WO1989000292A1

WO1989000292A1 - A color-coded solid phase analysis method

Info

Publication number: WO1989000292A1
Application number: PCT/US1988/002275
Authority: WO
Inventors: Mats Gustaf Ranby; Nils Arvid Bergsdorf
Original assignee: Cytrx Biopool Ltd.
Priority date: 1987-07-06
Filing date: 1988-07-01
Publication date: 1989-01-12
Also published as: EP0324008A1; AU2077588A; JPH02500861A; EP0324008A4

Abstract

In accordance with the present invention, an improved method of detecting an antigen in a sample is provided comprising the steps of coating a first solid phase (18) and a second solid phase (22) with an antigen-specific antibody, adding a first dye to the first solid phase (18), exposing the first solid phase (18) to a standard solution with a known amount of the antigen, exposing the second solid phase (22) to a solution with an unknown amount of antigen, and determining the amount of antigen bound to the first solid phase (18) and to the second solid phase (22). In another embodiment of the present invention, a second dye with a color different from the first dye can be added to the second solid phase (22).

Description

"A COLOR-CODED SOLID PHASE ANALYSIS METHOD"

Technical Field

The present invention relates to an improved solid phase analysis method, and more particularly, relates to a method of color-coding assays for the detection of antigens or antibodies by immunological solid phase methods.

Background

The term "t-PA", as used herein, means tissue plasminogen activator.

Clinical technicians frequently perform chemical assays in which a number of reagents are added sequentially to a container followed by the addition of a sample to be tested. The technician may utilize a rack of test tubes, a micro test plate or some other container as the solid phase. Aliquots of reagent or sample are normally added one at a time to each tube or microtest plate well from a larger vessel. Normally, the reagents are first added to all of the wells and then an aliquot of the sample solution is added individually to each well. If the technician is interrupted or otherwise distracted during the process of adding sample, a tube or well can be neglected resulting in an erroneous test result. The technician is normally unable to determine whether any particular well contains sample because the reagent solutions are nearly identical to the sample solutions due to the fact that all are normally colorless or lightly colored liquids.

Some technicians attempt to monitor the tube or well to which a sample has last been added by placing a finger or pipette onto the rim of that well, but this procedure often results in cross- contamination and is unsuccessful if the technician moves away from the immediate vicinity of the microtest plate.

What is needed is a visual method of determining whether or not a solution contains a sample.

An improved solid phase analysis method was disclosed in U.S. Patent Application No. 7/038,740 which is incorporated herein by reference and in the article by Ranby, et al., "Age Dependence of Tissue Plasminogen Activator Concentrations in Plasma, as Studied by an Improved Enzyme-Linked

Immunosorbent Assay, Clinical Chemistry, 32, 2160 (1986) which are incorporated herein by reference. The improved solid phase analysis method described above utilizes two microtest plate wells for each sample. Both wells are coated with an antigen-specific antibody, and the second well is exposed to the same antigen- specific antibody solution while the first well is exposed to a non¬ specific antibody. The sample containing antigen is then added and quantitative antigen values obtained. The value obtained for the sample in the second well is subtracted from the value obtained for the same sample in the first well so that the portion resulting from non-specific adsorption (or bonding) cancels out.

A technician performing an assay according to this method must add the same sample to two or more wells, thus increasing the probability of neglecting to add sample to any particular well. Therefore, what is needed is a method of easily and quickly distinguishing two or more otherwise identical wells from one another and also an easy and rapid method for distinguishing a well containing sample from a well to which sample has not yet been added. Summary of the Invention

In accordance with the present invention an improved method of detecting an antigen in a sample is provided comprising the steps of coating a first solid phase and a second solid phase with an antigen-specific antibody, adding a first dye to the first solid phase, exposing the first solid phase to a standard solution with a known amount of the antigen, exposing the second solid phase to a solution with an unknown amount of antigen, and determining the amount of antigen bound to the first solid phase and to the second solid phase. In another embodiment of the present invention, a second dye with a color different from the first dye can be added to the second solid phase.

