WO1997023774A1 - Particle assisted immunoassay - Google Patents

Abstract

The sensitivity of particle based immunoassays is improved by including a second, larger type of particle with a smaller particle. The technique is particularly useful to extend the sensitivity range of colloidal gold immunoassays and can be used in a convenient strip format. In one embodiment, larger selenium particles are included in a sandwich type assay that uses smaller gold particles. The larger selenium particles improve the color development of the gold particles. The technique is applicable to other particle based assays where an improvement in assay sensitivity is desired.

Description

PARTICLE ASSISTED IMMUNOASSAY

Field of the Invention

This invention relates generally to diagnostic assays and specifically to methods for improving the sensitivity of particle-based immunoassays. Preferred embodiments of this invention provide improved gold particle-based immunoassays that are carried out in strip formats.

Background of the Invention

Diagnostic assays play an indispensable role in the treatment and management of disease. Many different types of diagnostic assays have been developed over the years in response to the need for detecting water borne substances in environmental samples, food and drink samples, and clinical samples such as blood, serum, plasma, urine, saliva, tissue biopsies, stool, sputum, and skin or throat swabs. Advantageously, such assays should give a quick result to allow timely treatment of a disease. In reality, however, many of these assays are limited by their speed. Another important parameter of assays is sensitivity. Developments in test technology have led to increasingly more sensitive tests that allow detection of newer analytes in trace quantities as well as for detecting disease indicators at the earliest time possible. Still, increases in sensitivity are desired for some assays.

A third limitation is convenience. Many assays require training of the test operator and complicated instrumentation. These assays cannot be used easily for rapid testing in the field, where electricity or instrumentation are sometimes difficult to obtain. Yet another limitation is the size of test sample required to obtain a test result and the amount of diagnostic test material required to obtain a test result. This limitation is a natural consequence of assay sensitivity. A test method that is more sensitive usually will require less test reagent and less sample to reach a result.

These limitations were addressed in part by the development of a gold particle (also called "gold sol") immunoassay technique as reported by Horrisberger et al . in J . Hiεtochem . & Cytochem . 25: 295 (1977) . Horrisberger describes a gold particle immunoassay for annan wherein aggregation of colloidal gold particles causes a change in light absorption. A similar metal particle immunoassay is described by Leuvering in U.S. Patent No. 4,313,734 (1982). Yost et al. , in U.S. patent 4,954,452 showed that nonmetal colloids such as selenium also can be used for colloid immunoassays. Unlike many other immunoassay techniques, the gold particle immunoassay technique does not require instrumentation and can be read by eye. This characteristic makes the gold particle technique particularly suitable for use in third world countries and other countries such as Russia where electricity-based instrumentation based devices are not always easily purchased or used. Colloidal gold particles can be prepared by reduction of a gold chloride with sodium citrate in an aqueous solution as described by Frens, Nature, 241: 20 (1973) . Gold particle size can be varied by changing the concentration of sodium citrate used to make colloidal gold.

Although gold particle assays can be made very specific and sensitive by the appropriate choice of antibody, much more needs to be done to make these assays more sensitive. For example, the gold particles used in the assay exist in a colloidal dispersion, which exhibits complex behavior that is difficult to predict. A colloidal dispersion is a two phase system wherein each particle is separated from its liquid medium by a separation space. The particle interface has characteristic adsorption and electrical potential properties which influence the behavior of binding reactions at the particle surface. Per unit weight, smaller particles have a greater surface volume and, under some circumstances react faster in binding reactions. To more fully exploit the advantage of non- instrumentated detection for gold sol assays, one can combine the gold particle approach to a strip method, such as that shown by Grubb et al., in U.S. patent 4,168,146. This patent discloses porous test strips to which antibodies have been immobilized. In one embodiment the strips are contacted with a solution containing target analyte which diffuses across the porous test strip. An immobilized antibody then captures the target analyte. An enzyme labeled antibody which is specific for the target analyte is added and the target analyte is indirectly detected by formation of color after a colorigenic substrate is added.

