AUGMENTED AGGLUTINATION ASSAY
The present invention relates to agglutination assays and methods of detecting the presence of test substances in a sample solution. The invention further relates to methods and apparatus for performing such assays.
Agglutination assays are used to screen solutions for the presence of an antigen, antibody or some other recognisable substrate. For example, agglutination assays have been long used in ABO blood typing, testing for the presence of antibodies in serum, serotyping bacteria and analysing solutions for microbial contamination.
Agglutination assays generally comprise a particle upon whose surface are adsorbed affinity ligands. These surface affinity ligands bind to substrate sites on molecules that may be present in the test solution. When a solution contains the particular substrate being tested for, the affinity ligands on the particle surface bind the substrate. This in turn facilitates a crosslinking of the particles with each other and they aggregate together to form a consolidated matrix. This results in an often visible clumping or agglutination of the particles which can either be measured and scored visually or by more quantitative methods utilising the increase in optical density that occurs on agglutination.
Known assays of this type are commonly used in clinical applications as they tend to be fast, inexpensive and can be highly specific. However, they are not generally regarded as being particularly sensitive especially when compared to other techniques such as ELISA. This is a significant drawback in the use of agglutination assays, especially with regards to their use in parts of the world where laboratory facilities suitable for ELISA are unavailable and a cheaper, less rigorous assay would be of great benefit.
A further disadvantage of known assays is that towards the limit of sensitivity the signal weakens, making it difficult to determine if there has or has not been any agglutination. There is hence a desire for an agglutination assay with increased sensitivity or increased signal for a given level of analyte.
It is an object of the invention to provide an improved agglutination assay.
Accordingly, a first aspect of the invention provides a reagent for an agglutination assay comprising a synergistic combination of affinity ligand coated particles of different sizes.
The invention thus provides a combination of particle sizes whose combined effect facilitates an improved assay compared with that seen for assays based upon each particle size alone. This synergistic combination comprises both large and small particles which become crosslinked together in an agglutinated mixture when in the presence of the test substance, also referred to as the analyte.
In an example of the invention in use, which is described in more detail below, a combination of a plurality of small and large particles generated an assay with sensitivity down to a level where less than one hundred analyte bacteria were detected in a sample of liquid. Both small and large particles were coated with the same affinity ligand. When each particle size alone was used in a similar assay no analyte bacteria were detected. This example illustrates the increased sensitivity which can be achieved according to the present invention and places the reagent of that embodiment of the invention well within the sensitivity range of even the most sensitive ELISAs.
In further examples of the invention in use, also described in detail
below, the synergistic combination of differently sized particles provides for a greater signal strength compared with the signal obtained in similar assays using just one particle size. In some examples the reagent of the invention registers a positive signal that is between 20 and 40% stronger than that seen with corresponding single size particle preparations. This is of particular benefit when there is doubt as to whether a result is indeed positive or not and thereby allows for more certainty of results within the normal range. Generally, the invention may provide either improved sensitivity or increased signal compared with known assays, and may also provide both.
It is known in the art to use particles of a single size for agglutination assays and most agglutination assay compositions specifically comprise latex beads of the same size. Indeed, manufacturers of such beads emphasise the homogeneity of size in bead preparations and generally state a maximum deviation from the average bead diameter when advertising the product. For example, Dynal" manufactures bead preparations that are stated as having less than 10% variation in the size of the beads within the preparation (see table entitled "characteristics of Dynabeads™" on page 8 of the Dynal™ Biosciences
Product Catalogue 2000).
Suresh and Arp (Avian Diseases ( 1 993) 37: pp.767-772) describes a typical agglutination assay of the art, in which 3.03 micron latex antibody coated beads are used to test for a specific bacterial pathogen .
Thus, the assay composition of Suresh and Arp, like the majority of compositions in the art, is based on a highly homogenous population of latex bead particles whose size varies little if at all from the mean.
