CA2040913C - Method and element for measuring analytes in biological fluids using immobilized binder-analyte labeled complex - Google Patents

Abstract

The method of measuring analytes in biological fluids is disclosed wherein a specific binder to a given analyte is covalently immobilized onto a solid support to which a labeled analyte is pre-reacted and stabilized to form a binder-labeled analyte complex. A sample is contacted with said immobilized complex wherein an analyte in the sample, if present, competes with the labeled analyte bound to the immobilized binder for binding sites on said binder thus displacing a given amount of the labeled analyte which is directly proportional to the amount of analyte present in the sample. The affinity of the labeled analyte to the analyte's specific binder is lower than the affinity of the unlabeled analyte to the same binder.

Description

v BACKGROUND OF THE INVENTION
Competitive isotopic and non-isotopic immunoassays or binding assays with solid phase or second antibody separations for measuring analytes in biological fluids, have been described in the literature for over two decades.
Numerous United States and foreign patents have been issued dealing with one or more aspects of these basic techniques.
Competitive enzyme immunoassays for various analytes were disclosed in U. S. patent No. 3,654,090 to Wilhelmus et al and 3,850,752 i~o Wilhelmus et al.
In enzyrne immunoassays the enzyme label is prepared by one of several methods in which the analyte is covalently attached to the enzyme and the free unreacted analyte is separated from the: enzyme labeled analyte either by dialysis and/or chromatography.
In such methods the free unconjugated enzyme is, in most cases, not separated from the conjugated enzyme for two practical reasons:
1. It requires tedious affinity purification and if accomplished produces a very unstable conjugated enzyme-analyte label since the latter constitutes a very low ratio of the unconjugated enzyme.

2. The analyte specific binder bound enzyme, and not the free unbound enzyme, is the fraction that is quantitated after exhaustive washing of the free unbound enzyme.

t r It follows, therefore, that in the aforementioned type assays the zero dose concentration has the highest signal since there is no analyte to compete with the labeled analyte for binding sites on the analyte's specific binder.
In competitive enzyme immunoassays the absence of a given analyte in a sample produces the highest color while the presence of a given analyte in a sample will produce progressively less color as compared to the zero dose depending on the concentration of said analyte in a given sample. For qualitative, "on site" type assays whereby a "yes" "no" answer is needed for the detection of a given analyte in biological fluids, the type of competitive enzyme immunoassays described in U.S. Patent No. 3,654,090 and 3,850,752 are unsuitable for obvious reasons: (1) the decrease in color from a reference zero dose is difficult to detect by the naked eye, and (2) washing is required to separate the free unbound enzyme from the bound enzyme.
It is the object of the present invention to reverse such a trend since' it is more logical to observe the presence of color in samples containing a given analyte while negative samples, samples devoid of a given analyte, produce no color.
U. S. Patent No. 3,817,837 to Rubinstein et al and U. S. Patent No. 3,852,157 to Rubinstein et al and other follow-up patents disclose homogenous type enzyme amplification immunoassay for haptens where the separation of _.. i , antibody bound enzyme from free unbound enzyme is not required. In said enzyme amplification assay system the antibody-hapten enzyme labeled complex inhibits the enzyme from reacting with its substrate since the active site on the enzyme molecule is sterically hindered by the antibody to the hapten. By contacting the antibody's hapten-enzyme complex and the enzyme complex and the enzyme substrate with a sample containing a given hapten to the antibody it competes with the hapten-enzyme label for antibody sites thus allowing the enzyme to react with its specific substrate. This technique is limited to few enzymes specifically glucose-6-dehydrogenase (U. S. Patent No. 3,875,011) malate dehydrogenase (U. S.
Patent No. 4,191,613) and U. S. Patent Nos. 4,203,802 and 4,067,774.
U. S. Patent No. 4,434,236 to Freytag discloses a method for the rapid determination of analytes in biological specimens by usinc3 an analyte-analogue immobilized on a solid phase wherein a displaceable labeled antibody to the analyte is found. In thi:a disclosed method the antibody has a greater affinity for the analyte than for the analyte-analogue. The presence of an analyte in a sample specific for said antibody will easily displace the labeled antibody. Consequently, the amount of displaced labeled antibody is related to the amount of analyte present: in the sample.