A convenient strip technique could be combined with gold particle detection by visual means to expand the use of immunoassays to regions of the world where instrumentation and electricity are not available. However, if the sensitivity of this combined method could be improved over current technology, then analyte detection could be quickened and analytes could be detected at lower concentrations. That is, an increase in sensitivity would directly lead to earlier clinical treatment and thereby potentially decrease human suffering.

Accordingly, there exists a need for sensitive assays which are capable of visual reading. Similarly, there also exists a need for gold particle assays that exhibit improved sensitivity or provide other advantages over existing assays.

Summary of the Invention

It is an object of the present invention to improve sensitivity of particle based immunoassay assays and thereby allow faster detection of analytes and for their detection at lower concentrations.

It is a further object of the present invention to facilitate the use of smaller amounts of test reagent to obtain an assay result. This provides for smaller and less expensive test devices.

In order to accomplish at least some of the foregoing objects, the present inventors have provided a method for determining the presence or amount of an analyte in a sample. The method comprises the first step of contacting the sample with a reagent. The reagent comprises a first group of particles having bound thereto a binding component capable of specifically recognizing the analyte, and a second group of particles having bound thereto a binding component capable of specifically recognizing the analyte. The average diameter of the particles in the second group is smaller than the particles in said first group. The second step of the method is determining the presence or amount of analyte- particle complexes as a detection or measure of the amount of analyte in the sample.

In one embodiment of the invention, the presence or amount of analyte-particle complexes is determined after filtration through a membrane. In another embodiment of the invention, the presence or amount of analyte-particle complexes is determined after chromatographic movement through a porous support.

In another embodiment of the invention, a method is provided for enhancing the sensitivity of a gold particle immunoassay by employing less than an equivalent mass of selenium particles of larger size in the assay.

In yet another embodiment of the invention, a method is provided for an improved assay in which dissimilar particles, such as particles of dissimilar composition

(e.g., metallic and non-metallic), size and/or charge, are used in the assay.

The foregoing objects, as well as other objects, will be readily apparent to the skilled artisan from the detailed description of the preferred embodiments as summarized below.

Detailed Description of the Preferred Embodiments

Although poorly understood, the present inventors believe that manipulation of the particle interface of the colloidal dispersion containing the particles might lead to more sensitive immunoassays. Indeed, while studying gold particle immunoassays the present inventors surprisingly found that inclusion of a different particle type such as colloidal selenium particles imparted new, desirable properties to the immunoassay. In fact, the present inventors surprisingly found that an immunoassay which used a combination of colloidal gold particles and colloidal selenium particles performed much better than the same immunoassay using only selenium particles or only gold particles. Indeed, the new combination allowed more than a ten fold improvement in assay sensitivity.

It was further surprisingly found that improved immunoassay performance stemmed from combining one larger type of particle with a smaller type of particle. Both particle types are coated with reagent and participate in the same binding reaction. The larger particle type is present in equal to or smaller quantities (based on total mass) than the smaller particle type. A particle useful for the present invention can be comprised of any material as long as it can be coated with a binding member and can stay in suspension. Examples of suitable materials are metal colloidal particles such as gold, silver, platinum, iridium, ruthenium and the like as well as non-metals such as selenium, sulfur and tellurium. Although particles made from plastic and glass can be employed by one skilled in the art, particles comprised of a metal or other naturally colored substance are preferred because their aggregation can be more readily seen by the naked eye. Preferred are particles of colored latex, methacrylate, silica, and metal colloids such as colloidal gold, silver, or nonmetal colloids such as selenium, sulfur, tellurium. Particles of gold and selenium are especially preferred because colloidal suspensions of these two substances can be readily made from their salts and form strong colors upon aggregation.

