WO2014012210A1

WO2014012210A1 - Immunoassay method based on absorbance measurement

Info

Publication number: WO2014012210A1
Application number: PCT/CN2012/078717
Authority: WO
Inventors: 李久彤
Original assignee: Li Jiutong
Priority date: 2012-07-16
Filing date: 2012-07-16
Publication date: 2014-01-23

Abstract

An immunoassay method based on absorbance measurement comprises steps of: providing a mixed reaction solution containing first and second conjugates for a subject to be detected, adding the subject to be detected into the solution to form a mixed solution containing a ternary complex "first conjugate-subject to be detected-second conjugate", measuring an absorbance value A1 of the solution, separating the ternary complex from the mixed solution by liquid-phase separation, measuring an absorbance value A2 of the liquid phase obtained after separation, and determining, by comparing A1 with A2, the presence and/or quantity of the subject to be detected.

Description

Immunoreactivity analysis method based on measuring absorbance

Technical field

The present invention relates to the field of in vitro detection technology, and in particular to an immunoreactivity analysis method based on measuring absorbance. Background technique

A variety of immunoreactivity analysis methods have been developed based on "double-anti-sandwich", such as: radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence assay, time-resolved fluorescence assay and fluorescence immunoassay (Cl in. Chem. 1978, 24: 342-344), etc., can be used to determine pathogenic microorganisms, quantitative detection of specific proteins in the human body, thereby assisting diagnosis or monitoring of diseases, etc., and is widely used. The usual practice is: immobilize the capture antibody on a solid phase carrier, then react with the antigen (target protein), wash it, then react with the labeled antibody, wash, and add a substrate to detect signals such as photoelectricity. These methods are heterogeneous reactions between the solid phase and the liquid phase, and one experiment reports the results of a project, so there is a further increase in the reaction rate and the actual demand for multiple tests in one test.

The liquid phase chip technology that has emerged in recent years allows multiple tests to be performed in one test without the need for washing. The principle is to covalently crosslink differently encoded microspheres to probes, antigens or antibodies directed against a particular test substance. In the application, the coded microspheres for different detection materials are first mixed, and then a small amount of the sample to be tested is added, and the molecules of the target molecule and the surface of the microspheres are specifically combined in the suspension, and can be simultaneously completed in one reaction system. A variety of different biological reactions. Finally, the flow cytometry instrument is used for analysis. The instrument identifies the coded microspheres and the fluorescence intensity of the reporter molecules on the microspheres by two lasers, and reports the results of multiple test items. Because of the heterogeneous reaction, the results are reported. The time is generally 1-1. 5 hours, can not meet the requirements of clinical rapid report results (Chinese Journal of Laboratory Medicine, 2006, 29 (5): 431-432; Analytical Instruments, 2008, No. 1, 22-24) .

Therefore, there is an urgent need in the art to develop a detection method that is simple to operate (e.g., does not have to be washed) and that is uniform and/or rapid. Summary of the invention

SUMMARY OF THE INVENTION It is an object of the present invention to provide a detection method which does not require manual washing during the detection process, is simple to operate, and has a uniform reaction and/or rapidity. In a first aspect of the invention, there is provided an immunoassay method for detecting the presence or absence and/or quantity of a test substance, comprising the steps of:

(a) providing a test mixture, wherein the test mixture contains a first conjugate for the test substance, and a second conjugate for the test substance, wherein the second conjugate is labeled with a light absorbing substance And the first conjugate and the second conjugate can be simultaneously bonded to the analyte;

(b) adding a test object or a sample solution containing the analyte to the test mixture to form a mixed solution containing the "first conjugate - test substance - second conjugate" ternary complex;

(c) irradiating the mixed solution of step (b) with light, allowing the light to pass through the mixed solution, and measuring the transmitted light to obtain the absorbance of the mixed solution, which is recorded as the first absorbance A1;

(d) capturing or separating the ternary composite from the mixed solution of the previous step such that the ternary composite is separated from the liquid phase of the mixed solution;

(e) irradiating the mixed solution of the step (d) with light, allowing the light to pass through the mixed solution, and measuring the transmitted light to obtain the absorbance of the mixed solution, which is recorded as the second absorbance A2;

(f) comparing the first absorbance A1 with the second absorbance A2 to determine the presence or absence and/or quantity of the analyte.