Although this method of U. S. Patent No. 4,434,236 is an improvement over the previously cited patents it relies on two important factors; namely, (i) the use of an analyte-analogue that has a lower affinity to the analyte's labeled antibody, and (ii) the immobilization of analyte-analogues, especially small compounds (such as haptens), on a solid support is not easily achieved and requires specific functional groups on the analyte-analogue in order to affect immobilization. Furthermore, since the affinity of the analyte-analogue to the analyte's antibody is purposely low, the changes of labeled antibody leaking off the solid phase is quite probable.
U. S. Patent No. 4,446,232 to Liotta, is similar in context to that disclosed in U. S. Patent No. 4,434,236, wherein a given antigen is impregnated in a given matrix in the first zone of the disclosed device. In said matrix containing a given antigen, an enzyme-linked antibody is reacted to said antigen. In the presence of antigen in a biological specimen the antibody is displaced into a second zone which contains materials capable of reacting with the enzyme linked antibodies to produce a color. Determination of antibodies in biological fluids is also disclosed by Liotta wherein the antibody is impregnated in first zone and reacted with enzyme-linked, antigen. Although this approach is a simple modification of Freytag's approach, it suffers several v technical drawbacks; (1) the impregnation of antigens or antibodies in the first zone of Liotta's device will be prone to antigen or antibody "leakage" in the absence of patient antigens or antibc>dies, (ii) the affinity of enzyme-linked antibody or antigen is not defined. The competition between sample antigen and impregnated antigen in the first zone to the enzyme-linked antibody is by no means instantaneous because of steric hindrance and the ability of the sample antigen, like hCG in Example 1 of said patent, to dislodge the enzyme-linked antibody from the impregnated antigen is highly improbable because: of steric hindrance and equilibrium considerations.
It is well established in the art that large molecules (greater than 20 kilo daltons), require longer incubations with their specific binders to reach equilibrium.
When the specific antibody (molecular mass 150 kilo daltons) is linked to an enzyme (molecular mass greater than 50 kilo daltons), i.e., the effective molecular mass of the enzyme-linked antibody is approximately 200 kilo daltons, and said enzyme-linked or antigen is not defined. The competition between sample antigen and impregnated antigen in the first zone to the enzyme-linked antibody is by no means instantaneous because of steric hindrance and the ability of the sample antigen, like hCG in Example 1 of said patent, to dislodge the enzyme-linked antibody from the impregnated S T
antigen is highly improbable because of steric hindrance and equilibrium considerations.
It is well established in the art that large molecules (greater than 20 kilo daltons), require longer incubations with their specific bonders to reach equilibrium.
When the specific antibody (molecular mass 150 kilo daltons) is linked to an enzyme (molecular mass greater than 50 kilo daltons), i.e., the effective molecular mass of the enzyme-linked antibody is approximately 200 kilo daltons, and said enzyme-linked antibody is pre-reacted with an antigen (now molecular mass is approximately 220 kD) the patient antigen (20 kD) will require time to compete with the pre-reacted antigen bound to t:he enzyme-linked antibody and displace it.
This is quite obvious from the following equilibria.
kl E-Ab + Agl ~ E-Ab=Agl k2 and k3 E-Ab + Agl + Agl ~--. E-Ab~~Agl + Agl k4 where kl = k2; k3 - k4 and kl » k3 because of steric hindrance and dish>lacement of Agl from E-Ab Agl by Agl is not easily achieved as disclosed in U. S. Patent No. 4,446,232.
Furthermore, k3 > k4 only if the concentration [Agl] » [Agl]
that displacement will occur. This means that the amount of Agl measured will only be at high concentrations; therefore, low sensitivity assay.
This faces is exemplified in European Patent Application No. 0 279 097 in which Fuerstenberg shows that Liotta's disclosure in U. S. Patent No. 4,446,232 is quite insensitive as shown in Example 1 of EPA 0 279 097 for theophylline were the reflectance difference between theophylline levels of 10.4 ug/ml (therapeutic threshold) and 19.4 ug/ml (toxic threshold) is 0.55 - 0.43 or 0.12 units.
Similarly, in Example 2 of EPA 0 279 097, 200 mIU hCG was required to produce a change in color and displace the enzyme-linked hCG antibody. By all analytical standards the Liotta disclosure and the examples cited in EPA 0 279 097 using Liotta's method show that said method as disclosed in U. S.
Patent No. 4,446,.?32 is not sensitive enough to be a reliable analytical tool.
Other United States and foreign patent specifications and applications dealing with elements for the determination of biological fluids are cited here for completion.