The particles of the invention participate in one or more binding reactions between a binding component and an analyte, in which the analyte is a corresponding bindable substance in an aqueous test sample. The binding component can be, for example, one or more of the following: antibody, protein A, protein G, avidin, lectin, nucleic acid, a naturally occurring binder, and a synthetic binder. The analyte can be any one of a large number of antigens, antibodies, or ligands of interest. The binding component is preferably a protein and most preferably an antigen or antibody capable of specifically recognizing the corresponding analyte. Skilled artisans will recognize many possible binding components that can be used. Formation of analyte-particle complexes causes a color or color intensity change whereby the presence of an analyte can be visually determined. A wide variety of binding assay formats can be used to link one or more binding reactions to this color or color intensity change.

One format is a sandwich assay in which, for example, analyte binds two different particles or analyte binds an immobilized binding member (such as antibody bound to a membrane support) and a particle. Another format is a competitive inhibition assay in which analyte ligand competes with another substance for binding. In this format the presence of analyte modulates particle to particle binding or particle to immobilized binding member binding. Yet a third basic format is a displacement assay in which analyte in a test sample is detected by its ability to compete and displace a binding member. Such competition can, for example, be used to modulate the movement of particles within a moving stream of liquid such as sample fluid that has been taken up by an absorbent solid. Other assay formats are known to those skilled in the art and can be used.

Both larger and smaller particles are coated with the binding component. Conjugation of the binding component to the particles can be carried out using a number of methods known in the art including physical immobilization, covalent bonding, hydrophilic bonding, hydrophobic bonding, and ionic interaction. Most preferably an antibody capable of specifically recognizing the analyte of interest is adsorbed onto the particle surface, followed by washing and blocking with another protein such as bovine serum albumin.

Both larger and smaller particles are small enough to stay in suspension long enough to participate in a binding reaction and are smaller than l um in their longest axis. In accordance with this embodiment of this invention, the larger particles will have an average diameter that is larger than the average diameter of the smaller particles. Generally, it is contemplated that the larger particles will have an average diameter that is greater in diameter by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 100% or more, than the average diameter of the smaller particles.

In one advantageous embodiment, the larger particle is bigger than 50 nm, on average, in its largest dimension, and the smaller particle is smaller than 50 nm, on average, in its largest dimension.

In one advantageous embodiment, the larger particle is most preferably a selenium colloid particle prepared by boiling a solution of selenium dioxide in the presence of ascorbate. The smaller particle is most preferably a gold colloid particle prepared by boiling a solution of gold chloride in the presence of sodium citrate.

The larger and smaller particles are used together as one suspension. The larger particle can be present in a greater, equal or lesser amount (expressed as gm/liter) than the smaller particle.

In one advantageous embodiment, the larger particle is present in an equal amount or in lesser amount than the smaller particle. In such an embodiment, the larger particle comprises between 10% to 50% of the total particle mixture and the smaller particle comprises between 50% and 90% of the total particle mixture. The optimum ratio of larger to smaller particle for the particular assay employed is easily determined by one skilled in the art and can depart from these stated percentages, based on the mean particle size, degree of derivitization with binding component, total concentration of particles, pH, ionic strength, temperature, and other factors. In practice, one can determine the optimum use ratio by varying the ratio of large to small particles over the range 0.05 to 20 within a given set of reaction parameters.

The method of the present invention involves contacting a liquid sample having or suspected of having an analyte of interest, with the mixture of two particle types that are coated with at least one binding component. The particle mixture may be present as a dried material that is re-wetted by the sample. More preferably the particle mixture is dried within an absorbent such as porous plastic, porous glass, glass fiber, paper, and the like. Preferably the particle mixture is reconstituted by application of an aqueous sample suspected of containing the analyte to the absorbent. After an appropriate incubation time, (i.e. sufficient time to allow the binding component to bind, be displaced, or have its binding inhibited by analyte) , the presence or amount of analyte/particle labelled constituent complexes is determined as a measure of the amount of analyte in the sample. The relative amount of particle aggregation, or dispersal (in the case of a displacement assay) is determined by a color or color intensity change.

