The present invention pertains to a quantitative assay for determining the amount of a biological substance in a liquid sample. The invention also pertains to a kit comprising constituents needed for performing the assay.

It is common to apply quantitative assays to determine the amount of a biological substances in a liquid sample, for example an enzyme immunoassay (EIA), and in particular an enzyme linked immunosorbent assay (ELISA) are well known. The biological substance quantified is for example a molecule, a particle, a micro-organism or a composition comprising one or more of these, which can each be of substantially biological or biotechnological origin. In particular such assays are used to detect proteins, viruses or virus-like particles. The known EIA's are based on a specific antigen-antibody binding, i.e. an 'immunological binding'.

An EIA can be used for most types of liquid biological samples, such as serum, liquor, tears, saliva, urine, or extracts or homogenates of tissues or cells. Such assays commonly use a test-plate being a flat plate having multiple wells used as small test tubes, e.g. a microtitration plate. The wells are coated either with the antigen (the target biological substance), or with a primary antibody which recognizes the antigen and binds with it. Next the antigen-antibody complex is in turn recognized by a secondary antibody which is bound to a marker molecule. The marker is then used to perform a reaction in an incubation mixture in the well, in order to allow the detection of the secondary-, and thus of the primary antibody and the antigen. Commonly the marker is an enzyme (although other marker molecules can be used as well) which can convert a substrate to generate a specific colouring reaction. By measuring the intensity (the optical density, OD) of this colour by spectrophotometry, the presence and quantity of a specific substance can be determined and quantified.

Many different types of EIA's are described, for example "Direct-", "Indirect-" and

"Sandwich-" ELISA (see: Essential Immunology, by Ivan Roitt, Blackwell publ., seventh edition, 1991 , pages 96-101); "Competitive" ELISA (see "ELISA: Theory and Practice", in: Methods in Molecular Biology, 1995, Vol. 42, p. 177-205) and "Reverse" ELISA (see EP 1.499.894).

Although the known immunosorbent assays are adequate to quantify the amount of a biological substance in a liquid sample, a major disadvantage is that such assays require antibodies to obtain a specific binding to the biological substance. These antibodies need to be generated in an animal and must then be purified from its serum. This inherently is a labour intensive, complex and expensive procedure, which may also raise ethical concerns.

A further disadvantage is that it is not always possible to raise the required antibodies, as some biological substances may simply not be immunogenic enough to induce the required (level of) antibodies, and others may be too toxic for the animal that is used to produce the antibodies.

The present invention has as object to at least mitigate the disadvantages of known quantitative immunological assays.

It has now surprisingly been found that an assay could be devised to quantify the amount of a biological substance in a liquid sample, which assay does not rely on the specific binding by an antibody.

Therefore in one aspect the invention relates to a method to quantify the amount of a biological substance in a liquid sample, comprising the steps of: adding heparin to a sample; incubating the sample in order to allow the biological substance to bind to the heparin to form a complex therewith; and after that: determining the amount of the complex being formed, using a detectable marker that is operatively bonded to the heparin via a non-immunological binding.

For the invention, the biological substance is dissolved and/or dispersed in the liquid to be tested.

The 'binding' between heparin and biological substance to form the 'complex' can be via hydrogen bridges, Vander Waals forces, electrostatic forces, entanglements, etc., and/or a combination of various types of covalent bonds, all common for the binding between biological molecules.

Similarly, the marker is 'operatively bonded' to the heparin in any covalent or non- covalent way that allows the effective performance of the method according to the invention. The marker can be bound to the heparin directly or via an intermediary molecule or structure. For the invention, a 'non-immunological binding' is a binding not involving the specific binding by an antibody. Therefore, a non-immunological binding is an inter-molecular interaction which does not involve the binding of an immunoglobulin molecule.

Ofcourse an antibody molecule may be present in the sample or the test liquid as it comes from a biological source, however the antibody should not be a critical part of the quantitative assay.

