FI84863C - SEMIKVANTITATIV ELLER KVALITATIV IMMUNOMETRISK TESTMETOD OCH REAGENSFOERPACKNING. - Google Patents

Description

1 84863

Semiquantitative or qualitative immunoassay method and reagent kit

Semi-quantitative or qualitative immunometric test methods and reagent packaging

The invention relates to a semi-quantitative or qualitative immunoassay method in which the detection of an immunological reaction in an antibody or antigen-coated tube or well utilizes a colored end product resulting from the reaction. The invention also relates to reagent kits suitable for carrying out the assay according to the invention.

Immunometric assays utilize antibodies specific for the antigen or antibody to be determined, i.e., the analyte. A monoclonal antibody is a homogeneous antibody produced by a single cell and an identical cell line derived therefrom. It recognizes only one specific site on an antigen, i.e., an epitope, its affinity, protein chemical purity, isoelectric point, and immunoglobulin subclass can be determined. Compared to a polyclonal antibody, its main advantages are absolute and unchanged specificity, unlimited availability and homogeneity. Immunometric assays have become more common with the proliferation of monoclonal antibodies.

Immunometric determination is possible if the antigen has at least two epitopes far enough apart. The antigen to be determined in the sample adheres to a surface coated with a specific antibody (plastic tube, sphere or microtiter plate). Another antibody (or the same monoclonal antibody if the antigen has repetitive epitopes) labeled in some way is added to the solution either simultaneously with the sample or after washing. The labeled antibody adheres only to the antigens in the sample that have adhered to the immobilized specific antibodies. If the label is radioactive, 2 84863 is an immunoradiometric (IRMA) test, if the label is an enzyme, it is an immunoenzymometric (IEMA) test, if the label is fluorescent, if it is an immunofluorometric (IFMA) test and if the label is luminescent, it is an immunoassay. luminometric (AIR) determination. Correspondingly, the signal to be measured at the end of the assay is radioactivity measured with a gamma or liquid scintillation counter, light absorbing (colored) product of the enzyme substrate measured with a photometer, fluorescence measured with a fluorometer, or luminescence measured with a luminometer.

Fluorescence and luminescence can be derived directly from the labeled antibody or from a fluorescent or luminescent substrate measured in opaque wells to quantify the enzyme-labeled antibody. Such fluorescent substrates sensitize the corresponding enzyme immunometric assay up to several orders of magnitude.

These principles are well known and abundant in the literature (see, e.g., Practical Immunoassay. The State of the Art. Ed. Wilfrid R. Butt, Marcel Dekker Inc., 1984, New York; Hemmilä, I., Dakubu, S ., Mukkala, V.-M., Siitari, H., Lövgren, T., (1983), Europium as a label in time-resolved immunofluorometric assays, Anal Biochem 137: 335, and Shalev, A., Greenberg , GH, McAlpine, PJ, (1980),

Detection of “otograms of antigen by a high sensitivity enzyme-linked immunosorbent assay (HS-ELISA) using a fluorogenic substrate, J. Immunol. Methods, 38: 125).

When measuring fluorescence or luminescence, white or black tubes or microtiter plates are used and special devices measure either directly the fluorescence of the solution or, for example, the reflectance. All such devices are different devices and are intended for quantitative testing.

Correspondingly, the colored product of the enzyme substrate is measured as the absorbance of the solution in transparent microtiter plates or li 3 84863 tubes. Photometric devices are also used to measure absorbance and are also intended for quantitative testing. Quantifying the colored solution visually is difficult because the clear transparent wall is made into a cuvette and transmits light well, as it should be if the absorbance of the solution is to be measured.

There are also assays based on white filters in which the antigen adheres to the antibody bound to the white filter, adds another antibody labeled with the enzyme, performs a wash, and adds a substrate that precipitates on the white filter in the reaction. The resulting spot can be quantified by measuring the reflectance with a suitable device.

In addition, there are assays that use colored antibody or colored latex particles that pass through or through a white filter and stop at the antibody-containing site if the sample has contained antigen. The result of such a test is visually legible.

