Conglutinin was purified according to the method developed by Krogh-Meibom et al. [9] with some modifications. Bovine serum derived from Holstein cattle was batch-incubated with TSK-gel beads (Toyopearl HW-75F; Tosoh Bioscience, Japan) for 2.5 h at 37℃ in a glass flask. This enabled deposition of iC3b on the beads and subsequent binding of conglutinin to iC3b. The serum-treated TSK-gel beads were packed under flow in a fast protein liquid chromatography (FPLC) system; (BioRad, USA), washed with Tris base saline [(TBS)-Ca-1M NaCl; (10 mM Tris, 140 mM NaCl, pH 7.4 containing 5 mM CaCl₂ and a total of 1 M NaCl] and eluted with TBS-EDTA (TBS with 5 mM EDTA). In the second step of purification, the EDTA eluate was applied to an ion exchange column (Macro-Prep High-Q Support; Bio-Rad, USA) and the proteins retained were separated by running a linear gradient of NaCl. Chromatographically purified conglutinin was analysed by SDS-PAGE [10] and Western blotting [17] using a monoclonal antibody against bovine conglutinin (mAb 263-01; Abcam, UK). diluted 1 : 1,000 and secondary polyclonal peroxidase-labelled rabbit anti-mouse IgG or goat anti-rabbit IgG diluted 1 : 5,000. To induce a colour reaction, 1.4 chloronaphthol was used as a substrate.

The binding of conglutinin to mannan-coated wells was measured by an ELISA using anti-bovine-conglutinin monoclonal antibodies. Microtiter plates (MaxiSorp; Nunc, USA) were coated with different concentrations (0.37~100 µg/mL, 50 mL/well) of mannan (Sigma-Aldrich, USA) or N-acetyl-D-glucosamine (Sigma-Alrich, USA) in coating buffer (100 mM Na₂CO₃/NaHCO₃, pH 9.6) overnight at 4℃. The plates were then blocked with TBS (10 mM Tris, 140 mM NaCl, pH 7.4) containing 3% bovine serum albumin (Sigma-Aldrich, USA) for 2 h at room temperature. Next, the plates were washed with TBS-Ca (TBS with 5 mM CaCl₂) containing 0.05% Tween-20 (TBS-Ca-Tween) and incubated for 2 h at room temperature with conglutinin (50 mL/well) diluted (final concentration 50 µg/mL) with TBS-Ca containing 1% BSA (TBS-Ca-1% BSA). After washing with TBS-Ca-Tween, monoclonal anti-conglutinin antibodies (Abcam, UK) diluted in TBS-Ca-1% BSA (1 : 1,000) were added to the wells (50 µL/well). The plates were incubated overnight at 4℃, washed with TBS-Ca-Tween, and then incubated with rabbit anti-mouse Ig conjugated with horseradish peroxidase (HRP, 50 µL/well; Jackson Immuno Research, UK) diluted in TBS-Ca-1% BSA (1 : 13,000). Following 2 h of incubation, the plates were washed with TBS-Ca. Enzyme activity of HRP was detected using a 3,3', 5,5'-tetramethylbenzidine) peroxidase ELISA substrate kit (Bio-Rad, USA) according to the producer's recommendations. The reaction was read on a microplate reader (Bio-Rad, USA) at 655 nm.

Blood was collected via jugular venipuncture from four clinically healthy Holstein cows aged 2~3 years and in late lactation. PMNs were isolated from heparin-treated blood by hypotonic lysis as previously described [19]. Cells were centrifuged at 400 × g for 10 min at 4℃, washed, and resuspended in Hanks balanced salt solution (HBSS) without phenol red (Sigma-Aldrich, Poland). The leukocyte preparation consisted of 80~90% granulocytes as determined by hematological staining using staining kit (Hemacolor; Merck Chemicals, Germany) according to the procedure recommended by the producer. Cell viability exceeded 95% based on trypan blue staining.

The assay was performed according to the method described by Pick [16]. PMNs (8 × 10⁶ cells/mL) transferred to conglutinin-coated or non-coated wells (50 µL/well) of 96-well microplates were covered with 50 µL/well of nitroblue tetrazolium (NBT; Sigma-Aldrich, USA) solution (2 mg/mL) in phenol red-free HBSS. The wells received 5 µL of conglutinin (non-coated plate, final concentrations ranged from 0.125 to 5 µg/mL) or 5 µL of PMA (final concentration 200 ng/mL, positive control; Sigma-Aldrich, USA), or 5 µL of HBSS. Wells without conglutinin served as the negative control. Reference wells (blank) contained cells suspended in NBT (1 mg/mL) with iodoacetamide (10 mM). The plates were incubated at 37℃ in 5% CO₂ for 60 min. The amount of formazan was measured with a microplate reader (Bio-Rad, USA) at 550 nm. The results are expressed as the optical density per well after subtracting the values for the blank.