r r U. S. Patent Nos. 4,144,306; 4,366,241; 4,740,468;
4,774,192; 4,632,901; 4,774,174; 4,769,333; 4,769,216;
3,811,840 and 4,042,335.
European Patent Specification Nos. 0 042 755; 0 070 300 and European Patent Applications 0 281 201; 0 284 232 and International Applications W084/029193 and W088/06723 SD~ARY OF THE INVENTION
Briefly, the present invention comprises a method for measuring analytes in biological fluids wherein a specific binder (Ab) to a given analyte (Agl) is covalently immobilized on a solid phase support to which a labeled analyte (Agl*) is prereacted to saturate almost all binding sites on said specific binder to form an immobilized specific binder-analyte labeled complex, ~ - Ab ~ Agl*, which method comprises contacting a sample of biological fluid to be analyzed with said immObiliZed complex wherein an analyte (Agl), if present in said sample, competes with the labeled analyte (Agl*) bound to the immobilized binder for binding sites on said binder thus displacing a given amount of labeled analyte (Agl*) which is directly proportional to the amount of analyte (Agl) present in the sample.
More specifically, the present invention provides a method for measuring analytes in biological fluids which comprisess (1) covalently immobilizing a specific antibody binder to a given analyte on a solid phase supportf (2) saturating the binding sites on said specific antibody binfi3er With a labeled analyte to the extent steric hinderance permits to form an immobilized specific antibody binder-analyte labeled complex (3) saturating remaining unoccupied binding sites on the immobilized specific antibody binder-analyte labeled complex with unlabeled analyte prior to contacting said complex with a sample of biological fluid - g -(4) contacting a sample of biological fluid to be analyzed for the presence of the given analyte With said immobilized complex, said sample of biological fluid as contacted with said immobilized complex being in an untreated form as obtained from the donors and (5) allowing an analyte, if present in said sample, to compete with the labeled analyte bound to the immobilized binder for binding sites on said binder thus displacing a given amount of labeled analyte Which is directly proportional to the amount of analyte present in the sample, wherein the affinity of the analyte to the analyte~s specific binder is at least about 10~ 1/mol and is higher than the affinity of the labeled analyte to the same binder and Wherein the analyte has a molecular Weight greater than 20 kD.
The present invention also provides a method for measuring analytes in biological fluids which comprisess (1) covalently immobilizing a specific lectin binder to a given analyte on a solid phase support (2) saturating the binding sites on said specific lectin binder with a labeled analyte to the extant steric hinderance permits to form an immobilized specific lectin binder-analyte labeled complex (3) saturating remaining unoccupied binding sites on the immobilized specific lectin binder-analyte labeled complex Wlth unlabeled analyte prior to contacting said complex with a sample of biological fluid (4) contacting a sample of biological fluid to be analyzed for the presence of the given analyte with said _ 9a _ immobilized complex, said sample of biological fluid as contacted with said immobilized complex being in an untreated form as obtained from the donors and (5) allowing an analyte, if present in said sample, to compete with the labeled analyte bound to the immobilized binder for binding sites on said binder thus displacing a given amount of labeled analyte which is directly proportional to the amount of analyte present in the sample, wherein the affinity of the analyte to the analyte~s specific binder is at least about 10~ 1/mol and is higher than the affinity of the labeled analyte to the same binder, and wherein the analyte has a molecular weight greater than 20 kD.
The present invention also provides a diagnostic device for measuring analytes in samples of biological fluids which comprises:
(1) a column-type assembly defining a fluid pathway having an open end adapted to receive a sample of biological fluid to be analyzed, said fluid pathway being bridged by a first solid phase support, and an effluent discharge point on the lower and of said column-type assembly, opposite said open end f (2) a sleeve-type container having an open end and a closed end, said column type assembly being received in said open and of said sleeve-type containers (3) a specific antibody binder covalently immobilized to a given analyte on said first solid phase support, the binding sites on said specific antibody binder being saturated with a labeled analyte to the extent steric hinderance permits to - 9b -form an immobilized specific antibody binder-analyte labeled complex, remaining unoccupied binding sites on the immobilized specific antibody binder-analyte labeled complex being saturated with unlabeled analyte prior to contacting said complex With a sample of biological fluid, said solid phase support being adapted to have displaced therefrom a given amount of labeled analyte which is directly proportional to the amount of analyte present in the sampler and (4) a second solid support, spaced apart from said first solid phase support, housed at the closed end of said sleeve-type container and in proximity to said effluent discharge point, said second solid support, when contacted by the displaced labeled analyte, being adapted to produce a visible color either directly or after the addition of a substance capable of reacting with the labeled analyte to produce a visible color.
The present invention also provides a diagnostic device for measuring analytes in samples of biological fluids which comprises:
(1) a column-type assembly defining a fluid pathway having an open end adapted to receive a sample of biological fluid to be analyzed, said fluid pathway being bridged by a first solid phase support, and an effluent discharge point on the lower end of said column-type assembly, opposite said open end, (2) a sleeve-type container having an open and and a closed end, said column type assembly being received in said open and of said sleeve-type container, - 9c -(3) a specific lectin binder covalently immobilized to a given analyte on said first solid phase support, the binding sites on said specific lectin binder being saturated with a labeled analyte to the extent steric hinderance permits to form an immobilized specific lectin binder-analyte labeled complex, remaining unoccupied binding sites on the immobilized specific lectin binder-analyte labeled complex being saturated with unlabeled analyte prior to contacting said complex with a sample of biological fluid, said solid phase support being adapted to have displaced therefrom a given amount of labeled analyte which is directly proportional to the amount of analyte present in the samplef and (4) a second solid support, spaced apart from said first solid phase support, housed at the closed and of said sleeve-type container and in proximity to said effluent discharge point, said second solid support, when contacted by the displaced labeled analyte, being adapted to produce a visible color either directly or after the addition of a substance capable of reacting with the labeled analyte to produce a visible color.
This invention also comprises a diagnostic device for measuring analytes in samples of biological fluids which comprisess a column-type assembly defining a fluid pathway having an open end adapted to receive a sample of biological fluid to be analyzed, said fluid pathway being bridged by a first - 9d -solid phase support, and an effluent discharge point on the side of said support opposite said open end, a sleeve-type' container having an open end and a closed end, said assembly being received in said open end of said sleeve-type container, a specific binder (Ab) covalently immobilized on said solid phase support to which an analyte label (Agl*) is pre-reacted to saturate almost all binding sites on said binder to form a first solid phase specific binder-analyte label complex, ~Ab~Agl*, said solid phase complex when contacted with a biological fluid sample containing a specific analyte (Agl), being adapted to have displaced therefrom labeled analyte (Agl*) in an amount directly proportional to the concentration of Agl, a second solid support, spaced apart from first solid phase support, housed at the closed end of said sleeve-type container and in proximity to said effluent discharge point, said second solid support, when contacted by the displaced labeled analyte (Agl*) form the effluent discharge point said first solid phase complex, being adapted to produce a visible color on said second solid support either directly or after the addition to said second solid support a substance capable of reacting with the analyte label to produce a visible color.

In accordance with the present invention a method and diagnostic device are disclosed for the determination of analytes in biological fluids wherein a specific binder for a specific analyte is covalently immobilized onto a solid support, preferably microparticles but not restricted to same, to which a labeled. specific analyte is pre-reacted whereby all binding sites on the specific binder are completely occupied with the labeled a.nalyte and in certain instances the binding sites are saturated with a combination of the labeled analyte and unlabeled analyte. Determination of a given analyte proceeds according to the following, preferred but not restricted to, analytical steps: (a) admixing a biological sample, suspected of containing a given analyte, with a diluent in a separate tube; (b) pouring the analyte/diluent mixture onto a diagnostic device, as illustrated in the drawings, whereby the analyte/diluent is contacted with the immobilized binder-analyte label complex; (c) the resultant reaction mixture flowing through the solid phase microparticle bed is contacted with a substrate specific for the displaced label and a colored product is developed in which is directly proportional to th.e amount of analyte present in the sample.
The affinity of a specific analyte to its specific binder is higher than the affinity of the specific analyte-label to the given binder.

