JP2834950B2 - Sensitive optical immunoassay using enzyme labeling reagents - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for detecting a small amount of a substance derived from a subject by an enzyme labeling means.
High sensitivity, thin film, optical immunoassay
assay (OIA)).

[0002] Immunoassays based on thin film optical effects have a long history dating back to the work of Giaever. Such assays rely on, but are not necessarily limited to, an increase in thickness at the surface to provide an optical effect that can be viewed, for example, as a color change visually or using an instrument. If the analyte is large enough, it may bind to an optically suitable surface, resulting in a clearly observable thickness change on its own, but many analytes have particulate reagents added to the assay system. By making a larger final thickness change, it is more easily detected. Particulate reagents selected for this purpose include polystyrene latex, which can readily adsorb antibodies on the particle surface, thereby acting to amplify the detection of the corresponding antigen. Although such polymer latexes are available in a wide range of particle sizes, from a practical point of view, their usefulness in the assay is a trade-off between particle size giving increased thickness and surface area contributing to sensitivity. It is necessary to take into account that the smaller the particles, the less the increase in thickness, and the larger the particles, the smaller the surface area for binding the antibody. That is, the particulate reagent used in the thin-film optical immunoassay limits the sensitivity and scope of the assay.

The present invention overcomes the limitations associated with the prior art using particulate reagents. Furthermore, the use of enzyme labeling reagents (eg, antibody-enzyme conjugates) in place of latex reagent particles, especially when used with a substrate for the enzyme to provide an insoluble precipitation product, is unexpectedly higher. It has been found that sensitive thin film optical immunoassays can be performed. The present invention uses such enzyme-labeled antibody methods in thin-film assays to detect low concentrations of polysaccharide antigens from bacterial groups that are common causes of bacterial infections in humans, such as meningitis and streptococci. About doing. Detection and identification of such antigens is important in diagnosing a critical group of diseases caused by these bacteria and in selecting effective treatments.

[0004]

BACKGROUND OF THE INVENTION Many diagnostic methods and components useful for optical immunoassays rely on latex-based particles, some of which have been found to require the use of enhancers. In some of these methods, the agglutination immunoassay relies on antibodies that produce particles of sufficient size to produce a visible signal with the assembly of the particles. Visualization is achieved by light scattering. In other methods, highly crosslinked rigid copolymers provide a solid support for capturing and separating analytes. It is well known that the enzyme-linked immunosorbent assay (ELISA) is well accepted and widely used in both qualitative and quantitative assays of biomolecules. Many researchers have developed a method in which an enzyme is conjugated to an antibody to the molecule to be measured (ie, the analyte) and used in combination with a chromogenic substrate specific for that enzyme to produce a visible colored reaction product. We are using. In these methods, a test tube or a filter membrane is used as a solid phase on which the molecule to be measured is immobilized. More generally, in membrane-based assays, a secondary antibody is attached to a colored latex or metal particle for color development. Reactions involving such color changes are described in the Journal of Immunoassay (J. Immunoassay), 2
(3 & 4), 187-204 (1981). Once such a colored product is obtained, it can be qualitatively observed or quantitatively measured by various techniques, including spectrophotometry. There are numerous variations of such assay techniques, all of which depend exclusively on the rate and extent of color development, which is proportional to the presence and concentration of the antigen.

[0005] WOiszwillo is
Reported optically detectable color formation by ELISA using various enzymes such as horseradish peroxidase (HRP) (US Pat. No. 50136).
No. 46). Bishop et al. Also used a specific binding compound labeled with peroxidase in the form of an antibody against human chorionic gonadotropin (HCG),
A method for measuring the amount of dye present as an indicator of the amount of ligand present has been reported (US Pat. No. 5,024,935).
McClune, a dye-producing peroxidase reaction system, measures ligands by methods involving the formation and detection of immunological complexes of ligand and receptor (US Pat. No. 5,017,474). Furthermore, a similar method has been reported by Satoshi et al. (US Pat. No. 4,921,791). Furthermore, Tung uses the horseradish peroxidase enzyme as an antibody label bound to an insoluble support via a complex formed by the antigen and at least two antibodies (US Pat. No. 4,788,138). ).

[0006] The use of enzyme-labeled binding reagents in immunoassays or components thereof as in the present invention to detect antigens optically with high sensitivity has not been reported in the prior art. In this way, the insoluble precipitate containing the immunological complex formed on the support surface increased the optical thickness and improved the sensitivity to the presence of an ellipsometrically or visually measurable analyte. The present invention provides a very sensitive, accurate, reliable, simple and inexpensive thin film optical immunoassay and devices for use therein compared to the prior art.

[0007] It is an object of the present invention to provide an improved optical immunoassay with increased sensitivity over assays using latex particle amplification.

Another object is to provide a sensitive, reliable, accurate and simple optical system for detecting the presence of infectious bacterial analytes such as Neisseria meningitidis A, C, Y, W _135, etc. It is to provide an immunoassay method.

Yet another object is to provide an insoluble optically detectable thickness or mass (m) on the surface of a non-particulate, non-latex support in the presence of very low concentrations of a particular antigen.
(Ass) is to utilize an enzyme substrate and an enzyme-labeled reagent (for example, an antibody-enzyme conjugate or an enzyme-labeled antibody) capable of giving a change in ass).