Either the first dye or the second dye or both dyes can have the capability of changing colors when the sample to be tested is added. For example, if bromphenol blue is added to a solid phase to which a sample containing blood or plasma is to be tested, the bromphenol blue will change from purple to blue when the plasma sample is added. This aids the technician who is running the test to monitor the addition of the samples. The present invention is especially useful in assays utilizing microtest plates wherein large numbers of samples are added to many wells during the typical assay. Because the wells in the microtest plates are color-coded, it is much easier for the technician to tell which wells have sample added. Accordingly, it is an object of the present invention to provide a method of reducing sampling error in immunological assays.

Another object of the present invention is to provide a method for distinguishing a solution to which a sample has been added from a solution devoid of sample.

Another object of the present invention is to provide a method for distinguishing a solution to which a sample has been added from a solution devoid of sample by a color change.

Another object of the present invention is to provide a method for distinguishing lyophilized reagents from one another after reconstitution.

Another object of the present invention is to provide a method for distinguishing one solid phase from another by using various biochemical dyes. Another object of the present invention is to provide a color- coded immunoassay kit.

These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiment and the appended claims.

Brief Description of the Drawing

Fig. 1 is a top plan view of a microtest plate embodying the present invention.

Detailed Description of the Disclosed Embodiment

In accordance with the present invention, an improved method of detecting a substance in a sample is disclosed comprising the steps of immobilizing an antigen-specific antibody to the surface of a solid phase, adding a dye to the solid phase, adding sample containing unknown amounts of the antigen and then determining the amount of antigen bound to the surface of the solid phase. Determining the amount of antigen bound to the surface of the solid phase is well known to those of ordinary skill in the art. The present invention also includes an improved method of detecting an antigen in a sample, comprising the steps of coating a first solid phase and a second solid phase with an antigen-specific antibody, adding a first dye to the first solid phase, exposing the first solid phase to a standard solution with a known amount of the antigen, exposing the second solid phase to a solution with an unknown amount of antigen, and determining the amount of antigen bound to the first solid phase and to the second solid phase. In another embodiment of the present invention, a second dye with a color different from the first dye can be added to the second solid phase. Either the first dye or the second dye or both dyes can have the capability of changing colors when the sample to be tested is added. For example, if bromphenol blue is added to a solid phase to which a sample containing plasma is to be tested, the bromphenol blue can change from purple to blue when the plasma sample is added. This aids the technician who is running the test to monitor of addition of the samples.

The sample may induce a pH change when it is added. In this case many of the pH indicator dyes may be suited for use in the present invention. The color change may be governed by a pH change or it may be governed by other factors such as ionic charge, concentration of a component, etc.

The same concept can be applied to immunological assays for measuring antigen-specific antibody. In these assays, antibody- specific antigen is coated on the solid phase. The sample solutions have known or unknown amounts of antibody in the solution. The present invention can be utilized to analyze either monoclonal antibodies or polyclonal antibodies which are antigen-specific.

The present invention is especially useful in assays utilizing microtest plates wherein large numbers of samples are added to many wells during the typical assay. Because the wells in the microtest plates are color-coded, it is much easier for the technician to tell which wells have sample added.

In another embodiment of the present invention, wherein all of the wells of a microtest plate is used, a first dye can be added to the first row of wells which are coated with either antigen or antibody, depending upon whether antibodies or antigens are being measured. A first dye is added to the first row of wells and a second dye with a color different from the first dye can be added to the second row of wells in the plate. The first dye is then added to the third row and the process is repeated until all of the wells are colored. This results in a microtest plate that has alternating rows of wells with alternating colors. Because the rows have alternate colors, it is much easier to monitor which wells have solution added to them. The improved solid phase analysis method disclosed in U.S. Patent Application No. 7/038,740 and described in Ranby, et al., "Age Dependence of Tissue Plasminogen Activator Concentrations in Plasma, as Studied by an Improved Enzyme-Linked Immunosorbent Assay", Clinical Chemistry, 32, 2160 (1986), which is incorporated by reference herein, is especially well suited for use with the present invention. The improved solid phase analysis method utilizes two microtest plate wells for each sample to account- for the amount of physical adsorption or coating of antibodies on the inner surface of the wells. Both wells are coated with an antigen-specific antibody, and the second well is exposed to the same antigen-specific antibody solution while the first well is exposed to a non-specific antibody. The sample containing antigen is then added and quantitative antigen values obtained. The value obtained for the sample in the second well is subtracted from the value obtained for the same sample in the first well so that the amount of non-specific adsorption (or bonding) is removed.

