Immunometric test for banknote specific antigens: development and applications

Preparation of antigen for immunization and analysis of cross-reactivity
Soluble antigens of Euro (EUR10), US dollars (USD20) and Russian roubles (RUR100) were prepared from 20 notes of each sort. The banknotes were thoroughly washed from external contamination three times in 0,5 liter of 10mM phosphate-buffered saline + 0,1% Tween 20, ðÍ 7.4 and then three times by 0,5 liter of distilled water. Then banknote were homogenized by kitchen blender in 0.5 l distilled water with 0.1% sodium azide; the homogenate was spun down 30’ at 10000 g to remove debris, the supernatant was concentrated by evaporation at 20-25C to 50 ml volume.

Preparation of antigen for analysis
Small portion of tested banknote (approximately 25% of total mass) was cut by scissors, washed thoroughly three times in 0,5 liter of 10mM phosphate-buffered saline + 0,1% Tween 20, ðÍ 7.4 and then three times by 10 ml of distilled water. Then the piece of banknote was homogenized by laboratory blender in 5 ml of distilled water with 0,1% sodium azide; the homogenate was spun down 5’ at 10000 g, the supernatant stored at 4C before analysis.

Immunizations of rabbits resulted in obtaining of medium affinity antisera showing specific reaction towards the extracts of Euro and Russian roubles in direct adsorption ELISA. Antisera to EUR and RUR shown very high degree of cross-reactivity; we suggest that these two currencies share common antigen(s). None of rabbits shown the immune response to USD extract. The extracts of other papers were used as controls in direct binding ELISA.

Competition ELISA kit was constructed using rabbit anti-EURO antiserum and microwell-coated EURO extract. A figure below shows the high homology of EUR and RUR extracts as calibration materials, and few examples of different sample extracts – both good and apparently fake.

World currencies in XEMA Euro ELISA
The use of XEMA Euro/Rouble ELISA not only allows to distinguish fake Euro and RUR banknotes, but also to compare different banknotes by the content of EUR/RUR antigen and make suggestions about their common or different recipes.
The data on cross-reactivity (“EURO exchange rates”) are summarized in attached file.

Immunomoneychemistry: immunostaining of banknotes by specific antiserum
The immunomoneychemistry was performed similarly to immunohistochemistry: the banknotes were incubated for 1 hour at 37 Ñ in Petri dishes in 25 ml of blocking solution for IHC, SH001. Then the blocking solution was removed by decantation; a control part of the banknote was incubated for 2 hours at 37 Ñ in 1:100 dilution of normal rabbit serum; the immunostained part was incubated in working dilution of specific antiserum. Unbound material was removed by 5x washing by 0.1M phosphate buffered saline. Sheep anti-rabbit IgG-HRP conjugate (AS301-HRP) in working dilution was then added as secondary antibody; after subsequent 5x washing a precipitating TMB substrate (Sigma) was added for 15’ at 25C. Immunostained banknotes were then washed by distilled water, dried and photographed (see below).
Two variants of banknote cutting were used to avoid possible heterogeneity of target antigen content throughout the surface of the note.

Upper OR right half - control
Lower OR left half - immunostaining