[0010] These and other objects achieved by the present invention are described in further detail below.

[0011]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides for the direct and selective isolation of analytes from bodily fluids such as serum or cerebrospinal fluid (CSF) for identification purposes or various other substances by whatever mechanism. A novel thin-film optical immunoassay using a binding device. The present invention utilizes, in the broadest sense, a non-polymeric, non-particulate, non-latex support having one or more coating layers containing an unlabeled acceptor. This support has a high receptive function and is suitable for interacting directly with the analyte. The enzyme-labeled secondary acceptor then interacts directly, which may or may not be the same as the unlabeled acceptor immobilized on the support. The test substance may be a polysaccharide that is an indicator of a surface antigen or cell component of various bacteria such as Streptococcus, Neisseria meningitidis, and Haemophilus.

FIG. 1 has various layers including a receptor (ligand) layer, an enzyme labeling reagent (eg, an antibody-enzyme conjugate or an enzyme-labeled antibody) layer, and a precipitate (including a substrate for the enzyme) layer. FIG. 1 is a longitudinal sectional view of a thin-film optical immunoassay device.

[0013]

FIG. 1 is a schematic cross-sectional view of a multilayer OIA device having a support 1 having an upper surface and a lower surface, on which various layers are indirectly mounted. Mediately and directly coated. These layers can include an optional layer 2 of silicon nitride immediately adjacent to the upper surface of the support (see U.S. Patent Application No. 408291 (corresponding to Japanese Patent Application Publication No. 5-500567)). On top of this optional layer, if any, and on the upper surface if not, a polymer layer 3 such as a polymeric siloxane.
Is coated or deposited. This polymer layer provides an intermediate surface-modifying layer that improves the adhesiveness to proteins and maintains the environment in order to retain the receptor substance layer 4. The polymer layer must be of sufficient thickness so that the layer of biomolecules held thereon is not affected by any detrimental effects from the support. The receptor layer is one component of a binding pair, such as an antigen-antibody, oligonucleotide / DNA, chelator / metal, enzyme-inhibitor, enzyme-substrate, bacteria-receptor, Virus-receptor, hormone-receptor, DNA / RNA or RNA / RNA
And combinations of any other type of binding system or reaction system, but are not limited thereto. The acceptor layer 4 supports an analyte layer 5, which in turn supports an enzyme-labeled reagent (eg, antibody-enzyme conjugate or enzyme-labeled antibody) layer 6. The antibody in the enzyme labeling reagent is an antibody specific to an antigen derived from or capable of being derived from a subject. Since the analyte layer 5 and the enzyme-labeled antibody layer 6 bonded in this manner become an optically integrated layer, they can be regarded as a complex. This complex attached to the device is a mass with enzymatic activity. By dropping a precipitant such as "TM Blue" onto this mass, a precipitate layer 7 (containing a substrate for the enzyme) as the uppermost layer is generated.

As an embodiment of the present invention, a method, device and kit embodying the present invention include a virus antigen,
Includes DNA, RNA, hormones and any other similar analyte.

Table 3 of Example 3 and the graph of FIG. 2 show an immunoassay device using a latex particle reagent according to the prior art and an OIA using an enzyme labeling reagent according to the present invention.
This shows the result of comparison with the device. As dilution reduces the concentration of antigen from the bacterial analyte, it becomes significantly more difficult for prior art latex reagent devices to provide sufficient signal due to the optical thickness of the antigen-antibody-latex layer. In contrast, the enzyme-labeled antibody-antigen complex with the device of the present invention increases the measurable sensitivity by at least 5-fold. Representative enzymes useful in the present invention are glucose oxidase, galactosidase, peroxidase, alkaline phosphatase, and the like. Of particular note is that too high a concentration of antigen present will result in an unstable thickness increase provided by the enzyme labeling reagent. This increase in thickness is not mechanically stable and is easily removed from the surface by washing and drying. This causes apparent saturation in the signal, but is merely a phenomenon caused by the precipitated product. However, before removing the excess liquid,
A dark blue color results in the sample spot, indicating a positive result.

In one embodiment of the present invention, a support having an upper surface and a lower surface on which at least one layer containing an immobilized unlabeled antibody is indirectly supported. Sensitive thin film optical immunoassay devices, including bodies, are provided. The unlabeled antibody present in the device is allowed to interact with the analyte and a peroxidase conjugate capable of specifically binding to the analyte. The peroxidase conjugate bound by the presence of the analyte interacts with the peroxide releasing reactive substance (substrate),
It can form an insoluble reaction product.
This insoluble reaction product, when combined with alginic acid, dextran sulfate, methyl vinyl ether / maleic anhydride copolymer or carrageenan, etc., is present as "TM Blue" (3,
It precipitates by the action of a precipitant such as (3 ', 5,5'-tetramethylbenzylidene), and also includes an immobilized antibody-antigen-antibody HRP complex. Other materials such as chloronaphthol, diaminobenzylidene tetrahydrochloride, aminoethyl carbazole, orthophenylenediamine and the like can also be used. These reagents are about 10-10
Use at a concentration of 0 mM. This method results in a measurable mass increase above the peroxidase conjugate layer and the unlabeled antibody layer. This mass change is unaffected or independent of the color development of the peroxide releasing reactive material.

