Assay

Abstract

Methods are provided for determining the presence of an analyte present in a liquid sample involving: contacting the liquid sample with a predetermined amount of an analogue of the analyte and an excess of a first binding moiety, wherein the first binding moiety is capable of binding each of the analyte and the analogue independently; contacting the mixture with a second binding moiety; and determining the level of a signal indicative of the presence of the analogue-first binding moiety complex bound to the second binding moiety, wherein if the level of the signal determined is lower than the level of a maximum signal determined when no analyte is present, then analyte is present in the sample. Corresponding kits are also provided.

Claims

1-15. (canceled)

16. A method of determining the presence of an analyte present in a liquid sample, the method comprising: a) contacting the liquid sample with a predetermined amount of an analogue of the analyte and a first binding moiety, wherein: the analogue comprises an amino acid sequence which has been recombinantly expressed or synthesized in vitro to include a binding region composed of amino acids that can be bound by a second binding moiety, such that the analogue incorporates only one of said binding region, wherein the binding region is not present in the analyte or is present in the analyte but not accessible to the second binding moiety; and the first binding moiety is capable of binding each of the analyte and the analogue independently, such that a mixture is formed comprising an analogue-first binding moiety complex, or, when analyte is present in the sample, either (i) an analogue-first binding moiety complex and an analyte-first binding moiety complex, or (ii) an analyte-first binding moiety complex and no analogue-first binding moiety complex; b) contacting the mixture with the second binding moiety, wherein the second binding moiety is an antibody capable of binding the analogue-first binding moiety complex but not the analyte-first binding moiety complex, and is immobilised or immobilisable on a solid phase; c) determining the level of a signal indicative of the presence of the analogue-first binding moiety complex bound to the second binding moiety; wherein if the level of the signal determined in step c) is lower than the level of a maximum signal determined when no analyte is present, then analyte is present in the sample.

17. A method of determining the presence of an analyte present in a liquid sample, the method comprising: a) contacting the liquid sample with a predetermined amount of an analogue of the analyte and a first binding moiety, wherein: the analogue comprises an amino acid sequence which has been recombinantly expressed or synthesized in vitro to include a binding region composed of amino acids that can be bound by a second binding moiety, such that the analogue incorporates only one of said binding region, wherein the binding region is not present in the analyte or is present in the analyte but not accessible to the second binding moiety; and the first binding moiety is capable of binding each of the analyte and the analogue independently but not both the analogue and analyte simultaneously, such that a mixture is formed comprising an analogue-first binding moiety complex, or, when analyte is present in the sample, either (i) an analogue-first binding moiety complex and an analyte-first binding moiety complex, or (ii) an analyte-first binding moiety complex and no analogue-first binding moiety complex; b) contacting the mixture with the second binding moiety, wherein the second binding moiety is an antibody capable of binding the analogue-first binding moiety complex but not the analyte-first binding moiety complex, and is immobilised or immobilisable on a solid phase; c) determining the level of a signal indicative of the presence of the analogue-first binding moiety complex bound to the second binding moiety; wherein if the level of the signal determined in step c) is lower than the level of a maximum signal determined when no analyte is present, then analyte is present in the sample.

18. The method of claim 1, wherein the sample is contacted with the first binding moiety prior to being contacted with the analogue.

19. The method of claim 1, further comprising a step of determining the amount or concentration of analyte present in the sample by comparing the level of signal determined in step c) with calibration data.

20. The method of claim 1, wherein the analyte is hCG or a fragment or portion thereof.

21. The method of claim 1, wherein the signal is derived from a label that is either directly or indirectly associated with the first-binding moiety.

22. The method of claim 6, wherein the label is selected from the group consisting of an enzyme, a fluorophore, a radionuclide, a colloidal sol, a chromophore and a luminescent compound.

23. The method of claim 1, wherein the analogue includes an amino acid sequence that is identical to either the full analyte sequence or a portion of the analyte sequence.