Particles of the invention comprise any particle that is suitable for use in an agglutination assay. Examples of such particles are beads of substantially spherical, cubic, ovoid, rhomboid, rod-like or any other
shape. The most suitable particles tend to be made of relatively stable and inert substances that provide a support for the coating of affinity ligand. Such substances would therefore not typically be soluble in the assay solution or highly reactive with any component likely to be found in an assay solution. Particles may be suitably made from polymers such as latex, crosslinked dextran, crosslinked agarose, gelatin, cellulose, acrylic, polyacrylamide, polystyrene or mixtures of the aforementioned polymers. Non polymeric particles are made for example from glass, carbon, bentonite, alumina, metal and silica or any other suitably inert material. Other particles of the invention may include live or dead eukaryotic and/or prokaryotic cells such as bacterial cells or erythrocytes. These cells may also be chemically or genetically modified so as to make them more suitable for use in the assay.
Particles of the invention may also comprise additive compounds that may alter the properties of the particle. Such additives include magnetite particles that impart paramagnetic characteristics, or coloured dyes which may be of the visible spectrum or fluoresce when excited by UV light.
It is a feature of the present invention that the reagent or composition containing that reagent includes a mixture of particles that are of different sizes. The term "different sizes" is preferably used herein to refer to a particles whose average diameter differs by a minimum ratio of 3:2, preferably at least 5:3, these being referred to respectively as the large and small particles. In a typical composition of the invention the maximum ratio of diameters is not more than around 50: 1 , preferably no more than 25: 1 and more preferably 15: 1 or less. In one specific embodiment of the invention the ratio is about 6: 1 and in a further embodiment the ratio is about 2: 1 . The size range of the particles of the invention is not believed to be crucial to the invention but is typically between 0.25 microns and 20 microns in diameter.
Examples of compositions of the invention comprising different sized particles are described in more detail below.
The reagent of the invention comprises a combination of large and small particles, with the amount or number of each being sufficient to provide a synergistic effect in increasing the sensitivity and/or increasing the signal compared with assays based on single size particles alone. To achieve such a combination, there is generally provided a number ratio of large particles to small particles, or small particles to large particles, between 1 : 10000 and 1 : 1 . Typically the number ratio is around 1 :5000 or less, preferably no more than about 1 :2000 and more preferably 1 :500 or less. In specific embodiments of the invention described in more detail below ratios of large to small particles that show positive synergistic effects are around 1 :9000, 1 :270, 1 :33 and 1 :8. In various embodiments of the invention in use there are two or three different particle sizes in the composition and there can be as many as ten different particle sizes.
Particles of the invention need not all be of the same shape. The invention provides for compositions where some of the particles are substantially spherical beads and others are rod shaped, cubic or rhomboid. In one example of the invention a composition comprises large diameter particles of an irregular globular shape and a plurality of smaller diameter particles of a spherical shape. A reagent of the invention can comprise a combination of bacterial cells and differently sized beads coated with affinity ligand, illustrating how the invention is not limited to spherical or substantially spherical particles.
The particles of the invention are coated with an affinity ligand. The term "affinity ligand" is hereby used to refer to any compound or substance capable of providing a recognisable binding site for interaction with another compound or substance. Such binding sites can
be of a structural, magnetic, electrostatic or hydrophobic nature or indeed a combination of all or some of these.
Affinity ligands suitable for the invention include: - antibodies and fragments thereof e.g. Fab, Fc, antigen binding light chain or antigen binding heavy chain fragments;
- antigens and haptens;
- avidin and streptavidin or the corresponding ligand biotin;
- ligand binding domains of receptor molecules, e.g. cell surface receptor molecules such as the CD4 receptor or a Tbp receptor;
- ligands for cell surface receptor molecules, e.g. protein signalling molecules such as the interleukins;
- coagulation factors e.g. heparin or sulphated dextran;
- cell binding molecules e.g. protein A, protein L.
