Investigating the ability of antibodies to recognize specific oxidized protein epitopes

Stuart Meredith, Corinne Spickett, Gita Parekh, James Schouten, Helen Griffiths, Paul Davis

Research output: Contribution to journalMeeting abstract

Abstract

There is a growing awareness that inflammatory diseases have an oxidative pathology, which can result in specific oxidation of amino acids within proteins. Antibody-based techniques for detecting oxidative posttranslational modifications (oxPTMs) are often used to identify the level of protein oxidation. There are many commercially available antibodies but some uncertainty to the potential level of cross reactivity they exhibit; moreover little information regarding the specific target epitopes is available. The aim of this work was to investigate the potential of antibodies to distinguish between select peptides with and without oxPTMs. Two peptides, one containing chlorotyrosine (DY-Cl-EDQQKQLC) and the other an unmodified tyrosine (DYEDQQKQLC) were synthesized and complementary anti-sera were produced in sheep using standard procedures. The anti-sera were tested using a half-sandwich ELISA and
the anti-serum raised against the chloro-tyrosine containing peptide showed increased binding to the chlorinated peptide, whereas the control anti-serum bound similarly to both peptides. This suggested that antibodies can discriminate between similar peptide sequences with and without an oxidative modification. A peptide (STSYGTGC) and its variants with chlorotyrosine or nitrotyrosine were produced. The anti-sera showed substantially less binding to these alternative peptides than to the original peptides the anti-sera were produced against. Work is ongoing to test commercially available antibodies against the synthetic peptides as a comparison to the anti-sera produced in sheep. In conclusion, the antisera were able to distinguish between oxidatively modified and unmodified peptides, and two different sequences around the modification site.
LanguageEnglish
Article numberPP36
PagesS31
Number of pages1
JournalFree Radical Biology and Medicine
Volume86
Issue numberSuppl.1
Early online date28 Aug 2015
DOIs
Publication statusPublished - Sep 2015
EventSFRR-E/SNFS Meeting Stuttgart 2015 - University of Hohenheim, Stuttgart, Germany
Duration: 1 Sep 2015 → …

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Epitopes
Peptides
Antibodies
Proteins
Serum
Post Translational Protein Processing
Tyrosine
Sheep
Oxidation
Pathology
Uncertainty
Immune Sera
Enzyme-Linked Immunosorbent Assay
Amino Acids

Bibliographical note

SFRR-E/SNFS Conference Abstracts, Stuttgart 2015

Cite this

Meredith, Stuart ; Spickett, Corinne ; Parekh, Gita ; Schouten, James ; Griffiths, Helen ; Davis, Paul. / Investigating the ability of antibodies to recognize specific oxidized protein epitopes. In: Free Radical Biology and Medicine. 2015 ; Vol. 86, No. Suppl.1. pp. S31.
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Investigating the ability of antibodies to recognize specific oxidized protein epitopes. / Meredith, Stuart; Spickett, Corinne; Parekh, Gita; Schouten, James; Griffiths, Helen; Davis, Paul.

In: Free Radical Biology and Medicine, Vol. 86, No. Suppl.1, PP36, 09.2015, p. S31.

Research output: Contribution to journalMeeting abstract

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T1 - Investigating the ability of antibodies to recognize specific oxidized protein epitopes

AU - Meredith, Stuart

AU - Spickett, Corinne

AU - Parekh, Gita

AU - Schouten, James

AU - Griffiths, Helen

AU - Davis, Paul

N1 - SFRR-E/SNFS Conference Abstracts, Stuttgart 2015

PY - 2015/9

Y1 - 2015/9

N2 - There is a growing awareness that inflammatory diseases have an oxidative pathology, which can result in specific oxidation of amino acids within proteins. Antibody-based techniques for detecting oxidative posttranslational modifications (oxPTMs) are often used to identify the level of protein oxidation. There are many commercially available antibodies but some uncertainty to the potential level of cross reactivity they exhibit; moreover little information regarding the specific target epitopes is available. The aim of this work was to investigate the potential of antibodies to distinguish between select peptides with and without oxPTMs. Two peptides, one containing chlorotyrosine (DY-Cl-EDQQKQLC) and the other an unmodified tyrosine (DYEDQQKQLC) were synthesized and complementary anti-sera were produced in sheep using standard procedures. The anti-sera were tested using a half-sandwich ELISA andthe anti-serum raised against the chloro-tyrosine containing peptide showed increased binding to the chlorinated peptide, whereas the control anti-serum bound similarly to both peptides. This suggested that antibodies can discriminate between similar peptide sequences with and without an oxidative modification. A peptide (STSYGTGC) and its variants with chlorotyrosine or nitrotyrosine were produced. The anti-sera showed substantially less binding to these alternative peptides than to the original peptides the anti-sera were produced against. Work is ongoing to test commercially available antibodies against the synthetic peptides as a comparison to the anti-sera produced in sheep. In conclusion, the antisera were able to distinguish between oxidatively modified and unmodified peptides, and two different sequences around the modification site.

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SN - 0891-5849

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M1 - PP36

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