In another preferred embodiment, the detection mixture is a liquid phase (solution), a suspension or a solid phase.

In another preferred embodiment, when the detection mixture is a solid phase, the step (b) further comprises adding a solvent (e.g., water or buffering). In another preferred embodiment, the analyte comprises: a protein and/or a nucleic acid.

In another preferred embodiment, the analyte comprises a specific protein such as a tumor marker.

In another preferred embodiment, the tumor marker is selected from the group consisting of alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), carbohydrate antigen 19-9 (CA19-9), total Prostate-specific antigen (PSA), free prostate specific antigen (f-PSA), neuron-specific enolase (NSE), sugar chain antigen (CA242), cancer antigen (CA15-3), human chorionic gonadotropin Hormone (β-HCG).

In another preferred embodiment, the light absorbing material refers to a substance having a molar absorption coefficient ε > 10 ⁸ L · πιοΓ ¹ · cm- ¹ .

In another preferred embodiment, the light absorbing material is selected from the group consisting of colloidal gold, nanogold bars, nanosilver bars, or combinations thereof.

More preferably, the light absorbing material is a nano gold rod. In another preferred embodiment, the nanogold bars have an aspect ratio of from 1.5 to 10, preferably from 1.5 to 5.

In another preferred embodiment, the nanogold rod has a length of 10 to 200 nm, preferably 20 to 100 nm. In another preferred embodiment, the colloidal gold is colloidal gold particles having an average particle diameter of 10 to 70 nm.

In another preferred embodiment, the first conjugate is labeled with a capture agent; more preferably, the capture agent comprises: biotin, magnetic microspheres, or a combination.

In another preferred embodiment, the step (f) further comprises: comparing the ratio of the first absorbance A1 to the second absorbance A2 to a standard curve or a comparison to determine the amount of the analyte.

In another preferred embodiment, the analyte is an antigen, and the first binder and the second binder are antibodies that bind to the antigen simultaneously; or

The test substance is an antibody, and the first conjugate and the second conjugate are antibodies (anti-antibodies) which can bind to the antigen of the antibody or the antibody at the same time.

In another preferred embodiment, the analyte is an antibody, the first binder is an antigen that binds to the antibody, and the second binder is directed to the antibody (antibody) Antibodies, and vice versa. For example, when the test substance is a human antibody, the first conjugate is the antigen against which the human antibody is directed, and the second conjugate is a mouse or rabbit-derived antibody against the human antibody.

In another preferred embodiment, in step (e), the light path transmitted through the mixed solution is

0.1-200 cm.

In another preferred embodiment, the optical path is from 0.2 to 100 cm, preferably from 0.5 to 50 cm, more preferably from 1.0 to 20 cm.

In another preferred embodiment, the light includes visible light and infrared light.

In another preferred embodiment, the light has a wavelength of from 300 to 1500 nm.

In another preferred embodiment, the method further comprises the step of making a standard curve by measuring with a known concentration of the test object standard.

In another preferred embodiment, the ternary complex described in step (d) is separated from the liquid phase of the mixed solution, meaning that the ternary complex is absent on the light path through the liquid phase.

In another preferred embodiment, the analyte comprises 2-10 different substances, and the detection mixture contains a first binder and a second binder for the different analytes, respectively.

In another preferred embodiment, the nano-gold rods having different aspect ratios are marked on the second combination of the different analytes. In another preferred embodiment, the first conjugates for the different analytes are labeled with the same capture agent.

In a second aspect of the invention, there is provided an immunoassay detecting apparatus for detecting the presence or absence and/or quantity of a test object which can be used in the method of the first aspect of the invention, the apparatus comprising:

(a) a container for placing a test mixture, wherein the test mixture contains a first conjugate for the test substance, and a second conjugate for the test object, Wherein the second conjugate is labeled with a light absorbing substance, and the first conjugate and the second conjugate are simultaneously bonded to the analyte;

(b) a detector for detecting absorbance; and

(c) A description of the use of the method of claim 1. It is to be understood that within the scope of the present invention, the above various technical features of the present invention and the technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Limited to the length, no longer one by one. DRAWINGS

Figure 1A shows that in the method of the present invention, the first antibody, the analyte, and the second antibody form a "ternary complex" in the mixed solution.