In a subsequent mode, either an antigen derived from the subject or the subject and the labeled antibody in the solution are directly reacted to form an antigen-labeled antibody complex,
The complex may then be reacted with an unlabeled antibody bound to a non-latex, non-polymeric, non-particulate support.

In still another embodiment, the above device is prepared, and a peroxidase conjugate bound to the device via a subject and a peroxide releasing reactive substance (substrate) are allowed to react with each other to form an insoluble reaction product. A method is provided for detecting the presence of an analyte by the step of forming an object. Next, in order to separate, discard, and remove all unreacted layer materials, washing and drying are performed, and thereafter, mass change is measured and recorded.
The measurement is performed by various means including visual observation of the increase in the thickness of the peroxidase conjugate layer, or by use of an instrument such as an ellipsometric method or a method of measuring a change in light intensity caused by the increase in the thickness. That is, the enzyme-labeled secondary acceptor can directly interact with the analyte, more specifically, the body of the analyte, such as an antigen, thereby obtaining a visible mass change in the thin film. I can do it. This change can be detected by measuring the optical thickness and does not necessarily depend on the light reflectivity of the support material. In addition, as an example of the above-mentioned equipment, US Pat.
No. 558012, No. 4647207 and No. 465
There is a Sagax Ellipsometer described in No. 5595.

Substances that can be included in the unlabeled receptor include toxins, antibodies, antigens, hormone receptors, parasites, cells, haptens, metabolites, allergens, nucleic acids, nuclear materials,
Examples include autoantibodies, blood proteins, cell lysates, enzymes, tissue proteins, enzyme substrates, coenzymes, neurotransmitters, viruses, virus particles, microorganisms, bacteria, metals, and various chemical species and substances derived therefrom. These are just a few of the many different materials that can be coated on a support surface to produce an optical immunoassay system with an enzyme-labeled secondary acceptor. Whatever the analyte, the receptor is designed to interact specifically with the analyte. The term "enzyme label" refers to an enzyme that has been conjugated or otherwise specially prepared to be accepted, such as an antibody-enzyme conjugate. The enzyme-labeled secondary acceptor is captured only on the immobilized unlabeled acceptor in the presence of the antigen.

In the embodiment, a silicon wafer is used as a support, but various other materials can be used instead. The support can be made from silicon crystals,
This is cut with a diamond to make a wafer, KO
Anisotropic etching is performed in H and then isotropically etched to create a smoother surface. One surface of such a wafer is polished to provide a smooth mirror finish with a mirror-like surface. Opposite surface is 200-300 nm
With a certain degree of undulation, leaving it slightly irregular, it creates a scattering reflective surface. Although both surfaces of the wafer can be used, mirrored surfaces are preferred for instrument detection. The present invention allows the use of silicon wafers that are not acceptable in the semiconductor industry, thus reducing manufacturing costs. The amount and type of dopant in the silicon wafer is not relevant to the present invention.

As shown in FIG. 1, the support has an upper surface adapted to support additional material. This upper surface has the properties necessary to generate a signal. An interlayer material is a material that creates a favorable microenvironment for the receptive material, thereby forming a usable dense layer of the receptive material.

The intermediate layer can be made by applying one or more substances that can adhere to the support by various mechanisms (US Pat. No. 6,530,644).
506314))). The siloxane covalently modifies the support, while other substances may simply adhere to the surface. However, it must not cause delamination and must be stable in mechanical operation. Methods for coating the polymeric material on the support are known to those skilled in the semiconductor art, and it is contemplated that the polymeric material may be applied by spin coating, aerosol spraying, dipping, and the like. The coating method can be selected according to the kind of the interlayer material to be used.

[0023] Although not required, additional materials that impart the desired properties may be deposited on the intermediate coated support. Such a layer can improve the orientation of the receptor, similar to Protein A or Protein G used to orient the antibody. Other materials that can be used include avidin-biotin, synthetic or recombinant peptides, and the like.

In a preferred embodiment, the interlayer material is uniformly coated by spin coating or aerosol spraying. The various interlayer materials, when coated on a support at a thickness of 5 to 500 ° (even greater thicknesses), provide an assay test surface with the advantages listed above. The layers perform the following functions and can be formed of any material having the following characteristics: creating a favorable environment for the receiving material;
Tightly binding the acceptor in the active position in a cost-effective manner; low non-specific interaction; covalently or strongly adhering to the support; However, it is not always continuous and can be coated. The interlayer material can be disposed on the support in various ways.

As shown in FIG. 1, the interlayer material has a lower surface that adheres to the upper surface of the support, the lower surface being prepared to be compatible with the support. The polymer layer material also has an upper polymer surface adapted to adhere the immobilized reciprocal receiving material. Test surfaces coated with interlayer material are usually stable and can be stored until coated with the receiving material.

After the interlayer material is coated on the support, a curing period may be required. For example, in the case of T-polymer siloxanes, it has been found that a curing period before application of the acceptor can be used successfully.

The immobilization chemistry for attaching the receptive material to the interlayer material is chosen based on the properties of both the coated support and the receptive material. The acceptor can be covalently or passively attached to the interlayer material.

