WO2006090146A1

WO2006090146A1 - Assay

Info

Publication number: WO2006090146A1
Application number: PCT/GB2006/000622
Authority: WO
Inventors: Keith Griffiths; Attila Turkes
Original assignee: Eden Research Plc
Priority date: 2005-02-23
Filing date: 2006-02-23
Publication date: 2006-08-31
Also published as: GB0503741D0

Abstract

There is provided a method and kit for assaying for prostate specific antigen and insulin-like growth factor-1 in a sample of blood or a blood-derivative which comprises contacting the sample with a specific binding partner for PSA and, under conditions in which IGF-1 is decoupled from IGF-1:IGFBP complexes but in which both IGF-1 and IGFBP are in solution, with a specific binding partner for IGF-1, and detecting conjugates of PSA and said binding partner for PSA and conjugates of IGF-1 and said specific binding partner for IGF-1.

Description

Assay

This invention relates to an assay method for the detection of prostate specific antigen (PSA) and insulin-like growth factor-1 (IGF-I) in blood or blood-derived body fluids, e.g. serum or plasma.

PSA is an enzyme, a protein that is produced in the mammalian body, that is found for example in the prostate. In the event of prostate cancer (more particular prostatic adenocarcinoma) , PSA appears to leak into the blood and in the presence of elevated blood levels of PSA has been accepted as a sensitive marker for prostate cancer, and blood tests for PSA are routinely carried out for males over the age of fifty. Serum PSA levels in the normal healthy male are generally below 4ng/mL. Where the level is above 30 ng/mL the probability of prostate cancer is about 90%. Between 10 and 20 ng/mL the likelihood of prostate cancer or benign prostatic hyperplasia is high. There is however a "grey-zone" for levels between 4 and 10 ng/mL where the cause may be unrelated to prostate cancer.

As part of routine screening for prostate cancer, current practice is generally to screen for serum PSA and to carry out a digital rectal examination. If either is indicative of prostate pathology, the patient may be referred for further examination or testing, e.g. using ultrasound imaging or ultrasound-guided prostate biopsy.

As with any diagnostic screening system, a primary goal is to have an initial screening technique which may be used to filter out patients not requiring treatment (which mat be expensive and unpleasant) , and thus the avoidance of both false positives and false negatives is important. The recognition of a "grey-zone", as is the case with PSA testing, is an acknowledgement of the occurrence of undesirably large numbers of false positives and false negatives.

It has therefore been proposed also to screen for IGF-I in blood (see for example WO 99/38011 and Djavan et al in Urology _54_; 603-606 (1999)). IGF-I, also known as somatomedin, is a single chain, growth-promoting polypeptide hormone that is structurally, functionally and genetically related to insulin.

IGF-I is a relatively small peptide, having about 70 amino acid residues and molecular weight of 7649 Dalton. Growth hormone induces the generation of IGF-I in the liver, which is highest during puberty, and regulates the paracrine production of IGF-I in the prostate and many other tissues. The majority of IGF-I however is synthesis in the liver, released into the blood and thereby transported to other tissues. In the serum, almost 99% of IGF-I is complexed by a family of binding proteins, the IGFBs which modulate the availability of biologically active free IGF-I to the tissues. At least six IGFBPs are known, and in humans almost 80% of circulating IGF-I is carried by IGFBP-3. Since IGF- 1 is relatively small protein, an antibody-based assay for total IGF-I (i.e. both free and bound IGF- 1) will generally involve exposing the serum sample to conditions which decouple (dissociate) the bound IGF-I and precipitate out the IGFBP. To this end organic solvent extract and acid precipitation of the IGFBP has been proposed (being larger that IGF- 1, IGFBPs precipitate out leaving the IGF-I in the free form) . Since however precipitation of the IGFBPs in this manner will also cause PSA (which is larger that IGF-I) to precipitate, where assays for both PSA and total IGF-I (tIGF-1) have been proposed, this has been in the context of carrying out separate assays for PSA and tIGF-1. Clearly it is more efficient to carry out a single assay and it is the problem of assaying for both PSA and tIGF-1 that is addressed by the present invention.

