مشخصات

عنوان: تهیه و تولید کیت الایزا به منظور تشخیص عفونت آنفلوانزای ناشی از ویروس تحت سروتیپ H9N2 در طیور

گروه تخصصی:  دامپزشکی

سازمان مجری:  واحد تهران 

گروه پژوهشی: دامپزشکی

پژوهشگران: 
وصفی مرندی مهدی (مسئول طرح)
میرزایی حمید (همکار طرح)

تاریخ خاتمه:  شهریور 1382

کارفرما: معاونت پژوهش و فناوری جهاد دانشگاهی

خروجی طرح: 

دانش فنی تولید کیت الایزا تهیه و قابل واگذاری است.


نوع: کاربردی

 
تلفن: 66495417-021

نشانی سازمان مجری: تهران، خیابان انقلاب، خیابان 16آذر، سازمان مرکزی دانشگاه تهران، طبقه پنجم، صندوق پستی: 186-13145
 

چکیده:

1- تولید آنتی ژن:
بذر ویروس آنفلوانزایی که در این طرح مورد استفاده قرار گرفت سال 1377 از اپیدمی استان تهران A/chick/Iran/ZMT-101/98 (H9N2) در دانشکده دامپزشکی دانشگاه تهران جدا شده بود. ابتدا به منظور تولید آنتی ژن مورد نیاز، تخم مرغهای جنین دار 7-5 روزه از گله های مادر گوشتی سالم خریداری شده و به دستگاه جوجه کشی موجود در آزمایشگاه ویروس شناسی بخش طیور انتقال می یافت. پس از این تخم مرغها تا روز نهم بطور مرتب نور بین شده و جنین های تلف شده حذف می گردید. تخم مرغهای جنین دار 9 روزه سالم با استفاده از سرنگهای استریل به شکل کاملا آسپتیک بوسیله بذر اولیه تزریق می شد. جنین های تلف شده در فاصله زمانی 24 ساعت بعد از تزریق حذف می شدند. بعد از 48 ساعت انکوباسیون تخم مرغها به مدت 24 ساعت در یخچال قرار داده می شد تا جنین ها کشته شوند. سپس در زیر هود میکروبیولوژی و کنار شعله و شرایط کاملا آسپتیک مایع آلانتوئیک تخم مرغها جمع آوری شده و به داخل ظروف درب دار استریل انتقال داده می شد و از نظر وجود آلودگی میکروبی توسط آزمایش کشت در Nutrient agar در دمای 37˚C به مدت 48 ساعت مورد ارزیابی قرار می گرفت و در صورت عادی بودن از هر گونه آلودگی میکروبی با استفاده از فرمالین غیر فعال می شد. مجددا به منظور ارزیابی غیر فعال بودن، مایع آلانتوئیک حاصله به تخم مرغهای جنین دار 9 روزه تزریق می شد که در صورت غیر فعال بودن، هیچگونه تلفاتی در جنین ها ایجاد نمی کرد. علاوه بر آن مایع آلانتوئیک این تخم مرغها بعد از 4 روز جمع آوری و توسط آزمایش HA مورد ارزیابی قرار می گرفت. در صورت منفی بودن آزمایش HA جهت اطمینان از عدم تکثیر ویروس غیر فعال شده، پاساژ مجدد به عمل می آمد.



کلیدواژگان:

 
 
Title: Development of an Indirect Enzyme-linked Immunosorbent Assay for detection of h9n2 avian influenza antibodies in chicken sera

Abstract:

Influenza viruses belong to the family orthomyxoviridae and genus orthomyxovirus. These viruses are classified into types A, B and C on the basis of the antigenic character of their internal nucleoprotein and the matrix protein. Both antigens are regarded as common to all strains of influenza viruses of the same type. Influenza A viruses are further classified into subtypes on the basis of two surface glycoproteins including hemagglutinin and neuraminidase. Avian influenza virus (AIV) belong to type A and all 15 hemagglutinin and 9 neuraminidase subtypes have been reported from domestic and wild birds. Avian influenza due to H9N2 subtype was occurred in Iran in 1998 and caused significant economic losses in poultry industry (Vasfi Marandi and Bozorgmehri Fard). Attempts has been made to control AI disease by biosecurity programs and vaccination, but from 1998 to 2001 numerous outbreaks have been reported in different provinces. Serological and virological surveillance for avian influenza virus (AIV) of poultry is essential for early detection of virus and disease control and eradication. The routine surveillance and diagnosis of AIVs rely on the agar gel precipitin (AGP) test and the hemagglutination inhibition (HI) test. The AGP test detects precipitating antibody produced primarily against the ribonucleoprotein of the virus, and is therefore type specific. The HI test detects antibodies produced against the hemagglutinin surface glycoprotein of the virus, and is therefore subtype specific and different antigen is required to test each of the 15 hemagglutinin subtypes. The HI test is more sensitive and more rapid than the AGP test but because there are 15 hemagglutinin subtypes, the test setup can be very laborious and expensive to detect antibodies against AIV. The indirect enzyme-linked immunosorbent assays (ELISA) has been previously demonstrated to be an effective assay for detection of AIV antibodies in sera prepared from domestic poultry as well as other species including humans. The Elisa test was reported to be more sensitive and specific than the AGP and HI tests for detection of AIV antibodies in poultry sera. The aim of this study, was to develop an indirect ELISA test for detection of specific antibody against H9N2 subtype of AIV in chickens sera.
A/chicken/Iran/ZMT-101/1998 (H9N2) has been isolated from layer chicken in Tehran province of Iran, in jun 1998. This strain caused sever clinical disease in a very hot summer season. However, it was unable to cause mortality and clinical disease, in the laboratory pathogenesis test both in Avian Virlolgy Lab. Fac. Vet. Med – Univ. TehranIran and National Avian Influenza Lab. (Weybridge) in LondonUK. Therefore, This strain was designated as low pathogenic avian influenza virus (LPAI) or non-highly pathogenic avian influenza virus (nHPAI)
.
A total of 180 serum samples prepared from commercial layer flocks were used to the indirect ELISA using tow different kits including KJD (kit jehad Daneshgahie) and KPL. These sera were originally received for hemagglutination inhibition antibodies titration by HI test.
The A/Chick/Iran 101/98 virus was propagated in 10-day-old embryonating eggs from chickens known to be free of AIV, Mycoplasma and salmonella antibodies. Eggs were inoculated with each influenza virus strain by the intra-allantoic route. After 72 hours, infective amnioallantoic fluid was harvested and inactivated with 0.1% formalin. Inactivation of H9N2 antigens pool was confirmed by inoculating 0.2 ml of imnioallantoic fluid was clarified by centrifugation at 6000 X g for 10 min at 5 C. H9N2 antigen was purified as described by Kodiballi et al. purified antigens were resuspended in phosphate-buffered saline (PBS; pH 7.2) solution.
Optimal concentrations of the reagents for ELISA including purified antigens, positive and negative control sera, goat anti-chickens IgG conjugate, substrate and stopping solution were determined by checkerboard titration. In each ELISA plate test, the positive control sera were always placed in A1, A3 and H11 wells, and the negative control sera were placed in A2, H10 and H12 wells.
Optimally diluted inactivated H9N2 antigens in carbonate buffer was coated in 96 wells microtitration plates, and incubated overnight at 4 C. After washing five times with PBS-T (0.05% Tween 20 in PBS), positive and negative control sera as well as field sera samples diluted in diluent buffer containing 2% fetal bovine serum and 100 ml were placed in each well and incubated 30 min at 37 C. Plates were then rinsed 5 times with washing buffer. The goat anti-chicken immunoglobulin conjugated with horseradish peroxidase were diluted 2000-fold in diluent buffer and added 100 ml each well followed by incubating for 30 min at 37 C. After a final wash (5 X) with PBS-T the well were filled with 50 ml of a substrate ABTS (2, 2' Azinobis 3-ethylbenzthia-zoline sulfonic acid in hydrogen peroxide solution. After a 20 min incubation at 25 C, the reaction was stopped by addition of all wells were measured at 405 nm (OD 405) by an ELISA reader. The results of ELISA test using following defined procedure were compared with an ELISA kit coated similarly with H9N2 anteng (KPL.Co). Each serum samples was tested three time and then the average OD titers were compared. The hemagglutination inhibition (HI) test was carried out according to the standard beta procedure, using 4 hemagglutinating unit. The sensitivity and specificity of ELISA assay was obtained by determining the % of detectable positive and negative sera samples in KPL, KJD and HI tests. The results obtained in this study showed an existence of 95 percent correlation between KJD and KPL kits. Besides, the absolute correlation between their results was 86.67 percent. Furthermore, the correlation between KPL results and those of HI test was 95 percent. Finally, the absolute correlation between KPL and HI results as well as KJD and HI results were respectively 86.7 and 91.1 percents. These results indicate indicate that KJD ELISA kit developed in this study is sensitive and specific as well as KPL ELISA kit. However, it is necessary to examine a large number of field sera before commercialization of this kit.



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