CA2236134A1

CA2236134A1 - Method for the detection of an analyte by immunochromatography

Info

Publication number: CA2236134A1
Application number: CA002236134A
Authority: CA
Inventors: Gary W. Rheinheimer; Meitak Teresa Yip
Original assignee: Bayer Corp
Current assignee: Bayer Corp
Priority date: 1997-06-30
Filing date: 1998-04-29
Publication date: 1998-12-30
Also published as: AU730348B2; DE69809640T2; EP0889327A1; EP0889327B1; AU7391598A; US5895765A; DE69809640D1; JPH1164336A

Abstract

Disclosed is an improved method for the detection of an analyte in a fluid test sample using a strip of a negatively charged matrix material having a zone containing mobile, labeled binding partner for the analyte and a separate zone for capturing the labeled binding partner as it is carried through this zone by the fluid test sample. The improvement involves combining the fluid test sample with a polyalkoxylated amine surfactant to control non-specific binding of the labeled binding partner to the negatively charged matrix material.

Description

METHOD FOR THE DETECTION OF AN
ANALYTE BY IMMUNOCHROMATOGRAPHY

Backqround of the Invention There is a need for simple diagnostic tests for common diseases which can be carried out by untrained personnel.
Such tests facilitate home or doctor's office testing as op-posed to more complicatel~ procedures which require that the analysis be carried out in an outside reference laboratory. A
common format for these tests is the immunostrip format.
Typically, this format involves a matrix of a material through which a fluid test sample can flow by capillarity. The ma-trix, typically in the form of a strip, contains an analyte specific antibody which bears a detectable label so that the presence and/or concentration of the analyte in the test fluid can be determined by detection of the signal emitted from the detectable label. A classical format for such a device, some-times referred to as an immunochromatographic strip, is illus-trated by Fig. 1. Referring to Fig. 1, strip 10, bears a la-beled antibody specific for the analyte under investigation in zone 13 which binds with the analyte in the fluid test sample applied to the wicking zone 12 of the strip 10 and flows along the! strip to form an immunocomplex which further migrates due to capillary action through the capture zone of the strip 14 and the optional detection zone 16. In the capture zone 14 there is immobilized the analyte or a derivative thereof which is immunoreactive with t;he labeled antibody and is able to capture labeled antibody which has not reacted with analyte in the fluid test sample. The signal from the labeled antibody captured in the capture zone is measured and related to the concentration of analyte in the test fluid in an inverse rela-tionship since the greater the concentration of analyte in the test sample, the amount labeled antibody which is unbound and thereby free to specifically bind with analyte immobilized in the detection zone is diminished. Detection zone 16 is op-tional but can contain immobilized anti-mouse IgG to bind the analyte/labeled binding partner complex and thereby serve as a means for verifying that the test has been carried out cor-rectly.

A problem with this sort of test device involves the ten-dency of labeled antibody and its conjugate to engage in non-specific binding (NSB) with the matrix material forming the strip. When such non-specific binding takes place, the la-beled antibody binds to t.he matrix material before it reaches the capture zone and the assay fails because the movement of labeled antibody is either completely stopped or diminished such that the signals in the capture zone and detection zone are! greatly reduced.

In order to correct this bias, the strip can be treated wit.h a blocking solution such as 1% casein in phosphate buff-ered saline (PBS), washed with water and dried after deposi-tion of the reagents ont:o the capture and collection zones.
This blocking step, however, is problematic since there is re-quired extensive development effort to optimize the blocked system.

In U.S. Patent 5,451,507 there is described the prepara-tion of a blocked nitrocellulose membrane for use as an immu-nochromatographic strip in which the nitrocellulose membrane is incubated in a soluti.on of 1 mg/mL bovine IgG in sodium sulfate buffer for 30 minutes before being incubated with glu-taraldehyde and bovine IgG. This reference also mentions the desirability in some instances of including from about 0.05 to 0.~1 weight percent of a non-ionic detergent with the fluid test sample.

It wouId be desirable, and it is an object of the present invention to provide a means for reducing or eliminating non-specific binding of labeled specific binding partner in the capture and detection z;ones of the type of immunochroma-tographic strip under consideration.

