A versatile synthetic peptide-based ELISA for identifying antibody epitopes.
Abstract: A simple, versatile and very inexpensive procedure for cross-linking synthetic peptides to the polystyrene surfaces of micro-well assay plates for use in ELISA was developed. The method is based on the use of poly-L-lysine (PLL) as the anchor protein for synthetic peptides which were then easily and covalently linked to the PLL using glutaraldehyde. The synthetic peptides used for the study were based on the amino acid sequence of the equine infectious anemia virus (EIAV) envelope sequence and evaluated as antigens in an ELISA designed to detect antibodies in serum of EIAV-infected horses and ponies. The ELISA using cross-linked peptides proved to be significantly more sensitive when compared to assays where passively coated peptides were used. In one instance, a peptide was identified that was not recognized by any of our antisera and appeared not to bind to the assay plates. However, once this peptide was cross-linked to the assay plate it proved to be very useful for detecting EIAV-specific antibodies. This cross-linking approach functioned equally well with peptides of various charges and sizes and did not appear to alter epitopes contained in the peptides.
Publication Date: 1994-05-02 PubMed ID: 7513733DOI: 10.1016/0022-1759(94)90226-7Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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The research aims to develop an inexpensive and versatile process for attaching synthetic peptides to the surfaces of micro-well plates in ELISA tests, using poly-L-lysine and glutaraldehyde. The method was evaluated using synthetic peptides based on the sequence of equine infectious anemia virus (EIAV), and it proved significantly more sensitive than previous assays.
Research Procedure
- The study aimed to create a convenient, flexible, and cost-effective approach for linking synthetic peptides to polystyrene surfaces of micro-well plates in ELISA (enzyme-linked immunosorbent assay) tests.
- The researchers employed poly-l-lysine (PLL) as an anchor protein for the synthetic peptides. PLL can attach to various substances, making it advantageous for use in assays.
- The synthetic peptides were covalently bound to the PLL using glutaraldehyde. This forms a strong link that is resistant to several conditions, providing a robust and reliable bond between the peptides and the micro-well plates for the assay.
The Equine Infectious Anemia Virus Context
- The study specifically used synthetic peptides derived from the amino acid sequence of the EIAV (Equine Infectious Anemia Virus) envelope sequence.
- The peptides were evaluated as antigens in an ELISA, which was designed to detect EIAV-specific antibodies in the serum of horses and ponies.
Advantages of the New Method
- The new ELISA technique using cross-linked peptides outperformed other assays that used passively coated peptides. This indicates that the method of linking peptides to assay plates enhances the assay’s sensitivity.
- In one case, a peptide that initially did not bind to the assay plates and was not recognized by antiserum showed significant potential for detecting EIAV-specific antibodies once it was cross-linked to the assay plate using the new method.
- The cross-linking method worked equally well with peptides of different charges and sizes, highlighting its versatility and applicability in several ELISA assay types.
- The research implies that the method did not alter any epitopes contained in the peptides—-an important conclusion since any alteration might affect the assay’s performance.
Cite This Article
APA
Ball JM, Henry NL, Montelaro RC, Newman MJ.
(1994).
A versatile synthetic peptide-based ELISA for identifying antibody epitopes.
J Immunol Methods, 171(1), 37-44.
https://doi.org/10.1016/0022-1759(94)90226-7 Publication
Researcher Affiliations
- Division of Molecular Virology, Baylor College of Medicine, Houston, TX.
MeSH Terms
- Amino Acid Sequence
- Animals
- Antibodies / analysis
- Antibodies / immunology
- Antigens
- Chemical Phenomena
- Chemistry, Physical
- Cross-Linking Reagents
- Enzyme-Linked Immunosorbent Assay / methods
- Epitopes / analysis
- Glutaral
- Horses
- Microchemistry / methods
- Molecular Sequence Data
- Peptides
- Polylysine
- Reproducibility of Results
- Sensitivity and Specificity
Grant Funding
- AI25850 / NIAID NIH HHS
Citations
This article has been cited 10 times.- Nardini R, Autorino GL, Issel CJ, Cook RF, Ricci I, Frontoso R, Rosone F, Scicluna MT. Evaluation of six serological ELISA kits available in Italy as screening tests for equine infectious anaemia surveillance.. BMC Vet Res 2017 Apr 14;13(1):105.
- Gearhart TL, Montelaro RC, Schurdak ME, Pilcher CD, Rinaldo CR, Kodadek T, Park Y, Islam K, Yurko R, Marques ET Jr, Burke DS. Selection of a potential diagnostic biomarker for HIV infection from a random library of non-biological synthetic peptoid oligomers.. J Immunol Methods 2016 Aug;435:85-9.
- McClintock SD, Warner RL, Ali S, Chekuri A, Dame MK, Attili D, Knibbs RK, Aslam MN, Sinkule J, Morgan AC, Barsoum A, Smith LB, Beer DG, Johnson KJ, Varani J. Monoclonal antibodies specific for oncofetal antigen--immature laminin receptor protein: Effects on tumor growth and spread in two murine models.. Cancer Biol Ther 2015;16(5):724-32.
- Schroeder ME, Hostetler HA, Schroeder F, Ball JM. Elucidation of the Rotavirus NSP4-Caveolin-1 and -Cholesterol Interactions Using Synthetic Peptides.. J Amino Acids 2012;2012:575180.
- Belloc CG, Aguirre M, Peña C, Aparicio JL, Vega MD, Dormois S, Retegui LA. Properties of antibodies to a synthetic peptide representing an epitope shared by receptors of the type I cytokine family.. Clin Exp Med 2013 Feb;13(1):49-57.
- Aparicio JL, Peña C, Retegui LA. Autoimmune hepatitis-like disease in C57BL/6 mice infected with mouse hepatitis virus A59.. Int Immunopharmacol 2011 Oct;11(10):1591-8.
- Xainli J, Cole-Tobian JL, Baisor M, Kastens W, Bockarie M, Yazdani SS, Chitnis CE, Adams JH, King CL. Epitope-specific humoral immunity to Plasmodium vivax Duffy binding protein.. Infect Immun 2003 May;71(5):2508-15.
- Aucan C, Traoré Y, Fumoux F, Rihet P. Familial correlation of immunoglobulin G subclass responses to Plasmodium falciparum antigens in Burkina Faso.. Infect Immun 2001 Feb;69(2):996-1001.
- Aucan C, Traoré Y, Tall F, Nacro B, Traoré-Leroux T, Fumoux F, Rihet P. High immunoglobulin G2 (IgG2) and low IgG4 levels are associated with human resistance to Plasmodium falciparum malaria.. Infect Immun 2000 Mar;68(3):1252-8.
- Tian P, Ball JM, Zeng CQ, Estes MK. The rotavirus nonstructural glycoprotein NSP4 possesses membrane destabilization activity.. J Virol 1996 Oct;70(10):6973-81.
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