DETAILED DESCRIPTION OF THE INVENTION
Turning to the drawings, Figure T_ is a side plan view of the column assembly present in the device of this invention.
Figure 2 is a sectional view of the sleeve-type container used in the device of this invention.
Figure .3 is a sectional view of the device of this invention showing the elements of Figures 1 and 2 as fully assembled and ready to receive a sample of biological fluid at the top or open end.
Figure ~6 is a top plan view of the device of Figure 3.
Turning to the drawings in more detail, the method of the present invention is conducted in a novel self-contained diagnostic device in which a specific binder is covalently immobi7.ized on microparticles and reacted with a specific labeled analyte and placed in a column assembly 10 which includes a chromatographic-type compartment. The assembly 10 has an open end 12 to receive the sample of biological fluid t:o be analyzed. The fluid pathway 14 runs longitudinally of the assembly 10. The solid phase support 16 bridges the assembly 10. An effluent discharge point 18 is at the lower end. The solid phase support 16 separated by two O-ring type frits 20 and 22 made of a porous substance. The assembly of Figure 1 has a funnel-shaped cross-section and is inserted in an opaque outer-sleeve type container 24 shown in Figure 2 that houses a lcm porex*-type material 26 inserted at the bottom of the outer-sleeve 24. The porex*-type material 26 is used to absc>rb excess sample/diluent volume. The base 28 of the outer-sleeve 24 is also opaque but contains in the center of it a thin (approximately 10 mm) clear white absorbent element 30 wherein the displaced labeled analyte is concentrated either through simple absorption and/or through a secondary immunological reaction involving label-antilabel or analyte-ligand anti-ligand. The colored product of the label is thus formed on the surface of clear absorbent 30 either through direct reading of the label, in the case where the label is a colored! dye or a colored latex particle, or through the reaction of the label with its substrate, in the case where the label is~ an enzyme, or through the reaction of the label with its enzyme, in the case where the label is a substrate.
It is to be understood that the open end 32 of the outer sleeve 24 ma.y be provided with a sealing cap or closure held by a force or interference fit.
*Trade-mark Certain conditions have to be met in order to achieve maximum sensitivity for a given analyte when measured by the method of the instant invention.
The following conditions are set forth to fulfill the requirements of the present invention: Reactions (1) and (2) below describe the equilibria involve in such a method kl ~Ab + Agl* ~ ~Ab Agl* (1) k 92 and k3 ~Ab + Agl * + Agl ~- ~Ab Agl * + Agl ( 2 ) k4 where ~-Ab is the specific analyte binder covalently immobilized on solid support Agl* is the labeled analyte Agl is the unlabeled analyte to be measured.
The affinity constant for reaction (1) above is K1; K1 = k2 where k k2 > kl, i.e. the labeled analyte should have a fairly high affinity to the specific analyte binder and the reaction~is always favored to the direction of the binder-analyte label complex where K1 = k2 kl The affinity constant for reaction (2) above is K1; K1 = k4 where k3 k3 > k4, i.e. the analyte should have a higher affinity to the specific binder than does the labeled analyte, thus k3 > kl.
In other words, the affinity of the unlabeled analyte to the analyte's specific: binder is at least about 10~ 1/mol and should be greater than the label analyte has to the same binder.
For large molecular mass analytes (>20 kD) k3 could be made greater than k4 by two different analytical manipulations, either separately or combined:
1) Increas'i.ng the concentration of the enzyme labeled analyte [Agl*] that is bound to immobilized specific binder in reaction (1), supra, to a point of maximum saturation wherein there exists no binding sites unoccupied on the specific binder. Therefore, shifting the equilibrium to the right in reaction (1), supra, where kl > k2. This should be accomplished withc>ut creating a steric hindrance situation on the solid phase. This is possible by diluting the solid phase, e.g. sepharose, with sepharose that does not contain the specific binder thus spacing the immobilized specific binder entities within said solid phase matrix far apart to avoid steric hindrance.
The addition of a minimal concentration of Agl should easily displace the label analyte Agl* from the immobilized specific binder; therefore, k3 > k4 and a signal is produced which is directly proportional to the concentration of added analyte [Agl].
2) Binding sites unoccupied on the immobilized specific binder ~Ab by the labeled analyte Agl* could cause low sensitivity assays because the addition of analyte, Agl, from a biological specimen will first bind to these unoccupied sites on the specific binder and not displace the labeled analyte. By saturating these sites with "cold" analyte prior to the assay, the addition of analyte from a biological specimen will dish>lace enzyme labels analyte at low concentrations, thus a high sensitivity assay especially~since kl > k2 as explained, supra. A combination of 1 and 2, supra, could even yield sensitivities in the sub-nanogram range as will be shown in t:he instant examples of the invention.
Multiple analytes could be screened using the method and diagnostic device of the present invention by admixing several immobilized specific binders to various analytes in their respective appropriate dilutions; thus, the solid phase support will contain multiple specific binders to which a specific analyte-labeled has been pre-reacted and stabilized ~Abl ", Agl*
~~2 ~ Ag2 ~Ab3 ,~, Ag3 ~Abn ~" Agn*
where ~Abl to ~P,bn are various immobilized specific binders to which a specific analyte-labeled Agl* to Agn* have been pre-reacted and stabilized. The mixture containing the various immobilized pre-reacted specific binders-analyte labels could now serve as one reagent for screening several analytes in one given specimen without any loss of sensitivity. This is particulary useful for drug screening programs where multiple drugs could be screened on a given specimen. A positive result using said multiple approach could then be confirmed using the single analyte approach.
Screening for other types of analytes using the disclosed invention should be obvious to those skilled in the art.
In the following examples the conjugation and labeling methods used are well-known in the art and are presented here for' illustrative purposes only and should not be restrictive to the practice of the instant invention.
Other conjugation and labeling procedures could easily be used by those skilled in the art. Furthermore, the types of solid phase used to immobilize the analyte's specific binder and the type of enzyme or other label used are not restrictive and are obvious to those skilled in the art.
The analyte's specific binders used in the following examples were antibodies, polyclonal or monoclonal, raised against various analytes. The analyte's specific IgG from these various antibodies was routinely purified by Protein-A
chromatography using immobilized recombinant Protein-A
(Repligen, Cambridge, MA 02139) following well-known established procedures.