Su~mary of the Invention The present invention involves a method for the determi-nal;ion of the concentration of an analyte in a fluid test sam-ple which comprises the steps of:

a) providing a mat.rix through which the fluid test sam-ple can flow by capillarity which matrix comprises a negatively charged polymeric material and has a first region containing mobile specific binding partner for the analyte which bears a detectable la-bel and can react with the analyte to form an ana-lyte/labeled binding partner complex and a second region which contains immobilized analyte or an im-mobilized binding partner which is specific for an epitope of the analyte different from that to which the labeled binding partner is specific;

b) combining the fluid test sample with a cationic sur-factant which i, a polyalkoxylated amine; and c) developing the matrix by the application of the fluid test sample suspected of containing the ana-lyte and allowing the fluid to contact the mobile specific binding partner so that analyte present in the fluid test sample forms an analyte/labeled spe-cific binding partner complex and leaves excess, un-reacted labeled binding partner free to further re-act whereby the fluid test sample carries the ana-lyte/labeled binding partner complex and unreacted labeled specific binding partner along the strip by capillarity to the second region containing the im-mobilized analyte in which region unreacted, labeled specific binding partner is bound to immobilized analyte in an inverse relationship to the concentra-tion of the analyte in the fluid test sample or is bound to the immobilized specific binding partner in direct relationship to the concentration of analyte in the fluid test sample. The surfactant results in non-specific binding of the labeled specific binding partner to the test strip being reduced.

De~scription of the Invent;ion The present invention is practiced by first providing a test strip in the form of a matrix through which the test sam-pl~e can flow by capillarity. Typically, the matrix will be in the form of a strip through which the test fluid flows hori-zontally, although the matrix could be set up in layers through which the test fl;~id could flow vertically from top to bottom or vice-versa. The following discussion focuses on the strip format.

The strip can be prepared from any negatively charged ma-trix material through which the test fluid and the analyte con,tained therein can flow by capillarity. Accordingly, suit-able matrix materials include nitrocellulose, polysulfones and polycarboxylic acids. Nitrocellulose is a preferred material from which to fabricate the strip. These materials are re-lat.ed in that they bear a negative charge.

A particularly suitable immunochromatographic strip for-mat. for use in relation t;o the present invention is that for-mat. which is disclosed in U.S. Patent 4,446,232 wherein there is described a device for the determination of the presence of ant:igens, which device comprises a strip of a matrix material having a first zone in which there are provided immobilized ancl enzyme linked antibodies specific to the analyte to be de-termined. The labeled antibodies can flow to a second zone when reacted with analyte introduced into the first zone along wit;h the test fluid but will not so flow in the absence of analyte in the test fluid due to their being bound in the first zone by interaction with the immobilized analyte. The analyte is typically an antigen although the format can be de-siqned to detect the presence of antibodies as analyte. An all;ernative to this format is that in which the detection zone contains an immobilized binding partner which is specific for an epitope of the analyte different from that to which the la-be:Led binding partner is specific. This provides a means for capturing the labeled binding partner using the so-called sandwich format. In anolher modification, there is disposed in a separate region of the strip an immobilized binding part-ner for the conjugate such as anti-mouse IgG to thereby cap-ture the complex formed between the labeled specific binding partner and the analyte. Thus, by immobilizing the conjugate in a discrete detection zone located downstream on the strip from the zone in which lhe labeled binding partner for the analyte is bound, there are provided two zones from which the physically detectable property of the detectable label can be measured to determine its intensity and hence the concentra-tion of the detectable label in a particular region of the strip. By measuring the ,ignal from the physically detectable property of the detectable label in the second zone containing the immobilized analyte or binding partner specific to a de-fined epitope of the analyte as the capture means and the physically detectable property of the label in the third zone, in which the immobilized antibody against the labeled binding partner is the capture rneans, and determining the ratio of these signals, the accuracy of the test for analyte concentra-tion can be increased.

Regardless of the selection of the format for the assay, the accuracy of the final result can be skewed by non-specific binding (NSB) of the labeled binding partner to the matrix ma-terial and it is the goal of the present invention to reduce or eliminate this problem without the necessity of carrying out the extra step of applying a separate blocking layer to the negatively charged membrane. This is accomplished by com-bining the appropriate polyalkoxylated amine surfactant with the fluid test sample which can be accomplished by various met.hods such as by mixing the fluid test sample with the sur-factant before contacting it with the strip. An alternative method is to treat the strip's wicking pad with the surfac-tant, so that it is rehydrated upon contact with the fluid test sample and flow with the test sample to the labeled bind-ing partner zone (13 in Fig. 1), capture zone and detection zone. A third and preferred method is to combine the surfac-tant with the labeled binding partner and apply the combina-tion to zone 13 thereby causing it to become part of the test strip whereby rehydration occurs upon contacting the strip with the fluid test sample resulting in the surfactant flowing with the labeled binding partner and labeled binding part-ner/analyte complex to the capture and detection zones. Any one of these methods pro~ides the necessary dispersion of the surfactant in the test sample. Preferred surfactants are available under the tradename Tetronic~. The Tetronic surfac-tants are tetrafunctional block copolymers derived from the sequential addition of propylene oxide and ethylene oxide to ethylenediamine. The am:ine moiety in these surfactants pro-vides the surfactants with slightly cationic properties and contributes to their thermal stability. The Tetronic surfac-tan,ts have the poly-pro~poxy groups bonded directly to the amine nitrogens with the polyethoxy groups comprising the pen-dent portions of the surfactant molecule. This is in contrast to the less effective (for purposes of inhibiting non-specific binding) Tetronic~ R surfactants which are produced by the se-quential addition of ethylene oxide and propylene oxide to ethylenediamine resulting in a surfactant having the poly-ethoxy groups interspersed between the amine nitrogens and the po].y-propoxide groups.

