Preparation of Monoclonal Antibody for Akabane Disease and Establishment of c-ELISA Antibody Detection Method

doi:10.7668/hbnxb.20193929

Abstract

Abstract:

To establish a competitive ELISA method for the rapid detection of antibodies to Akabane disease virus. Balb/C mice were immunized with AKV virus,and their splenocytes were immunofused with SP2/0 cells to obtain monoclonal antibodies against AKV. Using the SBV N protein expressed by Bac-to-Bac baculovirus as a diagnostic specific antigen,and goat anti-murine HRP-IgG as a secondary antibody,a competitive ELISA method for AKV antibody detection was established and optimized. A hybridoma cell line that could secrete monoclonal antibody AKV nucleoprotein antibody was obtained with continuous and stable reproduction,and the monoclonal antibody subtype was identified as:heavy chain IgG1 and light chain kappa,which could only react positively with AKV virus,and did not react specifically with viral antigens such as BTV,FAMD,and EHDV virus.The established detection method for AKV antibody ELISA showed that the optimal coating concentration of diagnostic antigen was 0.5 μg/mL,1∶1 000 antibody dilution ratio,1∶50 serum dilution ratio,1∶2 000 secondary antibody dilution ratio,5% BSA,37 ℃ blocking for 2 h,and it was determined that the serum suppression rate was positive when it was greater than or equal to 44%,and it was negative when it was less than or equal to 44%. The established ELISA method sensitivity and specificity identification results are consistent with the ID Screen AKV Competition assay.A hybridoma cell line secreting AKV N protein was successfully prepared,and the established ELISA detection method can be used to detect AKV antibodies in animals,which lays a foundation for further research and industrialization of AKV antibody diagnostic reagents.

Key words: Akabane disease, Monoclonal antibodies, Competive ELISA

HU Shixiang, YE Lingling, XIAO Yan, ZHUO Na, WANG Tengfen, WANG Lin, PU Jing, CHEN Peifu, AI Jun. Preparation of Monoclonal Antibody for Akabane Disease and Establishment of c-ELISA Antibody Detection Method[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(S1): 452-456. doi: 10.7668/hbnxb.20193929.

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Figures/Tables 6

References 21

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OD₄₅₀吸光值 Light absorbance of 450 nm	1	2	3	4	5	6	7	8	9	10	11	12
A	0.759 5	0.257 1	0.516 8	0.458 6	0.470 3	0.519 1	0.553 8	0.533 5	0.571 8	0.476 1	0.493 8	0.431 5
B	0.434 8	0.505 5	0.678 8	0.358 3	0.517 9	0.428 5	0.436 8	0.426 5	0.560 9	0.558 9	0.322 7	0.545 9
C	0.328 1	0.488 1	0.486 1	0.582 4	0.477 1	0.524 2	0.451 9	0.430 7	0.382 8	0.484 2	0.465 6	0.582 5
D	0.527 2	0.460 0	0.417 0	0.351 0	0.585 5	0.622 2	0.631 2	0.526 5	0.494 4	0.360 2	0.540 6	0.491 0
E	0.378 6	0.562 4	0.519 0	0.459 8	0.662 2	0.430 1	0.451 8	0.435 3	0.499 1	0.545 1	0.631 6	0.527 3
F	0.475 4	0.435 0	0.451 6	0.789 5	0.436 6	0.626 8	0.525 3	0.545 5	0.675 5	0.434 5	0.565 3	0.420 4
G	0.329 1	0.647 8	0.548 5	0.445 7	0.662 3	0.418 1	0.420 9	0.631 9	0.506 3	0.478 4	0.428 4	0.443 3
H	0.672 3	0.488 9	0.498 1	0.575 2	0.576 5	0.518 2	0.544 5	0.434 3	0.566 3	0.473 5	0.461 9	0.485 3

OD₄₅₀吸光值 Light absorbance of 450 nm	1	2	3	4	5	6	7	8	9	10	11	12
A	0.759 5	0.257 1	0.516 8	0.458 6	0.470 3	0.519 1	0.553 8	0.533 5	0.571 8	0.476 1	0.493 8	0.431 5
B	0.434 8	0.505 5	0.678 8	0.358 3	0.517 9	0.428 5	0.436 8	0.426 5	0.560 9	0.558 9	0.322 7	0.545 9
C	0.328 1	0.488 1	0.486 1	0.582 4	0.477 1	0.524 2	0.451 9	0.430 7	0.382 8	0.484 2	0.465 6	0.582 5
D	0.527 2	0.460 0	0.417 0	0.351 0	0.585 5	0.622 2	0.631 2	0.526 5	0.494 4	0.360 2	0.540 6	0.491 0
E	0.378 6	0.562 4	0.519 0	0.459 8	0.662 2	0.430 1	0.451 8	0.435 3	0.499 1	0.545 1	0.631 6	0.527 3
F	0.475 4	0.435 0	0.451 6	0.789 5	0.436 6	0.626 8	0.525 3	0.545 5	0.675 5	0.434 5	0.565 3	0.420 4
G	0.329 1	0.647 8	0.548 5	0.445 7	0.662 3	0.418 1	0.420 9	0.631 9	0.506 3	0.478 4	0.428 4	0.443 3
H	0.672 3	0.488 9	0.498 1	0.575 2	0.576 5	0.518 2	0.544 5	0.434 3	0.566 3	0.473 5	0.461 9	0.485 3

包被的抗原 Coated antigens	OD₄₅₀
赤羽病病毒AKV	0.42	0.78	0.68	0.39	0.73	0.52
幼年仓鼠贤细胞BHK cell	0.17	0.09	0.16	0.14	0.20	0.15

包被的抗原 Coated antigens	OD₄₅₀
赤羽病病毒AKV	0.42	0.78	0.68	0.39	0.73	0.52
幼年仓鼠贤细胞BHK cell	0.17	0.09	0.16	0.14	0.20	0.15

包被的抗原 Coated antigens	OD₄₅₀
赤羽病病毒AKV	0.77	0.75	0.79
幼年仓鼠贤细胞BHK cell	0.11	0.13	0.15

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