The Protein-A purified specific IgG was covalently bound to cyanogen bromide activated sepharose* 4B by the' method of March, C'.S. et al., 1974. Anal. Biochem. 60:149-152.
Macromolecular antigens (molecular mass greater than 20 K.D.) were enzyme (horseradish peroxidase) labeled using the periodate method of Boorsma, D.M., et al (1979). J.
Immunol. Meth. 30:245-255.
Haptens were coupled to horseradish peroxidase enzyme using the carbodiimide method of Staros, J.V. (1986).
Anal. Biochem. 156:220-222.
The device can be built as an integral unit or alternatively elements 10 and 24 can be assembled at the time of use.
The following Examples are illustrative of the invention, and are not intended to be limiting in any way.

MORPHINE ASSAY IN URINE
Morphine-3-glucuronide was conjugated to horseradish peroxidase enzyme using a modification of the method of Staros et al, 1986, supra, as follows:
(1) 2.95 mg of morphine-3-glucuronide (6.4 x 10-3) mmol) were dissolved in 0.5 ml of normal saline to which 20 mg of horseradish peroxidase (RZ > 3) dissolved in 3.0 ml of normal saline were added.
*Trade-mark (2) 15 mg oi= N-hydroxysuccinimide was added to the mixture in (1) and stirred until it was dissolved to which 40 mg of EDC (N-ethyl-N-(3-dimethylaminopropyl) carbodiimide) dissolved in 0.5 ml normal saline was added dropwise over 30 minutes.
(3) The reacaion mixture in (2) was stirred for an extra 60 minutes at ambient temperature and a further 10 mg EDC
added as powder. The reaction mixture was stirred overnight.
(4) The enzyme conjugate was then dialyzed for 48 hours against 2 x 5 liters of phosphate-buffered saline and finally charcoal absorbed twice using 20 mg activated charcoal in an ice bath and filtered through 0.22 micron filter. 40 mls of morphine antibody (polyclonal) was purified on Protein-A
column as described, supra, to yield 378 mg IgG which was then conjugated to cyanogen bromide activated sepharose 4B as indicated to yield 3.6 mg IgG/ml of gel, and diluted at an appropriate dilution in unreacted sepharose 4B in the ratio of 1:100 (1 part morphine antibody coated gel to 100 parts unreacted sepharo~;e gel). The working gel could be stored in phosphate-buffered saline solution containing 0.025 (w/v) sodium azide at 4°C for extended time periods without any loss or "leakage" of Ig~G.

Binding of morphine-horseradish peroxidase conjugate to the solid phase morphine antibody was accomplished as~
follows:
The diluted morphine antibody (IgG)-sepharose gel was washed with a diluent prepared in 0.05 M phosphate buffer pH 7.0 containing 0.01 (w/v) thimerosal, 0.2~ (w/v) alkali treated casein, O,.l~s (w/v) charcoal absorbed human serum albumin and 20 ug/ml gentamicin sulfate, and was reconstituted such that 2 ml of gel suspension in said buffer contains 1 ml of settled gel volume. The morphine-enzyme conjugate was then added to the morphine-antibody gel suspension to give a final concentration of 7.:1000 and the mixture was incubated for 60 minutes at ambient: temperature on a rotary mixer. This process causes the' binding of morphine-enzyme label to the immobilized morphine specific antibody on the sepharose.
Unbound or free enzyme conjugate is washed off the solid phase with the 0.05 M phosphate buffer, pH 7.0 diluent. The washed gel containing they immobilized morphine antibody-morphine horseradish peroxi.dase complex is transferred to the diagnostic device (Figure 1) as described, supra, and each device now contains 250 ul of settle gel (3, Figure 1). Few millilitres of diluent are passed through the gel to ensure that no more enzyme elutes from the gel. The gel containing the immobilized antibody-analyte label complex could be stored at 4°C either lyopholized or suspended in the 0.05 M
phosphate, pH 7.0, diluent in the diagnostic device until used.
The affinity constant of morphine to the morphine antibody was calculated by the method of J. D. Teale ("Radioimmunoassa;r". In David Williams, Ronald Nunn, Vincent Marks (eds), Scientific Foundations of Clinical Biochemistry, Vol. 1, 1978:299-.322, Pub. William Heinemann, London) and found to be 4.a x 1011 1/mol. The affinity constant of morphine-3-glucuronide to the same antibody was also calculated by the same method and found to be 8 x 1010 1/mol.
Description of the Tnlorkiag Model The diac3nostic device once assembled will contain 250 ul of suspended gel (16) containing the immobilized antibody-analyte label complex in the column assembly 10 part of the device, Figure 1, and protected on each side by two porous frits 20 and 22. The inner part of device, the column assembly 10, is housed in the outer-sleeve 24 shown in Figure 2, as described, supra. 126 samples were analyzed to determine the presence or absence of morphine and/or opiates using the described diagnostic device and reagents as follows:

1. 2 drops of urine were added to 900 ul of sample diluent (0.05 mg/rnl of tetramethylbenzidine in phosphate buffered saline, pH 7.4) to give an approximate 1:10 (v/v) dilution of sample'.
2. The contents of sample plus diluent were poured onto the top 12 of the diagnostic assembly 10.
3. The: device was inverted and one drop of peroxide at a concentration of 0.02% in citrate/phosphate buffer, pH 5.0, was added to the clear white absorbent element (7). After 5 minutes color was observed and recorded: white =
negative; blue = positive.
The results of the above assays showed that out of the 126 samples analyzed 71 samples were negative and 55 were positive. When compared against a sensitive RIA assay for morphine (Coat-a-C'ount* Morphine kit Diagnostic Products Corporation, Los P.ngeles, CA 90045) 100% agreement was achieved. The positive samples had morphine RIA values ranging from 162 n.g/ml to 116,200 ng/ml. All 71 negative samples were from known volunteers not taking any drugs.
The Coat-a-Count* RIA assay had a cut-off of 25 ng/ml for free morphine. The morphine assay of'the present invention is set at a cut-off of 300 ng/ml for morphine-3-*Trade-mark glucuronide and 50 ng/ml cut-off for free morphine. Levels below 300 ng/ml of morphine-3-glucuronide or 50 ng/ml free morphine will not produce any visible color.
The fol7.owing compounds did not produce any visible color when assayed in the morphine method of the present invention at concentrations of 10,000 ng/ml:
oxazepam cotinine caffeine acetaminophen PCP
acetylsalicylic acid secobarbital amphetamine cocaine fentanyl LSD
bupreneorphine lidocaine ibuprofen fenfluramine D-propoxyphene methaqualone benzoylecogonine methadone PHENCYCLIDINE (PCP) ASSAY IN URINE
Phencyclidine (PCP) derivative, 1-[1-(phenyl-3-0-car-boxymethyl eth.er)-cyclohexyl] piperidine, synthesized according to the methods of Kalir, A., et al, 1969, J.Med.
Chem. 12:473 and R.ao, P.N. et al, 1980, J. Steroid Biochem, 13:1291, was conjugated to horseradish peroxidase by the method of Staros, et al 1986, supra, as outlined in Example 1, supra, except that the PCP derivative was dissolved in dimethylformamide instead of normal saline.
mls of PCP antibody (polyclonal) were purified on Protein-A column as described, supra, to yield 254 mg IgG
which was then conjugated to cyanogen bromide activated sepharose 4B as indicated to yield l.3 mg IgG/ml of gel and diluted at an appropriate dilution in unreacted sepharose 4B
in the ratio of 1:5 (1 part PCP antibody coated gel to 5 parts unreacted sepharose gel). The diluted gel is stored as 20 indicated in Example 1, supra. PCP-horseradish peroxidase conjugate was bound to the PCP antibody diluted gel after a dilution of 1:1000 in the phosphate buffer, pH 7.0, diluent as described under example l, supra. The gel containing the immobilized PCP antibody-PCP horseradish peroxidase label was first washed with 0.02 M citrate/acetate buffer, pH 5.O,~then rewashed with phosphate buffer, pH 7.0, diluent to remove unbound or free enzyme conjugate. The washed pre-reacted gel was transferred to the diagnostic devices, as in Example 1, supra.
The affinity constants of PCP and horseradish peroxidase PCP to the PCP antibody were calculated by the method of Teale, 1978, supra, and were determined to be 1.4 x 1012 1/mol for PCP and 3.0 x 1010 1/mol for horseradish peroxidase-PCP conjugate.
Seventy-one urine specimens were analyzed for PCP
using the described reagents and diagnostic device of the present invention, as indicated for morphine in Example 1, supra, and compared to a sensitive RIA PCP method (Coat-a-Count PCP, Diagnostic Products Corporation, Los Angeles, CA
90045) .
50 samples were from known PCP addicts and were RIA
positive at a cut-off of 25 ng/ml. All 50 samples were also positive by the method of the instant invention at a cut-off of 50 ng/ml. All 21 negative samples were correctly identified.

The positive samples had PCP RIA values ranging from 151 ng/ml to 2672 ng/ml.
The following drugs gave negative results, no visible color, when assayed in the PCP method of the present invention at concentrations of 10,000 ng/ml Ethyl morphine morphine methaqualone cotinine secobarbital lidocaine normorphine diazepan D-propoxyphene phenobarbital acetominophen acetylsalicylic acid amphetamine benzoylecgonine bupreneorpine caffeine ecgonine codeine.

The folJ.owing drugs gave positive results (equivalent to 100 ng/ml PCP) at the concentrations indicated:
1- [1- (2-Thienyl) -c:yclohexyl] piperidine 100 ng/ml 1-(1-Phenylcylcohexyl)-4-hydroxypiperidine 1000 ng/ml N-Ethyl Phencyclidine 10,000 ng/ml URINARY HUMAN ALBUMIN ASSAY
Human albumin was conjugated to horseradish peroxidase by the periodate method of Boorsma et al, 1979, supra.
Monoclonal antibody raised against human albumin was prepared according to the method of Galfre, G. and Milstein, C. (Preparation of Monoclonal Antibodies: Strategies and procedures. In Methods of Enzymology, Immunochemical Techniques, vol. .'3, Langone, J. and Van Vunakis, H., eds.
Academic Press (1981) pp. 3-46).
The asci.tes fluid was purified on Protein-A column as described, supra, and the affinity of the monoclonal antibody to human albumin was 2.3 x 107 1/mol as determined by the method of Adri.on, R. F. 1982, Clin. Chem. (lett); 28, p.
717. The monoclonal specific IgG was coupled to Sepharose 4B
by the cyanogen bromide activation procedure of March et al, 1974, supra, to yield 0.763 mg IgG/ml of gel. The IgG coupled gel was diluted with casein-coupled Sepharose 4B (4.92 mg casein/ml of gel), using the same coupling procedure as that for IgG, in various ratios described below. The diluted gel containing the human albumin monoclonal antibody was then reacted with human albumin-horseradish peroxidase conjugate for 1 hour at ambient temperature and washed with the phosphate buffer, pH 7.0, diluent as described in Example 1, supra. The washed gel now containing immobilized human albumin monoclonal. antibody-human albumin-horseradish peroxidase ( ~Ab~~HA - E) was then reacted with 300 ug/ml human albumin equivalent (30 ug albumin per 250 ul of gel) to saturate all binding sites on the albumin monoclonal antibody.
The gel is rewashe:d with the phosphate buffer, pH 7.0, diluent to remove any unreacted "cold" albumin and transferred to the diagnostic device of the present invention. To check the effective minimum detection level for measuring albumin in urine using the above-described method, urinary albumin calibrators containing 10, 20, 30 and 40 ug/ml of albumin were diluted 1:10 in the sample diluent (0.05 mg/ml of tetramethylbenzidi.ne in phosphate buffered saline, ph 7.4) as described under Example 1, supra, and applied to diagnostic devices containing the following ratios of reagents as shown in Table 1.