Figure 1B shows that in the method of the present invention, after the "ternary complex" is captured or separated from the mixed solution, a portion of the second antibody remains in the mixed solution.

Figure 2 shows the visible/infrared spectra of nanorods with different aspect ratios.

Fig. 3 shows a standard curve of CEA obtained in an example of the present invention, wherein the X axis is a logarithmic concentration and the y axis is a logarithm of the ratio of A1/A2.

Fig. 4 shows a standard curve of the AFP obtained in an example of the present invention.

Fig. 5 shows a standard curve of CEA obtained in an example of the present invention.

Fig. 6 shows a standard curve of the AFP obtained in an example of the present invention.

Fig. 7 shows a CEA standard curve obtained in an example of the present invention.

Fig. 8 shows an AFP standard curve obtained in an example of the present invention.

Fig. 9 shows a P S A standard curve obtained in an example of the present invention. detailed description

After extensive and intensive research, the inventors have for the first time developed a measurement of the same mixed solution in 2 no The same as the absorbance of the reaction phase, and then the ratio is determined to determine the presence or absence and/or quantity of the analyte. In the detection method of the present invention, the reaction is uniform and rapid; the whole process does not require washing, and multi-indicator joint inspection can be performed. In addition, the detection sensitivity can be conveniently adjusted according to actual needs, and the quantitative is accurate. The present invention has been completed on this basis. Fundamental

The basic principle of the detection method of the present invention will now be described with reference to the accompanying drawings.

(a) Principle of single indicator detection

Based on the "double antibody sandwich" method, the "first antibody" is appropriately labeled to have an separation function, and the second antibody is labeled with a substance having strong absorbance (such as colloidal gold, Nanorod), "first antibody", "second" The antibody "mixes with the sample to form a "first antibody-antigen-second antibody" sandwich complex. The absorbance at the maximum absorption peak of the mixed solution was measured and recorded as A1;

After capturing or separating the "first antibody-antigen-second antibody" sandwich complex formed by the reaction, the absorbance of the mixed solution at the peak position is measured as A2;

The ratio of A1/A2 is taken as the effect of the amount of antigen to be tested in the sample. Since the sandwich complex is separated, A1 A2, BP : A1/A2 value 1, and the more the amount of antigen to be tested in the sample, the larger the A1/A2 value.

Referring to Figures 1A and 1B, Stdl-5 is the standard line of the antigen to be tested, and there is no antigen to be tested in Stdl, and no sandwich complex is formed.

Std2-Std5 symbolically shows 2, 4, 6, and 8 analytes. Therefore, the number of sandwich complexes in Std2-Std5 increases with the concentration of the antigen to be tested, but the amount of the second antibody. Not changed. At this time, the absorbance of Stdl-5 was measured and recorded as Ai l (i = l-5).

The free primary antibody and the "sandwich complex" containing the first antibody were separated and removed by a suitable method, and the absorbance of the mixed solution was measured and recorded as Ai2 (i = l-5), which was compared with FIG. 1A. When the antigen was increased, the second antibody labeled with the light-absorbing substance in Std2-5 decreased, the absorbance decreased in turn, and Ai l/Ai2 (i = l-5) increased in turn.

(b) Multi-indicator joint detection principle

Different "first antibodies" for different antigens can use the same labeling method to facilitate separation or capture.

The different "second antibodies" directed against these antigens are each labeled with Nanorod having a different aspect ratio. For example, three kinds of "second antibodies" against three antigens were labeled with Nanorod (three Nanorods 2, 3, 4 in Figure 2), respectively, to make these Nanorod-labeled "second antibodies" spectrally Different from each other.