Affinity ligands may be attached to the surface of the particles of the invention by a variety of means that are known to the art, such as via covalent bonding to the particle substrate, electrostatic attraction to particle functional groups or merely passive adsorption to the particle surface. Some examples of typical attachment regimes are outlined below:-
Naked/Non functionalised - used mainly for passive adsorption via methods such as borate/bovine serum albumin (BSA) binding and sodium phosphate/phosphate buffered saline (PBS) binding techniques;
Amino functionalised - used in covalent coupling procedures and utilises glutaraldehyde as a chemical linker. The amino functionalised particles are incubated in glutaraldehyde solution overnight prior to addition of a protein affinity ligand solution. The beads are then incubated with a quenching solution to
quench any unreacted amino groups on the particle surface;
Carboxylate functionalised - used in covalent coupling procedures and utilises a chemical linker such as 1 -ethyl-3-(3- diethylaminopropyl) carbodiimide (EDC);
Chloromethyl functionalised - used in covalent coupling procedures and involves dialysing both chloromethyl functionalised particles and a protein affinity ligand solution against a wash buffer (phosphate buffer and anionic detergent).
The particles and protein solution are then mixed together for 18 hours and the particles are quenched with glycine solution;
Avidin coated - the avidin/biotin interaction is one of the strongest non-covalent bonds known and is stable over a wide range of pH and temperature. Each of avidin's four identical subunits can bind a single molecule of biotin or biotinylated ligand. Antibodies, antigens and proteins can be biotinylated with relative ease using commercially available biotinylation reagents. Biotin-N-hydroxysuccinimide linkers react with primary amines
(preferentially on the Fc portion of immunoglobulin) . Binding involves mixing avidin or streptavidin coated particles with a biotinylated affinity ligand and incubating for 40 minutes. The coated particles can then be harvested by magnet or centrifugation; and
Protein A coated - protein A coated particles can specifically bind to certain immunoglobulins at the Fc portion of the antibody, resulting in a higher activity due to correct orientation of the antibody affinity ligand. The antibody is bound to the particles via a passive adsorption technique.
Particles suitable for the invention also comprise eukaryotic and/or prokaryotic cells. One embodiment of the invention provides a composition that tests for the presence of antibodies in a solution, for example in serum, wherein the first particles are the bacterial cells bearing a bacterial cell surface antigen and the second particles are beads coated with the same bacterial cell surface antigen. In the presence of an appropriate antibody in the solution, the particles agglutinate to form a consolidated matrix. This embodiment demonstrates a situation where the particles may be of different shapes as well as of different sizes, since many eukaryotic and prokaryotic cells are non-spherical.
In a further embodiment a third particle is added to the composition of first and second particles wherein said third particle is also coated with the same affinity ligand as the other particles but is of a different size to the first and second particles and thereby contributes further to the synergistic nature of the composition.
In still a further embodiment, a composition of the invention comprises two or more different particle sizes, wherein at least one particle size comprises particles that contain magnetite or that respond to a magnetic field. Isolation of an agglutinated mixture from the test solution can then be achieved by a magnetic separation technique.
A second aspect of the invention provides a method of carrying out an agglutination assay. This method comprises the steps of:
(a) mixing a sample solution that may contain a test compound with a reagent comprising a synergistic combination of affinity ligand coated particles of different sizes wherein the affinity ligand binds to the test compound if present; and
(b) analysing the mixture of (a) for subsequent agglutination; wherein, in the case of an agglutination, the sample solution is considered to have tested positive for the presence of the test compound.
In one embodiment of the method of the invention the presence of an agglutinated mixture is analysed visually. Typically this is done by reference to a control agglutination mixture or to a reference scale. The level of agglutination seen also indicates to some extent the amount of test substance present in the sample solution.
In a further embodiment of the invention the analysis for agglutination is performed using a mechanised process. A positive agglutination necessarily results in an increase in the opacity of a solution as the particles and analyte crosslink into a consolidated matrix. This can be measured by various techniques known to the art, for example by spectrophotometry, digital image analysis techniques or by use of a fixed or variable orifice particle counter of the Coulter counter (RTM) type.