24. The method of claim 1, wherein the first binding moiety is an antibody.

25. The method of claim 1, wherein the first binding moiety is a Fab or F(ab).sub.2.

26. The method of claim 1, wherein the first binding moiety binds specifically to hCG.

27. The method of claim 1, wherein the first binding moiety is an hCG 3-loop-specific monoclonal antibody.

28. The method of claim 1, wherein the sample is derived from or consists of a physiological source including blood, serum, plasma, interstitial liquid, saliva, sputum, ocular lens liquid, sweat, urine, milk, ascites liquid, mucous, synovial liquid, peritoneal liquid, transdermal exudates, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, cerebrospinal liquid, semen, cervical mucus, vaginal or urethral secretions or amniotic liquid.

29. A test kit for detecting an analyte of interest comprising i) an analyte analogue; ii) either (a) a labelled first binding moiety or (b) an unlabelled first binding moiety and a labelled third binding moiety; and iii) an immobilisable second binding moiety wherein: the analogue comprises an amino acid sequence which has been recombinantly expressed or synthesized in vitro to include a binding region composed of amino acids that can be bound by the second binding moiety, such that the analogue incorporates only one of said binding region, wherein the binding region is not present in the analyte or is present in the analyte but not accessible to the second binding moiety; and the first binding moiety is capable of binding each of the analogue and the analyte of interest independently, the second binding moiety is an antibody capable of binding a complex of the analogue and the first binding moiety but is not capable of binding the analyte or a complex of the first binding moiety and the analyte, and the third binding moiety is capable of binding the first binding moiety or the analogue-first binding moiety complex.

30. The test kit of claim 14, wherein the first binding moiety is capable of binding each of the analogue and the analyte of interest independently but not both the analogue and analyte simultaneously.

Description

DESCRIPTION OF THE FIGURES

[0147] FIG. 1 represents a schematic description of a hybrid competitive immunoassay according to an embodiment of the invention, and indicates possible cross reaction that might occur between the target analyte and another species present or potentially present in the sample. In an exemplary embodiment where the target analyte is hCG, the cross reacting species is LH.

[0148] FIG. 2 represents an inhibition curve, indicating the degree with which a selected monoclonal antibody for an hCG assay, moc1, that has been raised against the conserved 3-hCG loop region, cross reacts with LH. The data indicate cross reactivity with LH is 0.27% compared with hCG, based on the quantity of ligand needed to saturate 50% of the antibody.

[0149] FIG. 3 provides further characterising data that demonstrate the specificity of the monoclonal antibody, moc1, for binding to hCG. The graph represents number of counts on the y-axis due to I125 radiolabelled moc1 that has bound tagged hCG, and the x-axis indicates the level of dilution from 1000.sup.th to 512000.sup.th. The data represent a series of curves that demonstrate the ability of the moc1 antibody to distinguish between hCG and either hCG or LH. The buffer control, 569 free alpha and LH all follow a similar profile, with high counts at low dilution, declining as sample is diluted. However, when the antibody and tagged hCG are incubated with either free hCG, nicked hCG, intact hCG, nicked hCG or core hCG (unlabelled target), the signals are significantly suppressed, even at low dilution, because the unlabelled target has successfully outcompeted the tagged-hCG for binding to the antibody.

[0150] FIG. 4 represents a comparison of recombinant moc1 Fab.sub.1 (monomer) and Fab.sub.2 (dimer).

[0151] FIG. 5 shows a comparison between the serum TAG assay and the RIA assay based on samples with low to high hCG concentrations. The x-axis displays the hCG concentration (IU/L) measured via the TAG assay. The y-axis displays the hCG concentration (IU/L) measured via the RIA assay.

[0152] FIG. 6 shows a comparison between the serum TAG assay and the Immulite assay based on samples with low to high hCG concentrations. The x-axis displays the hCG concentration (IU/L) measured via the TAG assay. The y-axis displays the hCG concentration (IU/L) measured via the Immulite assay.

[0153] FIG. 7 shows a comparison between the serum TAG assay and the RIA assay based on samples with mid to high hCG concentrations. The x-axis displays the hCG concentration (IU/L) measured via the TAG assay. The y-axis displays the hCG concentration (IU/L) measured via the RIA assay.

[0154] FIG. 8 shows a comparison between the serum TAG assay and the Immulite assay based on samples with mid to high hCG concentrations. The x-axis displays the hCG concentration (IU/L) measured via the Immulite assay. The y-axis displays the hCG concentration (IU/L) measured via the TAG assay.

