Preparation of Avian leukosis virus p27 Protein Polyclonal Antibody and Estab lishment of POCT Detection Method

doi:10.7668/hbnxb.20192997

Abstract

Abstract:

It aimed to develop a polyclonal antibody to Avian leukosis virus (ALV)and to establish a simple and accurate method for the detection of ALV.pGEX-6p-1-p27 and pET-32a-p27 protein vectors were constructed by amplifying the target gene of ALV p27.The proteins were immunized in BALB/c mice and New Zealand white rabbits,and mouse-derived polyclonal antibodies and rabbit-derived polyclonal antibodies were prepared.The mouse-derived polyclonal antibody coupled fluorescent microspheres were prepared,and the mouse-derived polyclonal antibody IgG was sprayed onto the sample pads using a three-dimensional spray point platform;the purified rabbit-derived polyclonal antibody IgG and goat anti-rabbit antibody dilution were scribed onto the NC membrane using a scribing instrument as T and C lines for the assembly of test strips,and a preliminary Point-of-care testing(POCT)immunochromatographic assay was established.The test strips were assembled and a preliminary point-of-care testing(POCT)method was developed for the detection of ALV by fluorescent microsphere immunochromatography.The results showed that the prokaryotic expression vectors for pGEX-6p-1-p27 and pET-32a-p27 were successfully constructed and the expression of p27 recombinant protein was induced.After mixing the purified p27 fusion protein with the adjuvant as an immunogen immune BALB/c mouse and a New Zealand white rabbit,a mouse-derived and rabbit-derived polyclonal antibody of the p27 protein was successfully prepared,and the immunogen response of the multi-antibody was shown to be good by serum potency and Western Blot identification.The optimal pH of the fluorescent microsphere-coupled murine polyclonal antibody was 6.2.POCT strips showed that positive samples for ALV bound well to the fluorescent microsphere-coated murine polyclonal antibody,with higher T/C data than other samples,and that positive samples bound to the fluorescent microsphere-coated polyclonal antibody showed fluorescent bands in the C line(quality control line)and T line(detection line)under UV light,which the results were consistent with clinical diagnostic results.It can be concluded that the POCT fluorescent microsphere immunochromatographic detection method for ALV established provides a simpler method for the detection of Avian leukosis virus and facilitates clinical detection for primary veterinarians.

Key words: Avian leukosis virus, p27, Prokaryotic expression, Polyclonal antibody, Fluorescent microsphere immunochromatography

SHAO Ying, HUANG Yan, YANG Yan, GONG Liufei, SONG Xiangjun, TU Jian, QI Kezong. Preparation of Avian leukosis virus p27 Protein Polyclonal Antibody and Estab lishment of POCT Detection Method[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(6): 201-209. doi: 10.7668/hbnxb.20192997.

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References 21

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血清稀释度 Serum dilution		包被抗原稀释度Dilution of coated antigen
血清稀释度 Serum dilution		1∶100	1∶200	1∶400	1∶800	1∶1 600	1∶3 200
1∶100	+	2.53	1.83	1.70	2.33	2.21	2.55
	-	0.06	0.06	0.07	0.06	0.05	0.06
	P/N	41.29	30.53	25.86	40.92	42.18	40.62
1∶200	+	2.17	2.11	2.14	1.95	1.91	2.43
	-	0.06	0.06	0.06	0.05	0.05	0.07
	P/N	38.08	38.07	38.27	38.31	38.65	33.68
1∶400	+	3.04	2.26	2.45	2.62	3.30	3.25
	-	0.05	0.06	0.05	0.06	0.06	0.06
	P/N	56.43	40.89	45.01	46.23	59.62	52.14
1∶800	+	2.14	1.83	2.34	2.20	2.50	2.98
	-	0.08	0.05	0.05	0.06	0.05	0.06
	P/N	28.18	34.22	44.62	37.88	50.32	48.19

血清稀释度 Serum dilution		包被抗原稀释度Dilution of coated antigen
血清稀释度 Serum dilution		1∶100	1∶200	1∶400	1∶800	1∶1 600	1∶3 200
1∶100	+	2.53	1.83	1.70	2.33	2.21	2.55
	-	0.06	0.06	0.07	0.06	0.05	0.06
	P/N	41.29	30.53	25.86	40.92	42.18	40.62
1∶200	+	2.17	2.11	2.14	1.95	1.91	2.43
	-	0.06	0.06	0.06	0.05	0.05	0.07
	P/N	38.08	38.07	38.27	38.31	38.65	33.68
1∶400	+	3.04	2.26	2.45	2.62	3.30	3.25
	-	0.05	0.06	0.05	0.06	0.06	0.06
	P/N	56.43	40.89	45.01	46.23	59.62	52.14
1∶800	+	2.14	1.83	2.34	2.20	2.50	2.98
	-	0.08	0.05	0.05	0.06	0.05	0.06
	P/N	28.18	34.22	44.62	37.88	50.32	48.19

血清稀释度 Serum dilution	阳性对照 (P)OD值 Positive control (P) OD value	阴性对照(N) OD值 Negative control (N) OD value	P/N
1∶400	3.715	0.057	65.35
1∶800	3.172	0.047	66.98
1∶1 600	2.780	0.047	59.15
1∶3 200	2.197	0.048	46.19
1∶6 400	2.287	0.045	50.64
1∶12 800	2.105	0.046	45.31
1∶25 600	2.020	0.050	40.19
1∶51 200	1.770	0.048	37.22
1∶102 400	1.527	0.049	30.94
1∶204 800	1.086	0.049	22.27
1∶409 600	0.408	0.048	8.53
1∶819 200	0.272	0.047	5.81
1∶1 638 400	0.055	0.049	1.10

血清稀释度 Serum dilution	阳性对照 (P)OD值 Positive control (P) OD value	阴性对照(N) OD值 Negative control (N) OD value	P/N
1∶400	3.715	0.057	65.35
1∶800	3.172	0.047	66.98
1∶1 600	2.780	0.047	59.15
1∶3 200	2.197	0.048	46.19
1∶6 400	2.287	0.045	50.64
1∶12 800	2.105	0.046	45.31
1∶25 600	2.020	0.050	40.19
1∶51 200	1.770	0.048	37.22
1∶102 400	1.527	0.049	30.94
1∶204 800	1.086	0.049	22.27
1∶409 600	0.408	0.048	8.53
1∶819 200	0.272	0.047	5.81
1∶1 638 400	0.055	0.049	1.10

血清稀释度 Serum dilution		包被抗原稀释度Dilution of coated antigen
血清稀释度 Serum dilution		1∶100	1∶200	1∶400	1∶800	1∶1 600	1∶3 200
1∶100	+	2.43	2.42	2.48	2.96	2.90	2.36
	-	0.21	0.22	0.23	0.24	0.20	0.19
	P/N	11.73	11.25	10.67	12.35	14.64	12.16
1∶200	+	2.30	2.78	2.73	2.89	3.12	2.29
	-	0.12	0.13	0.13	0.14	0.11	0.12
	P/N	18.53	22.06	21.80	20.39	29.35	19.77
1∶400	+	2.13	2.79	2.60	3.22	3.00	2.88
	-	0.10	0.09	0.09	0.10	0.08	0.08
	P/N	21.13	30.16	27.91	32.82	36.47	33.92
1∶800	+	2.65	2.76	2.63	3.26	3.08	2.01
	-	0.08	0.08	0.09	0.08	0.08	0.07
	P/N	31.32	36.59	30.24	41.36	40.21	27.04

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