A third aspect of the invention provides for an agglutinated mixture comprising affinity ligand coated particles of different sizes and the substrate for said affinity ligand. This agglutinated mixture corresponds to the end point of a positive agglutination assay, indicating for example that the sample solution does indeed contain the test substance. This agglutinated mixture can be used as a positive control in a test kit.
Further aspects of the invention provide the use of a composition comprising a synergistic combination of affinity ligand coated particles of different sizes for detection of microbial organisms, antibodies or fragments thereof or antigens or other analyte in a sample solution.
A still further aspect of the invention provides a kit for the detection of test compound in a solution via an agglutination reaction, comprising affinity ligand coated particles of different sizes which bind to the test compound. In one embodiment of the invention the kit further comprises a reference scale for assessment of the level of agglutination. It is considered that such a kit may also comprise directions for the use of the agglutination assay as well as any buffer solutions to be used therein. The term "reference scale" is herein used to include control reactions that show positive and negative results of the assay of the invention and also graphical representation of such results.
In a further aspect the invention provides for a method of enhancing an agglutination assay comprising the following steps:-
(a) mixing a sample solution that may contain a test compound with a composition comprising a first affinity ligand coated particle wherein the affinity ligand binds to the test compound if present;
(b) adding to the mixture of (a) at least a second affinity ligand coated particle that is of a different size to the first particle wherein the affinity ligand of said second particle also binds to the test compound if present;
(c) analysing the mixture of (b) for subsequent agglutination; wherein, in the case of agglutination, the sample solution is considered to have tested positive for the presence of test compound.
Thus, the synergistic combination of particles can be brought into effect by the addition of a second or subsequent particles to a standard agglutination assay, wherein the second or subsequent particles are different in size to the particles already present in the standard agglutination assay.
This two stage augmented agglutination assay is of particular advantage where the sensitivity of a standard agglutination assay needs to be extended for specific isolated cases but not in general applications.
The invention is now illustrated in specific examples below which are accompanied by the drawing in which:-
Fig. 1 shows a scanning electron microscope image of an augmented agglutination reaction, the particles are silica beads of 3/γm and 0.4//m in diameter both of which are coated with an antibody specific for pneumococcal capsule, the agglutinated pneumoccal cells are indicated by black arrows.
Example 1 A first experiment was carried out to demonstrate that the protocol used to coat particles with antibody was successful. This was achieved by examining whether antibody-coated particles gave good or poor agglutination with the homologous antigen. After this, blends of different sized antibody coated particles were tested for adverse or augmentary effect.
Materials and methods Latex particles Protein A coated latex particles used in this experiment were supplied by Bangs Laboratories Inc (U.S. A). Details of the particles used are shown in Table 1 .
Table 1 . Particle codes and details
Antibody The antibody used for coating particles in the agglutination experiments was Pneumococcal Type 2 typing antiserum (Statens Serum Institut, Denmark, Lot N° - T214B) .
Bacterial growth, storage and counting The bacterial whole cells used in these experiments were Streptococcus pneumoniae of serotype 2 (strain D39). The bacteria were grown on standard blood agar and incubated at 37°C in a humidified environment containing 5% v/v CO2. The viability of stock suspensions of bacteria was assessed by a standard colony forming unit (cfu) determination method. Briefly, 100μl aliquots of various dilutions of bacteria were spread across the surface of blood agar plates. After incubation under the conditions described above for 1 6-1 8hrs, colonies were counted and used to determine cfu/ml in stock solutions.
For use of bacteria in agglutination tests, a stock of bacteria was made up and a viability test carried out on the suspension. Immediately after initiating the viability test, the stock was preserved by adding 2% v/v formaldehyde. Dilutions of the preserved stock bacteria were also made in 2% v/v formaldehyde.
Latex particle coating method
Pneumococcal type 2 antiserum was bound to latex particles (used without prior cleaning) via the method detailed below.