EXAMPLE 1

Protocol for Serum TAG Assay

[0155] Reagents

[0156] Standard diluentSerasub (SS)

[0157] Sample pre-diluentNormal sheep serum (NSS)

[0158] moc110 ul stock (1:1000) in 4 ml 3% BSA/1% NSS/PBS

[0159] TAG 10 ul stock (1:1000) in 4 ml 3% BSA/1% NSS/PBS

[0160] Microtitre platepre-coated with anti TAG

[0161] Sample Preparation

[0162] 0-100 IU/Ldilute 1:2 in SS.

[0163] 100-1000dilute 1:5 in NSS then further in SS as required.

[0164] >1000dilute 1:50 in NSS then further in SS as required.

[0165] Assay [0166] Prepare standard curve by serial diluting top hCG standard (100 IU/L) six times in SS to generate 50, 25, 12.5, 6.25, 3.125 and 1.56 IU/L standards. Include SS alone to give a zero standard. [0167] Dilute serum samples as appropriate (detailed above). [0168] Using a multichannel pipette, add 35 l of moc1 solution to wells of the microtitre plate. [0169] Add 35 l of standards and samples. [0170] Using a multichannel pipette, add 35 l of TAG solution to all wells. [0171] Shake plate for 1 hour at room temperature. [0172] Remove the content of the wells by tipping and blotting onto tissue paper. [0173] Wash 6 with 0.1% Tween/PBS. [0174] Add 100 l femto substrate and read on BMG Fluostar luminometer. [0175] Calculate results using BMG Omega Mars data analysis software.

EXAMPLE 2

Protocol for Urine TAG Assay

[0176] Reagents

[0177] Standard diluentUrisub (US)

[0178] Sample diluent10% Normal sheep serum (NSS)/US

[0179] moc110 ul stock (1:1000) in 4 m1 3% BSA/1% NSS/20%FCS/PBS

[0180] TAG2.3 ul stock (1:1000) in 4 m1 3% BSA/1% NSS/PBS

[0181] Microtitre platepre-coated with anti TAG

[0182] Sample Preparation

[0183] 0-100 IU/LNeat

[0184] >100 IU/Ldilute in 10% NSS/US

[0185] Assay [0186] Prepare standard curve by serial diluting top hCG standard (100 IU/L) six times in US to generate 50, 25, 12.5, 6.25, 3.125 and 1.56 IU/L standards. Include SS alone to give a zero standard. [0187] Dilute urine samples as appropriate (detailed above). [0188] Using a multichannel pipette, add 35 l of moc1 solution to wells of the microtitre plate. [0189] Add 35 l of standards and samples. [0190] Using a multichannel pipette, add 35 l of TAG solution to all wells. [0191] Shake plate then incubate for 2 hours at 37 C. [0192] Remove the content of the wells by tipping and blotting onto tissue paper. [0193] Wash 6 with 0.1% Tween/PBS. [0194] Add 100 l femto substrate and read on BMG Fluostar luminometer (see settings data). [0195] Calculate results using BMG Omega Mars data analysis software (see analysis data).

EXAMPLE 3

Comparison of moc1 Fab Variants

[0196] Recombinant mod Fab.sub.1 (monomer) and Fab.sub.2 (dimer) were produced in E. coli by conventional methods known in the art by the skilled person. Each Fab variant (monomeric variant and dimeric variant) was purified and conjugated to horseradish peroxidase (HRP) using Lightning Link HRP kits (Innova Bioscience) according to the manufacturer's instructions. The molecular weight of each Fab variant was taken into account for the conjugation process so that the monomeric Fab.sub.1 variant and dimeric Fab.sub.2 variant were subjected to comparable levels of labelling with HRP.

[0197] Following labelling with HRP and initial activity testing, the labelled Fab variants were assessed in a competition assay using TAG and hCG in order to derive a standard curve following the general method provided in Example 1.

[0198] The results of the assays are given in Table 1 and FIG. 4.

