Development of an Immunochromatographic Strip for Rapid Detection of H7 Subtype Avian Inuenza Viruses

Background: H7N9 avian inuenza virus (AIV) including highly and low pathogenic viruses have been detected in China since 2013. H7N9 AIV has a high mortality rate after infection in humans, and most human cases have close contacted with poultry in the live poultry market. Therefore, it is necessary to develop a rapid point-of-care testing (POCT) technique for H7N9 AIV detection. Methods: The H7N9 AIV was inactivated and puried, and was used as the antigen to immunize BALB/c. Twelve H7-HA specic monoclonal antibodies (McAbs) were produced through the hybridoma technique. The McAb 10A8 was conjugated with colloid gold as detecting antibody; McAb 9B6 was dispensed on the nitrocellulose membran as the capture test line and the Goat-anti mouse IgG antibody was dispensed as control line respectively. The immunochromatographic strip was prepared. Results: The analysis of ELISA and virus neutralization test showed that the obtained McAbs specically recognized H7 HA. Based on the prepared strip, the detection of H7 AIV was achieved within 10 min. No cross-reaction occurred between H7 AIVs and other tested viruses. The detection limit of the strip for H7 was 2.4 log 10 EID 50 /0.1 mL for chicken swab samples. Conclusion: The McAbs were specic for H7 and the immunochromatographic strip developed in this study was convenient, rapid and reliable for the detection of H7 AIV. The strip could provide an effective method for the rapid and early detection of H7 AIV.


Introduction
Human infected with H7N9 avian in uenza virus (AIV) was rst reported in the spring of 2013 in China [1,2]. As of 24 th June 2019, a total of 1568 laboratory-con rmed human cases and at least 615 related deaths have been reported [3,4]. The main source of these human cases is thought to be infected live birds or contaminated environments, particularly in live poultry markets [5,6]. During the fth wave of epidemics, the H7N9 AIV was genotyped into two independent lineages, the Yangtze River Delta lingage and the Pearl River Delta lineage [4]. Highly pathogenic (HP)-H7N9 variants appeared during the 5th wave, in which the isolates had 2-3 additional basic amino acid residues insertion at the hemagglutinin (HA) cleavage site (CS) [7][8][9][10], resulting in high morbidity and mortality among poultry. The highly pathogenic H7N9 virus has posed a serious threat to public health and poultry farming [4].
Early diagnosis and management are crucial to controlling H7N9 infection. Therefore, it is necessary to develop a rapid point-of-care testing (POCT) technique for H7N9 AIV detection. Serological and molecular methods have been used for detecting H7N9 AIVs [11,12]. The National Avian In uenza Reference Laboratory (NAIRL) has established serological diagnostic techniques including hemagglutination (HA) and hemagglutination inhibition (HI) assays, agar gel immunodiffusion (AGID) assays, neuraminidase inhibition (NI) assays and indirect enzyme-linked immunosorbent assays (ELISA). Molecular diagnostic techniques include reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR [13,14]. However, these traditional detection methods are not only time-consuming, laborious with complicated operations, but also prone to false positive results. In addition, traditional diagnostic methods usually require special equipment, which limits the rapid detection for large number of samples.
Compared with other detection methods, the immunochromatographic test strip labled with colloidal gold is more attractive because it is rapid and does not require extra equipment for detection [15]. Therefore, in this study McAbs were prepared using the inactivated H7N9 virus as an immunogen. An immunochromatographic strip speci c for AIV H7 subtype was then developed using two H7-HA speci c McAbs, which can detect clinical samples within 10 min with high speci city and sensitivity.

Monoclonal antibodies production
McAbs against H7N9 were developed following a standard procedure. Six-weeks-old female BALB/c mice were immunized with the inactivated H7N9 AIV (A/Chicken/Huizhou/HZ-3/2016) puri ed by differential centrifugation at an immunization dose of 20 μg/mouse in Freund's adjuvant twice with a 3-week interval followed by nal immunization with 20 μg H7N9 antigen at 3 days before cell fusion. Splenocytes from the immunized mouse were fused with Sp2/0 myeloma cells, and the hybridoma cells were screened by immunoperoxidase monolayer assay (IPMA) and enzyme-linked immunosorbent assay (ELISA) and cloned by the limiting dilution method. The ascitic uids from the positive hybridomas were produced in mice.