'Heparin' is a naturally occurring mixture of highly sulphated mucopolysaccharides, and commonly known for its affinity to a wide range of structures such as proteins, viruses and virus-like particles; see i.a.: J. of Chromatogr. A., 2010, vol. 1217, p. 3489-3493;

Biochimica et Biophysica Acta, 2003, vol. 1620, p. 225-234; Glycobiology, 2007, vol. 17, p. 1094-1102; Clinica Chimica Acta, 2008, vol. 388, p. 173-178; J. of Virology, 2001 , vol. 75, p. 1565-1570; Virus Research, 2004, vol. 105, p. 107-112; J. of Virology, 2006, vol. 80, p. 3487-3494; J. of Biotechnology, 2007, vol. 131 , p. 309-317; J. of Chromatography B, 2007, vol. 846, p. 184-194.

Since heparin binds to various and very different substances, the art has never recognized that heparin can be used as the critical compound to quantify the presence of a specific biological substances in a sample; in particular in combination with a marker that is operatively bonded to the heparin via a non-immunological binding, i.e. via a binding process that does not involve the specific binding of an antibody to an antigen.

Due to its broad binding properties, heparin has never been contemplated as an entity useable for quantification in an assay (either directly or indirectly), in order to determine the amount of a known biological substance in a sample.

It was applicant's recognition that a quantitative assay can be devised using heparin that binds to a biological substance, and a marker operatively bonded to the heparin.

This recognition is based on a combination of insights. The first insight being that the binding of heparin to a biological substance, is particular and repeatable, i.e. it is a specific process, clearly distinct from an a-specific, or random-like process.

Secondly, applicant recognised that in a sample where heparin binds to a biological substance that needs to be quantified, part of the heparin will form a complex with the biological substance to be quantified, and part of the heparin will not be complexed, and the balance between the complexed and the non-complexed heparin depends on the amount of the biological substance that is present in the sample.

Combined this led to the advantageous application of this discovery in a quantitative assay for a biological substance, which does not involve binding by an antibody but by heparin, which is then detected via an operatively bound marker. For the present invention, the heparin that is complexed to the biological substance to be quantified is referred to as the "complexed heparin", and the heparin not complexed with that substance is referred to as the "non-complexed heparin". Of course the non- complexed heparin can during the course of the assay become complexed with a compound other than the biological substance that is to be quantified, but it remains non-complexed in respect of the biological substance to be analysed.

Not only is heparin much cheaper than antibodies, and is generally available, it has a further advantageous characteristic: given it's versatility in binding properties, heparin can be used for many completely different types of quantitative assays. For example: heparin is known to bind differently to active and inactive forms of the same proteins (Heger et al., 2002, Thrombosis Research, vol. 106, p. 157-164). The current invention therefore enables a quantitative determination of the presence of both forms in a mixture.

Also, heparin is known to bind differently to correctly and incorrectly formed virus like particles (Rommel et al., 2005, J. of Med. Virology, vol. 75, p. 1 14-121). The current invention thus enables quantification of the presence of such particles and even allows discrimination between correctly and incorrectly formed particles. Indeed, many embodiments of the present invention can be devised and put to practice by routine techniques by a person skilled in the art; for example as based on the ELISA methods as mentioned here-above: "Direct", "Indirect", "Competitive", "Reverse", etc..

It is noted that heparin has been used in quantitative assays before, but only as a mere a-specific coating to immobilise a biological substance to be quantified. For example, Najjam et al. (Cytokine, 1997, Vol. 9, p. 1013-1022), used heparin to bind interleukin-2 (IL- 2) to a test-plate, whereafter the amount of interleukin was quantified. However this applied the classic ELISA approach wherein a mouse monoclonal anti-IL-2 antibody was used to provide a specific immunological binding, which was thereafter made detectable by incubation with 'ABTS' substrate.

In the known EIA's, in contrast with the present invention, one relied on markers that were operatively bonded to the biological substance via a specific antibody-antigen interaction (i.e. an immunological binding), as a way to quantify the biological substance.