However, special readings are usually required to quantify the immunometric test. Still, there are numerous tests, in which the exact quantitative determination of the result with a special device is not essential, but a semi-quantitative or even qualitative determination would suffice for the test result. In particular, it would be necessary to provide a quick and easy semi-quantitative assay that could be used in laboratories or even at home where the specialized equipment required by conventional methods is not available.

Accordingly, in accordance with the invention, an immunoassay method as defined in the appended claims and a reagent kit for its application have been developed.

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The invention thus relates to a semiquantitative or qualitative immunoassay method in which an immunological reaction is performed in a tube or well coated with an antibody or antigen or a binding agent thereof, wherein the labeled antibody used in the reaction binds to the analyte to be determined and the relative concentration of the final product. This assay is characterized in that the coated tubes, well plates or microtiter plates used in the assay are light and opaque, preferably white, and that the detection of the reaction is performed visually at the visible wavelength of the solution from the absorbent final product.

According to the invention, it has surprisingly been found that if the test conventionally used to measure absorbance is carried out in an opaque rather than a transparent cuvette, the preferred! in a white tube or white microtiter plate, an assessment of the color intensity of the solution is possible without special measuring devices visually.

In particular, the invention is applicable to an enzyme-linked immunosorbent assay using white tubes, well plates or microtiter plates, or portions thereof, coated with a substance specific for the analyte to be determined. If the analyte is an antigen, the coating agent is an antibody, preferably a monoclonal antibody, and accordingly, if the analyte to be determined is an antibody, the coating agent is an antigen. It is also possible to coat the tubes or wells with the substance to which the antibody or antigen first binds. Such agents include, for example, the biotin and avidin systems, with one in the wall of the tube and the other coupled to the desired coating antibody or antigen that is added to the tube during or prior to the test. The end product of visual detection preferably uses a colored solution oxidized by the peroxide reaction of horseradish peroxidase (HRP) conjugated to a specific antibody.

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For example, from oxidized 2,2'-azinodi [3-ethylbenzothiazole-Solin-6-sulfonate] or ABTS solution (using a peroxidase label in the assay) in a white opaque well, absorbances of about 0.04 and about 0.06 can be distinguished, while in a clear tube or plate, an absorbance of about 0.30 is required to be able to visually separate the solution from 0.04.

The opaque tube or microtiter plate used in the assay of the invention prevents conventional absorbance measurement. However, the absorbance can be measured with a slight distortion if a part of the solution is transferred to a standard clear cuvette or the solutions of two parallel determinations are combined and half of this is quantified in the cuvette.

The reagent kit of the invention uses an opaque, preferably white tube, well plate or microtiter plate or strip thereof coated with an antibody, antigen or binding agent, or a strip thereof, a so-called "strip plate", and reagents which provide a visible light wavelength absorbing end. . In an enzyme immunometric assay, a labeled antibody specific for the analyte to be determined and the reagent required to visualize the label are used as reagents. Alternatively, an immunological test can be performed in which the antibody label directly produces a colored reaction without enzyme mediation.

It is essential in the invention that the analyte contained in the test sample provides a solution containing the end product absorbing visible light at a wavelength so that the analyte concentration can be detected visually from the solution in the well directly against a light background of the well surface without any special equipment.

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With the reagent kit according to the invention, it is thus possible to perform a rapid test, the result of which can be visually evaluated.

Thus, for example, a white microtiter plate for fluorescence assays (e.g., Dynatech Microfluor) is coated with a mono- or polyclonal antibody in the same manner as a corresponding conventional clear optically readable microtiter plate (e.g., Nunc Maxisorp) and subjected to an IEMA assay using enzyme-labeled HRP. horseradish peroxidase) or AFOS (alkaline phosphatase) conjugated antibodies. If desired, the substrate reaction can be stopped and the test result, i.e. the analyte content of the sample, can be assessed visually by comparing the degree of darkness of the sample substrate to the standards made in the same assay.