~IgG ~ CASEIN Albumin- "Cold" Minimum gel gel HRPO Albumin Detection Dilution ug per Limit 250 ul ug/ml gel DEVICE 1 1 part: 25 parts 1:100 30 20 2 1 part: 50 parts 1:25 30 40 3 1 part: 50 parts 1:50 30 20 4 1 part: 25 parts 1:200 30 20 1 part: 50 parts 1:100 30 20 6 1 part: 50 parts 1:50 0 320 7 1 part: 25 parts 1:25 0 340 Thus, saturating the unoccupied binding sites by manipulating the albumin-enzyme conjugate and the addition of "cold" albumin enable the system to detect 20 ug/ml of urinary albumin. Without the added "cold" albumin the detection limit is approximately 33 ug/ml. Published studies (Mogensen, 1984, supra) based on highly sensitive RIA for albumin have established the upper limit of normal for adults as approximately 15 ug/minute or approximately 17 ug/ml (based on 1600 mls of urine is excreted in 24 hour period) and a range extending from 20-~30 ug/ml to about 150 ug/ml as an operational definition of microalbuminuria. The disclosed methods of the present invention allows the rapid detection of albumin in urine at levels only previously achieved with highly sensitive immunoassays.

URINARY HUMAN CHORIONIC GONADOTROPIN (hCG) hCG was conjugated to horseradish peroxidase by the periodate method of Boorsma et al., 1979, supra.
Monoclonal antibody raised against hCG was prepared according to the method of Galfre et al, 1981, supra.
The asci.tes fluid was purified on Protein-A column as described, supra, and the affinity of the monoclonal specific antibody to hCG was 1.18 x 109 1/mol as determined by the method of Adri.on, R. F. 1982, supra. The monoclonal specific IgG was coupled to Sepharose 4B by the cyanogen bromide activation procedure of March et al, 1974, supra, to yield 0.95 mg IgGfml. The IgG coupled gel was diluted with unreacted Sepharose 4B in ratio of 1:25 (1 part IgG gel to 25 parts unreacted Sepharose 4B). The diluted gel containing hCG

monoclonal antibody was then reacted with hCG-horseradish peroxidase conjugate dilute 1:25 in phosphate buffer, pH~7.0, diluent for 4 hours at ambient temperature and washed with phosphate buffer, pH 7.0, diluent as described in examples 1 and 3, supra.
The washed gel now containing immobilized hCG
monoclonal antibody-hCG-horseradish peroxidase ( ~AbhCG'~hCG -E) was then reacted with various amount of "cold" hCG to saturate all binding sites to the hCG monoclonal antibody as shown in Table 2 below. The gel was re-washed with phosphate buffer, pH 7.0, di:luent to remove any unreacted "cold" hCG and transferred to the diagnostic device of the present invention.
The minimum detection level for measuring hCG in urine using the above-described method, was checked by using urinary hCG calibrators containing 20, 30, 40, 50 and 60 mIU/ml of hCG diluted 1:10 in the sample diluent as described under Examples 1 - 3, and applied to diagnostic devices containing the following reagent ratios as shown in Table 2.

--IgG ge.l ~--CASEIN hCG- "Cold"hCG Minimum gel HRPO hCG mIU Detection Dilution per Limit 250 ul gel mIU/ml DEVICE 1 1 part: 25 parts 1:25 0 60 2 1 part: 25 parts 1:25 1 50 3 1 part: 25 parts 1:25 2 40 4 1 part: 25 parts 1:25 3 30 1 part: 25 parts 1:25 4 20 The effective minimum detectable limit for the urinary hCG assay is approximately 20 mIU/ml. Without the added "cold" hCG the detection limit is approximately 60 mIU/ml. Meticulous titering of "cold" hCG in the system could yield sensitivities even lower than 20 mIU/ml.
Having fully described the invention it is intended that it be limited solely by the lawful scope of the appended claims.