This problem is solved according to the present invention by the use of IGF-IrIGFBP decoupling conditions that are sufficient to achieve decoupling but do not precipitate the PSA.

Thus viewed from one aspect the invention provides a method for assaying for prostate specific antigen and insulin-like growth factor-1 in a sample of blood or a blood-derivative which method comprises contacting said sample with a specific binding partner for PSA and, under conditions in which IGF-I is decoupled from IGF-IrIGFBP complexes but in which both IGF-I and IGFBP are in solution, contacting said sample with a specific binding partner for IGF- 1, and detecting conjugates of PSA and said binding partner for PSA and conjugates of IGF-I and said specific binding partner for IGF-I, and optionally generating an indication of the content of PSA and IGF-I in said sample.

According to the invention, the decoupling of the IGFrIGFBP complexes may for example be achieved by disruption of the tertiary structure of the IGFBPs, i.e. by partial or full denaturing of the proteins. However, although decoupling can be achieved by significantly denaturing the proteins, e.g. by heating or addition of tertiary structure destroying chemical agents, the specific binding partner used must then be one which binds to the denatured protein (e.g. an antibody raised against the denatured protein or a fragment or immunogenic conjugate thereof - a technique analogous to that proposed in the literature for the detection of prions and other analytes) . Since current PSA specific binding partners are adequate, the need to generate new PSA binding partners in order to allow the detection steps to be simultaneous will not generally be justified. Accordingly therefore the decoupling is preferably effected using more gentle means, e.g. by gentle heating, application of a weakly acid pH or contacting the sample with a dilute surfactant, preferably an ionic surfactant, for example sodium dodecyl sulphate (SDS or any of the other surfactants employed in gel electrophoresis under denaturing conditions.

Such surfactants, at high concentrations, would serve to significantly denature proteins (i.e. to lose their normal tertiary structure) and it is thought that their action according to the present invention may be conceived as a partial denaturing sufficient to uncouple the IGF-I: IGFBP complexes but not sufficient to change the tertiary structure of the IGF-I (or indeed the PSA) to such an extent that their specific binding partners can no longer bind to them.

In gel electrophoresis under denaturing conditions, the denaturing surfactant is generally employed at a concentration of about 1% w/v. For the "partial denaturing" according to the present invention, a lower concentration is preferably used, e.g. 0.05 to 0.6% w/v, more preferably 0.08 to 0.25% w/v, especially 0.1 to 0.2% w/v. The precise concentration used will depend on the particular surfactant used and on whether a subsequent dilution step occurs before formation of the IGF-I: specific binding partner complexes. The concentration used should be high enough to decouple the IGF-I: IGFBP complexes but not so high as to so denature the IGF- 1 (or PSA) to such an extent that it will not bind to its binding partner. If dilution occurs (e.g. on a subsequent addition of the binding partner) , either this must not be to an extent that significant reformation of the IGF-I: IGFBP complexes occurs or significant reformation should be otherwise countered, e.g. by using high relative concentrations of the IGF-I specific binding partners or by addition of a material which completes for binding to IGFBPs, for example IGF-2 or, more preferably, insulin.

The specific binding partners used in the method of the invention may be any materials capable of binding to IGF-I (or PSA respectively) preferentially relative to any other material present in the sample. Where IGF-2 or insulin is added as mentioned above, the IGF-I specific binding partner sbp) should of course not be cross-reactive with IGF-2 or insulin. The sbps may be monoclonal or polyclonal antibodies, single chain antibodies, antibody fragments, derivatives or constructs, oligonucleotides, or other binding agents. The generation of sbps for assays for biological analytes is well-established field of technology. For the present purpose it should be noted that anti-PSA and anti-IGF-1 antibodies are available commercially (e.g. from R&D Systems Europe Ltd., Abingdon, UK and PeproTech EC Ltd, London, UK) and these may be used in the method of the invention.