Those skilled in the art will appreciate that the stability of antibody-coated wafers can be enhanced by various protecting agents such as, for example, gelatin hydrolysates. One of the problems to be solved is to obtain uniformity of the protective coating and uniform spreadability of the coating material. It has been found that rinsing the protective coating and then air-drying the surface before performing the immunoassay gives good results. However, in this case, some circular spots may remain, but this does not need to be taken into account as the assay is not spot or color dependent.

EXAMPLE 1 Description of Reagents and Assays Cell suspensions derived from cultures of Neisseria meningitidis A, C, Y, W ₁₃₅ were collected by caprylic acid precipitation from high titer serum collected from rabbits previously injected. Horseradish peroxidase (Sigma grade VI) was chemically coupled to the purified immunoglobulin. Coupling is performed using the reagent S-acetylthioacetic acid N-hydroxysuccinimide ester, Analytical Biochemistry, 13
2, 68-73 (1983).
The resulting conjugate was MOPS 50 mM, pH 7.
Buffer 0 contained peroxidase (104 μM) and immunoglobulin (35 μM). The peroxidase-immunoglobulin conjugate was converted to casein (5 mg
/ Ml) and mixed with the same volume of a diluted solution of the filtrate obtained by removing the cells from the Neisseria meningitidis culture solution. Mix the mixture (25 μl) with silicon nitride, t
-Pipette on the surface of a silicon wafer coated with a layer of purified immunoglobulin from the same rabbit antiserum against polymer siloxane and Neisseria meningitidis. The antibody was 10 μg in 50 mM MOPS, pH = 7.0.
/ Ml of antibody was coated onto a t-polymer / silicon wafer. Wafers placed in antibodies for 1 hour at room temperature were rinsed with deionized water and dried under a stream of nitrogen. The antibody-coated support was further treated with 50 mM MOPS, pH
= 7.0 mg / ml in hydrolyzed casein for 1 hour at room temperature, then rinsed and dried as described above. After 2 minutes, the droplets were washed off with water and the wafer was dried with a stream of nitrogen or drained with absorbent paper. TM Blue precipitation substrate ("TM Blue" (TM) is commercially available from Transgenic Science, Inc. and is disclosed in U.S. Pat. No. 5,113,646). Apply to 5
Let stand for minutes. The wafer was washed and dried. A purple spot was observed in the portion where the reaction occurred. This precipitate formed was read visually and with an ellipsometer, and Neisseria
The presence of Meningitidis was confirmed.

[0030]

[Table 1] * Dilute stock antigen preparation to 50 mM MOPS. A 1: 20,000 dilution of the antigen is visually distinct from the negative. The sensitivity cut-off of the Wellcogen kit is 1+ at a 1: 4,000 dilution of the same antigen preparation.
It is.

Reagent Kit The reagent kit contains all the components necessary to perform up to 50 rapid optical immunoassay tests. The kit includes a solid support test station designed to facilitate the appropriate washing and drying procedures required.
pport test station). A slide containing 1-5 unreacted test surfaces specific for the conjugated analyte is placed on the test station. After the end of the first reaction, the slide is tilted forward of the operator. The test surface (s) are rinsed vigorously with a cleaning solution, which is drained from the ramp into the lower container. The container contains a solid water-absorbing block of cellulose acetate treated with a biocide. The slide is then returned to the level position and a piece of water-absorbent paper is placed directly on the test surface and contacted for a few seconds to allow it to soak. The absorbent paper is placed in convenient locations in front of the reagent kit as individually cuttable pads, but the washing / drying operation can be performed by alternative means such as capillary action. Next, an appropriately buffered and diluted enzyme-labeled substance (ie, a solution of an enzyme-labeled antibody that is specific for the analyte (eg, antigen)) is also provided. Finally, a precipitating means, such as a commercially available container of TM Blue liquid, is provided in a suitable volume and one to three or more drops are added to cause a mass change to produce a precipitate. After that, it is washed. The secondary incubation begins when the substrate is added to the surface, and the test is terminated by repeated washing / drying.

Example 2 Two different types of silicon wafers were used: one was a gold silicon nitride coated silicon wafer and the other was a silver silicon wafer without silicon nitride coating.
The visible color observed with the peroxidase / precipitated substrate system on silicon nitride may be strictly due to absorption of the dye precipitated on the surface. This is the case, and if the precipitated dye does not function as a thin film, the silver silicon wafer should produce a visible signal that is the deep blue of TM Blue only. T-polymer coated wafers were treated with a commercially available production grade antibody used in the Welcogen (Trademark of Welcome Diagnostics) latex agglutination test. Five separate tests (N. meningitidis A, C, Y,
W ₁₃₅ , N. meningitidis B, Streptococcus B, H. influenza and Streptococcus
Pneumoniae). The first reagent manufactured for each test was the intermediate layer as described previously.
These were gold and silver wafers coated with (T-polymer siloxane). By immersing these wafers in a suitable solution for 1 hour at room temperature, 50 mM MOPS, pH =
In 7.0, all five antibodies were coated at an antibody concentration of 10 μg / ml. The wafer was rinsed, dried and blocked as described in Example 1.