When applied, the principle of multi-indicator detection is exactly the same as the principle of single-index detection. The "first antibody" and "second antibody" for different analytes are mixed with the sample to form a "first antibody-antigen-second antibody" sandwich complex. Generally, the absorption spectrum of the mixed solution at a certain wavelength (e.g., 500-1000 nm) is scanned, and the absorbance at each peak position is recorded. Al i = l - the number of different antibody species).

After capturing or separating the different "first antibody-antigen-second antibody" sandwich complex produced by the reaction, the absorbance of the mixed solution at each corresponding peak position is measured to obtain A2i = l-the number of different antibody species).

The ratio of each Ali/AS i is taken as the effect of the amount of antigen to be tested in the sample. Since the sandwich complex is separated, Ali ^AS i , and Ali/ AS i ^ l , the more the amount of antigen to be tested in the sample, the larger the A / A2 i value. First antibody label

There are a variety of labeling methods for the first antibody to have a separation function, such as: the antibody is directly immobilized to the solid phase carrier by chemical or physical means, wherein the solid phase carrier can be a slide, a membrane, various kinds of microplates used in the field. And various microspheres, etc.; more preferably, the first antibody is labeled with biotin (Biot in), the immune reaction occurs in a homogeneous environment, and then the magnetic microspheres or enzyme labels modified with Streptavidin The plate or membrane is joined for separation purposes. Light absorbing substance

As used herein, the term "absorbance material" refers to a material having a molar absorption coefficient ε > 10 ⁸ L - moF ¹ · cm ^-1 , and representative materials include, but are not limited to: colloidal gold, nano gold particles (including Nanorod) , nano silver particles.

As used herein, the term "nano-gold rod" or "Nanorod" refers to gold particles having a certain aspect ratio and having a horizontal axis and a longitudinal axis in the range of 5-200 nm. Figure 2 shows the visible/red spectroscopy spectra of nanogold rods with different aspect ratios (Biotechnology and Genet ic Engineering Revi ews - Vol. 25, 93-112, 2008), in which different nanogold rods have different visible/infrared spectra. The characteristics can be distinguished from each other.

Nanorod is a nanomaterial developed in recent years. Its absorption spectrum and fluorescence spectrum are related to the length of gold rod. Anisotropy and unique spectral characteristics can be applied to diagnosis, nanomaterial assembly, cancer cell imaging and DNA analysis, etc., has become one of the research hotspots in the medical field. Available in conventional methods Prepared or commercially available, for example, from Nanopartz Inc. (Colorado, USA).

The nanogold rod suitable for use in the present invention is not particularly limited, and the preferred nanogold rod has an aspect ratio of 1.5 to 10, preferably 1.5 to 5.

In another preferred embodiment, the nanogold rod has a length of 10 to 200 nm, preferably 20 to 100 nm. Second antibody labeling

In order to detect changes in the absorbance of the mixed solution due to the immune reaction shown in Figures 1A and 1B, the substance labeled with the second antibody must have strong light absorption characteristics, such as: molar absorption coefficient ε > 10 ⁸ L · mol - ¹ · cm - ¹ substance.

In a preferred embodiment, the second antibody is labeled with nanogold particles and a nanogold rod, the physical size of which is about

10-100nm, a stable colloidal solution with a maximum absorption wavelength of 500-1500nm, the labeled second antibody can form a sandwich complex with the first antibody and antigen in a homogeneous solution.

A particularly preferred light absorbing material is colloidal gold, especially colloidal gold having a particle size of 20-40 nm. standard curve line

In the present invention, the presence or absence and/or the number of the analytes can be determined directly by comparison of the first absorbance A1 and the second absorbance A2.

In a preferred embodiment, quantitative results can be obtained by comparison with a standard curve.

The standard curve can be obtained by the following method: the first point solution Stdl of the antigen standard to be tested is mixed with the labeled first antibody and the second antibody solution, and after the reaction, the absorbance of the mixed solution is measured, and the antibody is Al i = l- different. Number of species);

After separating or capturing the "first conjugate-test object-second conjugate" ternary complex", the absorbance of the mixed solution is again measured as A2 i = l - the number of different antibody species);

Find the value of Ai ASi.