Because the improved solid phase analysis method utilizes a second vessel or well in a microtest plate for each sample, it is even more likely that a mistake could be made in adding reagents or sample to the wells of the microtest plate. By adding different colored dyes to the various wells in the microtest plate according to the present invention, the likelihood of making a mistake while adding reagents or sample to be tested is greatly reduced. It is to be understood that the improved solid phase analysis method of the present invention includes any addition of a dye in any order to an immunological assay so that the addition of reagents and samples to be tested is made easier to monitor. It is within the contemplation of the present invention that dyes could be added individually to the various wells utilized in a typical immunoassay system.

In performing the improved solid phase immunoassay of the present invention, solid phases, such as a series of test tubes or, more preferably, wells in a microtest plate, such as that used in an ELISA type assay, are coated with an antigen-specific antibody. The invention will now be described with specific examples which are only given for illustrative and not limiting purposes.

Example I The microtest plates are coated with antibody by incubating in the wells of the plates between approximately 30-300 μl of a solution containing between approximately 1-100 mg/1 antibody and between approximately 0.1-1.0 M/l NaHCθ3 while shaking carefully for about two to four hours at 20-30^βC. The wells are then emptied and the contents washed with PBS Tween.

Dyes that can be used in the present invention include, but are not limited to, phenol red, bromphenol blue and, bromcresol green, alizarin red S, bromcresol purple, bromthymol blue, neutral red, brilliant yellow and methyl orange. Referring now to Fig. 1, the following description is one embodiment of the present invention. The typical microtest plate 10 is made up of approximately twelve vertical columns 12, each including approximately eight wells 14. Horizontal rows are often designated with letters 13 from A to H and vertical columns are often designated with numbers 15 from 1 to 12 as indicated in

Fig. 1.

For each sample or standard, two wells in adjacent vertical columns are used as follows. The left-most row 16, designated column 1 , is prefilled with between approximately 100-200 μl of normal, or non-specific antibody, 5-15 mg/ml, dissolved in PBS

Tween. The column 18, designated column 2, immediately adjacent to the left-most column 16 is prefilled with 100-200 μl of a specific antibody, 5-15 mg/ml, also dissolved in PBS Tween. The specific antibody is preferably the same antigen-specific antibody as used for the coating.

The next adjacent column 20, designated column 3, is prefilled with normal antibody and the next adjacent column 22, designated column 4, is prefilled with antigen-specific antibody. Thereafter the columns 12 are alternatively prefilled with normal antibody or antigen-specific antibody. A dye having a distinctive color, such as phenol red, is added to each well in the two left-most columns 16 and 18.

A dye having a different color is added to the next column 20. This dye can be selected from the group of dyes that change color when exposed to the fluid to be tested. For example, bromphenol blue changes color from purple to blue when a solution containing albumin is added. Thus, if plasma is being tested, bromphenol blue is an excellent dye to use.

A dye having a color that can be distinguished from the dyes in the wells of columns 16, 18 and 20 is added to the wells in column 22. Preferably this dye is methyl orange.

Thereafter, wells are alternatingly filled with the latter two biochemical dyes so that all wells which contain the non-specific antibody also contain the biochemical dye which changes color when exposed to a biological fluid.

Therefore, if phenol red, bromphenol blue and methyl orange are used as dyes, the color of the first two columns will be red and the remaining columns will alternate color with one column being purple and the adjacent column being yellow before the addition of any sample.

The left-most columns 16 and 18 are then filled with equal amounts standard plasmas containing increasingly larger concentrations of antigen in concentrations from 0 ng/ml to approximately 30 ng/ml. A biochemical filler solution, such as mannitol, can be added to all the wells. The filler serves the purpose of physically stabilizing the contents of the wells so that the contents will remain in the wells after the microtest plate is lyophilized. The mannitol filler also protects the protein components from denaturing during the lyophilization process. Other substances can be used as fillers including, but not limited to, albumin, glycine, polyethylene glycol and Pluronic® F-68 (BASF Corporation, Parsippanny, NJ).