As the required second reagent, Example 1 was used.
The same five antibodies were used for the production of antibody-horseradish peroxidase conjugates as described in.
Preserved conjugate was prepared using 50 mM MOPS, pH =
The actual conjugate was prepared by diluting it to 7.0 5 mg / ml casein to a final conjugate ratio of 1: 100. The conjugate solution actually used was mixed 1: 1 with the standard antigen preparation, and 20 μl was added to the corresponding antibody-coated wafer.

According to the rapid protocol, a first immunoincubation for 2 minutes followed by washing, drying and then a 5 minute substrate incubation allowed the product to accumulate on the wafer surface. Following washing and blot drying,
Samples were read visually and with an ellipsometer. Intense visible purple spots appeared on the gold silicon nitride coated wafer and gray spots were observed on the silver silicon wafer without the silicon nitride coating. The increase in thickness could be easily measured using an ellipsometer. The visible color on the silver silicon wafer, observed in all cases, indicates that the precipitated product functions as a true thin film and produces interference effects without an anti-reflective coating. The color produced does not depend on the absorption properties of the dye. In addition, the chromogen
Evidence was obtained in the following experiments that n) did not contribute to the development of the observed visual response. That is, TMB / H
Treatment of the ₂ O ₂ product with the stopping reagent H ₂ SO ₄ produces a yellow precipitate. If the visual response observed for the optical support in this experiment was simply due to the chromogen,
Treatment of the surface precipitate with the stopping reagent should produce a yellow spot. Treatment of the immobilized surface precipitate with sulfuric acid does not alter the intense purple or blue spot created on the silicon nitride coated wafer. Thus, the signal obtained is entirely due to the formation of a thin film. Additional proof was obtained by using an adhesive strip to remove the precipitate from the surface of the silicon nitride. The precipitate removed by the adhesive was light gray / blue with no red component. The observed interference effect shows a strong reddish bright violet / blue. This emphasizes that thin film formation is the cause of the observed color effect.

[0035]

[Table 2] * It was later found that the batch of antisera used for OIA could not be used for the production of antibody-latex for the wellcogen kit, which could explain the relatively poor results observed here. ** Indicates a relative increase in sensitivity achieved with OIA as compared to latex aggregation. Notes: 1) For OIA, the dilution is the final dilution giving a visually positive result. 2) The cell supernate described here was prepared by adding a heavy cell suspension made with 0.5% formalin in saline overnight at +4
Supernatant removed after standing at ℃. They have a high content of polysaccharide and therefore require considerable dilution. 3) Latex testing was performed on Welcogen products that did not expire the warranty period.

Example 3 Comparison with Latex Particle-Enhanced Optical Immunoassay A new enzyme labeling assay was used for the detection of antigen from Streptococcus A, and amide-modified surface-activated latex 0.161 μm (Rhone Poulin) on the same silicon wafer. ). Assays using enzyme antibodies were 5 times more sensitive than those using latex antibodies.

The increase in sensitivity was based on a visual analysis of a 1: 1600 dilution of the antigen control in the enzyme technique versus a 1: 320 dilution for the latex based on the OIA technique. Both techniques cut at 1:80 dilution of antigen-
The sensitivity was better than the latex agglomeration technique showing off. A comparison of instrumental measurements of the two technologies is shown below.

[0038]

Table 3 Antigen dilution ratio m volt / latex m volt / enzyme 0 3.0 11.0 1: 320 32.0 203.0 1: 160 63.0 290.0 1:80 113.0 272. 0 1:40 195.0 194.0 1:20 316.0 168.0 1:10 428.0 258.0

The graph in FIG. 2 shows that the enzyme system in this assay tends to saturate more quickly than the latex system,
This is a qualitative test system, indicating that it is desirable to maximize the signal generation at the cut-off level of sensitivity. Overall, OIA of Streptococcus A using the enzymatic method shows very good performance.

Example 4 Multi-item test method Using the method described above, several peroxidase-antibody conjugates are combined in one reagent (about 3 drops). This was combined with an equal volume (75 μl) of the CSF sample and mixed, and one drop (25 μl) was added dropwise with a pipette onto a 5 antibody spot with the appropriate specificity. Incubation was performed for about 2 minutes, after which the wafer was washed and dried. One drop (25 μl) was added to each specific antibody spot and then incubated for 5 minutes. Thereafter, the wafer was washed and dried before reading. This method allows for easy analysis of the presence of one or more analytes in a single sample. No visual response occurred in the antibody region corresponding to the antigen not present in the sample. A positive response to the added antigen produced a signal comparable to that generated by the single item test method.

[0041] The performance of Example 5 an optical immunoassay (OIA) and enzyme-linked immunosorbent assay (ELISA), N. meningitidis A, C, Y, and W ₁₃₅ as the target, enzymatic amplification O
IA was compared with an ELISA using TMB as substrate.

The same experiment was performed using OIA and ELISA techniques. These experiments demonstrate the advantage of OIA over ELISA in sensitivity and readability, as can be seen in Tables 4, 5 and 6 below and FIGS. 3, 4 and 5 attached to each table.