However, relying on the non-immunological type binding of heparin, and detecting that via a marker bound to the heparin, was never described nor suggested. The marker for the invention can be any molecule that can serve as a marker in the method according to the invention, either as such, or after processing. The marker can for example be one of the known enzymes used in known ELISA methods, such as a peroxidase or an alkaline phosphatase.

The marker can be bound to the heparin after the complex between biological substance and heparin has been formed, by binding to the complexed heparin, or to the non-complexed heparin, or to both.

However, in an embodiment of the present invention the marker is already bound to the heparin before the complex between heparin and biological sample is formed.

Not only does this remove one process step from the assay, but in addition this improves the reliability of the assay since the heparin-marker binding can be formed outside the scope of the assay being performed, under conditions that are optimal to the forming of that bond.

The assay according to this embodiment, employing pre-marked heparin, is tentatively called: enzyme linked binding assay, or: "ELBA". In an embodiment the marker bound to heparin is an enzyme, for example horse-radish peroxidase such as is commonly used in known EIA's.

Applicant surprisingly found that it is more convenient to detect the marker bound to non- complexed heparin, than it is to detect the marker bound to complexed heparin. A reason for this is that the non-complexed heparin can conveniently be immobilised by a reactant, which allows the complexed heparin to be removed.

Since the amount of non-complexed heparin directly depends on the amount of the complex being formed, this is an advantageous way to determine the amount of a biological substance present in a sample.

Therefore, in a further embodiment allowing the determination of the amount of complex between heparin and biological sample being formed, the non-complexed heparin is detected.

This can conveniently be done by separating non-complexed heparin from the complexed heparin, by using a reactant that binds the non-complexed heparin, and coating that reactant to a well of a test-plate; after an incubation of test sample and heparin in a well coated in such a way, the complexed heparin, which does not bind to the reactant, can be washed away. In a further embodiment the complex of heparin and biological substance is being formed in a separate reaction vessel, after which at least part of the sample is transferred to a container comprising a wall having attached thereto a reactant that binds the non- complexed heparin. Such a container might for example be a microtitration plate having wells coated with the said reactant.

The separate and prior formation of the complex has the advantage that it allows for a (virtually) complete complex formation, which adds to achieving a reliable quantification.

In yet a further embodiment, the container is rinsed after the non-complexed heparin is bound to the reactant, to remove unbound complexed heparin. With this additional step, it is ensured that in the detection step, only the amount of the non-complexed heparin is determined, via the marker operatively bound to the heparin.

The reactant can in principle be any molecule suitable for the intended purpose, with the exception of an antibody.

In an embodiment the reactant is in essence the same as the biological substance to be quantified. This effectively enables a non-immunological assay, which is the equivalent of the known "Competitive" ELISA, without the need for a specific antibody and its inherent disadvantagous.

By performing the methods and their steps according to the invention one performes a biochemical test that embodies the quantitative assay that is within the scope of the invention.

Therefore another aspect of the present invention relates to an assay to quantify the amount of a biological substance in a liquid sample comprising performing the methods according to the invention.

In practice the assay according to the invention will also incorporate steps to the collection of the results of the method performed, as well as the subsequent analysis and interpretation thereof.

Conveniently such interpretation of results can be done by the incorporation in the methods of standardised reference samples.

The skilled artisan will appreciate that the different biological samples that will be tested with the methods of the assay as described herein, may require individual optimisation of the details of the assay. However, with the details and examples provided herein, it is well within the routine capabilities of the skilled person to make such adaptations on the methods described. For example variations to the incubation conditions can be fine-tuned by adapting temperature, duration, and buffer; similarly, alternatives to the marker and reactant as described can be tried and tested by adapting their concentration or form.

In another aspect, the invention relates to a kit to perform the method of the quantitative assay as described here-above. The kit comprises heparin, a marker for the heparin, which marker can be operatively bonded to the heparin via a non-immunological binding, and optionally instructions how to perform the method.

In an embodiment the kit may comprise a container having multiple wells, such as a microtitration plate.

