Viruses and cells
All the viruses used in this study are shown in Table 1. All the viruses were obtained from the virus repository in our laboratory [20, 36–39]. AIVs were stored at −80 °C and propagated at 37 °C using 10-day old chicken embryos, as described previously [20]. Influenza B virus, avian paramyxovirus 4 (APMV-4), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), and infectious bronchitis virus (IBV) were also acquired from our laboratory virus repository. All viruses were determined using hemagglutinin (HA) and tissue culture infectious dose 50 (TCID50) assays according to standard methods [40].
Table 1
The specificity and sensitivity of antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and immunochromatographic test (ICT) strip methods against different viruses.
Virus
|
Subtype
|
HA
|
AC-ELISA
|
Strip
|
|
|
titer
|
OD value
|
Test Limitation
(HA titer)
|
Result
|
Test Limitation
(HA titer)
|
A/duck /Zhejiang/D4/2018
|
H9N2
|
26
|
1.095
|
22
|
+
|
22
|
A/chicken/Zhejiang/1026138/2016
|
H9N2
|
25
|
0.986
|
22
|
+
|
23
|
A/chicken/Zhejiang/13163/2016
|
H9N2
|
26
|
1.254
|
23
|
+ a
|
23
|
A/chicken/Zhejiang/221/2016
|
H9N2
|
27
|
1.311
|
22
|
+
|
22
|
A/chicken/Zhejiang/C1/2013
|
H9N2
|
27
|
1.526
|
22
|
+
|
22
|
A/pigeon/Zhejiang/2P4/2013
|
H9N2
|
25
|
1.321
|
23
|
+
|
22
|
A/chicken/Zhejiang/4C91/2013
|
H9N2
|
25
|
1.452
|
23
|
+
|
22
|
A/quail/Zhejiang/D485/2013
|
H9N2
|
27
|
1.421
|
22
|
+
|
22
|
A/chicken/Zhejiang/C7195/2013
|
H9N2
|
25
|
1.321
|
22
|
+
|
22
|
A/chicken/Zhejiang/C497/2013
|
H9N2
|
27
|
1.256
|
22
|
+
|
22
|
A/chicken/Zhejiang/C55/2013
|
H9N2
|
27
|
1.236
|
22
|
+
|
22
|
A/chicken/Zhejiang/329/2011
|
H9N2
|
27
|
1.526
|
22
|
+
|
22
|
A/duck/Zhejiang/D1/2013
|
H1N2
|
26
|
0.055
|
-b
|
-
|
-
|
A/chicken/Zhejiang/2CP25/2014
|
H1N3
|
25
|
0.046
|
-
|
-
|
-
|
A/duck/Zhejiang/473/2013
|
H1N4
|
26
|
0.078
|
-
|
-
|
-
|
A/duck/Zhejiang/6D10/2013
|
H2N8
|
24
|
0.069
|
-
|
-
|
-
|
A/duck/Zhejiang/4613/2013
|
H3N2
|
26
|
0.065
|
-
|
-
|
-
|
A/duck/Zhejiang/5/2011
|
H3N3
|
27
|
0.028
|
-
|
-
|
-
|
A/duck/Zhejiang/D1-3/2013
|
H3N6
|
26
|
0.047
|
-
|
-
|
-
|
A/duck/Zhejiang/727145/2014
|
H4N2
|
24
|
0.054
|
-
|
-
|
-
|
A/duck/Zhejiang/409/2013
|
H4N6
|
25
|
0.054
|
-
|
-
|
-
|
A/goose/Zhejiang/97/2014
|
H5N1
|
26
|
0.039
|
-
|
-
|
-
|
A/duck/Zhejiang/6DK19/2013
|
H5N2
|
27
|
0.033
|
-
|
-
|
-
|
A/duck/Zhejiang/6D2/2013
|
H5N6
|
27
|
0.024
|
-
|
-
|
-
|
A/duck/Zhejiang/W24/2013
|
H5N8
|
27
|
0.027
|
-
|
-
|
-
|
A/chicken/Zhejiang/1664/2017
|
H6N1
|
26
|
0.065
|
-
|
-
|
-
|
A/duck/Zhejiang/727038/2014
|
H6N2
|
24
|
0.055
|
-
|
-
|
-
|
A/chicken/Zhejiang/727018/2014
|
H6N6
|
25
|
0.032
|
-
|
-
|
-
|
A/duck/Zhejiang/DK16/2013
|
H7N3
|
25
|
0.034
|
-
|
-
|
-
|
A/chicken/Jiangxi/C25/2014
|
H7N7
|
27
|
0.062
|
-
|
-
|
-
|
A/chicken/Zhejiang/ ZJU01/2013
|
H7N9
|
27
|
0.035
|
-
|
-
|
-
|
A/duck/Zhejiang/6D20/2013
|
H10N2
|
25
|
0.041
|
-
|
-
|
-
|
A/chicken/Zhejiang/8615/2016
|
H10N3