Solutions of corresponding reagents, the concentrations of which are, for example, for the pregnancy test, are preferably used as standards, and correspondingly, for the detection of a bacterial infection, they consist of a series of solutions whose concentrations rise from zero to the presumed peak value.

In the enzyme immunoassay, the end result is an antibody-enzyme conjugate adhering to the wall of the tube or well, which is detected by a substrate solution whose darkness (absorbance) is a function of the amount of analyte in the original sample. This method is applicable in principle to all rapid tests in which the result of the reaction is read visually. Examples of such tests are pregnancy test (HCG test), ovulation test (LH test), TSH test for neonatal thyroid hormone screening, C-reactive protein test (CRP test) for the detection or exclusion of bacterial infection, microbial tests, e.g. substance tests for, for example, viral or bacterial infections.

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The best visually readable colors are bluish tones. Suitable oxidizing substrates for the peroxide reaction of the HRP enzyme are, for example, blue 3 ', 3', 5''-tetramethylbenzidine (TMB), green 2,2'-azinoide (3-ethylbenzothiazoline-6-sulfonate] (ABTS), alkaline phosphatase the substrate is blue indoxyl phosphate, and the degrees of darkness of the reddish-brown color caused by ortho-phenylenediamine (OPD) are well visible against a white background, and other colors and / or color differences are more pronounced in the white well or tube.

The blue color of TMB turns yellow under the action of the strong acid used to quench the reaction. Against the white background of the pit, the intensity of the yellow color is difficult to assess. Instead, the yellow color can also be detected visually, for example, from a light blue well. However, in the rapid test, the reaction may not be stopped at all, in which case a blue semi-quantitative result can be obtained visually from the white tube formed by the TMB.

In an immunological test, in some cases a colored reaction can also be induced without the aid of an enzyme. In this case, the antibody label itself produces a colored end product that is visually detectable. The assay method of the invention can also be performed with such reagents.

The following examples illustrate the invention without, however, limiting it in any way.

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Example 1: Pregnancy test White polystyrene microtiter plates were coated with HCG antibody diluted in buffer (5008, Medix Biochemi-ca) and this solution was dispensed at 150 μΐ / well. The plate was allowed to stand overnight at room temperature.

The next day, the plates were washed 3 times and the remaining binding sites were covered with 0.5% BSA (bovine serum albumin) solution. For storage, the sheets were dried in a stream of warm air and packed in airtight laminate bags. Plates coated in this manner could be used to perform the test.

Assay Performance: 1. Pipette 50 μΐ of a 1:40 diluted anti-HCG label (5503-HRP label, Medix Biochemica).

2. Pipette 50 μΐ of sample or standard (0 and 50 IU HCG / 1 into the appropriate wells).

3. Allow to incubate for 5 min at room temperature.

4. Wash the wells 3 times with water.

5. Pipette 100 μΐ of ABTS substrate solution (Kirkegaard-Perry, 1-component ABTS, Cat. No. 50-66-00) into each well.

6. Allow to incubate for 5 minutes at room temperature.

7. Stop the reaction by adding 50 μΐ of 4% oxalic acid.

8. The darkness of the samples compared to the standards was visually inspected and their HCG content was assessed. A color darker than 50 IU HCG / 1 indicates a positive sample, a colorless solution is negative, and a slightly colored solution of 9,84863 is likely to indicate the onset of pregnancy, but the test should be repeated after one week, when the result should be> 50 IU HCG / 1 positive.

Example 2: CRP Assay White polystyrene microtiter plates were coated with diluted CRP antibody (6405, Medix Biochemica) and 150 μΐ / well of solution was dispensed. The plate was allowed to stand overnight at room temperature. The next day, the plates were washed 3 times and the remaining binding sites were covered with 0.5% BSA solution. For storage, the sheets were dried in a stream of warm air and packed in airtight laminate bags. Plates coated in this manner could be used directly to perform the test.

Assay Performance: 1. Pipette 80 μΐ 1: 5 diluted anti-CRP label (6405-HRP label, Medix Biochemica).