TABLE-US-00002 TABLE 1 Averaged data showing assay output and % Signal relative to negative hCG standard hCG standard Chemiluminescence (RLU) % signal (IU/L) Monomer Dimer Monomer Dimer 0 1202503 2572679 100 100 1.56 1165822 2346433 97 91 3.125 1080330 2162202 90 84 6.25 961748 1900875 80 73.9 12.5 687755 1340029 57 52 25 440407 848495 36.6 33 50 197693 504683 16.4 19.6 100 49539 130589 4 5 200 11774 23302 1 0.9

[0199] Table 1 and FIG. 4 demonstrate that the Fab.sub.2dimer variant has a significant, approximately two-fold, increase in chemiluminescent output relative to the Fab.sub.1 monomer variant under comparable assay conditions. In addition, the Fab.sub.2 dimer variant has a greater sensitivity to hCG concentration in the sample over the hCG concentration range 1.56-25 IU/L relative to the Fab.sub.1 monomer variant as indicated by the more acute decrease in % signal across this range (see FIG. 4).

EXAMPLE 4

Clinical Studies Comparing the TAG Assay with Immulite and RIA Assays

[0200] Studies were performed to compare the ability of the TAG assay employing the Fab.sub.2 dimer variant as described herein and known assays, the Immulite assay and RIA assay, to detect hCG in clinical samples.

[0201] Clinical serum samples were analysed for the presence and concentration of hCG by each assay method. The TAG assay was performed as described in Example 1. Immulite assays (Siemens) were performed according to the manufacturer's instructions. RIA assays were performed according to protocol instructions provided by the National Health Service, UK. In short, the assay uses a purified polyclonal antibody and iodinated hCG wherein sample is diluted and mixed with antibody and labelled hCG for 24 hours prior to measurement.

[0202] The results of the assays is given in Tables 2 and 3 and FIGS. 5-8.

TABLE-US-00003 TABLE 2 hCG detection in clinical serum samples via the TAG assay, Immulite assay or RIA assay method whereby the hCG concentration range across the samples was from low to high hCG concentrations. hCG concentration (IU/L) Sample TAG RIA Immulite 1 17 13 16 2 5 4 5 3 70 46 100 4 18 5 7 5 7 5 8 6 104 75 176 7 26 14 33 8 397 321 398 9 14 10 11 10 1163 1148 1279 11 82 70 95 12 76 75 120 13 1391 1336 1834 14 726 779 875 15 15 5 5 16 949 1305 1200 17 23 11 28 18 34040 49380 58146 19 96546 122920 177465 20 154 108 156 21 14 12 13 22 175265 194800 164721 23 24615 22330 25561 24 1545 1067 1425 25 3 <4 <1 26 2 <4 <1 27 12 9 13 28 1405 1063 1402 29 8 9 11 30 1 <4 <1 31 33 32 39 32 2 <4 <1 33 9 6 10 34 10 9 13

TABLE-US-00004 TABLE 3 hCG detection in clinical serum samples via the TAG assay, Immulite assay or RIA assay method whereby the hCG concentration range across the samples was from mid to high hCG concentrations. hCG concentration (IU/L) Immulite RIA TAG 164721 194800 175265 25561 22330 24615 21884 17700 19028 721139 676000 640363 21884 17290 12977 721139 735400 675028 182140 166750 164552 39602 30430 36581 26551 19230 23276 58258 51900 44120 36871 41600 38110 25546 35000 23997 17112 13350 12132 154163 147200 150430 17494 17200 14079 14202 10210 14459 14230 9700 19655 220085 193900 314760 69034 41860 48667 435384 264200 333333 19546 19670 15863 22642 14285 21467 27591 25800 14364 156336 106600 86333 376837 273500 148267 28086 18550 15583 66289 46560 33289 114379 59160 114315 28862 16770 26918

[0203] FIGS. 5-8 demonstrate that the detection and measurement of hCG concentration in clinical serum samples via TAG assay correlates strongly with the known Immulite and RIA assays with R.sup.2 values greater than 0.9 in all cases. Some variation between assays methods exists due to differences in antibody recognition of the target epitopes on hCG, however all assays can accurately detect hCG in clinical sera.

[0204] Furthermore, further clinical studies have demonstrated that the key benefit of the TAG assay is its ability to report specifically with good sensitivity. It has been shown to detect very low levels of hCG and detect increases in patient samples before the known RIA and Immulite assays were able to do so which is invaluable in monitoring patients undergoing therapy, particularly when the levels of hCG are very low. In addition to the superior sensitivity of the TAG assay over known assay methods, the TAG assay is laboratory based and can be completed in a much shorter period of time (4 hours) than the known assays using simple laboratory facilities and equipment.