In a preferred embodiment of the invention, gold particles and selenium particles are coated with a first antibody for one epitope of an analyte molecule. The particles are deposited onto one end of an absorbent material. A second antibody that binds a separate epitope of the analyte is immobilized in a line on the absorbent material at some distance from the deposited particles. A test fluid suspected of containing the analyte is contacted with an end of the absorbent material near the deposited particles. The aqueous sample fluid wicks along the absorbent material and, while doing so, re-wets and suspends the particles.

While the absorbent is imbibing sample fluid, the first antibody on particle surfaces binds its epitope of the analyte. Sometime later the second antibody which has been immobilized to a separate region more distal to the sample application end encounters the particles. Particles that have bound analyte on their surfaces adhere to the immobilized antibody and form a color in a line in response to the presence of analyte.

In another preferred embodiment particles are coated with antibodies that are targeted for various epitopes of the sample analyte. During use, test fluid is applied to a porous material in which particles have been deposited. The particles are re-wetted and aggregate in response to analyte in the test sample. The wetted particles contact one side of a membrane that has on its other side an absorbent which pulls fluid across the membrane. A suitable membrane will have a porosity that is at least five times the mean diameter of the larger particles. A preferred membrane is 5 um porosity nitrocellulose. Particles that have not reacted with analyte go through the membrane but particles that have become crosslinked by analyte are held back and accumulate on the membrane surface. Analyte is detected as the formation of color on the membrane. Most preferred in the context of this second embodiment is a membrane that has a second binding component which binds to ligand and which can capture particles. Another embodiment of this invention provides particle assays in which the particles employed are dissimilar in composition an /or charge. That is, particles which are made from dissimilar metals (e.g. gold and platinum) , or from metals and non-metals (e.g. platinum and selenium) , or particles of different electrostatic charge (e.g., selenium and plastic), are used in the assay. As above, both particle types are coated with reagent and participate in the same binding reaction. The particles and assay formats which may be used to prepare such assays are described above.

Other binding combinations and particle combinations are possible and readily understood by an average skilled worker in this field.

EXAMPLE

In this example, selenium and gold particles are used to detect the presence of V. cholera 01.

Preparation of mouse monoclonal V. cholera antibody: Purified V. cholera antibody was commercially obtained from Global Diagnostics, Gainesville, Fl. The antibody was dialyzed against 0.002 M borax buffer pH 8.2, filtered through a 0.2 micron cellulose acetate filter, and diluted in the same buffer to a final concentration of 100 ug per milliliter.

Preparation of particles: One hundred milliliters of a 0.03% w/v selenium dioxide solution were heated to boiling. Then, 2.25 milliliters of a freshly prepared solution of 2% ascorbic acid were added to the 0.03% Se0₂solution. The admixture was boiled until its volume evaporated to 50 milliliters and then was cooled to room temperature. The optical density of the cooled solution at 520 nm and 580 nm was 1.457 and 1.167 respectively. The colloidal selenium that was prepared by this treatment was centrifuged at 10000 x g for 15 minutes. The supernatant was discarded and the pellet was resuspended in 100 ml of distilled water.

A 4% solution of gold chloride was prepared by dissolving 360 mg of gold chloride (tetrachloroauric acid trihydrate) into 9 milliliters of deionized water. A 1% solution of sodium citrate was prepared by dissolving 1.0 gram of sodium citrate into 100 milliliters of deionized water. Three liters of deionized water were placed into a 4 liter beaker and brought to a boil on a hot plate. Then 7.5 milliliters of the 4% gold chloride solution were added to the boiling water. Seventy two milliliters of the 1% sodium citrate solution were added to the beaker and the solution was boiled until its volume was reduced to 2.2 liters. A colloidal gold solution was removed from the hot plate and allowed to cool to room temperature. The cooled colloidal gold solution was filtered through a 0.2 micron cellulose acetate filter unit into a clean amber bottle. Optical densities at 520 nm and 580 nm of the resultant filtrate were 1.54 and 0.455 respectively.