It has also been discovered that the labeled binding partner such as a gold sol labeled antibody (GSA) can be caused to more readily release from the first region of the strip, combine with the test sample and to flow along the strip to the capture and detection zones by the introduction of a saccharide into the assay. The saccharide, which may in-clude but is not limited to trehalose, sucrose, fructose or maltose, is typically combined with the labeled binding part-ner in an amount of from 0.2% to 5% by weight per OD of the labeled binding partner.

Typically the test fluid is urine although other body fluids, such as whole blood, plasma, serum, sweat or saliva can be tested. Many clinically significant analytes are pres-ent in urine and other ~body fluids and are determinable by means of the present invention. Among these analytes are deo-xypyridinoline (DPD), hunnan serum albumin, prostate specific ant.igen, drugs of abuse, TDM drugs, cancer markers, cardiac markers, hCG, strep A and Helicobacter pylori. The detectable label for the analyte may be any moiety which is detectable by reproducible means. Thus, the label can be an enzyme, a radio isotope, a chemillumiscent material or, preferably, a visible particulate label such as gold sol.

The method of practi.cing the invention is further illus-trated by the following examples:

Exc~ple I

A strip (2.54 cm x 43.18 cm) of nitrocellulose membrane was used to prepare a test strip similar to that depicted in Fig. 1 except that the strip had three capture zones 14. Rea-gents were deposited onto the nitrocellulose membrane in the following manner: One band of anti-mouse IgG (1 mg/ml of PBS) was deposited onto the matrix at about 3 and 3.5 cm from the bottom in amounts of 2 ~I, and 1 ~L respectively after which 3 ban,ds of DPD-PEG conjugate (1 mg/mL of PBS) were deposited on the nitrocellulose membrane at about 0.5, 1 and 1.5 cm from the bottom at 2 ~L/cm to provide three capture zones. The treated membrane was dried at 40~C for about 17 minutes.

A gold sol-anti DPD antibody (GSA) suspension was pre-pared having the following composition: GSA (10 OD) in 2 mM
borate at pH 9, 14.6% (1.46% per OD of GSA, OD is an optical density unit at 530 nm) trehalose, 0.5% bovine serum albumin (BSA) and 1.26% (0.126% per OD of GSA) Tetronic 1307 as sur-factant. An aliquot of 3 ~L of GSA suspension was pipetted ont:o a GSA pad (0.2'' x 0.2'', Whatman glass fiber F075-07) ancl air dried. The nitrocellulose strip containing the cap-ture zone and detection zone was assembled on a polystyrene bac:king using an acrylic based adhesive. The GSA pad was as-semble adjacent to the nitrocellulose with a 0.04'' overlap.
The wicking pad (0.5'' x 0.2'', Whatman glass filter F075-07) was then assembled adjacent to the GSA pad with 0.04'' over-aE~ .

For testing, the strips were dipped into a test tube con-ta.Lning the test solution, i.e. urine containing a measured amount of DPD, for about 3 seconds, removed from the solution and placed on the specimen table of a CLINITEK~ 50 reflectance meler which measured and recorded the % reflectance of each of the capture and detection, bands. A linear dose response curve was obtained for seven concentrations of DPD ranging from 0 to 250 nM.

