fa طراحی روش ELISA برای سنجش آنتی بادی های IgG و IgM انسانی علیه SARS-CoV-2 ايمونولوژي Immunology پژوهشي-کامل Research(Original) <span style="font-size:12pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:90%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><span lang="FA" style="line-height:90%"><span b="" koodak="" style="font-family:"><span style="color:black">سابقه و هدف:</span></span></span> <span b="" lang="FA" style="font-family:" zar=""><span style="color:black">بیماری </span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black">کووید-19، یک بیماری تنفسی است که به وسیله‌ی گونه‌ای از کروناویروس تحت عنوان </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">SARS-CoV-2</span></span></span><span style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> ایجاد می‌شود</span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">.</span></span></span><span style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> این مطالعه با هدف طراحی یک روش الایزای غیرمستقیم برای سنجش آنتی‌بادی‌های </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">IgM</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> و </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">IgG</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> علیه </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">SARS-CoV-2</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black">، انجام پذیرفت.</span></span></span></span></span></span></span></span></span></span><br> <span style="font-size:12pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:90%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><span lang="AR-SA" style="line-height:90%"><span b="" koodak="" style="font-family:"><span style="color:black"><span style="letter-spacing:-.1pt">مواد و روش</span></span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"><span style="letter-spacing:-.1pt">‌</span></span></span></span><span lang="AR-SA" style="line-height:90%"><span b="" koodak="" style="font-family:"><span style="color:black"><span style="letter-spacing:-.1pt">ها:</span></span></span></span> <span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black">در این مطالعه تجربی، توالی پروتئین نوترکیب نوکلئوکپسید </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">SARS-CoV-2</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> در باکتری</span></span></span><br> <span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">E. coli BL21</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> بیان شد. پروتئین تولیدی با استفاده از </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">SDS-PAGE</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> و وسترن بلاتینگ تایید شد. در ادامه، از پروتئین نوترکیب نوکلئوکپسید تولید شده، روش </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">ELISA</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> غیرمستقیم برای سنجش آنتی‌بادی‌های </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">IgG</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> و </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">IgM</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> علیه </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">SARS-CoV-2</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> طراحی شد. سپس از این روش برای سنجش آنتی‌بادی‌های </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">IgM</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> و </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">IgG</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> در 61 بیمار مبتلا یا بهبود یافته از بیماری </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">COVID-19</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> به همراه 31 نمونه سرم فرد سالم استفاده شد. در انتها نتایج به‌دست آمده از روش </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">ELISA</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> طراحی شده بر روی نمونه‌ها با کیت تجاری مورد تایید وزارت بهداشت مقایسه شد.</span></span></span></span></span></span></span></span></span></span><br> <span style="font-size:12pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:90%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><span lang="AR-SA" style="line-height:90%"><span b="" koodak="" style="font-family:"><span style="color:black">یافته</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"><span style="letter-spacing:-.2pt">‌</span></span></span></span><span lang="AR-SA" style="line-height:90%"><span b="" koodak="" style="font-family:"><span style="color:black">ها:</span></span></span> <span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black">پروتئین نوترکیب نوکلئوکپسید بیان و تخلیص شد و سپس به کمک </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">SDS-PAGE</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> و وسترن بلاتینگ</span></span></span> <span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black">مورد تایید قرار گرفت. میزان جذب نوری به‌دست آمده از روش </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">ELISA</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> طراحی شده در 61 بیمار و 31 فرد سالم در مقایسه با کیت تجاری هم راستا بوده است. حساسیت و ویژگی روش </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">ELISA</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> طراحی شده برای سنجش </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">IgG</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> 100 درصد تعیین شد، این در حالی است که حساسیت 96/72 و ویژگی 96/77 برای سنجش </span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:90%"><span style="color:black">IgM</span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> گزارش شد.