1 had higher titer of anti-ferritin Ab (Figure 1). After fusion between splenocytes of mouse 1 and Sp2/0 cells, supernatants of growing hybridoma cells were screened based on reactivity with ferritin by ELISA. In this screening assay, among 20 positive clones, a clone was characterized as 2F9-C9 with high reactivity with ferritin (OD: 1.438). The isotype of 2F9-C9 mAb was determined to be IgG2a (Figure 2) and its affinity was calculated to be 2.34×10⁹ M ^-1 (Figure 3) and (Table 1).

Known concentrations of ferritin were used to plot ELISA standard curve for 2F9-C9 mAb (Figure 4A). ELISA standard curve for the commercial ferritin measurement kit was also plotted according to the manufacturer's instructions (Figure 4B). As shown in Table 2, the concentrations of ferritin in 5 human sera which were measured with our ELISA test using 2F9-C9 mAb and the commercial kit were found to be very similar.

Since Hela cell line expresses ferritin intracellularly, 2F9-C9 mAb was applied for intracellular staining of ferritin in this cell line. Figure 5A shows that 2F9-C9 mAb recognized intracellular ferritin molecules in 79.4% of Hela cells, while the ferritin negative HepG2 cell line showed no ferritin expression by flow cytometry using 2F9-C9 (Figure 5B).

Western blot analysis demonstrated that 2F9-C9 mAb recognized ferritin as an approximately 20 kDa protein band in human liver, mouse liver and K-562 cells (Figure 6A). Additionally, blocking of the antibody with human ferritin resulted in abrogation of its reacting, hence no band was detected in western blot (Figure 6B).