Development and production of antibodies against gamma inactivated pathogenic bacterial spores

Sporulation of B. anthracis and characterization of spores

The supplied bacterial stock was cultured on typical nutrient agar plates prepared with 10 g/L biological peptone, 3 g/L beef extract, 5 g/L NaCl, and 15 g/L agar and incubated at 37°C overnight to obtain a single colony. Subsequently, a single colony was grown in Tryptic Soy Broth (TSB). The bacterial suspension achieved from single colony cultured in sporulation media prepared with 0.5 % peptone, 0.3 % beef extract, 0.5 % NaCl, and 1.5 % agar, which had relatively reduced nutritional properties, and was incubated for 48 h at 28 °C. Then, plates were incubated at room temperature for 24 h. Visible spore colonies on the plates were collected and suspended in dH₂O. Suspension was incubated for 48 h at room temperature to remove forms that had not transformed into spores. After 48 h, the spores were incubated at 60 °C for 1 h and washed thrice with cold 1x phosphate buffer saline (PBS). Centrifugation was done at 7000 rpm for 10 min.

To control the quality of the spores before inactivation, 50 µL of the spore suspension was spread on a glass slide and allowed to dry. Then, malachite green dye (Sigma, cat no: 2437-29-8) was added and held over the fire with forceps. The dye was heated until steam came out without boiling. As the dye evaporated, it was added again, and this process was repeated three times. Then, the coverslip was washed with dH₂O. Safranin dye (Sigma, cat no: 477-73-6) was added and left for 1 min. After washing abundantly with dH₂O, it was dried, and a coverslip was covered by dropping immersion oil and examined under a microscope.

Inactivation of spores by gamma irradiation

The spore suspension was washed three times with cold 1×PBS before irradiation, and counting was performed using the culturing method [25]. This method created stocks of different concentrations by serial dilution of the spore suspension. Spores at each concentration were cultured three times repetitively on nutrient agar plates, and the stock was determined by calculating over two consecutive concentrations. Twenty milliliters of spore samples were divided into falcon tubes to be 10⁷ spores/mL and irradiated with five different gamma doses: 5, 10, 15, 20, and 30 kGy (Nordion JS9600, GammaPak Sterilization Company, Tekirdağ, Türkiye). Thus, inactivation control was performed with the long-term culturing method, and deformation control of the antibody-binding surface of the spore was performed with the indirect ELISA test by using commercially obtained anti-B. anthracis spore polyclonal antibody (NB110-2502 Novus Biologicals, USA).

Monoclonal antibody (mAb) production by hybridoma technology and cross-reactivity-test

10⁶ pure inactivated B. anthracis spores were mixed 1:1 with Freund’s complete adjuvant (Sigma, cat no: F5881) and injected subcutaneously into 6–8-week-old BALB/c mice in a volume of 200 μL. The following injections were performed with Freund’s incomplete adjuvant (Sigma, cat no: F5506) once a month, and one mouse was kept non-immunized as a control. Mice displaying a high immune response were selected for the fusion study. Spleen cells and F0 myeloma cells (American Type Culture Collection, USA) were combined at a ratio of 3:1 through fusion. After the 10-day incubation period, the hybrids were screened under a microscope, and the initial ELISA screening against the spores was performed. After all limited dilutions and screening ELISA tests, we generated a hybridoma cell line that produced monoclonal antibodies with high affinity against spores. A cross-reactivity test was performed as the last step to measure the specificity of the MAM02 hybrid. For this purpose, Bacillus cereus, Bacillus subtilis, and Bacillus thuringiensis bacteria, closely related strains of B. anthracis, were obtained, sporulated and an indirect ELISA test was applied.

mAb purification and characterization

The subisotype of the MAM02 was determined to choose which chromatographic method must be applied to purification. The commercially obtained kit (BD Pharmingen Mouse Immunoglobulin Isotyping ELISA kit) has been used for this. A high volume of culture supernatant was collected to purify the MAM02 cell line. After increasing the concentration through ammonium sulfate precipitation and desalting through dialysis, the protein mixture containing the target antibody was subjected to affinity chromatography on a Hitrap Protein A column (Cytiva, cat no: 17040201) using the NGC (Next Generation Chromatography, BIORAD) device by using the recommended buffers by Cytiva. The purity of the obtained antibody fractions was demonstrated using SDS-PAGE gel electrophoresis followed by silver staining. The SDS-PAGE has been performed by following the instructions of BIORAD Mini-PROTEAN Tetra Cell System. The silver staining procedure was applied by following the specified protocol [26]. Additionally, the affinity of the fractions to the antigen was confirmed through indirect ELISA testing. The fraction containing antibodies was purified through dialysis, and characterization processes were completed.