Screening antibodies speci c for HA protein
McAbs against the HA protein of H7 subtype AIV were screened by ELISA. The HA proteins of different in uenza virus subtypes (Table 1) diluted in carbonate buffer (CBS) at a concentration of 1 μg/mL were added into 96-well plates at 50 μL/well and incubated at 37 ºC for 2 h. After blocked with 5% skim milk at 37 ºC for 1 h; hybridoma supernatant of McAbs were added and incubated at 37 ºC for 30 min. The reactions were then detected by HRP-labled Goat anti-mouse IgG, and color was developed using TMB solution at room temperature for 10 min which was was stopped by stop solution. The OD 450 value of each well was read with a microplate reader for statistical analysis .

Identi cation of antigen epitopes recognized by monoclonal antibodies
The peptide scanning technique was used to identify the epitope recognized by the McAbs. According to the H7N9 subtype avian in uenza HA protein amino acid sequence (ARG44098.1), peptide was synthesized by GL Biochem Ltd (Shang hai, China). The peptide was coupled to the bovine serum albumin (BSA) carrier protein by Sulfo-SMCC, and the coupled peptide was spot-printed on the nitrocellulose membrane. The H7N9 positive serum was used as a positive control, and BSA was used as a negative control. The supernatant of McAbs 9B6 and 10A8 were used as primary antibody, and reactions were then detected by HRP-labled Goat anti-mouse IgG. Finally, ECL color reagent was used to detect the reactivity of McAbs and HA polypeptide.

Virus neutralization test
Neutralizing activities of McAbs were determined by HI assay and virus neutralization (VN) assays.
Brie y, 10-fold serial dilutions of McAbs from 10 3 were mixed with 200 TCID 50 virus and incubated for 2 h at 37 ºC. The mixture was then used to infect Madin-Daby canine kidney cells (MDCK) and incubated for 24 h at 37 ºC. Then the 100% VN titers of McAbs were determined by Reed-Muench. At the same time, 10fold serial dilutions of McAbs were mixed with virus to determine HI titers, and HI titers ≥ 4 were considered positive.
Preparation of colloidal gold and gold-labeled antibodies Preparation of colloidal gold by trisodium citrate method [16]. Brie y, 1 mL of 1% chloroauric acid and 99 mL of double distilled water was added into the erlenmeyer ask, stiring and heating, followed by the rapid addition of 1.6 mL of 1% trisodium citrate solution with rapid stirring. The reaction mixture was boiled until the color gradually changes from light yellow to deep red and no longer changes in color, with the above process taking about 20 min. The colloidal gold solution was cooled to room temperature. 12 McAbs were incubated with different pH colloidal gold solution for 30 min. The 10% BSA was added to the colloidal gold conjugation and incubated for 10 min. The mixture was then centrifuged at 13,000 rpm and 4 ºC for 30 min to remove any unbound antibody. The pellet was resuspended in boric acid buffer containing 1% BSA.

Selection of paired McAbs for the strip
Among the twelve positive clones, two H7-HA McAbs which showing higher binding a nity were selected to establish a rapid detective strip by sandwich Dot-blot. The sandwich Dot-blot was performed as following. Twelve capture antibodies was blotted on the nitrocellulose membrane (Table 2)

Detecting tissue samples from experimentally infected chickens
Three-weeks-old SPF chickens purchased from Beijing Boehringer Ingelheim Vital Biotechnology Co., Ltd, were inoculated intranasally with 10 6 EID 50 of H7N9 AIV (A/Chicken/Huizhou/HZ-3/2016) in a 0.2 mL volume (n = 6). In addition, the other two chickens were not inoculated with virus as negative control. After 60 hours, the virus-infected chickens began to die. In order to con rm H7N9 AIV infection, the tissues (brain, windpipe, heart, liver, spleen, lung, thymus, pancreas, bursa of fabricius and cecal tonsil) were dissected from each chicken, and these samples were tested using the H7 detection strip and RT-PCR, respectively.