There are two major differences between these techniques. First, OIA uses silicon wafers polished for solid phase adsorption of antibodies, while ELISA uses clear polystyrene microtiter plates. Second, and more importantly, the substrate used for the OIA reaction produces insoluble deposits on the surface of the polished silicon wafer, whereas the substrate for the ELISA uses a colored solution in the wells of a microtiter plate. It is a point to generate. OIA is more effective due to this important difference in the results obtained.

The deposition of a mass on a reflective surface changes the refractive index of light passing through the mass as compared to its surroundings. Therefore, the light reflected back by visual inspection or optical assay is:
Although recognized as a color change, this technique is not dependent on a dye system. The ELISA technique relies exclusively on the dye system, and the solution in the microtiter well must undergo a color change, otherwise the reaction is undetectable.

Surface Preparation One surface was a polished silicon wafer (OIA) and the other was a clear polystyrene microtiter plate (ELISA).

On both surfaces, a 10 μg / ml antibody solution was applied for 1 hour at room temperature, washed with deionized water, a 0.5 mg / ml casein block solution was applied for 10 minutes at room temperature, and finally washed with deionized water. .

Implementation of Assay Antigen Diluent: 1: 5,000 1: 10,000 1: 20,000 1: 40,000 1: 80,000 1: 160,000 0 Conjugate Solution: of HRP-labeled antibody 1: 100 dilution 5 mg / ml casein 50 mM MOPSO, pH 7.0

Immediately before use, each antigen dilution was combined 1: 1 with the conjugate solution and added to each surface. This was allowed to react for 2 minutes at room temperature, then each surface was washed with deionized water.

Next, a substrate was added to each surface. TM Blue was applied to the OIA silicon wafer, and TMB was applied to the ELISA plate. This was reacted at room temperature for 5 minutes.

At this point, the reaction was terminated and the silicon wafer was washed with deionized water and dried with nitrogen. The thinnest antigen diluent that can be visually distinguished from negative was determined, the sample containing the insoluble product deposited on the surface was placed on an ellipsometer, and the respective voltages were measured.

The ELISA plate was also read visually while reacting, the reaction was stopped with an acid solution and read again. The plate after the reaction was stopped was placed on a spectrophotometer, and the absorbance was measured. The results obtained are as follows.

Visual Results OIA can be read up to 1: 40,000 antigen dilution, whereas ELISA does not stop the reaction, 1: 10,000-1: 1: 20,000 dilution, 1: 5 when stopped It can be read only up to 10,000-1: 10,000 dilution.

Machine reading result

[Table 4] Table 4 Input data X-value Background Foreground Y-value 5,000 0.3802 0.7010 0.3208 5,000 0.3802 0.7374 0.3572 10,000 0.3802 0.5198 0.1396 10,000 0.3802 0.5492 0.1690 20,000 0.3802 0.4340 0.0538 20,000 0.3802 0.4442 0.0640 40,000 0.3802 0.3985 0.0184 40,000 0.3802 0.4080 0.0278 80,000 0.3802 0.3913 0.0111 80,000 0.3802 0.3960 0.0158 160,000 0.3802 0.3879 0.0077 160,000 0.3802 0.3941 0.0139 0.000 0.3802 0.3865 0.0063 0.000 0.3802 0.3860 0.0058 OBS X-value AVG-Y SD (N-1) CV (%) Y- / + 1SD Y- / + 1SD 2 0.000 0.006 0.000 5.4 0.01 0.01 0.01 0.01 2 5,000 0.339 0.026 7.6 0.31 0.36 0.29 0.39 2 10,000 0.154 0.021 13.5 0.13 0.18 0.11 0.20 2 20,000 0.059 0.007 12.2 0.05 0.07 0.04 0.07 2 40,000 0.023 0.007 29.0 0.02 0.03 0.01 0.04 2 80,000 0.013 0.003 24.8 0.01 0.02 0.01 0.02 2 160,000 0.011 0.004 40.6 0.01 0.02 0.00 0.02

[0054]

Table 5 Dilution Absorbance reading at 450 nm 1 2 3 4 0 0 0 0.013 0.003 1: 160,000 0 0 0.008 0.015 1: 80,000 0.006 0.036 0.037 0.045 1: 40,000 0.006 0.005 0.027 0.001 1: 20,000 0.015 0.012 0.012 0.041 1: 10,000 0.030 0.043 0.068 0.053 1: 5,000 0.081 0.085 0.097 0.123 1: 5,000 0.063 0.064 0.094 0.088

[0055]

Table 6 Dilution Absorbance reading at 450 nm 1 2 3 4 0 0 0 0 0 1: 160,000 0 0 0 0 1: 80,000 0.022 0.010 0.011 0.020 1: 40,000 0.030 0.023 0.045 0.050 1: 20,000 0.028 0.062 0.038 0.031 1: 10,000 0.035 0.039 0.040 0.040 1: 5,000 0.057 0.062 0.072 0.086 1: 5,000 0.040 0.048 0.070 0.070