Blood samples are collected in the conventional manner and are centrifuged within several minutes at 1000-3000 x g for several minutes. A 10 to 100 μl aliquot from each plasma sample to be tested is placed in each of two adjacent wells, starting at column 20, containing one of the biochemical dyes and pre-filled with normal or specific antibody, respectively, in a concentration of 5-15 mg/1 dissolved in PBS Tween. Therefore, an uppermost well 30 in the first adjacent row 20 will contain the same sample as an uppermost well 32 in the second adjacent row 22 of wells. Likewise pairs 40 of adjacent wells, containing solutions of distinguishable colors, will contain an aliquot of the same sample. When plasma is added to the wells containing normal antibody and bromphenol blue, the color of the solution changes from purple to blue enabling the technician to distinguish between wells which already contain a plasma sample and wells to which a plasma sample has not yet been added. Thus, the kit, as described hereinbefore, has the wells with the standard plasmas colored red, the wells with the non-specific antibody are colored purple, and the wells with the antigen-specific antibody are colored yellow. Such a kit is much easier to use and the likelihood of sampling error is greatly reduced because the technician conducting the tests can easily monitor addition of samples.

The standards and samples are incubated for 3 hours at 20-30°C while shaking carefully.

A 50 μl aliquot of HRP-conjugated antibody, 8 mg/1, dissolved in PBS Tween, is added to each well in the microtest plate. The microtest plate is then incubated for 2 hours at 20-30° C in the wells.

The wells are emptied of their contents and are washed with PBS Tween.

A 200 μl aliquot of peroxidase substrate dissolved in citrate- phosphate buffer, pH 5, is then incubated in the wells at 20-30°C in the dark for 5-40 minutes.

The substrate reaction is interrupted by adding 50 μl of 4-5 M/l of H₂Sθ4.

The absorbance at 492 nm is recorded. The difference in absorbance between the wells filled with normal antibody and the wells filled with specific antibody is calculated for the respective plasma samples. This represents the antigen-specific part of the response; this is compared with the standard curve wherein antigen is diluted in antigen depleted blood plasma.

Example 2

The following is a (2-step) method for determination of human tissue plasminogen activator antigen in plasma utilizing polyclonal and monoclonal antibodies against t-PA.

Proteins

Human single-chain tissue plasminogen activator (t-PA) is purified from spent melanoma cell culture medium by affinity chromatography on monoclonal antibody (PAM 1 Sepharose) and subsequent gel filtration on Sephacryl SA-200 superfine. The final product is concentrated to about 1 mg/ml and stored in liquid nitrogen until used. The final product gives a single band on sodium dodecylsulphate polyacrylamide gel electrophoresis under reducing conditions. Protein content is determined by amino acid analysis after acid hydrolysis.

Polyclonal antiserum against t-PA is raised by immunization of 8 goats with highly purified human single chain t-PA. The IgG fraction is isolated by ammonium sulfate precipitation and Protein A affinity chromatography. The IgG is dialyzed against 20 mmol/L phosphate buffer pH 7.2 containing 150 mmol/1 NaCI and concentrated to 10 mg/ml and stored at -20°C or colder until used. Protein content is determined using an absorptivity of 1.6 mg-1 ml-1 cm-1.

Non-immune goat IgG is isolated by ammonium sulfate precipitation and Protein A affinity chromatography. The IgG is dialyzed against 20 mmol/L phosphate buffer pH 7.2 containing 150 mmol/1 NaCl and concentrated to 10 mg/ml and stored at -20°C or colder until used. Protein content is determined using an absorptivity of 1.6 mg-1 ml-1 cm-ϊ. An IgGlyk monoclonal antibody with high affinity against single and two-chain t-PA (PAM 3) is purified from mouse ascites by ammonium sulfate precipitation and Protein-A affinity chromatography. The IgG is dialyzed against 100 mmol 1 NaHC03 pH 9.2 and concentrated to 20 mg/ml and stored at -20°C or colder until used. Protein content is determined using an absoφtivity of

1.4 mg-l m-ϊ cm-1.