2. Pipette 20 μΐ of sample or standard (0.20, 40, 80 mg / l CRP) 3. Allow to incubate for 5 minutes at room temperature.

4. Wash the wells 5 times with distilled water.

5. Pipette 100 μΐ of ABTS substrate solution into each well.

6. Allow to incubate for 5 minutes at room temperature.

7. Stop the reaction by adding 50 μΐ of 4% oxalic acid.

8. The darkness of the substrate solution in the sample wells was visually inspected and compared to the darkness of the standard wells. If the sample solution is darker than the standard 80 mg / l, there is a probable severe bacterial infection, between 40-80 mg / l is likely to be a clear bacterial infection, 20-40 mg / l is likely to be a mild bacterial infection and <20 mg / l is probably a normal finding.

Example 3: LH Ovulation Assay White polystyrene microtiter plates were coated with diluted LH antibody (5302, Medix Biochemica) and 150 μΐ / well of solution was dispensed. The plate was allowed to stand overnight at room temperature.

The next day, the plates were washed 3 times and the remaining binding sites were covered with 0.5% BSA solution. For storage, the sheets were dried in a stream of warm air and packed in airtight laminate bags. Plates coated in this way could be used directly to perform the test.

Assay Performance: 1. Pipette 75 μΐ 1:25 diluted anti-LH label (5301-HRP label, Medix Biochemica).

2. Pipette 25 μΐ of standards with LH concentrations of 0, 10 and 20 U / l or a sample to be determined.

3. After 30 minutes, the wells were washed 5 times with distilled water.

4. 100 μΐ ABTS substrate solution was added.

5. After 15 minutes, the reaction was stopped by the addition of 50 μΐ 4% oxalic acid.

8. The darkness of the substrate solution in the sample wells was visually inspected and compared to the darkness of the standard wells. If the sample solution is darker than the standard 20 IU LH / 1 is Π 84863 in case of probable ovulation peak, between 10-20 IU LH / 1 is likely to increase or decrease ovulation peak, less than 10 IU / 1 is not likely to ovulate.

Example 4: TSH Assay for Neonatal Screening White polystyrene microtiter plates were coated with diluted TSH antibody (5403, Medix Biochemica) and 150 μΐ / well of solution was dispensed. The plate was allowed to stand overnight at room temperature.

The next day, the plates were washed 3 times and the remaining binding sites were covered with 0.5% BSA solution. For storage, the sheets were dried in a stream of warm air and packed in airtight laminate bags. Plates coated in this manner could be used directly to perform the test.

Assay Performance: 1. Pipette 25 μΐ (1:25) anti-HCG label (5503-HRP label, Medix Biochemica) into each well. 2. Pipette 50 μΐ sample or standard into the appropriate wells.

3. Incubate for 10 min at room temperature.

4. Wash the wells 3 times with water.

5. Pipette 100 μΐ of ABTS substrate solution.

6. Incubate for 10 minutes at room temperature.

7. Stop the reaction by adding 50 μΐ of 4% oxalic acid.

i2 8 4 8 63 8. The darkness of the substrate solution in the sample wells was visually inspected and compared to the darkness of the standard wells. If the sample solution is darker than the standard 50 mIU TSH / 1, it is likely to be hypothyroidism, if the sample solution is lighter than the 50 mIU / Ι TSH standard, it is likely to be a normal finding.

Example 5: Pancreatic amylase assay White polystyrene microtiter plates were coated with diluted pancreatic amylase antibody (6103, Medix Bio-chemica) and a solution of 150 μΐ / well was dispensed. The plate was allowed to stand overnight at room temperature.

The next day, the plates were washed 3 times and the remaining binding sites were covered with 0.5% BSA solution. For storage, the sheets were dried in a stream of warm air and packed in airtight laminate bags. Plates coated in this manner could be used directly to perform the test.

Assay Performance: 1. 75 μΐ 1:40 diluted anti-pancreatic amylase label (6105-HRP label, Medix Biochemica) was pipetted into the wells of the plate.