Rabbit polyclonal antibody production

New Zealand rabbits were immunized with inactivated spores at 10⁷ spores/mL once a month, and antibody titration in blood serum was routinely checked using the indirect ELISA method. After reaching proper antibody titration, a high volume of blood serum was collected. Purification and characterization procedures were carried out by following the same protocol with mAb purification [27].

Horseradish peroxidase (HRP) – anti-BAs pAb conjugation

1 mg anti-BAs pAb antibody was conjugated with Horseradish peroxidase (HRP) (Roche, cat no: 11089153001). This process was carried out by following the relevant protocol steps [28]. As different from the protocol followed, Vivaspin 500 columns (Sartorius, cat no: VS0172) were used instead of dialysis procedures. After conjugation, the usage concentration of HRP-labeled antibody was tested by direct ELISA.

Indirect ELISA test

In the indirect ELISA test to detect the inactive spore, 10⁶ spores/100 µL were coated on the bottom of the 96 well plates in 1×PBS solution and incubated at 37 °C overnight. The wells were blocked with 1.5 % skim milk powder for 1 h at 37 °C, and an anti-spore polyclonal antibody commercially obtained was used as recommended at a ratio of 1:1000. After using HRP-labeled anti-rabbit antibody (Sigma, A0545) at a ratio of 1:2000, TMB (3,3′,5,5′-Tetramethylbenzidine) substrate was added, and absorbance was measured at a wavelength of 450 nm by using plate reader (BIOTEK, Synergy HT). The wells were washed three times with 1×PBST after each step and five times before the substrate by microplate washer (BIOTEK, 405 LS) [29]. This method was applied to control the interaction of the epitopic regions of the inactivated spore with the antibody, to control the response of the developed antibody to spores of other Bacillus species, and to all stages of the antibody development process.

Spore detection by sandwich ELISA assay

To be a primitive study on the use of the produced antibodies in diagnostic tests for spore detection, a sandwich ELISA assay was performed. For this purpose, anti-BAs MAM02 was coated as a capture antibody at +4 °C. After washing with 1×PBST and blocking with 1.5 % skim milk powder, different concentrations of inactive spores were used as intermediate antigens, and HRP labeled anti-BAs pAb was used as the detection antibody. After the TMB substrate was added, absorbance was measured at a wavelength of 450 nm [30].

Statistical analysis

Statistical analysis was performed using one-way and two-way ANOVA tests, and data are presented as mean±standard error. Data with a p-value less than 0.05 were considered statistically significant. GraphPad Prism 5 was used to visualize data.

Preparation of spores, staining, and inactivation

The supplied B. anthracis bacteria were sporulated, and 40 mL of spores were obtained at a concentration of 2.5×10¹⁵ spores/mL. Spores were stained with malachite green for characterization, and the remaining vegetative bacteria were stained with safranin. Spores were stained green and vegetative bacteria stained red (Figure 1A). The spores were radiated with 5, 10, 15, 20, and 30 kGy gamma irradiation doses respectively. As a result of inactivation control, it was observed that spore colonies did not occurred at any dose except for spores exposed to 5 and 10 kGy irradiation after incubation. An indirect ELISA test was performed, and it was determined that surface antigenic regions of the spores exposed to 15 kGy gamma dose best reacted with spore specific polyclonal antibody (Figure 1B). In another ELISA study, the antibody responses of spores inactivated with 15 kGy were tested at doses of 10³–10⁹ spores/well, and 10⁶ spores/well were selected as the coating concentration for future assays (Figure 1C).

Figure 1:

Examination of staining and antigenic properties of gamma irradiated spore samples by ELISA. (A) Staining with malachite green, the green dots present spore forms, and the vegetative forms stained with safranin dye are present as red chains. (B) The gamma irradiation doses that spores absorb, active Bacillus anthracis spores (aBAs) are non-inactivated spores. (C) Usage concentration of spores exposed to 15 kGy gamma irradiation. **Means both active BAs and gamma inactivated BAs show the same antigenic properties at the 10⁶ spores.

After obtaining inactive spores that retained their antigenic properties, they were used to develop antibodies.