In the same operation, the A1 ratios of other points of the antigen standard to be tested are respectively obtained, and the logarithm of the concentration of each of the standard strains of the antigens is plotted against the corresponding Ali/ASi logarithm to obtain i standard curves. Capture and separate ternary complexes

In the present invention, the "first conjugate-test substance-second conjugate" ternary complex can be captured and separated by a conventional method.

A common method is to capture and immobilize the complex on a solid phase carrier. The commonly used solid phase carrier has a slide and a membrane. Tablets, microplates and various microspheres. For example, the first antibody is labeled with biotin to form a "sandwich complex" which is then bound to a streptavidin-coated solid support to capture or separate the ternary complex from the liquid phase.

In addition, the ternary complex can also be captured or separated from the liquid phase by magnetic microspheres (in combination with magnetic fields) or other means.

In the method of the present invention, a light source is used to provide light of a certain emission wavelength to emit illumination and to pass light of the detection solution.

In the present invention, any light source that can provide a suitable wavelength can be selected. A suitable wavelength range is included in another preferred embodiment, wherein the light has a wavelength of from 300 to 1500 nm, preferably from 400 to 1000 nm. The main advantages of the detection method of the present invention include:

(a) The physical size of the antigen, antibody, etc. in the solution is 100 nm, so the reaction is carried out in a homogeneous reaction system of the sol, and the reaction is uniform and rapid;

(b) No washing is required throughout the test;

(c) Multiple indicator detection can be performed in the same detection system.

(d) High detection sensitivity and accurate quantitation. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Parts by weight and percentages are by weight unless otherwise stated. Example 1 : Detection of carcinoembryonic antigen (CEA) in serum

The paired antibodies against CEA A1/C9 were purchased from Shanghai Second Medical University;

CEA antigen was purchased from China National Institute for the Control of Pharmaceutical and Biological Products;

Sreptavi din coated ELISA plate, commercially available;

30nm colloidal gold is commercially available; Detector: USB4000-FL (US Ocean Optics)

Tungsten halogen light source, wavelength 350~2000 let

1. Preparation of gold standard antibody (second antibody)

1. 1 colloidal gold - antibody preservation solution

Sodium tetraborate 0. lg calf serum albumin (BSA) 0. 25g

Nag ₃ 0. 025g After adding water, the pH was adjusted to 7.4 with 6N HCL, hydrated to 250 ml, filtered with a 0.45 μm filter, and stored at 4-8 ° C;

Working fluid

Na ₂ HP0 ₄ · 12H ₂ 0 6. lg

NaCl 8. 5g

PVP40 (polyvinylpyrrolidone 40) 5. Og

2. lg

PEG l. Og

10% BSA 50ml

NaN ₃ 0. 2g

After adding water, the pH was adjusted to 7. 0-7. 5 with 6N HCL to 1500 ml, filtered with a 0.45 μm filter, and stored at 4-8 ° C;

1. 3 Preparation of gold standard antibody (second antibody)

1. 3. 1 Take 20-30nm colloidal gold solution 20ml, slowly add purified A1 antibody 1. 0ml (0. 6mg / ml) under magnetic stirring, stirring at room temperature for 30min;

1. 3. 2 Add 10% BSA 0. 8ml (final concentration 0.4%), stir at room temperature for 5min;

1. 3. 3 Add 10% PEG 0. 4ml (final concentration 0.2%), stir at room temperature for 5min;

1. 3. 4 12000-1500r/min centrifugation for 60-40min, carefully remove the supernatant, dissolve the precipitate in 20ml preservation solution, filter with 0. 45 μ πι filter, store at 4 °C for use;

1. 3. 5 Measure the 0. D value of the above gold standard antibody solution at 530 nm to 1. 5, and the equivalent of 36 μg of A1 antibody per ml of gold standard antibody solution.

2. Biotin label C9 antibody (primary antibody)

2. 1 C9 antibody pretreatment

The antibody contains sodium azide, glycine or the like and is thoroughly dialyzed with pure water. Small molecules of amino groups and other small molecules

The antibody contains a large Protein A column such as bovine serum albumin or other column purification.