</span></span></span></span></span></span></span></span></span></span><br> <span style="font-size:12pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:90%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><span lang="FA" style="line-height:90%"><span b="" koodak="" style="font-family:"><span style="color:black"><span style="letter-spacing:-.1pt">استنتاج:</span></span></span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black"> تست‌های سرولوژیک به تنهایی ابزاری مناسب برای تشخیص نیستند</span></span></span><span b="" lang="FA" style="font-family:" zar=""><span style="color:black">، با این حال همراهی با تست</span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black">‌</span></span></span><span b="" lang="FA" style="font-family:" zar=""><span style="color:black">های مولکولی موجب افزایش صحت و حساسیت در تشخیص بیماری می</span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black">‌</span></span></span><span b="" lang="FA" style="font-family:" zar=""><span style="color:black">شود. هم</span></span><span lang="FA" style="line-height:90%"><span b="" style="font-family:" zar=""><span style="color:black">‌</span></span></span><span b="" lang="FA" style="font-family:" zar=""><span style="color:black">چنین این آزمایشات برای بررسی های اپیدمیولوژیکی بسیار ارزشمند می باشند.</span></span></span></span></span></span></span></span></span><br> &nbsp; <span style="font-size:12pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:140%"><span new="" roman="" style="font-family:" times=""><b><span style="font-size:10.0pt"><span style="line-height:140%"><span arial="" style="font-family:"><span style="letter-spacing:-.2pt">Background and purpose:</span></span></span></span></b> <span style="font-size:10.0pt"><span style="line-height:140%">Coronavirus disease 2019 (COVID-19) is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).&nbsp;In this study, an indirect ELISA method was designed to measure the human IgM and IgG antibodies against SARS-CoV-2.</span></span></span></span></span></span></span><br> <span style="font-size:12pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:140%"><span new="" roman="" style="font-family:" times=""><b><span style="font-size:10.0pt"><span style="line-height:140%"><span arial="" style="font-family:">Materials and methods:</span></span></span></b> <span style="font-size:10.0pt"><span style="line-height:140%">Protein sequence of nucleocapsid antigen from SARS-CoV-2 was expressed in <i>E. coli</i> BL21 and then was purified by chromatography. The purified protein was confirmed by SDS-PAGE and Western blotting. An indirect ELISA method was designed to measure the specific IgG and IgM antibodies against SARS-CoV-2 using recombinant N protein. The optimized ELISA method was then applied to measure the IgG and IgM antibodies in 61 infected or recovered COVID-19 patients and in 31 healthy controls. Finally, data obtained from the designed ELISA method were compared with those of a commercially approved ELISA kit.</span></span></span></span></span></span></span><br> <span style="font-size:12pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:140%"><span new="" roman="" style="font-family:" times=""><b><span style="font-size:10.0pt"><span style="line-height:140%"><span arial="" style="font-family:">Results: </span></span></span></b><span style="font-size:10.0pt"><span style="line-height:140%">The recombinant nucleocapsid protein was successfully expressed and purified which was confirmed by SDS-PAGE and Western blotting. The amount of optical densities obtained from the designed ELISA method was similar to those of the commercial kit in 61 patients and 31 controls. The sensitivity and specificity of the designed ELISA method for IgG were 100% compared with the commercial ELISA kit, while the sensitivity and specificity for IgM were 96.72 and 96.77, respectively.</span></span></span></span></span></span></span><br> <span style="font-size:12pt"><span style="line-height:140%"><span style="tab-stops:236.25pt"><span style="vertical-align:top"><span new="" roman="" style="font-family:" times=""><b><span style="font-size:10.0pt"><span style="line-height:140%"><span arial="" style="font-family:">Conclusion:</span></span></span></b> <span style="font-size:10.0pt"><span style="line-height:140%">Serological tests alone are not suitable for diagnosis; however, their combination with molecular tests increases the accuracy and sensitivity of the COVID-19 diagnosis. These tests are also vauable for epidemiological studies. </span></span></span></span></span></span></span> نوکلئوکپسید, IgG, IgM, الایزا, سارس کرونا ویروس-2 nucleocapsid, IgG, IgM, ELISA, SARS-CoV-2 26 36 http://jmums.mazums.ac.ir/browse.php?a_code=A-10-9381-2&slc_lang=fa&sid=1 Saeid Taghiloo سعید تقیلو saeid.taghiloo@yahoo.com 1003194753284600145839 1003194753284600145839 No PhD in Medical Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran دکترای تخصصی ایمونولوژی پزشکی، دانشکده پزشکی، دانشگاه علوم پزشکی مازندران، ساری، ایران Mina Dabiri مینا دبیری mina.dabiri@yahoo.com 1003194753284600145840 1003194753284600145840 No MSc in Medical Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran کارشناس ارشد ایمونولوژی پزشکی، دانشکده پزشکی، دانشگاه علوم پزشکی مازندران، ساری، ایران Reza Valadan رضا ولدان valadan.reza@gmail.com 1003194753284600145841 1003194753284600145841 No Assistant Professor, Department of Immunology, Molecular and Cell-Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran استادیار، گروه ایمونولوژی پزشکی، مرکز تحقیقات بیولوژی سلولی مولکولی، دانشگاه علوم پزشکی مازندران، ساری، ایران Hossein Asgarian Omran حسین عسگریان عمران asgarianhossein@yahoo.com 1003194753284600145842 1003194753284600145842 Yes Associate Professor, Department of Immunology, Gastrointestinal Cancer Research Center, Mazandaran University of Medical Sciences, Sari, Iran دانشیار، گروه ایمونولوژی پزشکی، مرکز تحقیقات دستگاه گوارش، دانشگاه علوم پزشکی مازندران، ساری، ایران