|
26
|
0.077
|
-
|
-
|
-
|
A/chicken/Zhejiang/2CP2/2014
|
H10N7
|
26
|
0.053
|
-
|
-
|
-
|
A/chicken/Zhejiang/102622/2016
|
H10N8
|
25
|
0.026
|
-
|
-
|
-
|
A/duck/Zhejiang/727D2/2013
|
H11N3
|
23
|
0.045
|
-
|
-
|
-
|
A/duck/Zhejiang/71750/2013
|
H11N7
|
23
|
0.035
|
-
|
-
|
-
|
Infectious bursal disease virus (IBDV)
|
NF8
|
25
|
0.067
|
-
|
-
|
-
|
Infectious bronchitis virus (IBV)
|
H120
|
25
|
0.044
|
-
|
-
|
-
|
Newcastle disease virus (NDV)
|
La Sota
|
25
|
0.032
|
-
|
-
|
-
|
Avian paramyxovirus 4(APMV-4)
|
ZJ-1
|
25
|
0.058
|
-
|
-
|
-
|
B/Massachusetts/2/2012
|
Yamagata
|
25
|
0.049
|
-
|
-
|
-
|
a“+”, positive result. |
b“-”, negative result. |
Madin-Darby canine kidney (MDCK) cells and SP2/0 mouse myeloma cells were maintained in our laboratory. Purified HA protein from the H9N2 (A/chicken/Zhejiang/329/2011) subtype AIV was purchased from Sino Biological (Beijing, China) [41].
Generation and purification of MAbs
BALB/c mice (9 weeks old) were immunized with the purified H9N2 subtype AIV HA protein mixed Freund’s adjuvant (Sigma, St. Louis, MO, USA) twice intramuscularly, 3 weeks apart. After 6 weeks, the mice were immunized once more with HA protein by tail vein injection. After 3 days, the spleen lymphocytes of the selected mice were fused with SP2/0 cells [41, 42]. The hybridoma cells were screened using a purified H9N2 HA protein-coated ELISA method. The positive monoclonal hybridoma cell line that was obtained after three consecutive limiting dilutions was continuously subcultured and then injected into mice intraperitoneally. To obtain MAbs, a Protein G column (GE Healthcare, Chicago, IL, USA) was used to purify ascites collected from the mice injected with the hybridoma cells [43].
Isotype and affinity of MAbs
Isotyping of the MAbs was performed using a Monoclonal Antibody Isotyping Kit (Bio-Rad, Hercules, CA, USA). The affinities of each MAb were measured using ELISA, as described previously [43]. In brief, the ELISA plate was coated with purified H9N2 HA protein (20 ng/well) overnight at 4 °C. MAbs were 2-fold serially diluted, starting with 1 mg/ml, and added to the plate. Incubation was carried out for 1 h at 37 °C. Then, goat anti-mouse IgG (Novus, St Charles, MO USA) was diluted by 10000 times and added as the secondary antibody. Incubation was carried out for 30 min at 37 °C. The color reaction was performed using the 3, 3’, 5, 5’-tetramethylbenzidine (TMB) reagent (KPL, Gaithersburg, MD, USA). After 10 min, the color reaction was stopped using the terminating reagent (KPL). Between each step, phosphate-buffered saline (PBS) with Tween 20 (PBST) was used to wash the plate five times. An ELISA plate reader (Bio-Rad) was read used to read the optical density (OD) at 450 nm, and the result of affinity was estimated as the minimum concentration of Mab required to provide a positive reaction. The variable genes of the heavy or light chains of Mabs were sequenced by Sino Biological.