The rate of H₂O₂ production was determined by peroxidase-dependent oxidation of phenol red as described by Pick [16]. A PMN suspension (8 × 10⁶ cells/mL) was added to conglutinin-coated or non-coated wells (50 µL/well) of a microplate. The cells were covered with 50 µL/well of assay solution, which was prepared on the day of the experiment and consisted of HBSS, phenol red (final concentration 0.2 g/L; Sigma-Aldrich, USA), and HRP (final concentration 20 U/mL; Sigma-Aldrich, USA). The wells received 5 µL of conglutinin (non-coated plate, final concentrations ranged from 0.125 to 5 µg/mL), 5 µL of PMA (final concentration 200 ng/mL, positive control), or 5 µL of HBSS. The plate was incubated at 37℃ in 5% CO₂ for 90 min. The reaction was then stopped by adding 10 µL/well of 1 N NaOH. After a 3-min equilibration interval, the plate was read at 620 nm by a microplate reader (Bio-Rad, USA). The amount of H₂O₂ in each well was calculated according to a standard curve and expressed as µM H₂O₂/90 min/4 × 10⁵ cells.

Data obtained from the NBT and H₂O₂ assays were analysed with Statistica (ver. 9.0; StatSoft, USA). The aim of the statistical analysis was to ascertain the effects of conglutinin (mannan-bound and unbound) on the generation of superoxide radicals and H₂O₂ by bovine granulocytes. Significance of differences between the control and conglutinin-treated cells were evaluated by a one-way ANOVA using the Dunnett test for post-hoc comparisons. Prior to the analysis, data not falling within a normal distribution (Shapiro-Wilk test at the 95% confidence interval) were transformed using the Box-Cox method. Homogeneity of variance was verified by the Brown-Forsythe test. As Box-Cox transformed data were not normally distributed or if date failed Brown-Forsythe test, we applied the Kruskal-Wallis test to perform a nonparametric analysis of variance followed by rank-based multiple comparisons. The results are presented as the mean ± SD of four independent experiments. p-values below 0.05 were considered significant.

Conglutinin purified using a two-step chromatographic method was analysed by SDS-PAGE under non-reducing conditions and Western blotting using monoclonal antibodies. Comparative characterization of the conglutinin protein profiles in the gel and on PVDV membranes indicated a high level of purity for the protein obtained. In the Western blot, this appeared as a ladder composed of many bands with molecular weights ranging from 34 to 630 kDa (Fig. 1).

Conglutinin was observed to bind more strongly to mannan than N-acetyl-D-glucosamine (Table 1). The highest value was observed for 25 µg/mL of mannan, so this concentration of mannan was selected for further experiments using microplates coated with conglutinin.

To determine whether conglutinin affects superoxide anion generation in bovine PMNs, we performed an NBT microplate assay. Inside the cells water-soluble, yellow NBT dye is rapidly converted into insoluble dark blue monoformazan by two O₂^- molecules. Monoformazan concentration (and therefore the concentration of O₂^-) is calculated based on spectrophotometric measurements at 550 nm [21].

Mannan-bound conglutinin was shown to stimulate oxidation processes in the PNMs. The average absorbance of the sample with conglutinin was 0.227 ± 0.062 and the difference in absorbance compared to the control (wells without mannan and conglutinin) was statistically significant. The mean absorbance of the samples with PMA (standard receptor-independent stimulus of ROIs) was 0.208 ± 0.017, similar to that of the sample with conglutinin (Fig. 2).

In the other variant of the experiment, conglutinin at concentrations ranging from 0.125 to 5 µg/mL was added directly to a suspension of PMNs. The average absorbance of the samples containing different concentrations of conglutinin was lower than the absorbance of the control samples. The inhibitory effect of conglutinin on PMNs metabolism depended on the concentration of the protein; the higher the concentration of protein, the stronger the inhibitory effect. Statistically significant differences were observed for all PMN samples treated with conglutinin at concentrations greater than 2 µg/mL (Fig. 3).

The phenol red colorimetric method was used to assess the effect of conglutinin on H₂O₂ production by bovine PMNs. The dye undergoes oxidation in the presence of H₂O₂ released from the cells. The concentration of H₂O₂ is directly proportional to the absorbance of wells.

The results showed that mannan-bound conglutinin in the microplate wells stimulated the production of H₂O₂ by the PMNs. The amount of H₂O₂ in the sample treated with conglutinin was more than twice as high (51.78 µM ± 12.8) as in the control (23.3 µM ± 11.0). PMA proved to be a very strong stimulator of H₂O₂ production; the average concentration of H₂O₂ produced by cells treated with this reagent was 130.9 µM ± 27.04 (Fig. 4).

In the other variant of the experiment, different concentrations of conglutinin (0.125~5 µg/mL) were added to cells suspended in HBSS with phenol red. An inhibitory effect on the production of H₂O₂ by PMNs was observed in the presence of all concentrations of conglutinin. However, statistically significant differences were observed at concentrations between 0.5 to 5 µg/mL (Fig. 5).