Hemagglutination, Bacterial Agglutination, Binding, and Encapsulation Assay

The blood of mouse was collected in a centrifuge tube containing sodium citrate. After washing 3 times with TBS buffer, the blood was re-suspended in TBS buffer at a concentration of 2% (v/v), and then transferred to a 96-well type V hemagglutination plate. Four groups were set up (rSpCTL6 with or without 10 mM CaCl₂ groups, TBS with 10 mM CaCl₂ group, and BSA with10 mM CaCl₂ group). rSpCTL6 was added at a series of dilution concentrations (from 0.75 μg/mL to 100 μg/mL). An equal volume of BSA (100 μg/mL) and TBS with 10 mM CaCl₂ were added as control groups. Hemagglutination was observed after incubation at 4°C for 1 h. Each group had three biological parallels. The independent experiment was repeated at least three times.

Gram-positive bacteria (Staphylococcus aureus), and Gram-negative bacteria (Pseudomonas aeruginosa, Pseudomonas fluorescens, Aeromonas hydrophila, Vibrio harveyi, Vibrio fluvialis, V. parahaemolyticus, and V. alginolyticus) were selected to investigate the agglutination activity of rSpCTL6. All the bacteria were cultured to the logarithmic growth phase, washed 3 times with TBS, and re-suspended in 0.1 M NaHCO₃ (pH 9.0). They were then incubated with a final concentration of 0.1 mg/mL FITC (Sigma, USA) for 30 min at room temperature, and washed 3 times with TBS to remove unlabeled FITC. The concentration of the bacteria was adjusted to 10⁸ CFU/mL. Four groups were set up (rSpCTL6 with or without 10 mM CaCl₂ groups, TBS with 10 mM CaCl₂ group, and BSA with10 mM CaCl₂ group). The FITC-labeled bacteria were incubated with 10 μL of rSpCTL6 (40 μg/mL) at room temperature in the dark for 1 h. An equal volume of BSA (40 μg/mL) and TBS with 10 mM CaCl₂ were added as control groups. The agglutination results were observed with an inverted fluorescence microscope (Zeiss, Germany). Each group had three biological parallels. The independent experiment was repeated at least three times.

A modified enzyme-linked immune sorbent assay (ELISA) was carried out as previously described (39). Briefly, LPS, peptidoglycan (PGN), lipoteichoic acid (LTA) and glucan (GLU) were diluted with an ELISA coating solution to a working concentration of 20 μg/mL, and then added to a 96-well ELISA plate. The plate was coated overnight at 4°C, blocked with 5% skim milk at 37°C for 2 h, and then added with a series dilution of rSpCTL6 (0~100 μg/mL) at 37°C for 2 h. There were 3 parallels for each concentration of protein. Mouse anti-His antibody (1:5000, prepared in 1% skim milk) was added and incubated at 37 °C for 2 h followed by incubation with goat anti-mouse HRP antibody (1:5000). TMB solution was added and incubated at 37 °C for 10-30 min. The reaction was stopped with 2 M H₂SO₄. The absorbance at 450 nm was measured using a microplate reader (TECAN GENios, GMI, USA). Each group had three biological parallels. The independent experiment was repeated at least three times.

In order to evaluate the encapsulation activity of rSpCTL6, the Ni-NTA agarose beads [Senhui Microsphere Technology (Suzhou) Co., Ltd, China] was used. Four groups were also set up (rSpCTL6 with or without 10 mM CaCl₂ groups, TBS with 10 mM CaCl₂ group, and BSA with10 mM CaCl₂ group). The beads were first equilibrated in TBS, then incubated with different amounts of rSpCTL6 (25 μg-200 μg) at 4 °C overnight, and washed with TBS 3 times. A 24-well culture plate was pre-coated with 1% agarose. The hemocytes were isolated from S. paramamosain as previously described (35), and added to the plate for at least 10 min to allow them settle down. The beads containing proteins were then added and incubated at 26 °C. Encapsulation was detected after 6 and 24 h under a light microscope (LEICA DMi1, Germany). Each group had three biological parallels. The independent experiment was repeated at least three times.