کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8298365 | 1536893 | 2018 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and β-enolases
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کلمات کلیدی
PBSpNPPSDSESIIgGUPLC-Q-TOF-MSTBSTPMSFPAGEHRPBSA - BSApara-nitrophenyl phosphate - para-nitrofenyl phosphateopd - UPDbovine serum albumin - آلبومین سرم گاوAlkaline phosphatase - آلکالین فسفاتاز یا فسفاتاز قلیاییSpecific antibodies - آنتی بادی های خاصpolyacrylamide gel electrophoresis - الکتروفورز ژل پلی آکریل آمیدortho-phenylenediamine - اورتو-فنیلین دی آمینimmunoglobulin G - ایمونوگلوبولین GELISA - تست الیزاEnzyme-linked immunosorbent assay - تست الیزاsodium dodecylsulfate - سدیم دودسیل سولفاتMass spectrometry - طیف سنجی جرمیPhosphate buffered saline - فسفات بافر شورphenylmethylsulfonyl fluoride - فنیل متیل سولفونیل فلورایدMethanol - متانولMeOH - مونCross-reactivity - واکنش پذیری متقابلWestern blotting - وسترن بلاتینگhorse radish peroxidase - پراکسیداز تربچه اسبEpitope prediction - پیش بینی حوادثliquid chromatography - کروماتوگرافی مایع
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Human α- and β-enolases are highly homologous enzymes, difficult to differentiate immunologically. In this work, we describe production, purification and properties of anti-α- and anti-β-enolase polyclonal antibodies. To raise antibodies, rabbits were injected with enolase isoenzymes that were purified from human kidney (α-enolase) and skeletal muscle (β-enolase). Selective anti-α- and anti-β-enolase antibodies were obtained by affinity chromatography on either α- or β-enolase-Sepharose columns. On Western blots, antibodies directed against human β-enolase, did not react with human α-isoenzyme, but recognized pig and rat β-enolase. To determine what makes these antibodies selective bioinformatic tools were used to predict conformational epitopes for both enolase isoenzymes. Three predicted epitopes were mapped to the same regions in both α- and β-enolase. Peptides corresponding to predicted epitopes were synthesized and tested against purified antibodies. One of the pin-attached peptides representing α-enolase epitope (the C-terminal portion of the epitope 3 - S262PDDPSRYISPDQ273) reacted with anti-α-enolase, while the other also derived from the α-enolase sequence (epitope 2 - N193VIKEKYGKDATN205) was recognized by anti-β-enolase antibodies. Interestingly, neither anti-α- nor anti-β-antibody reacted with a peptide corresponding to the epitope 2 in β-enolase (G194VIKAKYGKDATN206). Further analysis showed that substitution of E197 with A in α-enolase epitope 2 peptide lead to 70% loss of immunological activity, while replacement of A198 with E in peptide representing β-enolase epitope 2, caused 67% increase in immunological activity. Our results suggest that E197 is essential for preserving immunologically active conformation in epitope 2 peptidic homolog, while it is not crucial for this epitope's antigenic activity in native β-enolase.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochemistry and Biophysics Reports - Volume 15, September 2018, Pages 25-32
Journal: Biochemistry and Biophysics Reports - Volume 15, September 2018, Pages 25-32
نویسندگان
Jadwiga Pietkiewicz, Regina Danielewicz, Iwona S. Bednarz-Misa, Ireneusz Ceremuga, Jerzy WiÅniewski, Magdalena Mierzchala-Pasierb, Agnieszka Bronowicka-SzydeÅko, Edmund Ziomek, Andrzej Gamian,