Production of monoclonal antibodies against recombinant nucleoprotein of avian influenza virus, serotype H9N2

Document Type: Immunology


1 Department of Pathobiology, School of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran

2 Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

3 Department of Pathobiology, School of Veterinary Medicine, University of Lorestan, Khorram Abad, Iran


Background: Avian influenza viruses (AIVs) including the subtype H9N2 cause considerable financial losses to poultry industries. Rapid and accurate diagnosis of avian influenza (AI) infection is important in control and eradication programs. OBJECTIVES: The aim of this study was to produce monoclonal antibodies (MAbs) specific for the nucleocapsid protein )NP (of AIV H9N2 subtype to improve diagnostic assays. METHODS: Recombinant NP protein was expressed in Escherichia coli and purified using amylose resin chromatography column and used as an antigen for mice immunization. Spleen cells of the immunized mice were fused with SP2/0 myeloma cells. Next, culture supernatants of primary hybridoma clones were screened by indirect ELISA. After three rounds of sub cloning, the reactivity of the MAbs with recombinant and natural antigens was assessed by Western blotting. RESULTS: Six MAbs showed specific binding to recombinant and natural NP from AIV H9N2 in Western blot analysis, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay. Cross-reactivity with genetically non-related including Newcastle viruse (Paramyxoviridae family) was not detected. CONCLUSIONS: Based on the results, the MAbs generated in this study could be used for the development of rapid diagnostic assays for recognition of AIV.


Article Title [Persian]

تولید آنتی بادی منوکلونال بر ضد نوکلئوﭘروتئین نوترکیب ویروس آنفلوانزای ﭘرندګان، سروتیپ H9N2

Authors [Persian]

  • مسعودرضا صیفی آبادشاپوری 1
  • آزاده یکتاسرشت 2
  • مسعود قربانپور نجف آبادی 1
  • امین جایدری 3
1 گروه پاتوبیولوژی، دانشکده دامپزشکی دانشگاه شهید چمران، اهواز، ایران
2 گروه پاتوبیولوژی، دانشکده دامپزشکی دانشگاه شیراز، شیراز، ایران
3 گروه پاتوبیولوژی، دانشکده دامپزشکی دانشگاه لرستان، خرم آباد، ایران
Abstract [Persian]

ویروس‌های آنفلوانزای پرندگان شامل تحت تیپ H9N2سبب ضررهای اقتصادی قابل ملاحظه‌ای به صنعت
طیور می شوند. تشخیص سریع و دقیق عفونت آنفلوانزای پرندگان در برنامه‌های ریشه کنی و کنترل این بیماری بسیار مهم می‌باشد. هدف: هدف ازاین مطالعه تواید آنتی بادی‌های منوکلونال اختصاصی نوکلئوﭘروتئین ویروس آنفلوانزای پرندگان
تحت تیپ H9N2 برای بهبود و بیشرفت روش‌های تشخیصی بود. روش کار: ﭘروتئین نوترکیب NP در باکتری E.coli
بیان گردید و با استفاده از ستون کروماتوگرافی رزین آمیلوز خالص سازی و به عنوان
یک آنتی ژن برای ایمن سازی به موش تزریق شد. فیوژن سلول‌های طحال با سلول‌های میلوما انجام شد. در
مرحله بعد مایع رویی کشت سلولی کلون‌های هیبریدوما
اولیه به وسیله الایزای غیر مستقیم غربالگری شدند. بعد از سه بار کلونینگ واکنش
آنتی بادی‌های منوکلونال با آنتی ژن‌های طبیعی و نوترکیب به وسیله وسترن بلات تأیید شد. نتایج:
شش آنتی بادی منوکلونال اتصال اختصاصی به نوکلئوﭘروتئین نوترکیب و طبیعی در ویروس
آنفلوانزای برندگان تحت تیپ H9N2 در وسترن بلات٬ الیزا
و ایمونوفلورسانس را نشان دادند. واکنش متقاطع با ویروس‌های غیر مرتبط از لحاظ ژنتیکی از جمله ویروس نیوکاسل (خانواده
ﭘارامیکسوویریده) تشخیص داده نشد. نتیجه گیری نهایی: بر‌اساس نتایج آنتی بادی‌های منوکلونال
تولید شده در این مطالعه می‌توانند برای طراحی
آزمایشات تشخیصی سریع برای شناسایی ویروس آنفلوانزای ﭘرندگان استفاده شوند

Keywords [Persian]