Detection of the PSA: sbp and IGF-I : sbp conjugates in the method of the invention may be direct or indirect. Thus for example if the sbp is labelled with a reporter moiety, for example a fluorophore, chromophore or radiolabel, that moiety may be detected directly following separation of bound sbp from unbound sbp. Alternatively a labelled secondary binding partner may be used which binds to the analyte: sbp conjugate and the label on the secondary binding partner may then be detected. In the case of indirect detection, an analyte analogue capable of binding to the sbp may be added and a labelled analyte: sbp conjugate may then be detected (following separation from unbound analogue if the analogue is labelled or from unbound sbp if the sbp is labelled) . Where bound and unbound substances have to be separated before detection occurs, it will often be desirable for one of the substances to be immobilised on a substrate (e.g. a polymer sheet or bead or membrane) so that separation can be effected by rinsing the substrate. These techniques are all standard within the field of diagnostic assays.

Where PSA and IGF-I conjugates are to be detected simultaneously, it is of course necessary to use interdistinguishable labels, e.g. radionuclides having different characteristic emissions or fluorophores or chromophores having characteristics but non-overlappping emission or absorption wavelengths. Alternatively simultaneously detection is feasible even with non distinguishable labels if the conjugates being detected are physically separated, e.g. using a substrate plate carrying in one area binding agent which will immobilize one labelled material (e.g. the PSA: sbp conguate or a PSA: labelled binding partner conjugate) and in another area a binding agent which immobilizes the other (i.e. the IGF-I version).

The sample used in the method of the invention may be serum or plasma, or less preferably whole blood, in which case either the reporter moieties detected in the detection step must clearly be distinguishable from haemoglobin or red blood cells or the haemoglobin therein must be removed from the sample before the detection step. Red blood cells removed may be effected by filtration or by contacting the sample with a substrate-bound erythrocyte binding agent (e.g. an oligo-peptide with an RGD motif) .

The PSA sbp used in the present invention may be one specific for free PSA (fPSA) if fPSA is to be determined; however it is believed that total PSA (tPSA) is more clinically relevant and the PSA sbp is preferably one which binds to both fPSA and bound PSA (bPSA) . In general however, since the decoupling conditions may also decouple PSA from its binding proteins, it will be preferred that the PSA sbp is not one that binds to unbound PSA binding proteins (PSABPs) .

The assay results, as with most assays for biological analytes, will preferably be calibrated against known PSA and IGF-I standards. The results may be presented as concentrations, e.g. tPSA and tIGF-1 concentrations in serum, or as ratios, e.g. a ratio of tPSA to tIGF-1 concentrations, or simply as qualitative or semi-quantitative indications, e.g. that a concentration or ratio is within a certain band of values or above or below a preset threshold value.

The invention, in another aspect, provides a kit for use in a method of the invention, said kit comprising: a specific binding partner for PSA; a specific binding partner for IGF-I; an IGF-I: IGFBP decoupling agent; and optionally and preferably instructions for the performance of the method. If desired the kit may include buffer or rinsing solutions or secondary binding partners as described above, or cell lysing agents (e.g. for use when whole blood samples are used) . In a particularly preferred aspect, the kit is in the form of a reagent-containing cartridge, e.g. as described in WO 02/090995, for use at the point-of-care .

If desired the assay may be run without addition of the decoupling agent so as to determine fIGF-I, and optionally fPSA values and ratios. The running of the assay in both ways is a further aspect of the invention. Yet again, the assay may be run using a fIGF-1 sbp, and determining the fIGF-1 concentration before decoupling the bIGF-1 and determining tIGF-1.

The invention will now be described further with reference to the following non-binding Examples.

Example 1 Assay Reagents

Assay Buffer: 0.01 M Tris buffer, pH 8.6 containing 0.1% bovine serum albumin, 0.01% gamma globulin and 0.01% sodium azide.

