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Rational design of a low-affinity peptide for the detection of cystatin C in a fast homogeneous immunoassay

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Abstract

Immunoassays play an essential role in current research and diagnostics resulting in a variety of detection principles. Thereby, homogeneous assays are often used for a fast signal response as demanded for example in point-of-care diagnostics. These systems often rely on a competitive assay design where the sample analyte and the corresponding dye-labeled substance are competing for binding sites on an antibody present in limited amounts. Due to the similar affinities of the antibody towards the sample analyte and the competitor, both sensitivity and assay time are limited. As a consequence, a competitor with a slightly reduced affinity towards the antibody can potentially overcome these drawbacks. Here, we present the rational design of a low-affinity peptide (donor peptide) as a specific analyte competitor for a FRET-based homogeneous immunoassay for the analysis of the protein cystatin C. Thereby, the strategy of peptide-induced antibody generation was combined with the selective variation of the immunization sequence in order to achieve a lower affinity towards the antibody. We could show that shortened donor peptides improved the resulting quenching efficiency in the immunoassay. In addition, the substitution of small hydrophobic amino acids by those with a higher steric demand appeared to be the most promising strategy providing a fast assay response for cystatin C of only 90 s.

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Abbreviations

BCA:

Bicinchoninic acid

BHQ10:

Black Hole Quencher 10

BSA:

Bovine serum albumin

Cf:

Carboxyfluorescein

CSF:

Cerebrospinal fluid

DIC:

N,N′-Diisopropylcarbodiimde

DMEM:

Dulbecco’s modified Eagle medium

DMF:

N,N-Dimethylformamide

ELISA:

Enzyme-linked immunosorbent assay

FRET:

Förster resonance energy transfer

HOBt:

Hydroxybenzotriazole

HPLC:

High-performance liquid chromatography

HRP:

Horseradish peroxidase

K d :

Dissociation constant

mAb:

Monoclonal antibody

MALDI-TOF-MS:

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

PBS:

Phosphate buffered saline

PETIA:

Particle-enhanced turbidimetric immunoassay

QE:

Quenching effect

SD:

Standard deviation

TFA:

Trifluoroacetic acid

TMB:

Tetramethylbenzidine

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Acknowledgments

The authors thank Prof. Dr. Ralf Hoffmann for lab access, fruitful discussions and proof-reading. We gratefully acknowledge the technical assistance of Stefanie Langanke for synthesizing and purifying peptides. This work was supported by the Federal Ministry of Education and Research (BMBF) Go-Bio Project No. 031598870 to TZ.

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Author notes

  1. Thole Zuchner

    Present address: Octapharma Biopharmaceuticals GmbH, Im Neuenheimer Feld 590, 69120, Heidelberg, Germany

Authors and Affiliations

  1. Ultrasensitive Protein Detection Unit, Faculty of Chemistry and Mineralogy and Center of Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Universität Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany

    Kristin Dobslaff, Kristin Zscharnack, Thomas Kreisig & Thole Zuchner

Authors
  1. Kristin Dobslaff
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  2. Kristin Zscharnack
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  3. Thomas Kreisig
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  4. Thole Zuchner
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Correspondence to Thomas Kreisig.

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The authors declare no conflict of interest.

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Handling Editor: D. Tsikas.

T. Kreisig and T. Zuchner contributed equally to the study.

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Dobslaff, K., Zscharnack, K., Kreisig, T. et al. Rational design of a low-affinity peptide for the detection of cystatin C in a fast homogeneous immunoassay. Amino Acids 48, 479–486 (2016). https://doi.org/10.1007/s00726-015-2101-3

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  • DOI: https://doi.org/10.1007/s00726-015-2101-3

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