The definitions of the terms used above are shown below. OBS: number of measurements made at a given value of X. X-value: concentration tested, known Y-value: photodiode reading in volts of reaction area expressed as (foreground)-(background), unknown Avg- Y: average value (volt) of Y measured by the photodiode, ie, Y ₁ + Y ₂ +... Y _N / N (where N = OBS) SD (n−1): average value of Y , The standard deviation CV (%) calculated by the N-1 equation (where N = OBS): The coefficient of variation is calculated from the Y value and expressed as a percentage. SD divided by the average value of Y. Y +/- 1 SD: Calculate the range of possible Y values. The range of possible Y values is average +1 x SD vs. average -1 x SD.
Expressed as Y +/− 2 SD: Calculate the range of possible Y values, the range of possible Y values is average + 2 × SD vs. average−2 × SD.
Expressed as Background: Intensity reading of unreacted antibody-coated test specimen (volts) Foreground: Intensity reading of reaction area of test specimen (volts) X values given in the table are shown below: 0 Negative control, buffer only 5 Conserved antigen standard Indicates 1: 5,000 dilution of product 10 Indicates 1: 10,000 dilution of stock antigen sample 20 Indicates 1: 20,000 dilution of stock antigen sample 40 1: 40,000 dilution of stock antigen sample Indicates a 1: 80,000 dilution of the preserved antigen preparation 160 indicates a 1: 160,000 dilution of the preserved antigen preparation

In the software package in the photodiode data acquisition mode, the X value must be a concentration expressed in ng / ml, not a dilution factor (1: 5,000, etc.). (5,000
Etc.). All Y values (volts) for a known X value are collected and displayed in volts. The antigen preparation used in the experiments was prepared as follows: Bacteria were incubated in Todd-Hewitt broth at 37 ° C. for 2 hours.
The cells were cultured anaerobically for 4 to 48 hours, the culture was centrifuged to precipitate the cells, the cells were washed with a buffer, and centrifuged again.
(2x), cell pellets were resuspended in buffer and heat inactivated or formalin treated. The inactivated and lysed cell preparation was filtered to produce a filtrate of the antigen derived from the original cell-free microorganism (hereinafter referred to as "preserved antigen"). The stock antigen was diluted at 50 mM MOPS, pH = 7.0 to produce a standard used to evaluate test performance.

As a result of a positive reaction, a comparative ellipsometer fitted with a photodiode leads to an optical thickness change or an optical mass change, which is recognized as a light intensity change recorded as a voltage change at the detector. The voltage is a direct function of the light intensity change collected by the photodiode and is proportional to the increase in optical mass or thickness created by the interaction of the analyte / secondary reagent complex with the immobilized ligand on the slide surface. The method relies on the fact that when the reference and test surfaces are identical in optical thickness (thickness multiplied by refractive index), no light is detected at the detector and a true zero (0 volt) is observed. doing.
Even if there is a slight mismatch between the reference standard and the specimen, the background of the specimen is never truly zero. This can be easily corrected by measuring the background of the unreacted area of the test specimen and subtracting this value from the intensity of the reaction area of the test specimen.
The reaction zone may be with a positive or negative control or sample. As can be seen from the concentration of antigen applied to the surface in these examples, the observed value can be related to a particular concentration. The measurement is performed using a negative control; this is advantageously blank in the present system, and the residual signal resulting from the negative control may be subtracted from the positive sample if necessary. Thickness changes are not measured in terms of independent physical units such as Angstroms.

The enzyme-labeled antibody is not coated on the surface of the wafer. Only when the antigen is present in the sample, the enzyme labeling reagent has the opportunity to interact with the ligand on the surface of the wafer only through the antigen. The antigen acts as a filler between the immobilized ligand or acceptor and the enzyme-labeled secondary reagent. In particular embodiments, the ligand and the secondary reagent are both antibodies. The antigen / labeled antibody complex is captured by the immobilized ligand, creating a new layer that is a complex of layers 5 and 6. Layers 5 and 6 are not formed as separate layers, but rather are formed simultaneously.

The complex formed exists between the antigen in the subject and the two antibodies. One of the antibodies is bound on the support as a receptor intermediate layer, and the other is an unbound enzyme-labeled antibody. Part of this enzyme-labeled antibody passes through a complex formed between the analyte and the receptor,
Attach to the support. Subsequently, the insoluble reaction product precipitates, indicating a mass change indicating the presence of the analyte.

Example 7 Commercially available Becton Dickinson
The nitrocellulose membrane coated with the anti-Streptococcus A antibody contained in the reagent kit of ickinson) was reacted with the dilution of the positive Streptococcus A-specific antigen preparation used in Example 4. The antigen standard is 50 mM MOP
S, 1: 1 mixed with a 1: 100 dilution of anti-Streptococcus A antibody / HRP conjugate with 20 mg / ml casein in pH = 7.0. Streptococcus A-OI
As in the A enzyme assay, 80 μl of sample was added to the membrane and incubated for 2 minutes. The membrane is then rinsed with water,
50 μl of TM Blue was added and reacted for 5 minutes. The membrane was rinsed again and the assay sensitivity was determined visually. A 1:40 dilution of the antigen gave a clear triangle because the manufacturer's test was designed to do so with a positive reaction. Very weak triangles were formed at the standard 1:80 dilution. A comparable visual response was obtained at a 1: 1600 dilution in the Streptococcus A-OIA enzyme amplified samples of the present invention.