A one milliliter aliquot of colloidal selenium was mixed with an equal volume of colloidal gold. The pH of this mixture was 5.0. The combined selenium-gold mixture was centrifuged at 10000 x g for 1 minute to remove any aggregated material. The pH of the supernatant was adjusted to 8.0 by the careful addition of 0.2 M potassium carbonate.

Other mixtures of selenium and gold particles were similarly made as follows:

Vol. Colloidal Gold Vol. Colloidal Selenium milliliters mill:iliters

A. 0 2.0

B. 0.8 1.2

C. 1.0 1.0

D. 1.2 0.8

E. 2.0 0

Two hundred microliters of the anti ViJrio cholera monoclonal antibody (100 ug/ml) were added to each 2.0 milliliter colloidal selenium-colloidal gold admixture as shown in the above table. Then, 200 microliters of 20% bovine serum albumin were added to each aliquot. Each prepared mixture was incubated for 5 minutes at room temperature. The mixtures were then centrifuged at 16,000 x g for 5 minutes. Their supernatants were removed and pellets were resuspended in 0.02 M Tris buffer pH 8.2 that contained 1% bovine serum albumin. Each preparation was washed twice. After the last wash each pellet was resuspended in 100 microliters of .02 M Tris buffer pH 8.2 that contained 1% bovine serum albumin.

Protein protection studies were performed on each combination as follows: 1.0 ml colloid + 100 ul varying concentrations of anti-ViJrio cholera monoclonal antibody (80 ug/ml, 100 ug/ml, 120 ug/ml, 160 ug/ml, and 200 ug/ml) at various pH's (6.0, 7.0, 8.0, and 9.0) prepared by the addition of 0.2 M potassium carbonate. Minimum protein protection occurred at an antibody concentration of 10 ug per milliliter of selenium-gold mixture at pH 8.0 and pH 9.0.

Preparation of nitrocellulose strips: Mylar backed five micron nitrocellulose was cut into 22 mm x 4 mm strips (Grade 8980, Gelman Sciences, Ann Arbor, Michigan) . Rabbit anti-vibrio cholera affinity purified antibody (Louisiana State University, Baton Rouge) was diluted into 0.05 M sodium borate buffer Ph 8.2 to a concentration of 1.6 mg/ml. Then 1.5 microliters of antibody were spotted near the center of each nitrocellulose strip. The strips were dried in a vacuum desiccator.

A vinyl strip backed with acrylic adhesive was cut into 4 mm x 70 mm size portions. Each mylar backed nitrocellulose strip was affixed to a vinyl strip. An absorbent paper (type III 4 mm x 30 mm from Gelman Sciences) was affixed to each nitrocellulose/vinyl strip so as to overlap the top of the nitrocellulose strip. A glass fiber pad (Gelman Sciences, grade 8980) was affixed to overlap the bottom of each nitrocellulose strip. Assay for V. cholera

V. cholera 01 (ATCC strain #11628) and V. cholera non 01 (ATCC strain #14547) were grown in alkaline peptone water media overnight at 35 degrees C. The organisms were centrifuged at 5,000 x g for 5 minutes and each pellet was resuspended in phosphate buffered saline pH 7.2 to an optical density at 650nm of 0.6 (approximately 10* organisms per ml) . Log,₀ dilutions of each organism were made into an extraction buffer consisting of 1% Triton X-100 in 0.02 M Tris with 1% bovine serum albumin and O.l M NaCl at pH 8.0. One hundred microliters of each diluted organism suspension was placed into a separate 10 mm x 75 mm test tube. To each pair of tubes was added 50 ul of the following mixtures of colloidal gold/selenium antibody conjugates:

Colloidal gold Colloidal Selenium

% volume % volume

A. 0 100 B. 40 60

C. 50 50

D. 60 40

E. 100 0

A test strip was placed in each tube and the fluid in each tube was allowed to diffuse up and through the nitrocellulose strip. After 10 minutes each strip was examined for the presence of an orange-purple color in its middle zone where antibody had been mobilized. The results were as follows:

ViJrio cholera 01 Vibrio cholera nonoi

8AMPLE 10* 10⁷ 10⁶ 10⁵ 10* 10⁷ IO⁶ A 3 + 2+ N N N N N

B 3 + 2 + 1+ N N N N

C 4 + 3 + 2 + N N N N

D 4 + 3 + 2 + N N N N

E 2 + 1+ N N N N N

4+ very strong reaction

3+ strong reaction

2+ moderate reaction

1+ weak reaction

N no reaction

The following table summarizes the test results for various concentrations of colloidal selenium to colloidal gold when reacted with V. cholera 01 extracts at a concentration of IO⁶ organisms/ml.