Immunofluorescence analysis
An immunofluorescence assay (IFA) was used to visualize the binding of the MAbs to the virus‑infected MDCK cells [44, 45]. After incubation with the virus for 24 h, virus‑infected MDCK cells were fixed with paraformaldehyde. Thereafter, the MDCK cells were permeabilized using Triton X-100. Then, the MAbs 3G4 or 2G7 were added and incubated for 1 h at 37 °C. The goat anti-mouse IgG heavy plus light chain (H+L)-Alexa Fluor (Abcam, Cambridge, UK) was then added. The wells were washed with PBS three times between each step. The results were scored using an EVOS M7000 instrument (Thermo Fisher Scientific, Waltham, MA, USA).
Preparation of H9N2 AC-ELISA
The procedure for AC-ELISA (Fig. 1) was described previously [33, 46]. In brief, based on the results of MAb affinity measurements, MAb 3G4 was selected for capture and used to coat a 96-well ELISA plate at 80 ng/well in 100 µl of coating buffer at 4 °C. After 12 h, MAb 2G7, which was selected as the detection antibody, was labelled with horseradish peroxidase (HRP; Innoreagents, Huzhou, China). Then, the ELISA plate was washed and blocked with bovine serum albumin (BSA). After washing, samples were added into the ELISA plate and incubated for 1 h at 37 °C. Then, after washing the plate, 2G7-MAb-HRP (4 µg/ml) was added and incubated for 30 min at 37 °C. The plate was then washed and TMB solution was added at 100 µl/well. After 10 min, the TMB stop solution was added. The OD value (450nm) was then detected using an ELISA reader. The OD value greater than 2.1 times that of the negative control was considered to indicate a positive reaction.
ICT strip preparation
The procedure for using the ICT strip is showed in Fig. 1. The preparation of the colloidal gold solution was described previously [35, 47]. In brief, 0.01% HAuCl4 solution was heated to 100 °C, and then a trisodium citrate solution was added quickly with continuous vigorous stirring. Then, the colloidal gold solution was continuously boiled until the color changed to wine-red. After cooling, the pH of the solution was adjusted to 7.2 using potassium carbonate. Then, 10 ml of the colloidal gold solution placed into a glass bottle into which 100 µl MAb 3G4 (1 mg/ml) was added. Incubation was carried out for 30 min with gentle stirring. After blocking with BSA, the solution was centrifuged for 30 min at 4 °C. The colorless supernatant was discarded and the pellet was re-dissolved with 1 ml PBST (containing 1% BSA).
The ICT strip contained an absorbent pad, nitrocellulose (NC) membranes, a MAb-gold conjugated, pad and a sample pad. The NC membranes were coated with the test line (MAb 2G7) and the control line (goat anti-mouse IgG; Solarbio, Beijing, China).
Sensitivity, specificity, and repeatability of the H9N2 AC-ELISA and ICT strips
The specificity of the assays was tested using different subtypes of AIV (12 H9N2 subtypes of AIV and 25 other subtypes of AIV), and other viruses (IBDV, IBV, APMV-4, NDV, and influenza B virus). To determine the sensitivity, 2-fold serial dilutions of H9N2 AIV allantoic fluid and purified H9N2 proteins were used. A 2-fold serial dilution of H10N7 AIV allantoic fluid was used as the negative control. To evaluate repeatability, all samples were tested in triplicate, and all assays were repeated three times.
Assessment of the ability to rapidly detect actual poultry samples
To assess the clinical application of the two methods, 200 cloacal swabs collected from poultry in farms in Zhejiang Province were detected using multiplex qRT-PCR, AC-ELISA, and ICT strip methods. The Influenza A virus universal PCR kit (Liferiver Bio-Tech, Shanghai, China) was used to perform the qRT-PCR assay [35].