  • آنتی بادی
  • پرندگان
  • آنفلوانزا
  • منوکلونال
  • نوکلئوپروتئین



Blastocystis is the most common anaerobic protozoa in the large intestine of human and many animals (Stensvold and Clark 2016, Mirjalali, Abbasi et al. 2017). The frequency of Blastocystis is worldwide with the highest rate in the countries with inappropriate sanitation (Amin 2006, El Safadi, Gaayeb et al. 2014). Likewise, high prevalence of Blastocystis in the studied populations with close-contact to animals supports the importance of animal hosts as potential reservoirs of the parasite (Yoshikawa, Abe et al. 2004, Lee, Chye et al. 2012). Because of the frequent reports of Blastocystis from domesticated and pet animals like cattle, sheep, pig, dog, birds, etc., it is strongly suggested that the parasite can transmit to human subjects from animal sources (Cian, El Safadi et al. 2017). There are evidences of transmission of the parasite via unhealthy drinking water as well (Cian, El Safadi et al. 2017). The pathogenicity of Blastocystis is still unclear because of the frequent reports from both symptomatic and asymptomatic subjects (Roberts, Stark et al. 2014).The clinical symptoms associated with the parasite are often non-specific including diarrhea, bloating, nausea, abdominal pain (Stensvold, Nielsen et al. 2009, Alinaghizade, Mirjalali et al. 2017, Jalallou, Iravani et al. 2017). Although many studies have indicated potential linkage between Blastocystis and some clinical manifestations such as irritable bowel syndrome (IBS) (Jimenez-Gonzalez, Martinez-Flores et al. 2012), skin manifestations (Cassano, Scoppio et al. 2005) and gastrointestinal disorders (El Safadi, Meloni et al. 2013, Dagci, Kurt et al. 2014), the recent studies have shown a negative correlation between the presence of the parasite with some gastrointestinal disorders like inflammatory bowel disease (IBD) (Mirjalali, Abbasi et al. 2017).

IBD is a chronic inflammation including Crohn’s (CD) and Ulcerative colitis (UC) with unknown etiology. CD is a chronic inflammation that may involve full thickness of the layers of upper and lower gastrointestinal tract (GT) while UC is an inflammation restricted to colon and only affects surface epithelium and mucosal layer (Satsangi, Silverberg et al. 2006). Many factors are supposed to be potential causes of onset or relapse of IBD including genetic, climatic regions zones, vitamin D3 deficiency, high protein diet, diet high in omega 6 and microbial infections (Hunter 2014). However, some studies describe the significant role of microbiota at onset of the symptoms in IBD patients (Nourrisson, Scanzi et al. 2014). Furthermore, some researches proposed a protective role for some intestinal parasites against immunological disorders (Wang, Cao et al. 2008). Therefore, the aim of the current study was to determine the frequency of Blastocystis among IBD patients referred to the Research Institute for Gastroenterology and Liver Diseases during the period August 2016 to February 2017 and to evaluate the correlation of demographic data on the infection rate.


Material and Methods


Stool sampling: In the current study, a total of 80 stool samples were collected from IBD-proved patients who were referred to Gastroenterology Clinic of the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences from August 2016 to February 2017. A well-trained interviewer filled a questionnaire that consisted of demographic data, drug type and dosage consumed through one month before the sampling. The consumption of metronidazole during the last month was considered as exclusion criteria. The stool samples were immediately transferred to Parasitology lab of Foodborne and Waterborne Diseases Research Center located in the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences for further investigation.

Microscopic examination and stool cultivation: All the stool samples were examined directly by Lugol’s iodine staining. Furthermore, the samples were concentrated with routine formalin-ethyl acetate and checked by light microscopy for detection of enteric parasites. A portion of the samples was immediately cultivated in Dulbecco’s modified Eagle’s medium (DMEM) with 20% inactivated calf serum (Zhang, Qiao et al. 2012). All the cultivated samples were incubated at 37 °c for 72 h and 10 µL of the sediment of the medium was examined by light microscopy with magnification X400 for growth of Blastocystis. The samples were checked for any growth of Blastocystis every 48 h and the samples without any growth after 10 days were considered negative.



Table 1, Overview of demographic and laboratory findings in IBD patients

              Age group, Patient age, Gender










Total no.





































Positive   patients

Close-contact   to animal










aIBD: Inflammatory bowel disease; M: Male; F: Female.






The enrolled patients consisted of 52 (65%) men and 28 (35%) women. In this study, Blastocystis was detected in 16/80 (20%) of the samples by microscopical examination and culture method (Fig 1, 2). The parasite was seen among 12 (23.08%) and 4 (14.29%) men and women, respectively. Fisher’s exact test represented that there was no correlation between gender and the presence of Blastocystis (P value= 0.397). The mean age + SD of the IBD patients enrolled in this study was 38.07 + 11.11. Indeed, the mean age + SD of the IBD patients carrying Blastocystis was 37.06 + 12.35 while in the uninfected IBD patients it was 38.28 + 10.87.  Fisher’s exact test denoted that there was no statistical correlation between age and the presence of the parasite (p value= 0.130). Likewise, the frequency of Blastocystis in the defined age groups was as followed: 37.5% (3/8) in less than 25 years-old, 15.38% (4/26) in 26-35, 18.51% (5/27) in 36-45, 25% (3/12) in 46-55 and 14.28% (1/7) in more than 56 years-old. Fisher’s exact test also showed that there was no significant correlation between age group and Blastocystis (P value= 0.699). All the demographic data are summarized in Table 1.