Claims (18)

1. A method for measuring analytes in biological fluids which comprises:
(1) covalently immobilizing a specific antibody binder to a given analyte on a solid phase support;
(2) saturating the binding sites on said specific antibody binder with a labeled analyte to the extent steric hinderance permits to form an immobilized specific antibody binder-analyte labeled complex;
(3) saturating remaining unoccupied binding sites on the immobilized specific antibody binder-analyte labeled complex with unlabeled analyte prior to contacting said complex with a sample of biological fluid;
(4) contacting a sample of biological fluid to be analyzed for the presence of the given analyte with said immobilized complex, said sample of biological fluid as contacted with said immobilized complex being in an untreated form as obtained from the donor; and (5) allowing an analyte, if present in said sample, to compete with the labeled analyte bound to the immobilized binder for binding sites on said binder thus displacing a given amount of labeled analyte which is directly proportional to the amount of analyte present in the sample, wherein the affinity of the analyte to the analyte's specific binder is at least about 10' l/mol and is higher than the affinity of the labeled analyte to the same binder and wherein the analyte has a molecular weight greater than 20 kD.

2. A method for measuring analytes in biological fluids which comprises:
(1) covalently immobilizing a specific lectin binder to a given analyte on a solid phase support;
(2) saturating the binding sites on said specific lectin binder with a labeled analyte to the extent steric hinderance permits to form an immobilized specific lectin binder-analyte labeled complex;
(3) saturating remaining unoccupied binding sites on the immobilized specific lectin binder-analyte labeled complex with unlabeled analyte prior to contacting said complex with a sample of biological fluid;
(4) contacting a sample of biological fluid to be analyzed for the presence of the given analyte with said immobilized complex, said sample of biological fluid as contacted with said immobilized complex being in an untreated form as obtained from the donor; and (5) and allowing an analyte, if present in said sample, to compete with the labeled analyte bound to the immobilized binder for binding sites on said binder thus displacing a given amount of labeled analyte which is directly proportional to the amount of analyte present in the sample, wherein the affinity of the analyte to the analyte's specific binder is at least about 10' l/mol and is higher than the affinity of the labeled analyte to the same binder, and wherein the analyte has a molecular weight greater than 20 kD.

3. The method of claim 1 wherein the displaced labeled analyte is then detected by contact with a solid support which is adapted to produce a visible color directly or after the addition to said solid support of a substance capable of reacting with the labeled analyte to produce a visible color.

4. The method according to claim 1 wherein multiple immobilized specific antibody binders are pre-reacted with their respective analyte labels to form multiple immobilized specific antibody binder-analyte labeled complexes and admixed together to serve as a single solid phase complex for screening multiple analytes in one given specimen.

5. The method according to claim 1 wherein the specific antibody binder is a polyclonal antibody raised against a given analyte.

6. The method according to claim 1 wherein the specific antibody binder is a monoclonal antibody raised against a given analyte.

7. The method according to claim 1 wherein the label is an enzyme.

8. The method according to claim 1 wherein the label is a substrate.

9. The method according to claim 1 wherein the label is a colored dye.

10. The method according to claim 1 wherein the label is a colloidal gold.

11. The method according to claim 1 wherein the label is a colored dye entrapped in a liposome.

12. The method according to claim 1 wherein the label is a pH indicator.

13. The method according to claim 1 wherein the solid phase support is an activated insoluble cross-linked carbohydrate polymer gel.

14. The method according to claim 1 wherein the solid phase support is an activated polymer microsphere latex particle.

15. The method according to claim 1 wherein the solid phase support is an activated controlled pore-size glass particle.

16. The method according to claim 1 wherein the solid phase support is an activated cellulose particle or nitrocellulose particle.

17. A diagnostic device for measuring analytes in samples of biological fluids which comprises:
1) a column-type assembly defining a fluid pathway having an open end adapted to receive a sample of biological fluid to be analyzed, said fluid pathway being bridged by a first solid phase support, and an effluent discharge point on the lower end of said column-type assembly, opposite said open end;
2) a sleeve-type container having an open end and a closed end, said column type assembly being received in said open end of said sleeve-type container;
3) a specific antibody binder covalently immobilized to a given analyte on said first solid phase support, the binding sites on said specific antibody binder being saturated with a labeled analyte to the extent steric hinderance permits to form an immobilized specific antibody binder-analyte labeled complex, remaining unoccupied binding sites on the immobilized specific antibody binder-analyte labeled complex being saturated with unlabeled analyte prior to contacting said complex with a sample of biological fluid, said solid phase support being adapted to have displaced therefrom a given amount of labeled analyte which is directly proportional to the amount of analyte present in the sample; and 4) a second solid support, spaced apart from said first solid phase support, housed at the closed end of said sleeve-type container and in proximity to said effluent discharge point, said second solid support, when contacted by the displaced labeled analyte, being adapted to produce a visible color either directly or after the addition of a substance capable of reacting with the labeled analyte to produce a visible color.

18. A diagnostic device for measuring analytes in samples of biological fluids which comprises:
1) a column-type assembly defining a fluid pathway having an open end adapted to receive a sample of biological fluid to be analyzed, said fluid pathway being bridged by a first solid phase support, and an effluent discharge point on the lower end of said column-type assembly, opposite said open and, 2) a sleeve-type container having an open end and a closed end, said column type assembly being received in said open end of said sleeve-type container, 3) a specific lectin binder covalently immobilized to a given analyte on said first solid phase support, the binding sites on said specific lectin binder being saturated with a labeled analyte to the extent steric hinderance permits to form an immobilized specific lectin binder-analyte labeled complex, remaining unoccupied binding sites on the immobilized specific lectin binder-analyte labeled complex being saturated with unlabeled analyte prior to contacting said complex with a sample of biological fluid, said solid phase support being adapted to have displaced therefrom a given amount of labeled analyte which is directly proportional to the amount of analyte present in the sample; and 4) a second solid support, spaced apart from said first solid phase support, housed at the closed end of said sleeve-type container and in proximity to said effluent discharge point, said second solid support, when contacted by the displaced labeled analyte, being adapted to produce a visible color either directly or after the addition of a substance capable of reacting with the labeled analyte to produce a visible color.