SAMPLE %SELENIUM/ %GOLD REACTIVITY AT 10⁶/iuilliliter

A 100/ 0 N B 60 / 40 1+

C 50/ 50 2 + D 40/ 60 2+ E 0 / 100 N

These results show that the V. cholera assay test was at least 10 times as sensitive when a mixture of selenium particles and gold particles were used compared to when only selenium particles were used or when only gold particles were used.

It will be apparent to those skilled in the art that various modifications and variations can be made to the compositions and processes of this invention. Thus, it is intended that the present invention cover such modifications and variations, provided they come within the scope of the appended claims and their equivalents.

Claims

What Is Claimed Is:

1. A method for determining the presence or amount of an analyte in a sample comprising the steps of: A. contacting said sample with a reagent comprising a first group of particles having bound thereto a binding component capable of specifically recognizing said analyte, and a second group of particles having bound thereto a binding component capable of specifically recognizing said analyte, wherein the average diameter of said particles in said first group is larger than the average diameter of the particles in said second group; and

B. determining the presence or amount of analyte-particle complexes to detect the amount of analyte in the sample.

2. The method of claim 1, in which said larger particles comprise selenium and said smaller particles comprise gold.

3. The method of claim 1, in which the total volume of smaller particles exceeds the total volume of larger particles.

4. The method of claim 1, in which the mean diameter of said smaller particles is less than 50 nm and the mean diameter of said larger particles is greater than 50 nm.

5. The method of claim 4, in which said smaller particles are gold particles.

6. The method of claim l, wherein step B is carried out by determining the presence or amount of analyte-particle complexes as a measure of the amount of analyte in the sample by chromatographic movement through a porous support.

7. The method of claim 6, in which said porous support is comprised of a membrane coated with antibody at a detection zone.

8. The method of claim 1, wherein step B is carried out by determining the presence or amount of analyte-particle complexes as a measure of the amount of analyte in the sample by filtration through a membrane.

9. The method of claim 8, in which said membrane has a porosity of between 0.22 and 5 microns.

10. A method for enhancing the sensitivity of a gold particle immunoassay employing gold particles having bound thereto a binding component capable of specifically recognizing an analyte, said method comprising employing selenium particles in said immunoassay, said selenium particles having bound thereto a binding component capable of specifically recognizing said analyte.

11. The method of claim 10, wherein said selenium particles are present in less than an equivalent amount by volume as compared to the volume of said gold particles, and the average diameter of said selenium particles is greater than the average diameter of said gold particles.

12. A method for determining the presence or amount of an analyte in a sample comprising the steps of:

A. contacting said sample with a reagent comprising a first group of particles having bound thereto a binding component capable of specifically recognizing said analyte, and a second group of particles having bound thereto a binding component capable of specifically recognizing said analyte, wherein the particles in said first group are metallic and the particles in said second group are non-metallic; and

B. determining the presence or amount of analyte-particle complexes to detect the amount of analyte in the sample.

13. A method for determining the presence or amount of an analyte in a sample comprising the steps of:

A. contacting said sample with a reagent comprising a first group of particles having bound thereto a binding component capable of specifically recognizing said analyte, and a second group of particles having bound thereto a binding component capable of specifically recognizing said analyte, wherein the particles in said first group have an electrostatic charge that is different from the electrostatic charge of the particles in said second; and

B. determining the presence or amount of analyte-particle complexes to detect the amount of analyte in the sample.