The wells of the container may be treated such that non-complexed heparin can be bound (immobilised) directly to the wells, for example via hydrogen bridges, Vander Waals forces, and/or electrostatic forces, etc.. Alternatively, the wells may be treated with a coating comprising a reactant.

In an embodiment the marker is an enzyme, for example a peroxidase or an alkaline phosphatase.

The instructions optionally comprised with the kit according to the invention, may for example be written on a box containing the constituents of the kit; may be present on a leaflet in that box; or may be viewable on, or downloadable from, an internet website from the distributor of the kit, etc.

For the invention the kit may also be an offer of the mentioned parts (relating to commercial sale), for example on an internet website, for combined use in an assay comprising the methods according to the invention.

To demonstrate that a quantitative assay for a biological substance in a liquid sample can be based on methods comprising the specific binding of heparin in stead of an antibody, the invention will be further explained by the following embodiment as a detailed example. Legend of Figures:

Figure 1 : The linearity of an assay comprising the methods according to the invention is demonstrated by the graphical representation of the results of three runs of an ELBA in which the same sample was tested in five concentrations.

EXAMPLE Introduction

The biological substance to be quantified in these experiments was recombinant expressed ORF 2 protein of porcine circovirus type 2 (PCV2), such as is present in the commercially available vaccine Porcilis® PCV (available from Intervet/Schering-Plough Animal Health, Boxmeer, the Netherlands). The assay used was an enzyme linked binding assay (ELBA) using horse-radish peroxidase (HRP) marked heparin, which enabled quantification via optical density measurement.

Materials

Heparin had been obtained and purified from porcine intestinal mucosa. Such heparin is commercially available i.a. from Merck KGaA, Darmstadt, Germany.

Heparin-HRP was made by the method as described by Ueda et al. (1999, Natural

Science Report, Ochanomizu University, Vol. 50, No. 1. p. 31-45).

A solution comprising PCV2 ORF 2 protein as an antigen (hereafter called "PCV2 antigen") was obtained by a method as described in WO 2007/028823, in particular as described in Examples 2 and 3, on pages 8 and 9.

Also a commercial vaccine was used which comprises the PCV2 antigen in an oily emulsion: Porcilis® PCV. Methods

PCV2 ELBA

Test-plates were coated with PCV2 antigen as follows: PCV2 antigen was diluted 1 :640 in 0.05 M sodium carbonate buffer. 96 well Greiner E200 plates (available from Greiner Bio- One BV, Alphen a/d Rijn, Netherlands) were coated at 150 μΙ/well by incubation for 18-24 hours at 37°C. The coated plates were washed using a plate washer, with common wash buffer (0.04 M phosphate buffered solution, to which 0.05% v/v of a 10% w/v Polysorbate 20 solution was added). The PCV2 antigen that was coated to the plates this way, served as a reactant to ultimately bind heparin in the assay.

To perform the assay, another sample of the same PCV2 antigen was pre-diluted 1 :5 in dilution buffer (the same as the wash buffer, but containing 1 % w/v skimmed milk powder added) and 150 μΙ was dispensed to row 1 of the uncoated Greiner E200 plates. All samples were assayed in duplicate, in two adjacent wells. Next, samples were 2-fold serially diluted by transferring 75 μΙ from row A down to row H, which wells B - H already contained 75 μΙ of the dilution buffer. Heparin-HRP was diluted 1 :1 15 in the same dilution buffer and 75 μΙ was dispensed to all wells. Column 11 was used for Bmax readings (containing 75 μΙ dilution buffer and 75 μΙ heparin-HRP) and column 12 for Bmin readings (containing 150 μΙ dilution buffer). Plates were placed on a plate-shaker for 5 seconds and then incubated 30 minutes at 37°C to allow the PCV2 ORF 2 antigen to complex with the heparin HRP. After incubation, 100 μΙ/well from the uncoated plates was transferred to the plates coated with PCV2 antigen, and the coated plates were incubated 60 minutes at 37°C to allow heparin to bind to the coated antigen. Thereafter, the wells were washed with wash buffer to remove unbound (i.e. complexed) heparin. After washing, 100 μΙ of tetramethylbenzidine (TMB) was added per well. After 15 minutes the reaction between HRP and TMB was stopped by adding 50 μΙ of a 4 N sulphuric acid solution per well. Absorbances were read at 450 nm and results were calculated using a protocol based on 50% optical density (between Bmax and Bmin).

