The MAIIA technology
For rapid determination of protein isoforms


MAIIA and Isoforms

MAIIA (Membrane Assisted Isoform ImmunoAssay) is a novel proprietary technology for rapid and sensitive measurement of protein isoforms in biological specimens. Posttranslational modifications, like glycosylation, of a protein may result in a huge number of protein variants (isoforms) which might have high impact on its biological function. There are numerous reports on protein isoform distributions related to clinical effects, but there is a lack of suitable methods for measuring isoforms occurring at low concentration in blood and urine. The MAIIA technology seems to fulfil the requirements as a rapid and sensitive isoform determination method with ability to resolve and detect several types of posttranslational modified proteins even when they occur in femto-molar concentration.

 

Fig. 1: A glycoprotein with the peptide chain and the protruding carbohydrate structures can host and transfer large amounts of information. The carbohydrate structures are the fine-tuning regulators of protein activity.

 

 

Technology
An ion-exchange or affinity chromatographic separation of protein-isoforms and a sensitive immunoassay detection are combined on a few cm2 of a porous monolith chip. Thin lines of immobilized antibodies are used for specific capturing of target molecules, which then can be detected by the reaction with antibodies bound to carbon black nano-strings or other suitable labels. The bound label is quantified by the use of an image scanner. This technology can distinguish minor differences in protein carbohydrate structure and enables specific determination of proteins in a complex environment, requiring only a few femtogram of each isoform for its detection.

Fig. 2: An isoform separation zone containing ion-exchange or affinity ligands is combined with a capturing zone with immobilised specific antibodies. The separation and capturing process is performed on a few cm2 of a porous monolith chip during some minutes.

 

 

 

Fig. 3: There are different types of MAIIA chips. The 1D MAIIA (left) is a micro-column where isoforms interacting with the ligands in the separation zone are retarded. After having passed the separation zone, the weak binding isoforms will be captured and detected in the antibody zone. The result for the separation of EPO isoforms is shown in Fig. 5. The 2D MAIIA (right), with flow in various directions, can be used to measure isoform profiles as shown from the result for separation of transferrin isoforms in Fig. 6. The testing procedure only takes 10-20 min.


Applications

Several interesting analytes appear in low concentrations in biological specimens with extensive micro-heterogeneity. Determination of selected isoforms should give much higher diagnostic specificity. Prostate specific antigen (PSA), about 100 pM in serum, occurs mainly in complexes with other proteins. It appears as degradation products and as isoforms with differences in the carbohydrate chains and should be a better marker for prostate cancer if the aberrant glycosylated isoforms could be determined. Cardiac troponin I (cTnI), about 3 pM in serum, is released into the circulation after myocardial injury (MI). It appears in complexes with other proteins, and may undergo oxidation and phosphorylation as well as proteolysis after its release. The isolated measurement of recently released non-degraded isoforms should make possible earlier and more specific diagnosis. Erythropoietin (EPO), about 0.3 pM in urine, is a glycoprotein hormone with about 40% carbohydrate and several hundreds of isoforms appearing in samples from normal individuals. Methods for identification of aberrant EPO isoforms are useful for detection of doping with recombinant EPO, as the recombinant and endogenous iso-forms have different types of glycosylation.


Fig. 4: The components for the MAIIA tests, besides the chip with separation and capture zone, are chromatography using capillary flow; sandwich immunoassay with the use of antibodies bound to carbon black nano-strings as label; scanner for detection of bound carbon black intensity; and specially designed software.


1D MAIIA – measures the weak binding population
For EPO Doping tests it has been found that the 1D MAIIA test (see Fig. 3), using a MAIIA micro-column, rapidly can distinguish different types of EPO subpopulations. The separation zone can be a zone with anion exchange groups (see results in Fig. 5) or a zone with ligands like the lectin wheat germ agglutinin.

Fig. 5: A rapid and easy-to-use EPO doping test has been developed that can reveal the presence of aberrantly charged glycosylated EPO isoforms, like recombinant EPO, even when they appear in the low concentration range (down to 0.5 ng/L) of EPO found in urine specimens.


2D MAIIA – measures the isoform profile

The Carbohydrate deficient transferrin (CDT) population with asialo-, monosialo- and disialotransferrin constitutes about 3 % of the total transferrin population, but will increase after repeated too high alcohol intake. The major isoform population is tetrasialotransferrin which makes up 70-75% of all isoforms. The method, transferrin anion exchange MAIIA, with consequtive liquid flow in more than two directions (2D MAIIA), can efficiently separate the different transferrin isoforms in a chip of a few cm2. The results can be seen in Fig. 6.


Fig. 6: Partially purified transferrin isoforms were separated and specifically quantified by the anion-exchange chromatography MAIIA technology in less than 15 min.