Reagent preparation
Coating Buffer (0.1 M Sodium Phosphate Buffer) . Solution A: 27.6g Sodium Phosphate monobasic (NaH2PO4.2H2O) in 1 L of double distilled water (ddH2O).
Solution B: 28.4g Sodium Phosphate dibasic (Na2HPO4, anhydrous) in 1 L of ddH2O.
470ml of Solution A was added to 30ml Solution B. This was adjusted to pH 7.8 with 1 M NaOH and then to a total volume to 1 L with ddH2O.
Blocker buffer: Coating buffer was supplemented with 1 % w/v skimmed milk (Marvel).
Protein Solution: 200μg Pneumococcal type 2 antiserum in 1 6.7/vl was added to 4ml coating buffer.
Wash Buffer: 1 .56g NaH2PO4.2H2O and 9.00g NaCI were added to
800ml ddH2O. This was adjusted to pH 7.8 with 1 M NaOH and to a total volume of 1 L with ddH2O.
Coating particles with antibody Particles were centrifuged, the original buffer discarded, and then the particles were resuspended in coating buffer at room temperature (RT) at a concentration of 1 % w/v. Next, 0.4ml of particle solution was added to 4ml of protein solution. After incubation at RT for 1 hr on a rolling mixer, the particles were centrifuged at 5,500g in a microfuge for 5 minutes. The particles were then resuspended in 4 ml blocker buffer and incubated at RT for 1 hr on a rolling mixer. After centrifugation as above, the particles were resuspended in 1 ml blocker buffer. After
blocking, the particles were finally centrifuged at 1 9,OOOg for 1 0 minutes, and then resuspended to 1 % w/v in blocker buffer. They were then sonicated in a water bath for 2 minutes at 22°C to disperse any large clumps of beads resulting from the centrifugation steps.
Results
Individual bead agglutination
Initial agglutination tests were set up to determine whether the antibody coated particles would agglutinate bacteria when tested individually.
5 /I of antibody coated beads were placed on a black card. 5μl of stock bacteria (approximately 7.5E04 bacterial cells) were added and mixed with the beads using a plastic tip. The card was rotated for 5 minutes to allow the reaction to develop. The cards were then observed and scored manually (Table 2) . As shown in Table 2, all of the antibody- coated particles agglutinated homologous bacteria, and did not agglutinate in the absence of bacteria (negative control).
Table 2. Agglutination of single sized antibody coated particles
see Table 1
2 Score Description
0 no agglutination - test remained indistinguishable from control negative
1 hint of agglutination - slightly grainy, though still mainly opaque
2 mild agglutination - small aggregates in opaque fluid
3 medium agglutination -visible grainy aggregates in opaque fluid
4 strong agglutination - mainly aggregated with hint of opacity in fluid
5 very strong agglutination - large aggregates within clear fluid
Particle mixing experiments In the experiment above, a dose of 7.5E04 bacteria gave a maximum agglutination score for beads A, B and C. In order to examine the possible augmentary effects of size mixing, a series of dilutions of the bacteria were tested with combinations of different sized particles as well as single sized particles alone. The dilution range of S. pneumoniae used started at 10 times lower than in the above experiment
(approximately 7.5E03 bacterial cells) and ended at 10,000 times less (approximately 7.5 bacterial cells).
In all experiments described where combinations of particles were used, they were mixed in the following ratios: -
Combinations of two different sized particles were mixed 50:50 v/v. Combinations of three different sized particles were mixed 33:33:33 v/v.
In ail cases, 5 μl of bead suspension was added to 5μl of bacterial cells. 5μl of PBS was added instead of bacteria to the negative controls.
As can be seen in Table 3 by the number of italicised results (indicating an augmented result) in Column 1 , augmentation of agglutination, as a result of particle size mixing, occurred in 81 % (26/32) of the experiments carried out compared with the best result obtained with single size preparations. In no case was the result for a combination of sizes worse than the best result for a single size.