The experiment was repeated with other Tetronic and Tetronic R surfactants at; various concentrations of the gold sol-anti DPD suspension. In each case the release of the GSA
from the pad upon application of the test fluid was measured by the amount of GSA (red color) left on the GSA pad 3 minutes after the strip was dipped into the test solution. A ''-'' was used to indicate greater than 80% of GSA left on the pad.
One ''+'' indicates poor release with greater than 50% GSA
left on the pad and ''++~-+'' indicates good release with less than 10% GSA left on the pad. Two and three ''+'' ratings were given for intermedia,te release values. The non specific binding of the gold sol labeled anti DPD to the nitrocellulose strip was determined by the amount of GSA ~red color), after release from the GSA pad, bound to the nitrocellulose in the areas where neither capture reagent nor detection reagent was applied such as the area between the GSA pad and the first capture band and the area between the capture zone and the de-tec:tion zone. The GSA should not bind to these areas because they contain no capture or detection reagent to bind to the ant:ibody. When no surfactant was used in the formulation, greater than 90% of the (SA was non-specifically bound in the area between the GSA pad and the first capture band. A rating system for non-specific binding was established in which ''-'' indicates greater than 80% of the GSA engaged in non-specific binding, one ''+'' indicated very strong non-specific binding wil:h greater than 50% of the BSA bound in areas other than the capture and detection zones and ''++++'' indicates very little non-specific binding of less than 10%. Two and three ''+'' ratings were given for intermediate release values. The re-sults of these experiment:, are tabulated in Table 1.

Cb~ ~ ~q~ % r- -~ ~m NSBo~
r~ GS~-t T~m~c~ 70R-2 T~c . ~." i . i~ISOoi ~
-J 1;.14 ~ tI I I

U. I I I I
._ I I I I
ISO~ I 3~No ."I ~

The Tetronic surfactants used are produced by BASF.
There are two classes of Tetronic surfactants; Tetronic and Tetronic R. The Tetro:nic surfactants are tetrafunctional block copolymers derived from the sequential addition of pro-pylene oxide and ethylene diamine. The resulting polymeric surfactants carry the propylene group next to the amine group.
The Tetronic R surfactants are tetrafunctional block copoly-mers derived from the sequential addition of ethylene oxide and. propylene oxide to elhylenediamine resulting in polymeric surfactants which carry the ethylene group next to the amine group. Both of these classes of surfactant contain the amine functional group which provides a cationic property to the mol.ecule. Since nitrocellulose and various other matrix mate-rials are negatively charged, the positively charged surfac-tants tend to bind to the nitrocellulose surface thereby blocking the nonspecific binding of the gold sol-antibody con-juclate. Based on the da1:a presented in Table 1, the Tetronic surfactants are preferred over the Tetronic R surfactants both in terms of enhancement of GSA release and inhibition of non-specific binding. Certain of the higher molecular weight Tet;ronics beginning at above 7900 are particularly preferred since, with only minor exceptions, they provide four ''~'~
performance in both catec;ories. As indicated by the data re-lat:ing to Tetronic 1307, performance improves with increased concentration of the surfactant although the data suggest that concentrations above 0.7% are not necessary to achieve the de-sired results.

The desirable molecular weights for the Tetronic surfac-tants are greater than 5,000 and preferable greater than 10,000 with a molecular weight in the range of from 10,000 to 30,000 being particularly desirable. The concentration of the su.rfactant used is typically 0.5 to 10% and preferably 0.05%
to 1% by weight per OD of the GSA when the surfactant is in-clllded in the GSA formulation. Each strip is typically pre-pared with 30 OD of the gold sol labeled antibody. When the surfactant is included iLn the strip, a loading of 15 ~g to l,000 ~g per strip is usually sufficient. When the surfactant is added to the test sample, a concentration of 0.02% by 1.3%
by weight is used.

Despite the successful experiments with the Tetronic cationic surfactants, other cationic surfactants which were tested in a similar manner did not prove to be as successful as the Tetronics. The results of these experiments are tabu-lated in Table 2 wherein a ''-'' indicated that lack of GSA
release and non-specific binding were very severe.

C~ p-d ~t~on~c ~enz~i~on~um c~lor~ae 0. 6t - +
Benzyldm~etnyl~etr~aecy~- O. 8 t aroQnium ch~oride Decamethonau3 DrQm~ae 0. 8 t Benzyl~metnylhexaaccyi- O. 8~ - -ammonlum chloride Di~ethyldioc~adecy.L- O. 8 ~ - -am~onlu~ br~mide ~et~yltz~octyial lu~ 0.8~ - ~
ohloride ~enzyia~me~nyldoae,_y - O . 8 t ammon~um br ~ide Cety~pyrldinau~ cnioz:~e 0.8~ +
Cetyid~etllyietnylammor. um I 0. 8t ++ ++
brom de Exa~ple II

In order to demonstrate the advantage of combining the cat,ionic surfactants useful in the present invention with a sac:charide a study was carried out involving the following two formulations:

A: Gold sol antibody conjugate: 1.7 OD (@ 530 nm) Trehalose: 2.5%
Tetronic 1107: 0.17%
BSA: 0.06%
B: Gold sol antibody conjugate: 1.7 OD (@ 530 nm) Tetronic 1107: 0.17%
BSA: 0.06%

All percentages are based on weight, i.e. weight % of the com,ponent to weight of the GSA suspension. After drying the mem~brane, the gold sol antibody conjugate from formulation B
became aggregated and it, release and flow were found to be much worse than that of formulation A.