PAM 3/HRP conjugate for 1000 micro-test plates is performed as follows: 100 mg Horseradish peroxidase (Sigma type VI (St. Louis, MO., USA) is dissolved with 2 ml 1.25% glutaraldehyde (dissolved in 50 mmol/1 sodium phosphate buffer pH 7.2 containing 150 mmol/L NaCl). The solution is incubated 18 hours at 25°C in dark. After incubation, the solution is diluted to 9 ml with 100 mm NaHCθ3 and dialyzed 8 hours against the same buffer. 0.3 ml PAM 3 antibody (20 mg/ml) is added and the mixture is incubated 18 hours at 25°C in dark. 100 μl 2 mol/1 lysine moπohydrocloride is added to block out remaining active sites. After 90 minutes incubation, the conjugate is sterile filtered and applied to a protein. A sepharose column (75 mg protein A coupled to 25 ml Sepharose 6B FF) is equilibrated with 100 mmol/1 NaHCθ3. After washing the column with equilibration buffer, the column is eluted with 100 mmό^'1/1 sodium acetate pH 5.0. The conjugate fraction is collected, pH is raised to 7.2 and the eluate dialyzed 20 hours against 20 mmol/1 sodium phosphate pH 7.2 containing 200 mmol/1 NaCl. The absorbances at 403 nm and 280 nm is measured, and the A403/A280 ratio is calculated and found to be between 0.2 and 0.3. The conjugate is concentrated to a protein concentration of approximately 1 mg/ml and then stored at -20°C or colder until used. t-PA depleted human plasma. Human plasma found free of hepatitis and AIDS antibodies is t-PA depleted by immunoadsoφtion on sepharose bound polyclonal goat antibodies against human t-PA.

Bovine serum albumin (No. A-7638) is purchased from Sigma, St. Louis, MO., USA. Other Reagents Mannitol, Phenol red, Methyl orange a n d Polyoxiethylenesorbitanmonolaurate (Tween 20) are purchased from Kebo AB, Stockholm, Sweden.

Hydrogen peroxide solution (30%) is purchased from Labassco, Stockholm, Sweden

EDTA, Sodium Chloride, Sodium hydrogen carbonate, and o-phenylenedamine are purchased from Merck, Darmstadt, FRG

Micro-test plates. Micro-test plates (Immuno plate II) is purchasedjfrom Nune, Denmark. PBS-Tween buffer, 10 mmol/1 sodium phosphate buffer containing pH 7.4 containing 140 mmol/1 NaCl, and 0.05% Tween 20.

PET-buffer, 30 mmol/1 phosphate buffer pH 7.25 containing 5.5 mmol/l EDTA, 140 mmol/1 NaCl, and 0.05% Tween 20. 4.5 mol/1 H2SO4.

Ready to use substrate. 250 vials, 56.77 g Na2 HPO4 +

38.4 g citric acid is diluted to 625 ml with distilled water. When all is in solution 2.4 g 1, 2-phenylenedamine (O.P.D.) is added. When

O.P.D. is dissolved, the solution is sterile filtered and lyophilized in 2.5 ml portions.

It should be understood that the foregoing relates only to a preferred embodiment of the present invention and that numerous modifications or alterations may be made without departing from the spirit and scope of the invention as set forth in the appended claims.

Claims

ClaimsI Claim:

1. An improved method of detecting an antigen in a sample, comprising the steps of: a. immobilizing an antigen-specific antibody to the surface of a solid phase; b. adding a dye to the solid phase; c. adding a sample containing unknown amounts of the antigen; and d. determining the amount of the antigen bound to the surface of the solid phase.

2. The method of Claim 1, wherein the antigen-specific antibody is selected from the group consisting of polyclonal antibodies and monoclonal antibodies.

3. The method of Claim 1, wherein the dye changes color upon addition of the sample.

4. The method of Claim 1 , wherein a second solid phase is coated with the antigen-specific antibody.

5. The method of Claim 4, wherein a dye with a color different from the first dye is added to the second solid phase.

6. The method of Claim 5, wherein the second solid phase is exposed to a standard solution with a known amount of antigen.

7. The method of Claim 6, comprising the additional step of lyophilizing the first solid phase and the second solid phase containing the dyes prior to adding samples.

8. The method of Claim 7, comprising the additional step of adding a buffer solution before lyophilizing solid phases prior to adding samples.