Production of monoclonal antibodies

After immunizations periodically, indirect ELISA was performed to check the antibody titration, and after fifth immunization, second mouse was boosted intraperitoneally with a half dose of the spores (Figure 2A). 10 days after cell fusion, limited dilutions, subcultured hybridomas, and with continuous screening ELISA tests, it has been shown that one of the hybridoma preserved its affinity for spores (Figure 2B). The monoclonal antibody cell line named as anti-BAs MAM02 has been obtained. To determine the specificity of the anti-BAs MAM02, a cross ELISA test was performed against B. cereus, B. subtilis, and B. thuringiensis spores. The results showed that the produced antibody did not recognize any other spore and was specific to its antigen (Figure 2C).

Figure 2:

Immunizations of mice, immune response stability, and cross ELISA tests of candidate monoclonal antibody. (A) The immune responses of 5 mice after immunizations. The no-immune mouse is kept as a control group. p<0.05. (B) The antibody response stability of MAM02 cell line. (C) The cross-reaction of MAM02 cell line with other species of Bacillus group. p<0.0001.

After being developed and ensured that it does not respond to any related strain spores, characterization studies have begun for the use of the anti-BAs MAM02 antibody in immunological tests.

Purification and characterization of anti-BAs MAM02

The subisotype of the anti-BAs MAM02 was determined as IgG1, Kappa by commercially obtained ELISA kit (Figure 3A). As binding buffer 3 M NaCl, 1.5 M Glycine pH:8 and as elution buffer 0.1 M Glycine pH:2.7 were used for purification, and affinity chromatography was performed on the HiTrap protein A column using the NGC device. The purity was determined using SDS-PAGE gel electrophoresis followed by silver staining, and the affinity was confirmed through an ELISA test (Figure 3B). Following these controls, called anti-BAs MAM02, was obtained at a concentration of 2.2 mg/mL. The affinity of anti-BAs MAM02 at different concentrations to the antigen was evaluated, and it was shown that it can recognize even at 0.25 μg/mL concentration (Figure 3C).

Figure 3:

Characterization of anti-BAs MAM02. (A) IgG1 heavy chain, Kappa light chain determination of sub isotypes produced anti-BAs MAM02 p<0.0001. (B) The silver staining gel of the fractions containing purified antibodies. S, sample fraction; SW, sample wash fractions; E, elution fractions; EW, elution wash fraction. (C) The immune response of anti-BAs MAM02 at different concentrations against 10⁶ spores/well p<0.001.

The anti-BAs MAM02 antibody with a very high affinity for its antigen has been successfully developed and characterized.

Production of polyclonal antibody and HRP conjugation

Antibody titration of immunized New Zealand rabbit was checked by indirect ELISA and a high volume of blood serum was collected after the third immunization (Figure 4A). All the characterization steps, including cross-ELISA tests, were carried out (data not presented). As a result, anti-BAs pAb was obtained at a concentration of 2.4 mg/mL. The usage of HRP labeled anti-BAs pAb was checked at 1:250, 1:500, 1:1000, 1:2000, and 1:4000 ratios against two different spore concentrations, 10⁵ and 10⁶ spores/well. When the results were evaluated, a 1:1000 ratio was chosen (Figure 4B).

Figure 4:

Development of pAb against inactivated spores and its conjugation with HRP. (A) The antibody responses of the immunized rabbit sera at different immunization periods. (B) Choosing the proper concentrations of the HRP labeled anti-BAs pAb.

The anti-BAs pAb antibody has been developed successfully and conjugated with HRP. The studies have planned to use HRP-labeled anti-BAs pAb antibodies at the selected concentration as a couple with anti-BAs MAM02.

Inactive spore detection by sandwich ELISA as a preliminary diagnostic assay

To determine the potential usage in immunologic assays, both produced antibodies were tested as both capture and detection antibodies at different concentrations in the sandwich ELISA test (data not presented). In conclusion, it was shown that inactive spore detection could be achieved at different concentrations ranging from 10⁷ to 5×10⁴ spores/well, in the combination of which the 400 ng/well anti-BAs MAM02 was the capture antibody and at a ratio 1:1000 HRP labeled anti-BAs pAb was the detection antibody (Figure 5).

Figure 5:

Detection of inactive spores at different concentrations (10⁷−5×10⁴ spores/well) by generated anti-BAs MAM02 and anti-spore rabbit polyclonal antibody couple. p<0.0001.