In a variation on this protocol, an ELBA was performed wherein heparin was bound to the wells, and these were incubated with a mixture containing a fixed amount of HRP-labeled PCV2 antigen and a sample of PCV2 antigen to be quantified, in a series of dilutions. Pre-treatment of PCV2 vaccine

To apply the assay of the invention to the PCV2 vaccine, the oily emulsion was broken to release the antigen. For this, a volume of PCV2 vaccine was added to 1 volume of 40% w/v potassium phosphate, and 1 volume of PBS 0.04 M; test were done in duplo. The mixture was homogenized well and centrifuged for 5 to 10 minutes at 3.000 to 10.000 xg at 4°C. The aqueous bottom phase (containing the PCV2 antigen) was recovered with a syringe and needle. Calculations based on a standard PCV2 antigen ELISA of the two phases indicated that in essence all PCV2 antigen present in the vaccines had been recovered by this procedure.

Results

The linearity, reproducibility, and detection-limit of the assay of the invention was assessed, in particular of the ELBA assay using a PCV2 antigen.

Linearity was assessed by testing five concentrations of the PCV2 antigen (100%,

50%, 25%, 12.5%, and 6.25%) in two replicates, and in three runs. The results are depicted in Figure 1. The coefficient of variation (CV %) of the three-run-averages of each sample was calculated to provide an indication of reproducibility. These results were also used to estimate the detection limit of the assay. The results demonstrated an excellent linearity of the PCV2 ELBA assay (R ^A 2 > 0.99) between the 6.25% and 100% test points.

Based on the raw data, the highest calculated CV% of the three-run-averages was 6.4%, implying a good level of reproducibility.

In two of the three runs the lowest PCV2 concentration (6.25% of the antigen concentration) could still be detected. This low concentration is therefore a good indication of the detection limit of the assay.

To determine the reproducibility of an ELBA, two experiments were performed, each with four samples of PCV2 antigen solution. In each experiment, the samples were quantified in duplo (replicate 1 and 2). In Tables 1 and 2 below the amount of PCV2 ORF 2 antigen found for the two replicates is indicated as normalised values, whereby the value for replicate 1 for each sample was set to 100% to allow convenient comparison between the two replicates. Table 1: Reproducibility of four PCV2 samples quantitatively assayed with ELBA; first experiment

Table 2: Reproducibility of four PCV2 samples quantitatively assayed with ELBA; second experiment

Although the reproducibility appears not to be perfect for all samples, the above results shows that the reproducibility obtained is more than adequate for an assay according the the invention.

To determine the amount of PVC2 antigen in a vaccine, the aqueous antigen-containing phase of a PCV2 vaccine was recovered as indicated above, and quantitatively assayed using the ELBA, as well as by a known standardized PCV2 ELISA test. Four samples were used and assayed separately. The results, presented in Table 3, were determined in arbitrary units/ml of the test sample, as compared to a reference sample, and these were normalised to allow comparison. Table 3: Comparison of ELBA and ELISA tests of PCV2 vaccine samples

It was shown that ELBA values were consistently higher than ELISA values up to about 5%. The overall correlation between the new ELBA and the existing ELISA tests are strong; the only mismatch found was for sample 3 with an ELBA value that was much higher that the ELISA value, probably resulting from an experimental error.

Based on the above results it was concluded that a quantitative assay based on the specific binding of marked heparin, such as ELBA, is an effective alternative fro an immuno-assay, such as ELISA, for the quantification of the amount of a biological substance in a liquid sample.