Furthermore, history of close-contact to animal was investigated and showed that there was no statistically significant correlation between the presence of the parasite and keeping pet or domesticated animals. Accordingly, although there was missing data from 10 IBD patients, only 3 of Blastocystis-positive patients stated history of close-contact to animals.




In the current study, Blastocystis was detected in 20% of IBD patients. No statistical correlation was found between age, sex and animal contact with the presence of the parasite. Blastocystis is a common enteric protozoan in fecal samples of human, worldwide. The pathogenic role of the parasite is still debated. In spite of evidence of the positive-correlation between IBS and Blastocystis, the association between IBD and Blastocystis has mostly been found negative (Nagel, Traub et al. 2015, Mirjalali, Abbasi et al. 2017). In this respect, a lower frequency of Blastocystis in IBD patients with active phase compared with controls was reported by Rossen and colleagues (Rossen, Bart et al. 2015). Coskun et al. investigated the presence of Blastocystis among UC patients and stated that patients with active phase had lower frequency of the parasite in comparison with those patients who were in the remission phase (Coskun, Malatyali et al. 2016). The study by Dugruman et al. showed that IBD patients had lower frequency of Blastocystis in comparison with IBS patients (Dogruman-Al, Simsek et al. 2010). In another study, Peterson et al. reported lower prevalence of Blastocystis in IBD patients in comparison with the patients with other gastrointestinal disorders as well as healthy subjects and suggested that this observation might be due to the unfavorable condition of GT in IBD patient for colonization of Blastocystis (Petersen, Stensvold et al. 2013). Recently, a case-control study performed by Mirjalali and colleagues showed that the prevalence rate of the parasite in IBD patients was significantly lower than healthy subjects. This observation was supported by previous studies assuming the role of IBD in dysbiosis (Mirjalali, Abbasi et al. 2017). According to the current findings contrasting mentioned studies, a high prevalence of Blastocystis 20% (16/80) was observed. These results are similar to the surveys conducted by Yamamoto-Furusho and Cekin et al. among UC patients (Yamamoto-Furusho and Torijano-Carrera 2010, Cekin, Cekin et al. 2012). Cekin et al. showed higher prevalence of Blastocystis among IBS and IBD patients than those subjects without significant gastrointestinal disorders (Cekin, Cekin et al. 2012). Yamamoto-Furusho described higher prevalence of Blastocystis among IBD patient who suffered from active phase than those patients who were in intermittent or remission phases(Yamamoto-Furusho and Torijano-Carrera 2010).

The current study showed that, although the prevalence of the parasite in age group under 25 years-old was higher than other groups, no significant correlation was found between age group and the presence of the parasite. Moreover, there was no significant correlation between the mean age of infected patients and uninfected subjects. Some studies have declared a noteworthy association between age group and the presence of the parasite in general population. This correlation was also seen in some studies that have been conducted among IBD patients. Against the current study, the correlation between age and the presence of the parasite was seen in the study conducted by Mirjalali et al. (Mirjalali, Abbasi et al. 2017). In agreement with our findings, in the study performed by Muttiucci and colleagues there was no significant association between age group and the presence of the parasite (Mattiucci, Crisafi et al. 2016). Similar results were seen in the study implemented by Cekin and colleagues, where there was no statistical correlation between age group and Blastocystis (Cekin, Cekin et al. 2012).

As the results illustrate, statistically significant relationship was not seen between gender and the presence of the parasite that is in accordance with the studies performed by Mirjalali and Muttucci (Mattiucci, Crisafi et al. 2016, Mirjalali, Abbasi et al. 2017).

Although no correlation was found between animal contact and infection with Blastocystis, it has been suggested that pet or farm animals could be considerable reservoirs of zoonotic transmission (Cian, El Safadi et al. 2017). Considering the possibility of zoonotic transmission of this parasite, keeping pets as well as close-contact to animals should be a concern, particularly in the human subjects with immunity disorders.

Conclusion: In this study Blastocystis was found in 20% of enrolled patients who suffered from IBD. This infection rate was significantly higher than the studies that have previously described Blastocystis in this group of patients. Therefore, these findings showed the importance of stool examination for surveying the common intestinal protozoans like Blastocystis in IBD patients particularly in different phases (Flare up and remission) of these groups.




This study received financial support from Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. We thank all the colleagues of Foodborne and Waterborne Diseases Research Center and Behbood Research Center for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran for their laboratory corporations.


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