9. The method of Claim 1, wherein the dye is selected from the group consisting of phenol red, bromphenol blue, bromcresol green, alizarin red S, bromcresol puφle, bromthymol blue, neutral red, brilliant yellow and methyl orange.

10. The method of Claim 5, wherein the dye having a color different from the first dye is selected from the group consisting of phenol red, bromphenol blue, bromcresol green, alizarin red S, bromcresol puφle, bromthymol blue, neutral red, brilliant yellow and methyl orange.

11. The method of Claim 1 , comprising the further step of adding a filler to the surface of the solid phase.

12. The method of Claim 11 , wherein the filler is selected from the group consisting of mannitol, albumin, glycine, polyethylene glycol and Pluronic® F-68.

13. An improved method of detecting a substance in a sample, comprising the steps of: a. coating a first solid phase and a second solid phase with an antigen-specific antibody; b. adding a first dye to the first solid phase; c. exposing the first solid phase to a standard solution with a known amount of the antigen; d. exposing the second solid phase to a solution with an unknown amount of antigen; and e. determining the amount of antigen bound to the first solid phase and to the second solid phase.

14. The improved method of Claim 13, wherein a second dye with a color different from the first dye is added to the second solid phase.

15. The improved method of Claim 14, wherein the second dye changes color upon addition of the solution with the unknown amount of antigen.

16. The improved method of Claim 13, wherein the first dye changes color upon addition of the solution with the unknown amount of antigen.

17. The method of Claim 13, comprising the additional step of lyophilizing the first solid phase and the second solid phase containing the dyes prior to step (c).

18. The method of Claim 13, comprising the additional step of adding a buffer solution before lyophilizing the first and second solid phases prior to step (c).

19. The method of Claim 13, comprising the additional step of adding a filler before lyophilizing the first and second solid phases prior to step (c).

20. The method of Claim 13, wherein said first dye is phenol red.

21. The method of Claim 13, wherein said second dye is bromphenol blue.

22. The method of Claim 21, wherein said solution with the unknown amount of antigen is contained in blood.

23. An improved method for detecting an antigen in a sample, comprising the steps of: a. immobilizing an antigen-specific antibody on a first solid phase and a second solid phase; b. adding a first dye solution having a first color to the first solid phase and adding a second dye solution having a different color from the first dye to the second solid phase; c. adding a solution containing a non-specific antibody to the first solid phase; d. adding a solution containing the same antigen- specific antibody used to coat the first and second solid phases to the second solid phase; e. adding the sample to the first and second solid phase; f. determining the amount of antigen bound to the first and second solid phase; and g. quantifying the amount of antigen in the sample by subtracting the assay value for the second solid phase from the assay value for the first solid phase.

24. The improved method of Claim 23, wherein a third solid phase is coated with an antigen specific antibody and a third dye is added to the third solid phase.

25. The improved method of Claim 24, wherein a solution with a known amount of antibody is added to the third solid phase.

26. An improved method of detecting an antibody in a sample, comprising the steps of: a. coating a first solid phase and a second solid phase with an antibody-specific antigen; b. adding a first dye to the first solid phase and a second dye with a color different from the first dye to the second solid phase; c. exposing the first solid phase to a standard solution with a known amount of the antibody; d. exposing the second solid phase to a solution with an unknown amount of antibody; and e. determining the amount of antibody bound to the first solid phase and to the second solid phase.

27. An improved method for detecting an antibody in a sample, comprising the steps of: a. immobilizing an antibody-specific antigen on a first solid phase and a second solid phase; b. adding a first dye solution having a first color to the first solid phase and adding a second dye solution having a^" different color from the first dye to the second solid phase; c. adding a solution containing a non-specific antigen to the first solid phase; d. adding a solution containing the same antibody- specific antigen used to coat the first and second solid phases to the second solid phase; e. adding the sample to the first and second solid phase; f . determining the amount of antibody bound to the first and second solid phase; and g. quantifying the amount of antibody in the sample by subtracting the assay value for the second solid phase from the assay value for the first solid phase.

28. The improved method of Claim 28, wherein a third solid phase is coated with an antibody specific antigen and a third dye is added to the third solid phase.

29. The improved method of Claim 29, wherein a solution with a known amount of antibody is added to the third solid phase.