Molecule

Antibody concentration calibration Spectrophotometer measures OD280 (10D280 equivalent

0.7 mg/ml monoclonal antibody). Finally using 1XPBS,

The concentration of PH7.4 was set at 2 mg/ml (concentrated using Pall's desalting spin column).

2.2 Biotin Labeling C9 Antibody

The above pretreated C9 antibody was added, 25 μL of lmg/ml NHSS-Biotin DMSO solution was added, mixed, and protected from light for 2 hours in a refrigerator at 4 ° C, and dialyzed overnight for a final concentration of 550 μg/ml.

3. Drawing of standard curve

3.1 Preparation of CEA standard solution

The CEA antigen was diluted to a standard series with PBS (pH 7.4) at concentrations of 0 (Stdl), 10 (Std2), 30 (Std3), 100 (Std4), 350 (Std5) ng/ml;

3.2 Formation of sandwich composites and absorbance measurement

Take the second antibody solution 10 μ 1 , the standard solution Stdl 290 μ 1 , the first antibody solution 1 μ 1, mix, react at 37 ° C for 10 min, form a sandwich complex, measure the absorbance of the mixed solution at 530 nm, which is A1; The mixed solution was transferred to the well of the microplate, and after reacting at 37 ° C for 15 min, the absorbance of the mixed solution at 530 nm was measured as A2;

Find the value of A1/A2.

The same operation was carried out with other standard solutions, and the ratios of other points of the CEA standard, Ail/Ai2 (i=2-5), were obtained. The results are shown in Table 1.

Table 1: Test results of CEA standard system

The results of the above table are plotted and the standard curve is obtained (Fig. 3). 4, sample testing Replace the standard series of solutions with 6 unknown serum samples, repeat the steps of 3.2, substitute A1/A2 into the standard curve, and measure the CEA values of the six samples: 12.2, 43.9, 32.6, 78.8, 103.3, 211.6 ng/ml .

Example 2: Detection of alpha-fetoprotein (AFP) in serum

1, raw materials

Paired antibodies against AFP G4/C2 were purchased from Shanghai Second Medical University;

AFP antigen was purchased from China National Institute for the Control of Pharmaceutical and Biological Products;

Str printed tavidin coated ELISA plate, commercially available;

A nanorod with a horizontal axis of 10 and a vertical axis of 41 nm, with an aspect ratio of 4.1, commercially available;

Detector: USB4000-FL (US Ocean Optics)

Tungsten halogen light source, wavelength 350~2000 let

2, antibody labeling

2.1 Nanogold rod labeling Preparation of G4 antibody (second antibody)

200 w L of nano gold rod, adding 1 ml of 0. lmg / ml of the second antibody, shaking, standing for 30 min, washing with 3% BSA 3 times, the second antibody is fixed to the surface of the nanorod by non-specific physical adsorption;

The precipitate was dissolved in an appropriate amount of the preservation solution (prepared in the same manner as in the previous example), and filtered through a 0.45 μm filter to have an absorbance of -1.0 at -830 nm, and the G4 antibody solution labeled per nanometer gold rod was equivalent to about 30 μg. G4 antibody.

2.2 Biotin tag C2 antibody (primary antibody)

2.2.1 Pretreatment of C2 antibodies

The antibody contained sodium azide and glycine and was thoroughly dialyzed against pure water.

An amino group-containing small molecule such as an acid

Other small molecules

The antibody contains bovine serum white egg Protein A column or other column purification.

White and other macromolecules

0.7 mg/ml monoclonal antibody). Finally using 1XPBS,

The concentration was set at 2 mg/ml for PH7.4 (concentrated using a Pall desalting spin column). 2.2.2 Biotin labeling reaction

The above pretreated C2 antibody was added, 25 μL of lmg/ml NHSS-Biotin DMS0 solution was added, mixed, and protected from light for 2 hours in a refrigerator at 4 ° C, and dialyzed overnight for a final concentration of 650 μg/ml.