Coating Buffer: 0.01 M phosphate buffered saline, pH 7.2 or 0.01 M Bicarbonate buffer, pH 8.6.

Re-coating Buffer: 0.01 M Tris buffer, pH 8.6 containing 1% bovine serum albumin.

Wash Buffer: 0.01 M Tris buffer, pH 8.6 containing 0.1% TWEEN 20.

Europium and Samarium Labelling Reagents for the preparation of labelled IGF-I and PSA antibodies are available as Delphia Labelling kits from Perkin Elmer UK, Beaconsfield, UK

Enhancement Solution: 0.01 M Acetic acid. The ready made Assay and Wash Buffers and Enhancement Solution are also available from Perkin Elmer.

All individual reagents for the in-house preparation of buffers, mentioned above, are available form Sigma-Aldrich Company Ltd. , Poole, UK

Example 2 Assay Performance

The example given here, is on the basis of two-site immunometric assay for the simultaneous determination of IGF-I and PSA in serum or plasma samples. This principle relies on two antibodies (one for IGF-I and one for PSA) coated or attached onto plastic surface of the microtitre plate wells as capture antibodies and another two antibodies labelled with rare earth chelates for example, IGF-I antibody labelled with samarium and PSA antibody labelled with europium, and following incubation of the serum or plasma sample forming capture antibody: IGF-I or PSA: labelling antibody complexes. The amount of complex formation directly related to the IGF-I and PSA concentrations in the sample.

The coating of antibodies to plastic surfaces of microtitre plates is achieved by using of MaxiSorb plates available from Nunc A/S, Roskilde, Denmark. A PSA polyclonal antibody, raised in sheep, and a monoclonal IGF-I antibody, mouse origin, dilutes in coating buffer to a concentration (depending on the affinity of the antibody of 1-5 μg/ml. Bicarbonate buffer pH 8.6 is generally used for adsorbing proteins to plastic surfaces however, sometimes some monoclonal antibodies aggregate in alkaline pHs and reduce the coating efficiency. In these situations phosphate buffered saline, pH 7.2 can be used.

A 100 μL aliguot of antibody dilution (equivalent to 100-500 ng of antibody) in coating buffer is dispensed into mircotitre wells and the plates are sealed and incubated for 18-24h at 4 °C. The wells of the microtitre plate are washed three times with wash buffer by dispensing 250 μL wash in to each well and decanting. This procedure can also be performed with an automatic plate washer. After the final wash the plate is blotted on an absorbent paper and 250 μL of re-coating buffer is added into each well to block the uncoated plastic surface. The plate is incubated for Ih at room temperature or stored at 4 °C until it used for assay. The plate is then washed three times with wash buffer and made ready for an assay.

Aliquots of serum or plasma samples (25-100 μL) are diluted 20 fold with 0.1-1% solution of SDS to disassociate the IGFBP: IGF-I complexes. This step requires optimization according to the affinity of the antibodies used. The standards which are prepared in 0.01 M Tris, buffer pH 8.6 containing 1% bovine serum albmin to give concentration of 0-100 ng of PSA and 0-500 ng of IGF-I are also treated with SDS solution the same as the samples. The sample and standard dilutions in SDS are left to stand at room temperature for 5-10 min and 50 μL aliquots of each sample and standards dispensed into their respective wells. Into each well 100 μL of assay buffer containing 1 μg/100 μL insulin or 200 ng/100uL IGF to eliminate the re-formation of the IGFBP: IUGF-I complexes is dispensed. The plate is sealed with a tape and incubated for 60-90 min at room temperature on a plate-shaker. The plate is washed three times with wash buffer and 150 μL of labelled antibody solution in assay buffer is dispensed in to each well. Labelled antibody solution contained 25-100 ng/150 μL of Europium labelled PSA antibody and 25-100 ng/150 μL of Samarium labelled IGF-I antibody. The plate is the incubated another 60-90 min at room temperature on a plate shaker to form antibody: PSA: labelled antibody and antibody: IGF-I : labelled antibody complexes. The plate is washed three times with wash buffer (250 μL) and following addition of 200μL of enhancement solution the fluorescence activity in each well is measured using a Victor-2 time resolved fluorimetry instrument (Perkin Elmer) .