Detection of simulated clinical swab samples
Tracheal swabs and cloacal swabs (n = 30) were collected from healthy poultry in Henan Province. Swab samples were collected in 2 mL PBS, and the virus allantoic uids (HZ-3) were added into tracheal swabs and cloacal swabs to simulate clinical swab samples. The simulated clinical swab samples were 2-timediluted from 2 -8 to 2 -12 to evaluate the rapid detection strip by HA test and strip test.

Preparation and characterization of monoclonal antibodies
Twelve McAbs against H7N9 AIVs HA protein were produced, and only two McAbs 9B6 and 10A8 related to the strip were introduced in this article (Fig 1). The IPMA titers of both two McAbs were 10 -6 . The HI titers of the two McAbs were 13 log2 and 4 log2 , and 100% VN titers of the two McAbs were 1:16 000 and 1:1 000 , respectively. The epitopes recognized by McAbs 9B6 and 10A8 were determined as GVTSACRRSGSSFYAEMK (aa positions 142 to 159), and YKSTQSAIDQITGKLNRL (aa positions 381 to 398), respectively ( Table 3).

Establishment of a rapid detective immunochromatographic strip
The colloidal gold was obtained and labeled with twelve McAbs. After several comparable experiments, the optimum pH was found to be 4, and the optimum labeled McAbs dose was 4 μg/mL. It was validated that the McAb 9B6 used as capture antibody and McAb 10A8 used for detection showed the best performance (2c) in this assay (Fig 2). The colloidal gold conjugation of 10A8 was dispensed on the berglass pads as conjugated McAb. The dosage of colloidal gold-labeled 10A8 antibody mixture was 1.79 μL/cm. The McAb 9B6 was diluted to 0.8 mg/mL in physiological saline and dispensed on the nitrocellulose membran as the capture test line. Then the Goat-anti mouse IgG antibody was diluted to 1.3 mg/mL in physiological saline as control line to develop the immunochromatographic strip.
Broad reaction of the strip for H7 subtype AIVs H7N9 viruses isolated from 2013 to 2017 showed two red lines in the test control area, indicating that the strip could detect H7 subtype AIVs with broad reaction (Fig. 3).
Speci city evaluation of the rapid detective strip A rapid detective strip for the double antibody sandwich mode was established using H7-HA speci c mAbs 9B6 and 10A8 as capture and conjugation antibodies, respectively. The speci city test results showed that only H7 subtype had two red bands at the T and C lines, and other subtypes of in uenza virus and poultry virus had only one red band at the C line Fig. 4 , indicating that the rapid detective strip had high speci city for the detection of H7 AIVs.

Sensitivity evaluation of the rapid detective strip
The sensitivity of the rapid detective strip was tested using three strains of H7 AIVs (HZ-3, G1 and SW15154). The results of the strip were read by TSR-3000 Reader showed that the sensitivity of the strip could be 2 -11 (Fig. 5), which was 2-8 times higher than the HA titer of chicken embryo allantoic uid (Table 4), and 10 2.6 TCID 50 or 10 2.4 EID 50 could be detected by using the strip, indicating that the rapid detection strip had high sensitivity for the detection of H7 AIVs.

Stability evaluation of the rapid detective strip
The strips still had the same detection limit for H7 AIV (A/Chicken/Guangdong/G1/2013) as freshly produced strips after 6 months of storage, indicating that the gold immunochromatographic strip had high sensitivity for the detection of H7 AIVs (Fig. 6).