Summary

Posttranslational modification (PTM), like glycosylation, results in a huge number of protein variants (isoforms). The distribution of isoforms seems to have high impact on the biological function as well as being an indicator of pathological conditions, but the lack of suitable methods hampers the clinical use of isoform based diagnostics. The combination of chromatographic separations and a sensitive immunoassay detection miniaturised in a small chip constitutes a promising isoform determination methodology.

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Lateral Flow Test using Carbon Black Nano-Strings


Introduction

Lateral flow test, also called immuno-chromatographic test, is a rapid and sensitive form of immunoassay for analysis in complex media. The reaction takes place in a thin porous matrix layered on top of an optical clear polyester film, mostly formed as a 5 mm wide and 50 mm long strip (see Fig. 1). The analyte is transported from an application zone along the membrane and binds to an immobilised antibody in a combined capture and detection zone. Another antibody, directed towards a different epitope on the analyte, is labelled with a coloured particle, and then transported through the capture zone where it binds to the analyte in the immuno-complex. The non-bound particles are washed away with the passing liquid flow.

Carbon black particles of the type used in the printing industry have an intensive black colour and can be modified to bind antibodies with preservation of their activity. These particles can be used to construct tests with high sensitivity for visual detection and semi-quantitative determination. In combination with a common scanner the test can be made fully quantitative. MAIIA Diagnostics provides different types of specially selected and designed (modified) carbon black nano-string particles which show much higher sensitivity than commonly used coloured polystyrene or gold particles.

This technique can be used for rapid and extremely sensitive analysis of low concentrations of substances in complex media such as urine and serum. MAIIA Diagnostics’ lateral flow test for erythropoietin (EPO) shows a detection limit of 1.2 femtomolar, 0.035 ng EPO/L.

Fig. 1: The lateral flow strip with the application zone and the combined capture and detection zone is shown to the left. The same strip after performing the test using 25 µl of sample with a concentration of 1000 ngEPO/L is shown on the right.

 

 


Carbon Black Nano-Strings as a label

About 100 types of carbon black are currently found on the market, each with its own specific profile of characteristics. The particles differ in size, structure and surface groups. The particle consists of chained or cluster like branched aggregates of roughly spherical particles, the primary particles. These primary particles, with sizes ranging from 10-500 nm, are fused together in the production-process, so the final particle may occasionally consist of several hundreds of primary particles in a string.

On the surface of the carbon black particle there exists different functional groups containing oxygen such as quinones which can react with e.g. amino groups in the proteins under formation of covalent bonds. The surface also binds proteins via other interactions. The particles have very large surface area and can bind high amounts of antibodies.

The black intense colour, the high “molecular weight” and chainlike structure makes carbon black nano-strings an excellent label for lateral flow tests. MAIIA Diagnostics provides specially produced carbon black suspensions suitable for labelling with antibodies.

 

Fig. 2: Carbon black nano-strings covered with antibodies.


Detection with scanner

Scanners for image processing have during the past few years rapidly become very affordable and it is now possible to buy high quality scanners for less than $500. The technical specifications of interest in the scanners are the optical and greyscale resolutions as well as the quality of the CCD detectors. An optical resolution of 4,800 ppi will show 189 pixels/mm or 35,721 pixels/mm2. A greyscale resolution of 16-bits allows each pixel to be represented by 65,536 different intensity levels. MAIIA Diagnostics has developed a special software which locates the capture zone and calculates the intensity of the captured anti-EPO carbon black nano-string. With an Epson Expression 1680 it is possible to quickly scan surfaces and analyze the signal intensity with very good precision. It is in fact possible to detect 0.02 attomol carbon black/mm2 by the use of a scanner. This corresponds to 22 carbon black nano-strings molecules per 42 µm pixel.

Fig. 3: An ordinary image scanner can be used as a powerful detection instrument.


Software for quantification of signal intensity

Software developed for quantification of the signal on the lateral flow test is available from MAIIA Diagnostics (see Fig. 4). The program transfers the information from each pixel to blackness intensity values (between 0 and 65,535 for 16-bit resolution). It searches within the capture zone for the peak value as well as outside for a suitable base value and gives the signal intensity in delta blackness per pixel. It also displays the image of each strip together with its profile making it easy to verify the correctness of the automatic search as well as detecting any anomalies. The signal intensities of the bands from hundreds of strips can be quantified in minutes.

Fig. 4: The software quantifies the signal intensity for each strip and displays its image and zoomable profile. Also shown is the overview of all strips in the scan-serie to correlate location of found peak values. Each strip is added to the list and has a peak, base and delta value as well as real location and precision of the values, all easily transferred to Microsoft Excel for further calculations.

 

Dilution curve for EPO in buffer
The results when analyzing 25 µl samples of a dilution serie of 3-1000 ng/L of EPO in buffer is shown in Fig. 5. With MAIIA’s technique for lateral flow test it takes less then 20 min. to simultaneously process up to 60 strips.