2. 25 μΐ standards 0 and 250 nq / l or sample were added to the appropriate wells.

3. After 5 minutes, the wells were washed 5 times with distilled water.

4. 100 μΐ ABTS substrate solution was added.

5. After 5 minutes, the reaction was stopped by the addition of 50 μΐ 4% oxalic acid.

l! 13 84 863 6. The darkness of the substrate solution in the sample wells was visually inspected and compared to the darkness of the standard wells. If the sample solution is darker than the standard 250 ug / l, it is probable acute pancreatitis and <250 yg / l is likely to be a normal finding.

Example 6: Microbial test White polystyrene microtiter plates are coated with diluted Chlamydia antibody and a solution of 150 μ 150 / well is dispensed. Allow the plate to stand overnight at room temperature.

The next day, the plates are washed 3 times and the remaining binding sites are covered with 0.5% BSA solution. For storage, the sheets are dried in a warm air stream and packed in airtight laminate bags. Plates coated in this way can be used directly to perform the test.

Test procedure: 1 · Pipette 80 μΐ of 1:50 diluted Chlamydia antibody conjugated to HRP.

2. Pipette 20 μΐ of sample, negative and positive control into the appropriate wells.

3. Allow to incubate for 30 minutes at room temperature.

4. Wash the wells 3 times with water.

5. Pipette 100 μΐ of ABTS substrate solution into each well.

6. Allow to incubate for 10 minutes at room temperature.

7. Stop the reaction by adding 50 μΐ of 4% oxalic acid.

i4 84863 8. Visually inspect the darkness of the substrate solution in the sample wells and compare with the darkness of the standard wells. If the sample solution is darker than the positive control, it is likely to be a Chlamydia infection, and if the sample solution is as pale as the negative control, it is likely to be a normal finding. If the sample solution is darker than the negative control but lighter than the positive control, it is a possible Chlamydia infection.

Example 7 Microbial Antibody Assay Polystyrene microtiter plates were coated with diluted bacterial antigen solution and the solution was dispensed at 150 μΐ / well. The plate was allowed to stand overnight at room temperature.

The next day, the plates were washed 3 times and the remaining binding sites were covered with 0.5% BSA solution. For storage, the sheets were dried in a stream of warm air and packed in airtight laminate bags. Plates coated in this manner could be used directly to perform the test.

Test procedure: 1. A patient sample containing 20 μΐ of bacterial antibody was pipetted in serial dilutions of 1:10, 1: 100, 1: 1000, 1: 10000 and 1: 100000 or negative (buffer) control into the appropriate wells.

2. 80 μΐ assay buffer was added to all wells.

3. Allow to incubate for 1 hour at room temperature.

4. Wash wells 5 times with wash buffer.

5. Pipette 100 μΐ 1: 2000 diluted Rabbit anti-human immunoglobulin HRP label (Dako P212, peroxidase conjugated I: 154863 rabbit immunoglobulins to human IgA, IgG, IgM, kappa, lamda) into each well.

6. Incubate for 30 minutes at room temperature.

7. Wash wells 5 times with wash buffer.

8. Pipette 100 μΐ of ABTS substrate solution into each well.

9. Allow to incubate for 10 min at room temperature.

10. Stop the reaction by adding 50 μΐ 4% oxalic acid to all wells.

11. Visually compared the color given by the patient sample dilutions with the negative control. Depending on the gradual dilution, it could be inferred from the lightening color that the antibody titer was, for example, between 1: 100 and 1: 1000, i.e. the patient sample had then probably contained antibodies specific for the antigen in question.

Example 8: Comparative example of an oxidized ABTS solution on a white plate compared to the absorbance measured on a transparent plate

A CRP test was performed according to Example 2 on two parallel transparent microtiter plates. When the reaction was complete, the substrate solutions (300 μΐ) in two parallel wells were combined and transferred to a 150 μΐ white microtiter plate. The absorbances of the remaining solutions were measured, the solutions on both white microtiter plates and transparent microtiter plates were visually inspected and their degree of darkness was assessed.