Detection of tissue samples from infected chickens
RT-PCR test and strip test were performed on 60 infected tissue samples and 20 uninfected tissue samples, and the strip test results were compared with the RT-PCR test results. The results of the RT-PCR test showed that 100% (60/60) of infected tissue samples were positive for H7N9 AIV and 100% (20/20) of uninfected tissue samples were negative for H7N9 AIV, consistent with 100% (60/60) of infected tissue samples were positive for H7N9 AIV and 100% (20/20) of uninfected tissue samples were negative for H7N9 AIV of determined by strip test (  (Fig. 7). These data suggested that the rapid detection method was suitable for detecting H7N9 AIVs from infected samples.

Discussion
With the vigorous development of the breeding industry, bird u has always been a threat to our country's poultry breeding industry, and the requirements for the prevention and control of bird u have become more stringent. In uenza virus is a segmented single negative-stranded RNA virus with an envelope, which is prone to mutation [7]. Nowadays, the classic and commonly used methods for detecting in uenza virus include hemagglutination inhibition test and uorescent quantitative PCR [17]. However, the use of the above methods or the need for detection equipment, or the need to prepare fresh red blood cells, and has certain requirements for operation, is not convenient for large-scale rapid screening and testing of grassroots enterprises and institutions or farmers. Therefore, colloidal gold test strips have been widely used due to their huge advantages.
The biological characteristics of monoclonal antibodies are a key factor in determining the performance of colloidal gold immunochromatography strip. In this study, we developed a rapid detective immunochromatographic strip speci c for the H7 subtype by using two H7-HA speci c McAbs 9B6 and 10A8 as capture and conjugation antibodies. In order to improve the speci city and a nity of McAbs, the McAbs that targeted HA protein were selected develop a sandwish mode. Two H7-HA McAbs were developed, and identi ed by HI assay and VN assay. The HI and VN titers of different McAbs [18] were different, which may lead to differences in the speci city and a nity of McAbs. In addition, we identi ed the epitopes recognized by these two McAbs through peptide scanning technology. Among them, the McAb 9B6 recognized conservative epitope GVTSACRRSGSSFYAEMK that has been reported [19], and the McAb 10A8 recognized conservative epitope that has been reported YKSTQSAIDQITGKLNRL [20]. Although the in uenza strains change rapidly, the two McAbs recognize conservative epitopes and provide technical support for the long-term monitoring of in uenza viruses. At the same time, different batches of ascites may affect the performance of the test strips. For this reason, we prepared a large amount of ascites at one time and puri ed and packed them to ensure the availability of experimental data.
The colloidal gold immunochromatography technology is convenient to apply, and the results show rapid and intuitive, rapid diagnosis of diseases, high sensitivity, and rapid application in the eld of animal medicine. Manzoor [21] developed a Pensite test kit for rapid diagnosis of H7 highly pathogenic avian in uenza. The detection limit for swab samples and tissue homogenates was 4.5 log10EID 50 , and the sensitivity was lower than our strips (2.4 log10EID 50 ). Liu X [18] developed a colloidal gold-based immunochromatographic strip for rapid detection of H7N9 in uenza viruses with a limit of detection of 2.5 log10EID 50 , less sensitive than our strips (2.4 log10EID 50 ). Kang [22] developed a rapid immunochromatographic test for hemagglutinin antigen of H7 subtype in patients infected with novel avian in uenza A (H7N9) virus with a limit of detection of 10 3 TCID 50 , less sensitive than our strips (10 2.6 TCID 50 ). Although the rapid immunoassay was less sensitive than rRT-PCR or virus isolation, the strips could achieve POCT, which could be used as an indicator for H7N9 AIVs infections. The rapid spread of highly pathogenic H7N9 subtype avian in uenza viruses around the world may increase the risk of human infections. H7N9 subtype AIVs isolated from human and chicken were successfully detected by the strip, indicating a broad reaction for H7N9 AIVs. Morever, the strips did not detect the other subtypes of in uenza virus and poultry virus. Therefore, a rapid detection method based on double antibody sandwich mode H7 AIVs was successfully prepared in this study, which has good speci city, sensitivity and stability, and provided technical support for the diagnosis and monitoring of H7 AIVs.