The concentration of PSA and IGF-I in samples is then calculated from the dose-response curve achieved by plotting the standard concentrations versus relative fluorescent units using a data reduction software.

The example given above is designed to measure total PSA and total IGF-I in a sample of serum or plasma. However, exclusion of SDS treatment of samples with SDS provides and assay capable of measuring total PSA and free IGF-I.

Claims

1. A method for assaying for prostate specific antigen and insulin-like growth factor-1 in a sample of blood or a blood-derivative which method comprises contacting said sample with a specific binding partner for PSA and, under conditions in which IGF-I is decoupled from IGF- 1: IGFBP complexes but in which both IGF-I and IGFBP are in solution, contacting said sample with a specific binding partner for IGF-I, and detecting conjugates of PSA and said binding partner for PSA and conjugates of IGF-I and said specific binding partner for IGF-I.

2. The method of claim 1 comprising the step of generating an indication of content of PSA and IGF-I in said sample.

3. The method of claim 1 or 2 in which IGF-I is decoupled from IGF-I: IGFBP complexes by gentle heating, application of a weakly acid pH or contacting the sample with a surfactant.

4. The method of any preceding claim in which IGF-I is decoupled from IGF-I: IGFBP complexes by contacting the sample with an ionic surfactant.

5. The method of claim 4 in which the ionic surfactant comprises sodium dodecyl sulphate (SDS) or an other surfactants employed in gel electrophoresis under denaturing conditions.

6. The method of claim 4 or 5 in which the concentration of surfactant used is high enough to decouple the IGF-I: IGFBP complexes but not so high as to so denature the IGF-I or PSA to such an extent that it will not bind to its binding partner.

7. The method of any one of claims 3 to 6 in which the surfactant is SDS at a concentration of 0.05 to 0.6% w/v.

8. The method of claim 7 in which the SDS is at a concentration of 0.08 to 0.25% w/v.

9. The method of either claim 7 or 8 in which the SDS is at a concentration of 0.1 to 0.2% w/v.

10. The method of any proceeding claim in which a material which completes for binding to IGFBPs is added.

11. The method according to any preceding claim in which the specific binding partner is a monoclonal antibody, polyclonal antibody, single chain antibody, antibody fragment, derivative or construct or oligonucleotid.

12. The method according to any preceding claim in which PSA specific binding partner is one which binds to both free PSA and bound PSA.

13. The method according to claim 12 in which the PSA specific binding partner does not bind to unbound PSA binding proteins .

14. The method according to any preceding claim in which detection of the PSA: sbp and IGF-I : sbp conjugates is direct or indirect.

15. The method according to any preceding claim in which the PSA and IGF-I conjugates are detected simultaneously using interdistinguishable labels.

16. The method of claim 15 in which the interdistinguishable labels comprise radionuclides having different characteristic emissions.

17. The method of claim 15 in which the interdistinguishable labels comprise fluorophores or chromophores having characteristics but non-overlapping emission or absorption wavelengths.

18. The method of any proceeding claim in which the PSA and IGF-I conjugates are detected simultaneously and the conjugates being detected are physically separated.

19. The method of any preceding claim in which the sample is serum, plasma or whole blood.

20. The method of any proceeding claim comprising the additional step of preparing the assay without addition of the decoupling agent so as to determine fIGF-I, and optionally fPSA values and/or ratios.

21. A kit for use in a method according to claim 1, said kit comprising: a specific binding partner for PSA; a specific binding partner for IGF-I; and an IGF-I: IGFBP decoupling agent.

22. A kit as claimed in claim 21 comprising instructions for the performance of the method.