Application of BRV-R mAbs to detection of corresponding receptors

Abstract

Key Words: retavirus, antibodies, mono-clonal, IgG

INTRODUCTION

Rotavirus is a major pathogen of acute gastroenteritis in human infants and young animals. It has been the second cause of infant death especially in developing countries. The infections sometimes occur in adults too. We have used the BRV-R mAb to study the BRV and the BRV-R on MA104 cell surface in three aspects: (1) the conjugation test of the BRV-R mAb and rabbit anti-BRV IgG; (2) the a nti-infectious action of the BRV-R mAb; and (3) the immuno-spot test of the BRV-R mAb.

MATERIALS AND METHODS

Conjugation test of BRV-R mAb and anti-BRV IgG

Preparation of rabbit anti-BRV IgG Virosomes were extracted from the BRV (NCDV strain) suspension concentrated by PEG using ultracentrifugation and observed under electron microscope. Using the virus (10⁷/ mL), we prepared the rabbit anti-BRV im-mune serum. Antibody titre was determined by the complement fixation test. The rabbit anti-BRV IgG was extracted by salting-out method and DE52 chro-matographic analysis. Antibody protein was 7.2 g/L measured by Lowry method and was pre-served at -20 °C.

Preparation of ascities BRV-R mAb The concention-al procedure adopted in our lab was used.

Conjugation test of BRV-R mAb and rabbit anti-HBV IgG ELISA was employed for the detection. The main procedures were: The 96-pore polythylene plate (America Costor) coated with rabbit anti-BRV IGG (130 mg/L) was incubated for 24 h at 4 °C, washed and enclosed. Its horizontal rows were added with doubling diluted a scitic type BRV-RmAb. The first spot of each row was added with di-luted fluid as blank control, and the SP_2/0 ascities was used as a negative control. After that, the plate was incubated, washed and added with goat anti-mouse IgG-HRP. It was then put in substrate fluid for color reaction Bio-RAD (America) 2550 type enzyme linked immune measurer was used to detect A (the OD value, λ = 492). The titre of BRV-R mAb was determined and the IgG of BRV-R mAb was extracted by DE52.

Anti-infectious action of BRV-R mAb

MA104 strain culture and BRV (NCDV strain) suspension preparation were completed using our own procedure. We set up five control groups: (1) the normal MA104 strain; (2) BRV; (3) rabbit anti-BRV IgG; (4) BRV-R mAb; (5) fluid combined with the rabbit anti-BRV IgG and BRV. Test groups: (1) MA104 strain tube was first incubated with BRV-R mAb for 30 min at 37 °C and then added with BRV (NCDV strain) suspension. It was supplemented with maintenance media after 30 min at 37 °C; (2) The rabbit anti-BRV IgG and BRV-R mAb were quantitatively mixed, incubated for 30 min at 37 °C, and then put in the MA104 strain tube. After another 30 min at 37 °C, it was added to BRV (NCDV strain) suspension. Maintenance media was added in the tube after the third 30 min at 37 °C. The cells of the control and test groups were cultured at 37 °C and CPE was observed.

Immuno-spot test of BRV-R

Preparation for BRV-R After routinely cultured, amplified, digested, collected and washed with PBS, the MA104 strain cells were suspended in the 0.01 mol/L PB (pH 7.0), swollen thoroughly at 4 °C and mechanically splitted. Supernatant was collected after centrifugation 1000 ×g for 5 min and discarded after immersing cell membrane-via-cen-trifugation 200000 ×g for 1 h. Add 3 g/L sodium deoxycholate-Tris chlorhydric acid and incubate for 40 min at 4 °C. After centrifugation 100000 ×g for 45 min, supernatant was taken, which is BRV-R, and preserved at -20 °C.

Immuno-spot test After drying at room temperature, BRV-R was dropped on the pyroxylin mem-brane (British product) with a total dose of 30 μm, and then put in the 10% ovi albumin-0.1 mol/L glucine-Tris chlorhydric acid buffer solution for 1 h at 37 °C for enclosing. After washing, it was placed into the BRV-R mAb preparation for 1 h at 37 °C, rewashed and added with the goat anti-mouse IgG-HRP conjugate for 1 h at 37 °C. Substrate solution was added for color reaction and observed with naked eye.

RESULTS

The conjugation test of BRV-R mAb and rabbit anti-BRV IgG

The positive result was judged if the OD value of the sample was 0.1 higher than that of the negative control determined by ELISA. The conjugation titre of the ascitic type BRV-R mAb and rabbit anti-BRV IgG was 1:5120 and when the BRV-R mAb was extracted by DE52 chromatographic analysis it was 1: 2560.

Anti-infectious action of BRV-R mAb

The BRV was inoculated into the MA104 strain cells. The CPE of each group was observed at dif-ferent time points every day. The result is shown in Table 1.

Table 1 The anti-infectious action of the BRV-R mAb.

Groups	CPE at different time point (h)
	24	48	72	96
Control group 1	-	-	-	-
Control group 2	+	++	+++	++++
Control group 3	-	-	-	-
Control group 4	-	-	-	-
Control group 5	-	-	-	-
Test group 1	-	-	-	+/-
Test group 2	+	++	+++	++++

Immuno-spot test

The result indicated that the BRV-RmAb can com-bine with the BRV-R, and colored brown, while the control groups showed no color.

DISCUSSION

The conjugation reaction was produced by the rabbit anti-BRV IgG and BRV-R mAb when detecting the plate coated with rabbit anti-BRV IgG using ELISA. The cytoprotection test indicated that the BRV-R mAb was able to prevent the corresponding sensitive cell strain MA104 from being infected by the BRV. We inferred that the BRV-R mAb and BRV shared the correlative antigen determinants, which were related to the BRV-R on the cell surface. The immuno-spot test demonstrated that the antigenicity of the BRV-R on the bovine enteric mucosal cell surface was correlated with that of the BRV-R on the MA104 strain cell surface. To a certain extent, it has laid a basis for purifying the BRV-R on the MA104 strain cells by affinity chromatography, studing its property, and searching the correlative receptors on different tissues and cell surface in vivo utilizing labelled BRV-R mAb.