Detection of EPO injections using a rapid lateral flow isoform test.

Misuse of recombinant human erythropoietin (rhEPO) is a major concern in competitive sports, and the implementation of tests allowing for higher detection rates than what current tests are capable of is required. In this study, a novel lateral flow EPO isoform test kit, EPO WGA MAIIA, is evaluated on the basis of plasma and urine samples obtained from eight healthy males in connection with a 28-day rhEPO injection period. rhEPO was injected every other day during the first 14 days of the study, and the method proved to be 100% effective in detecting rhEPO in the concomitantly obtained samples. Seven days after the last injection, three positive (>99.99% confidence limit (CL)) subjects were found. When using 99% CL as the cut-off limit, six of the eight subjects (75%) were found to be suspected of doping. Samples obtained 14 and 21 days after the last injection showed no detectable trace of rhEPO. A previous study using indirect methods to determine EPO doping on the same samples indicated only that two of the subjects had suspicious values 7-21 days after the last injection. We propose implementing the easy to-use EPO WGA MAIIA test as an initial screening procedure in anti-doping work to (1) increase the detection rate of potential rhEPO doping athletes and (2) allow for a 10- to 20-fold higher analytical rate than what is possible today.

Lectin affinity chromatography as a tool to differentiate endogenous and recombinant erythropoietins.

This work exploits the combination of the lectin affinity chromatography (LAC) with an ultra-sensitive immunochromatographic assay to differentiate several types of erythropoietin (EPO). The chromatographic behaviours of different commercial types of recombinant human EPO (rhEPO), EPO analogues (Aranesp) and urine human EPO (uhEPO) from healthy individuals on eight lectin-Sepharose columns, have been worked out. Results show that when using wheat germ agglutinin (WGA)-Sepharose columns, a careful desorption regime starting with very low concentration (2mM) of the competitive sugar N-acetylglucosamine (GlcNAc) makes it possible to efficiently distinguish endogenous EPO from recombinant EPO and EPO analogues.

Chromatographic performance of a thin microporous bed of nitrocellulose.

Chromatography along thin (125 microm) porous beds of nitrocellulose, layered on top of an polyester backing, shows good separation efficiency with plate heights of 10-20 microm. Flow is controlled by capillary forces and shows low rate variations between the individual disposable devices. Positively charged groups were introduced into the nitrocellulose and efficient separation of transferrin isoforms, differing by only 0.1 pI units, was found after a short migration distance (1 cm). The upper surface is not covered, which allows sample and reagents to be added, and the clear backing permits detection. The chromatography can easily be combined on-line with sensitive immunoassay detection down to the microM (10(-12) M) range. This microscaled combination device should have a wide range of applications in analytical biochemistry.

Quantitative detection in the attomole range for immunochromatographic tests by means of a flatbed scanner.

This work describes the use of the combination of carbon black as an antibody label, a membrane-based immunochromatographic device, and a flatbed scanner as a quantitative test system. The scanner detected 0.4-345 ng carbon black/mm(2) on a nitrocellulose membrane (0.2-170 amol carbon black/mm(2)) with an imprecision (coefficient of variation, CV) lower than 2% for the carbon black determination and a detection limit of 0.04 ng carbon black/mm(2) (0.02 amol/mm(2)). The detection ability was compared to that obtained with alkaline phosphatase (ALP) using a substrate yielding a chemiluminescent signal (0.02 amol ALP/well), beta-galactosidase using a substrate yielding a fluorescent signal (0.3 amol beta-galactosidase/well), and horseradish peroxidase (HRP) using a substrate yielding a colored signal (5 amol HRP/microtiter well). The carbon black immunochromatographic test for immunoglobulin E (IgE) showed a detection limit of 0.13 pM IgE (0.01 kU/L) after a testing time of 10 min. The scanner detection imprecision for the IgE determination was 0.6% CV in the range 1-10 kU IgE/L when 2.3 mm(2) was used for detection and 1% CV when 0.19 mm(2) was used. A flatbed scanner is an inexpensive instrument with multiple uses, which now also includes the sensitive evaluation of immunoassays.

Membrane assisted isoform immunoassay. A rapid method for the separation and determination of protein isoforms in an integrated immunoassay.

Proteins often exist as isoforms with structural microheterogenity due to, for example, small variations in their carbohydrate structure. This can give rise to differences in biological activity. Measurements of low concentrations of such isoforms are usually performed by complicated methods, which demand discrete steps of separation (chromatographic or electrophoretic) and immunodetection. In this paper a new, highly specific and rapid (<15 min) analytical technique is described which permits the quantitative determination of several types of protein isoform. The method, which is called membrane assisted isoform immunoassay (MAIIA), is based on separation (by ion-exchange or affinity chromatography) and immunoassay detection of the protein isoforms in the same lateral flow, assisted by capillary forces in a membrane device. The technique is exemplified with a method for measuring carbohydrate-deficient isoforms of the glycoprotein transferrin, where the measured isoforms constitute a minimal part (<3%) of the total amount of transferrin. The time for the measurement was about 10 min and the correlation coefficient with an established, commercial two-step procedure, which takes 4-5 h to perform, was 0. 99. The detection limit was about 1 fmol of transferrin. The anion-exchange membrane used in the test device had the ability to separate transferrin isoforms with down to 0.1 unit difference in pI. The results indicate that the technique should be useful for rapid point-of-care testing of clinically interesting charged protein isoforms.