Using a dosage level of approximately 7500 organisms added to the card, all antibody coated beads showed mild agglutination regardless of their diameter (Table 3 columns 2, 3 and 4; experiments 1 -10) . In experiments 1 , 2, 7 and 8, good amplification (increase in highest single bead score of two or more) of agglutination and an increase in ease of detection was observed for the mixed beads when compared to the beads tested individually. Experiments 3, 6 and 9 also indicated that amplification was occurring in these bead size mixtures.
Using approximately 750 organisms added to the card, individual beads showed weaker reactions on their own (columns 2, 3 and 4, experiments 1 1 -20) than at the higher concentration of bacteria as detailed above. In all of the bead combinations at this bacterial dose, amplification was observed. Good amplification was observed in most of these combinations (column 1 , experiments 1 1 , 1 2, 14, 1 5, 1 6, 1 7, 1 9, 20), with only two combinations showing an increase in score of less than two (column 1 , experiments 1 3 and 18).
Results using approximately 75 organisms added to the card were very interesting since no single bead gave visible agglutination. All combinations except one (column 1 , experiment 29) showed signs of agglutination. Being beyond the sensitivity of single bead preparations, it was considered surprising to note a score of 3 in the A + C mixture
(column 1 , experiment 22) and a score of 2 in the A + B + C mixture (column 1 , experiment 27) .
At a dose of approximately 7.5 bacteria added to the card, no agglutination was observed. The lower limit of detection (around 75 organisms) is highly significant considering that the antibody coating regimes were not specifically optimised.
No agglutination was seen either with individual beads or with combinations in the absence of bacteria (negative control, experiments 33-42) .
Table 3 Effects of mixing different sized antibody coated beads on agglutination1
An agglutination score of 3 in the A + C particle mixture (column 1 , experiment 22) at a dose of approximately 75 organisms represents the greatest increase in agglutination score in any combination compared with individual beads. This combination (A and C) consistently amplified the score of the two individual beads at doses of bacteria between 75 and 7500 organisms. In this agglutination system, for the beads examined, it was apparent that a combination of beads sized 0.5μm and 3.2μm gave the greatest amplification of score and ease of detection although many other combinations showed good results as well.
The data in Table 3 can be used to estimate the degree of amplification observed by comparison of the agglutination scores of the two individual beads with the corresponding blend. The score of the individual beads were only as high as the A + C mixture (column 1 , experiment 22) at 100 times greater dose of S. pneumoniae (column 2, experiments 1 , 2, 3, 7, 8, 9 for A; column 3, experiments 2, 4, 9 and 10, column 2 experiment 6 and column 4 experiment 7 for C) . If a score of 1 or above is taken as a positive, then we can also see an increase in sensitivity of agglutination has occurred by combining two or three different bead sizes in 90% (column 1 , experiments 21 , 22, 23, 24, 25,
26, 27, 28, 30) of the experiments carried out
It can therefore be stated that these data show an increase in sensitivity of at least 10 fold (based on a score of 1 or above as positive) can be achieved for many different combinations of bead sizes and amplification (based on equivalent scores) of up to 100 times can be observed by mixing two different sizes of beads.
Example 2 Two sizes of silica particle (of 3μm and 0.4μm in diameter) with a ratio of diameters of about 6.5: 1 were coated with antibody specifically against the capsule of serotype 2 S. pneumoniae (Statem Serum
Institute, Denmark) . The antibody coated beads were adjusted to approximately 10% (w/v) then blended before being mixed 1 : 1 (v/v) with a suspension of S. pneumoniae of serotype 2. After 5 minutes incubation at room temperature the resultant agglutinated matrix was placed onto a glass coverslip and fixed with Osmium tetroxide. The fixed material was then coated in gold before examination by a scanning electron microscope (Phillips), the results are shown in Fig. 1 . The agglutinated matrix consisted of pneumococci, small antibody coated and large antibody coated beads. The capsular material produced by the pneumococci was also released into solution, thus allowing a synergistic agglutination reaction to take place both directly between beads and the bacteria and also via soluble capsular material and other antibody coated beads.
The invention thus provides reagents and methods for agglutination assays.