Claims

CLAIMS:

l. A method for the determination of the concentration of an analyte in a fluid test sample which comprises the steps of:

a) providing a matrix comprising a negatively charged polymeric material through which the fluid sample can flow by capillarity, said matrix having a first region which contains mobile specific binding partner for the analyte which binding partner bears a detectable label and can react with the analyte to form an analyte/labeled specific binding partner complex and a second region which contains immobilized analyte or an immobilized binding partner which is specific for an epitope of the analyte different from that to which the labeled binding partner is specific;

b) combining the fluid sample with a cationic surfactant which is a polyalkoxylated amine; and c) developing the matrix by applying the fluid test sample suspected of containing the analyte thereto and allowing it to contact the mobile specific binding partner so that analyte present in the fluid test sample binds to the specific binding partner to form an analyte/labeled specific binding partner complex, leaving excess, unreacted labeled specific binding partner free to further react whereby the fluid test sample carries the analyte/labeled specific binding partner complex and unreacted labeled specific binding partner along the matrix by capillary to the second region containing the immobilized analyte in which region unreacted, labeled specific binding partner is bound to immobilized analyte in inverse relationship to the concentration of the analyte in the fluid test sample or is bound to the immobilized specific binding partner in direct relationship to the concentration of analyte in the fluid test sample and the non-specific binding of the labeled specific binding partner to the matrix is reduced.
2. The method of Claim 1 wherein the matrix is in the form of a test strip through which the fluid test sample flows horizontally or vertically.
3. The method of Claim 2 in which the flow is horizontal.
4. The method of Claim 1 wherein the negatively charged polymeric material is nitrocellulose, polysulfone or a polycarboxylic acid.
5. The method of Claim 1 wherein the fluid test sample is combined with the surfactant before the fluid test sample is applied to the matrix.
6. The method of Claim 1 wherein the surfactant is applied to the matrix before contact with the fluid test sample and is rehydrated upon contacting the matrix with the fluid test sample.
7. The method of Claim 1 wherein the labeled binding partner is a gold sol labeled antibody.
8. The method of Claim 1 wherein the fluid test sample comprises urine.
9. The method of Claim 1 wherein the surfactant is a tetra-functional block copolymer derived from the sequential addition of propylene oxide and ethylene diamine resulting in a polymeric surfactant carrying a propylene group next to the amine group.
10. The method of Claim 9 wherein the polymeric surfactant has a molecular weight of from 10,000 to 30,000.
11. The method of Claim 1 wherein a saccharide is combined with the fluid test sample.
12. A method for the detection of an analyte in urine which comprises contacting the urine with a strip of nitrocellulose said strip having a wicking region for application of the urine sample, a region containing a labeled antibody specific for the analyte together with a tetrafunctional block copolymer derived from the sequential addition of propyleneoxide and ethylene diamine having a molecular weight of from 10,000 to 30,000 as surfactant and a capture zone in which there is immobilized a material capable of specifically binding with the labeled antibody.
13. The method of Claim 12 wherein the analyte is deoxypyridinoline.
14. The method of Claim 12 wherein the antibody if labeled with gold sol.
15. A test strip for the determination of an analyte in a fluid test sample which comprises a matrix of a negatively charged polymeric material having a first region containing mobile specific binding partner for the analyte which binding partner bears a detectable label and can react with the analyte to form an analyte/labeled specific binding partner complex together with a polyalkoxylated amine surfactant and a second region which contains immobilized analyte or an immobilized binding partner which is specific for an epitope of the analyte different from that to which the labeled binding partner is specific.
16. The test strip of Claim 15 which has a third region in which there is immobilized means for binding the analyte/labeled specific binding partner complex.
17. The strip of Claim 15 wherein the negatively charged polymer is nitrocellulose, polysulfone or a polycarboxylic acid.
18. The strip of Claim 15 wherein the labeled binding partner is a gold sol labeled antibody.
19. The strip of Claim 15 wherein the surfactant is a tetra-functional block copolymer derived from the sequential addition of propylene oxide and ethylene diamine.
20. The strip of Claim 19 wherein the surfactant has a molecular weight of from 10,000 to 30,000.