MAIIA Diagnostics
Rapid EPO MAIIA doping test


Introduction

Recombinant human erythropoietin (rhEPO) has been widely used for doping in endurance sports. In spite of the obvious need, there exists presently no easy-to-use method for EPO doping testing. Conventional immunoassays e.g. cannot distinguish the recombinant forms from the endogenous ones. rhEPO differs from the endogenous form by its aberrant glycosylation pattern and this is the basis for the presently used isoelectric focusing technique. However, this technique is time-consuming, expensive, and not suitable for EPO doping screening.

The novel MAIIA technology

The MAIIA technology, which combines chromatography (anion-exchange or affinity) with a sensitive EPO immunoassay in one device, seems to fulfil the requirements for an easy-to-use rhEPO doping test. It can also be used to rapidly measure several samples simultaneously, and thus be used for screening large numbers of specimen.



Presently used method for detecting EPO doping

The WADA-accredited EPO doping method combines isoelectric focusing (IEF) with double immunoblotting performed as described in F. Lasne, J. de Ceaurriz, 2000, Nature 405, 635.


Day 1:
  • Centrifugation, filtration and ultrafiltration to concentrate the urine ~1000 times (20 ml => 20 µl).
  • Measuring EPO concentration by EIA.
  • WGA sepharose purification, if required.
Day 2 (12 h):
  • Isoelectric focusing in 7 M urea.
  • EPO blotting and reaction with anti-EPO.
  • Anti-EPO blotting.
  • Reaction with biotin-anti-mouse IgG.
Day 3:
  • Reaction with streptavidin-peroxidase.
  • Reaction with chemiluminiscent substrate.
  • Chemiluminiscent detection of isoform distribution (expensive detection equipment).
Fig. 1: Results from the IEF based method for detection of EPO doping in which the EPO isoforms are separated due to their isoelectric point. In the left lane the band pattern for endogenous urine EPO is shown. In the right lane the less negatively charged rhEPO (alpha and beta mixture) with the bands found near the cathode is shown. Also the highly negatively charged darbepoetin alpha (Aranesp) with bands near the anode is shown in the right lane.


Procedure for EPO doping test

A MAIIA technology procedure that meets the requirements for the doping laboratories has been developed. By using the MAIIA micro-column strips, 56 samples can be processed in 20 minutes. The MAIIA technology can also be implemented into automatic large scale analyzers.
  1. Preparation of urine sample by anti-EPO affinity purification => Concentration of EPO from 5 ml urine to 200 µl eluate in buffer with concomitant removal of other urine proteins.
    Total time: ~ 20 min.
  2. The MAIIA micro-column strip is placed in a well with 25 µl affinity concentrated sample. All EPO is captured in the first few mm:s of the separation zone (see Fig. 3, 4).
  3. When the entire sample has been aspirated, the strip is moved to another well where the captured EPO is desorbed during 5 min, using a buffer that enables separation of selected EPO forms. When measuring the total EPO content (all isoforms), a buffer that desorbs all forms of EPO is used.
  4. The piece of the strip with the separation zone (the bottom part) is cut off and discarded.
  5. The remaining part (the top part) of the strip, with the antibody line, is moved to a well filled with carbon black nano-string anti-EPO, which is allowed to migrate along the membrane for 5 min.
  6. The strip is moved to a well filled with washing solution, which is allowed to migrate during 5 min.
    Total time (2-6): ~ 20 min.
  7. The intensity of the obtained gray to black band is quantified by use of a scanner. The total EPO concentration (obtained when using a buffer that desorbs all EPO) is calculated as well as the concentration for the selected isoforms after the MAIIA procedure. A ratio is calculated = (concentration of less binding EPO isoforms / concentration of total EPO isoforms) x 100 = % less binding EPO.





Fig. 2: Procedure for EPO doping test.



MAIIA micro-columns

The key component in the MAIIA test is the MAIIA micro-column (see Fig. 3, 4) which contains immobilized ligands. The micro-column shows excellent chromatographic performance and separate isoforms during their migration along the 125 µm thick and 5 mm wide porous bed. The isoforms are first chromatographically separated in the separation zone (containing charged groups or lectin ligands) and the isoforms that have passed the separation zone are then captured by the anti-EPO antibodies immobilized in the following detection zone and determined by the novel technology using anti-EPO Carbon Black Nano-Strings.

Results with the EPO MAIIA technology

Urine from healthy normal individuals and from patients receiving recombinant EPO injections due to anaemia was analysed with two varieties of the rapid and novel EPO MAIIA technology that can distinguish EPO isoforms (see Fig. 5, 6).
The MAIIA micro-column that separates due to charge (anion exchange) retards the endogenous forms more efficiently than the recombinant forms, as the carbohydrate groups on endogenous EPO contain more negatively charged structures like sialic acid and sulfonated groups. The micro-column with WGA in the separation zone interacts with other structures in the carbohydrate chains by which the rhEPO isoforms interacts to a higher degree than the endogenous isoforms.



Fig. 3: The EPO anion exchange MAIIA column has a 15 mm separation zone containing positively charged ligands where the migration rate for isoforms containing more negatively charged groups is slowed down.
Fig. 4: The EPO WGA MAIIA column has a 5 mm separation zone with the lectin, wheat germ agglutinin (WGA) immobilized. Interaction with the WGA ligands reduces the migration rate for isoforms containing more WGA binding carbohydrate groups.



Fig. 5: The EPO anion exchange MAIIA isoform test distinguishes recombinant from endogenous EPO due to their differences in charge. Recombinant isoforms are less negatively charged than endogenous ones.
Fig. 6: The EPO WGA MAIIA distinguishes recombinant from endogenous EPO due to their differences in interaction with the lectin wheat germ agglutinin (WGA). Endogenous isoforms interact less with WGA than recombinant ones.



Summary

The EPO MAIIA test can rapidly distinguish the endogenous EPO isoforms from recombinant isoforms like epoetin alpha, beta, omega, delta and EPO analogues like darbepoetin alpha and CERA (pegylated epoetin). Even when EPO is appearing in such low concentration in urine as 0.5 ng EPO/L it is possible to reveal the isoform characteristics.
The MAIIA technology, which allows chromatographic separation using several types of ligands, is very suitable for EPO doping testing and its flexibility is necessary to meet the test requirements for the new generations of different erythropoiesis-stimulating agents.