Example 8 A nitrocellulose membrane manufactured by Millipore was
Coated with anti-Streptococcus A polyclonal antibody according to the same protocol used for coating IA wafers. These membranes were tested with the protocol used above, except that the sample volume used was 30 μl of sample and 30 μl of TM Blue. As observed with Becton Dickinson membranes, a positive visual result compared to negative was only observed at a 1:40 dilution of the antigen preparation. A clear improvement in sensitivity is achieved by the device according to the invention, which combines the use of thin-film optical detection methods and the precipitation reagent TM Blue.

This example demonstrates that the device of the present invention has a much lower concentration of the target analyte than prior art devices that are neither a combination of HRP complexes or precipitation reagents nor a combination of non-HRPs such as alkaline phosphatase. Reveals that it is at least 40 times more sensitive in showing a signal proving the existence of

The test kits, immunoassay devices and basic coating and detection methods described herein may be performed according to the assay formats described herein, or for indicated volumes, concentrations or specific components, targets and detections for various reagents. It is not intended to be limited by a vessel. It should be understood that chemical analogs and other functional equivalents of the layers, layer coatings, or components used in the various reagents, additives, controls, calibrators, etc., may also be utilized within the scope of the present invention. is there.

It is to be understood that the invention concepts described herein are capable of further different embodiments and that the appended claims are intended to cover all alternative embodiments of this invention except as limited by the prior art. Should be construed as including.

[Brief description of the drawings]

FIG. 1 is a cross-sectional view of a multilayer OIA device including a support having an upper surface and a lower surface.

FIG. 2 shows a comparison of sensitivity between an antibody assay using an enzyme-labeled antigen of the present invention and an antibody assay using a latex antigen.

FIG. 3 shows the optical immunoassay sensitivity of rabbit anti-meningitidis.

FIG. 4 shows the sensitivity of rabbit anti-meningitidis by RxN reading at 450 nm (TMB substrate) after stopping the reaction (using Nunc ELISA polystyrene microtiter plate).

FIG. 5 shows the sensitivity of rabbit anti-meningitidis by RxN reading at 450 nm (TMB substrate) after stopping the reaction (using Dynatech ELISA polystyrene microtiter plate).

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Debbie Gables Clyder, 3895, Bosque Street, Boulder, Colorado, United States 80301 Allison Way No. 7819 (72) Inventor Gregory Robert Bogart Ash Drive 1107, Fort Collins, 80521 Colorado, USA (56) Reference International Publication 90/11525 (WO, A1)

Claims (17)

(57) [Claims] 1. A method for detecting an analyte in a sample, comprising the following steps: (a) a support, which is provided on an upper surface thereof and which can specifically bind to the analyte. Providing a thin-film optical immunoassay device having a thin film containing the acceptor; (b) allowing the acceptor to interact with the analyte and an enzyme-labeling reagent capable of specifically binding to the analyte; Interacting a precipitant capable of interacting with an enzyme present in the enzyme labeling reagent to increase the optical thickness of the thin film; and (c) the optical thickness of the thin film as an indicator of the amount of the analyte Measuring the increase in interference using interference or elliptic ellipsometry. 2. The method according to claim 1, wherein the receiving substance is fixed to the upper surface of the support via an adhesive intermediate layer. 3. The method according to claim 1, wherein the anti-reflection layer is provided on the upper surface of the support and below the receiving substance. 4. The enzyme-labeled reagent according to claim 1, wherein the enzyme-conjugated reagent is a conjugate of an enzyme and a substance capable of specifically binding to an analyte.
3. The method according to any one of 3. 5. The method according to claim 4, wherein the substance capable of specifically binding to the analyte is an antibacterial antibody. 6. The enzyme is alkaline phosphatase,
The method according to claim 4. 7. The method according to claim 1, wherein the enzyme labeling reagent is capable of reacting with a precipitant to form a precipitate. 8. The method according to claim 7, wherein the precipitant is a substrate for the enzyme. 9. The subject may be an antibody, an antigen, an allergen, an enzyme, an enzyme substrate, a coenzyme, a hormone, a hormone receptor, a protein, a blood protein, a tissue protein, a cell, a cell lysate, a nuclear material, a virus, a virus particle, 4. A metabolite, a neurotransmitter, a hapten, a drug, a nucleic acid, a metal, a microorganism, a parasite, a bacterium, an environmental substance, and various chemical species, and selected from the group consisting of substances derived therefrom. The method described in Crab. 10. The method according to claim 1, wherein the subject is a Strep A or B antigen. 11. The subject according to claim 1, wherein the subject is a causative organism for meningitis, Neisseria meningitidis, Streptococcus, Pneumoniae, Streptococcus group B or Haemophilus influenzae type B, or is derived therefrom. The method according to any of the above. 12. The method according to claim 1, wherein the increase in optical thickness is detected by use of an instrument. 13. The method of claim 12, wherein the instrument is an ellipsometer. 14. The method of claim 13, wherein the ellipsometer is a comparison ellipsometer. 15. The method according to claim 1, wherein the increase in optical thickness is detected visually. 16. The method according to claim 1, wherein in step (b), the acceptor is first reacted with the analyte. 17. The method according to claim 1, wherein in step (b), the acceptor is allowed to react with a complex of the analyte and the enzyme labeling reagent.