Fig. 5: Results from measuring samples of a dilution serie of 25 µl EPO in buffer, using the 15 min. lateral flow test procedure and scanner detection.

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Protein Isoform Determination


Protein isoforms

Posttranslational modification (PTM), like glycosylation, results in a huge number of protein variants (isoforms) showing micro heterogeneity of different kinds. The dominating heterogeneity is found in the carbohydrate moieties of glycoproteins. Other examples are genetic polymorphism, modified functional groups in amino acids by e.g. phosphorylation, acetylation, or limited proteolysis. No protein seems to be homogenous and many proteins show a huge number of different isoforms. The distribution of the isoforms might change due to physiological regulation and pathological processes and the distribution pattern seems to have high clinical significance. Production of a typical pattern from one cell-type may affect the whole isoform distribution in the circulation. The glycosylation of proteins seems to be an important regulator for the fine-tuning of biological activity. Isoforms show various interactions with receptors which also can affect their survival time. In cancer, inflammation or liver diseases, the protein glycosylation pattern is typically changed at an early stage of the disease. The need for protein isoform detection ranges from high abundance proteins like haemoglobin (10^-3 M) down to proteins like erythropoietin (10^-15 M).


Protein isoform determination

The distribution of isoforms seems to have high impact on the biological function as well as being an indicator of pathological conditions, but the lack of suitable methods hampers the clinical use of isoform diagnostics.
Few quantitative methods are available for determination of isoforms, especially for measurement of low concentrations in biological specimens. A combination of discrete steps of separation (electrophoresis or chromatography) and detection (immunological technique) is used. The quality of the test depends on the type of specific isoforms that can be distinguished, how efficiently these isoforms are separated (resolution), and how well they can be quantified (specificity and sensitivity). The electrophoretic or chromatographic separations in columns are gentle techniques which do not require extensive pre-preparation of the biological sample. The advantage with chromatographic separation is the possibility to use different ligands for isoform separation. Analysis of the fractions using immunoassay is quantitative, sensitive and specific, although quite expensive. Slab-gel and capillary electrophoresis separate isoforms due to charge differences but lack suitable means to quantify and specify correctly. Mass spectrometry detection does not solve these specificity and quantification problems, especially not when analysing low abundance glycoproteins. An affinity based pre-treatment step that specifically captures the protein of interest and concentrates it in relation to other proteins will enhance the possibilities to use these types of methods. None of the available techniques is suitable for high-throughput testing or for point-of-care applications. The chromatographic approach, utilizing selected ligands for separation, in combination with immunoassay seems to be the best way for quantitative isoform detection but a miniaturisation is needed.


Urgent need for better methods for isoform analysis

Several interesting and valuable analytes appear in low concentrations and show extensive micro-heterogeneity which causes variable results when using available test kits. Prostate specific antigen (PSA), about 100 pM in serum, appears mainly in complexes with other proteins. It shows degradation products and differences in the carbohydrate chains and should be a better marker for prostate cancer if the relevant isoform pattern, including glycoforms, could be determined. Cardiac troponin I (cTnI), about 3 pM in serum, is released into the circulation after myocardial injury (MI). It appears in complexes with other proteins, and may undergo both oxidation and phosphorylation as well as proteolysis after release. cTnI is a marker with wide diagnostic window, but abnormal levels appear late after the onset of MI. The isolated measurement of early released non-degraded isoforms will most probably result in earlier diagnosis. Erythropoietin (EPO), about 0.3 pM in urine, is a glycoprotein hormone with about 40% carbohydrate and probably several hundreds of isoforms appearing in normal samples, and as such, a challenging analyte. Methods for identification of aberrant EPO isoform profiles have been developed for identification of illegal use of recombinant EPO, which enhances the performance during sport competitions. Profiles of differently charged EPO isoforms can be shown by column electrophoresis combined with sensitive immunoassay quantification of the fractions. The presently accredited EPO doping test is using isoelectric focusing and a double immunoblotting technique. These tests take several days to perform and are very expensive.


Development of new methods

MAIIA (Membrane Assisted Isoform ImmunoAssay) is a novel lab-on-a-chip based technology for rapid and sensitive measurement of protein isoforms in biological specimens. The chromatographic zone can be provided with suitable ligands, such as lectins and receptors, as well as charged ones, and the porous monolith shows excellent chromatographic properties. The technology has been utilized to show the transferrin isoform profile, to measure carbohydrate-deficient transferrin as well as to distinguish low amounts of recombinant and endogenous EPO present in urine. The MAIIA technology seems to fulfil the requirements as a rapid and sensitive isoform determination method with potential to resolve and detect several types of PTM isoforms even when they appear in the femto-molar (10^-15 M) concentration range.

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