Development of an immunochromatographic strip for rapid detection of H7 subtype avian influenza viruses

Background H7N9 avian influenza 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 purified, and was used as the antigen to immunize BALB/c. Twelve H7-HA specific 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 specifically 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 log10EID50/0.1 mL for chicken swab samples. Conclusion The McAbs were specific 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 influenza virus (AIV) was first reported in the spring of 2013 in China [1,2]. As of 24th June 2019, a total of 1568 laboratory-confirmed 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 fifth 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 Open Access *Correspondence: zhanggaip@126.com † Ge Li and Xun Wang have contributed equally to this work 1 College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China Full list of author information is available at the end of the article 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 Influenza 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 transcriptionpolymerase 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 specific for AIV H7 subtype was then developed using two H7-HA specific McAbs, which can detect clinical samples within 10 min with high specificity 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) purified by differential centrifugation at an immunization dose of 20 μg/mouse in Freund's adjuvant twice with a 3-week interval followed by final 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 fluids from the positive hybridomas were produced in mice.

Screening antibodies specific for HA protein
McAbs against the HA protein of H7 subtype AIV were screened by ELISA. The HA proteins of different influenza 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 °C for 2 h. After blocked with 5% skim milk at 37 °C for

Preparation of colloidal gold and gold-labeled antibodies
Preparation of colloidal gold by trisodium citrate method [16]. Briefly, 1 mL of 1% chloroauric acid and 99 mL of double distilled water was added into the erlenmeyer flask, 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 °C 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 affinity 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 (

Broad reaction of the strip for H7 subtype AIVs
To evaluate the broad reaction of the rapid detective strip, H7N9 AIVs isolated from 2013 to 2017 and antigen strains including H7-Re-2 and H7-Re-3 were tested. 100 μL of each sample cotaining 10 5 TCID 50 allantoic fluid or original solution of H7-Re-2 and H7-Re-3 were added to the sample pad of the test strip and incubated for 10 min at room temperature.

Specificity evalution of the rapid detective strip
To evaluate the specificity of the rapid detective strip, the H1, H3, H5, H7, H9 subtype influenza viruses and other avian viruses including NDV, MDV, IBV and IBDV were simultaneously detected. 100 μL of each sample containing 10 5 TCID 50 virus was added to the sample pad of the test strip and incubated for 10 min at room temperature.

Sensitivity evalution of the Rapid rapid detective strip
Three viruses HZ-3, G1 and SW15154 of the H7 subtype were used to detect the sensitivity of the rapid detection strip. The virus was diluted 2 times with 0.01 M PBS from 2 −1 to 2 −15 and PBS was used as a negative control.

Stability evalution of the rapid detective strip
These strips were tested to determine their sensitivity in detecting the virus HZ-3 upon storage at room temperature for 6 months. The virus HZ-3 was diluted 2 times with 0.01 M PBS from 2 −1 to 2 −15 and PBS was used as a negative control.

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 h, the virus-infected chickens began to die. In order to confirm 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 fluids (HZ-3) were added into tracheal swabs and cloacal swabs to simulate clinical swab samples. The simulated clinical swab samples were 2-time-diluted 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) 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

Broad reaction of the strip for H7 subtype AIVs
H7N9 viruses isolated from 2013 to 2017 and antigen strain H7-Re2 showed two red lines in the test control area, indicating that the strip could detect H7 subtype AIVs with broad reaction (Fig. 3). Antigen strain H7-Re3 showed one red line in the test control area.

Specificity evaluation of the rapid detective strip
A rapid detective strip for the double antibody sandwich mode was established using H7-HA specific mAbs 9B6 and 10A8 as capture and conjugation antibodies, respectively. The specificity test results showed that only H7 subtype had two red bands at the T and C lines, and other subtypes of influenza virus and poultry virus had only one red band at the C line (Fig. 4), indicating that the rapid detective strip had high specificity 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 fluid (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).   (Table 5).  (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 flu has always been a threat to our country's poultry breeding industry, and the requirements for the prevention and control of bird flu have become more stringent. Influenza 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 influenza virus include hemagglutination inhibition test and fluorescent 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 specific for the H7 subtype by using two H7-HA specific McAbs 9B6 and 10A8 as capture and conjugation antibodies. In order to improve the specificity and affinity of McAbs, the McAbs that targeted HA protein were selected develop a sandwish mode. Two H7-HA McAbs were developed, and identified by HI assay and VN assay. The HI and VN titers of different McAbs [18] were different, which may lead to differences in the specificity and affinity of McAbs. In addition, we identified 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 the epitope that has been reported YKSTQSAID-QITGKLNRL [20]. The influenza strains change rapidly, and the strip composed of these two McAbs can identify H7-Re2 but not H7-Re3. The HI test and Dot test identified the reactivity of the two Mcabs with H7-Re2 and H7-Re3 respectively. The results showed that 9B6 can react with both H7-Re2 and H7-Re3, while 10A8 can only react withH7-Re2. Therefore, the strip developed in this study can be used to differentially diagnose H7-Re2 and H7-Re3 strains. In subsequent experiments, we will update the antibodies used on the test strips in time according to the variation of influenza strains to achieve the purpose of rapid detection. 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 purified 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 field of animal medicine. Manzoor [21] developed a Pensite test kit for rapid diagnosis of H7 highly pathogenic avian influenza. 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 goldbased immunochromatographic strip for rapid detection of H7N9 influenza 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 influenza 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 influenza 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 influenza 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 specificity, sensitivity and stability, and provided technical support for the diagnosis and monitoring of H7 AIVs.

Conclusions
H7N9 infected human and chicken and most human cases have close contacted with poultry in the live poultry market. In this study, 12 H7-HA specificMcAbs were produced and two McAbs 9B6 and 10A8 were used to develop an immunochromatographic strip for detecting H7 AIVs. The obtained McAbs and strip had a great potential in application of the rapid detection and surveillance of H7 AIV.