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Trastuzumab quantification in serum: a new, rapid, robust ELISA assay based on a mimetic peptide that specifically recognizes trastuzumab

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Abstract

Trastuzumab, a humanized monoclonal antibody directed against the epidermal growth factor receptor 2 (HER2), is a milestone in the treatment of HER2-overexpressing breast cancer patients. An enzyme-linked immunosorbent assay (ELISA) for trastuzumab has been developed for routine use in the laboratory to support clinical and pharmacokinetic studies to optimize therapy. The method relies on an antigen peptide linked to a 96-well plate via the streptavidin/biotin system. The peptide sequence mimics the extracellular portion of the HER2 receptor that is recognized by trastuzumab. The calibration range of the assay is 10 to 360 ng/mL per well, corresponding to a trastuzumab serum concentration from 5 to 180 μg/mL with a lower limit of quantification of 10 μg/mL. Validation results demonstrate that trastuzumab can be accurately and precisely quantified in human serum using this assay. The procedure was also tested in sera obtained from breast cancer patients to evaluate trastuzumab serum levels, confirming the applicability of method that could be a valid assay to use in daily laboratory practice.

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Abbreviations

BSA:

Bovine serum albumin

CV:

Coefficient of variation

ELISA:

Enzyme-linked immunosorbent assay

Fmoc:

9-Fluorenylmethoxycarbonyl

HER2:

Human epidermal growth factor receptor 2

IgG:

Gamma immunoglobulin

LLoD:

Lower limit of detection

LLoQ:

Lower limit of quantification

PBS:

Phosphate-buffered saline

pNPP:

p-Nitrophenyl phosphate

QC:

Quality control

ULoQ:

Upper limit of quantification

References

  1. Eisenhauer EA (2001) From the molecule to the clinic-inhibiting HER2 to treat breast cancer. N Engl J Med 344(11):841–842

    Article  CAS  Google Scholar 

  2. Leveque D, Gigou L, Bergerat JP (2008) Clinical pharmacology of trastuzumab. Curr Clin Pharmacol 3:51–55

    Article  CAS  Google Scholar 

  3. Maple L, Lathrop R, Bozich S, Harman W, Tacey R, Kelley M, Danilkovitch-Miagkova A (2004) Development and validation of ELISA for Trastuzumab detection in human serum. J Immunol Meth 295(1–2):169–182

  4. Damen CWN, de Groot ER, Heij M, Boss DS, Schellens JHM, Rosing HA, Beijnen JH, Aarden LA (2009) Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma. Anal Biochem 391(2):114–120

    Article  CAS  Google Scholar 

  5. Jiang B, Liu W, Qu H, Meng L, Song S, Ouyang T, Shou C (2005) A novel peptide isolated from a phage display peptide library with trastuzumab can mimic antigen epitope of HER-2. J Biol Chem 280(6):4656–4662

    Article  CAS  Google Scholar 

  6. De Silva B, Smith W, Weiner R, Kelley M, Smolec J, Lee B, Khan M, Tacey R, Hill H, Celniker A (2003) Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules. Pharm Res 20(11):1885–1900

    Article  Google Scholar 

  7. Food and Drug Administration (2001) Guidance for industry: bioanalytical method validation. http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf

  8. Bruno R, Washington CB, Lu JF, Lieberman G, Banken L, Klein P (2005) Population pharmacokinetics of Trastuzumab in patients with HER2+ metastatic breast cancer. Cancer Chemother Pharmacol 56(4):361–369

    Article  CAS  Google Scholar 

  9. Tokuda Y, Watanabe T, Omuro Y, Ando M, Katsumata N, Okumura A, Ohta M, Fujii H, Sasaki Y, Niwa T, Tajima T (1999) Dose escalation and pharmacokinetic study of a humanized anti-HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer. Br J Cancer 81(8):1419–1425

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The study was supported by AIFA (Italian Drug Agency) grant no. FARM6N78KN

Conflict of interest

All authors disclose no conflict of interest.

Author information

Author notes

  1. Andrea Armirotti

    Present address: Drug Discovery and Development, Italian Institute for Technology (IIT), 16163, Genoa, Italy

Authors and Affiliations

  1. Development of Innovative Therapies Unit, IRCCS AOU San Martino-IST, 16132, Genoa, Italy

    Barbara Cardinali, Andrea Armirotti, Giuseppina Iacono, Alessia Levaggi & Lucia Del Mastro

  2. Medical Oncology Unit, Sacro Cuore Don Calabria Hospital, Via Sempreboni, 5, 37024, Negrar, Verona, Italy

    Gianluigi Lunardi & Stefania Gori

  3. Department of Experimental Medicine (DIMES), Section of Biochemistry, University of Genoa, 16132, Genoa, Italy

    Enrico Millo & Gianluca Damonte

  4. Center of Excellence for Biomedical Research, University of Genoa, 16132, Genoa, Italy

    Enrico Millo & Gianluca Damonte

  5. Biopolymers and Proteomics Unit, IRCCS AOU San Martino-IST, 16132, Genoa, Italy

    Aldo Profumo

Authors
  1. Barbara Cardinali
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  2. Gianluigi Lunardi
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  3. Enrico Millo
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  4. Andrea Armirotti
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  5. Gianluca Damonte
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  6. Aldo Profumo
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  7. Stefania Gori
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  8. Giuseppina Iacono
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  9. Alessia Levaggi
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  10. Lucia Del Mastro
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Corresponding author

Correspondence to Gianluigi Lunardi.

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Cardinali, B., Lunardi, G., Millo, E. et al. Trastuzumab quantification in serum: a new, rapid, robust ELISA assay based on a mimetic peptide that specifically recognizes trastuzumab. Anal Bioanal Chem 406, 4557–4561 (2014). https://doi.org/10.1007/s00216-014-7842-4

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  • DOI: https://doi.org/10.1007/s00216-014-7842-4

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