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Score:

Visual evaluation on the plate Standard point Absorbance Transparent White 0 mg / 1 0.060 20 mg / 1 0, 120 - ± 40 mg / 1 0.202 - + 80 mg / 1 0.368 + ++

Example 9: Comparative example of ABTS solutions of different strengths

A series of ABTS solutions of varying darkness were made and pipetted onto a 150 μΐ white plate. 100 μΐ of the same solutions were taken for absorbance measurement on transparent plates.

Score:

Absorbance Visual estimate 0.044 0.043 0, 476 + + 0, 077 ± 0, 060 ± 0.043 0, 042 ϊ 0, 275 + 0, 058 ± 0, 058 t 0, 056 ± 0, 160 ♦ From these readings it could be concluded that the eye it is possible to distinguish between absorbances 0, 043-0, 044 and 0, 056-0, 060 when the measured volume is 100 μΐ.

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Claims (10)

1. Semi-quantitative or qualitative immunometric assay method in which the immunological reaction is carried out in a tube or well coated with antibodies, antigens or a substance which amplifies them, in which reaction the labeled antibodies used bind to the analyte to be determined and the content of a final product arising in the reaction is determined from the solution, characterized in that light and opaque, advantageously white tubes or wells are used, the coating of which contains mono- or polyclonal antibodies or antigens, and that the detection of the immunological reaction occurs visually. of an end product absorbing within the visible path range in the solution in the tube or well. A method according to claim 1, characterized in that in an enzyme-immunological assay, white tubes, wells or microtiter plates or portions thereof are used, the coating of which contains antibodies or substance which binds antibodies specific for the analyte to be determined. The final product for the visual detection is a colored ABTS, TMB or OPD solution oxidized by an HRP peroxide reaction. Method according to Claim 1 or 2, characterized in that the test to be performed is a pregnancy test (HCG determination), ovulation test (LH test), TSH test for saturation of newborn thyroid hormone (C reactive protein determination). CRP test) to detect or exclude bacterial infections, microbial or antibody tests to detect viral or bacterial infections. 4. Reagent package for an immunological test, which contains a tube, a well plate or a microtiter plate or a portion thereof coated with antibodies, antigens or a substance which is capable of binding them, and the reagents needed for the immunological reaction, - that the tubes or wells containing the package are light and opaque, advantageously white, and - that the package contains reagents with labeled antibodies specific for the analyte to be determined, and if necessary, reagents which visualize the label, whereby The reagents are so selected that due to the analyte in the sample to be tested, a solution containing an end product which absorbs light within the visible path length range is obtained. 5. Reagent packaging according to claim 4, characterized in that the color intensity of the reagent solution is dependent on the sample's analyte haze and that the package contains the standards needed for a semi-quantitative determination. 6. Reagent package according to claim 4 or 5, characterized in that the package contains labeled antibodies specific for an enzyme-immunological reaction and reagents that visualize the label and advantageously termination reagent for the reaction. 7. A reagent package according to claim 4, 5 or 6, characterized in that the package contains a white microtiter plate or part thereof which is coated with antibodies or antigens specific to the analyte to be determined. Reagent package according to claim 4, 5 or 6, characterized in that the well or wells containing the package are coated with a substance which increases the binding of antibodies or antigens specific to the analyte to be determined, the package advantageously also containing specific antibodies. or antigens that bind to the coating agent. 22 84863 Reagent packaging according to any one of claims 4 to 8, characterized in that the labeled reagent is HRP-labeled antibodies which are specific for the test to be performed and that the reagent providing the visually detectable final product is an ABTS, TMB. - or OPD solution. 10. Reagent packaging according to claim 7 or 9, characterized in that the wells are coated with specific antibodies selected from the following: HCG antibodies for pregnancy test, CRP antibodies for CRP test, LH antibodies for LH ovulation test, TSH antibodies for neonatal saline thyroid hormone screening, pancreatic amylase antibodies for pancreatic amylase testing, and Chlamydia antibodies for Chalmydia infection testing.