3. Drawing of standard curve

3.1 Preparation of AFP standard solution

The AFP antigen was diluted to a standard series with PBS (pH 7.4) at concentrations of 0 (Stdl), 10 (Std2), 20 (Std3), 100 (Std4), 400 (Std5) ng/ml;

3.2 Formation of sandwich composites and absorbance measurement

Take the second antibody solution 30 μ 1 , the standard solution Stdl 270 μ 1 , the first antibody solution 1 μ 1 , mix and react at 37 ° C for 10 min to form a sandwich complex, scan the absorption spectrum of the mixed solution at 750-900 nm, and take The maximum value is A1;

Transfer the mixed solution to the well of the microplate, react at 37 ° C for 15 min, scan the absorption spectrum of the mixed solution at 750-900 nm, and take the maximum value of A2;

Find the value of A1/A2.

Repeat the above procedure with the standard solutions Std2 to Std5 to obtain other points in the AFP standard system.

The ratio of Ail/Ai2 (i = 2 - 5) is shown in Table 2.

Table 2: AFP Standard System Test Results

The results of the above table are plotted and the standard curve is obtained (Fig. 4).

4, sample testing

Replace the standard series of solutions with 6 unknown serum samples, repeat the steps of 3.2, substitute A1/A2 into the standard curve, and measure the AFP values of the six samples: 14.2, 56.9, 31.6, 82.8, 123.3, 351.6ng/ml .

Example 3: Simultaneous detection of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) in serum

1, raw materials The paired antibodies A1/C9 against CEA were purchased from Shanghai Second Medical University;

Str printed tavidin coated ELISA plate, commercially available;

A nanometer gold rod having a horizontal axis of 25 nm and a vertical axis of 47 nm, an aspect ratio of 1.88, a commercially available product, a maximum absorption peak of -600 nm, hereinafter referred to as 1. 88 Nanorod;

The nanometer gold rod with a horizontal axis of 10 nm and a vertical axis of 41 nm has an aspect ratio of 4. 1, a commercial product, and a maximum absorption peak of -800, hereinafter referred to as 4. INanorod;

Detector: USB4000-FL (US Ocean Optics)

Tungsten halogen light source, wavelength 350~2500 let

2, antibody labeling

2. 1 1. Preparation of 88 Nanorod Marker A1 Antibody (Second Antibody)

</ br> </ br> </ br> </ br> N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N Surface

The sample is dissolved in an appropriate amount of the preservation solution (prepared in the same manner as in the previous example), and filtered with a 0.45 μππ filter to give an absorbance at -630 nm of -1. 0, 1.88 Nanorod-labeled A1 antibody solution per ml. Approx. 10 μg of A1 antibody, 1.88Nanorod-Al o

2. 2 4. Preparation of INanorod marker G4 antibody (second antibody)

The precipitate was dissolved in an appropriate amount of the preservation solution (prepared in the same manner as in the previous example), and filtered with a 0.54 μm filter to make it

The absorbance at -830 nm is -1. 0, and the G4 antibody solution labeled per nanometer gold rod is equivalent to about 10 μg of G4 antibody, resulting in 4. INanorod-G4.

2. 3 Biot in tag C9 antibody (primary antibody)

2. 3. 1 C9 antibody pretreatment

The antibody is thoroughly dialyzed with pure sodium such as sodium azide or glycine.

Small molecules of amino groups and other small molecules The antibody contains a large Protein A column such as bovine serum albumin or other column purification.

Molecule

0.7 mg/ml monoclonal antibody). The concentration was finally set at 2 mg/ml with 1X PBS, pH 7.4 (concentrated using a Pall desalting spin column).

2.3.2 Biotin Labeling C9 Antibody

Take the above pretreated C9 antibody, add 25 μL of 1 mg/ml NHSS-Biotin DMS0 solution, mix well, avoid the light reaction in the refrigerator at 4 °C for 2 hours, and dialyze overnight for use to obtain Biotin-C9 with a final concentration of 550 μg. /ml.

2.3.3 Biotin Labeling C2 Antibody (First Antibody)

In the same manner as above, Biotin-C2 was obtained at a final concentration of 650 μg/ml.

3. Drawing of standard curve

3.1 Preparation of CEA and AFP standard solutions

A mixed standard series of CEA and AFP antigens was prepared in PBS (pH 7.4) at concentrations of 0, 0 (Stdl), 3.5, 10.33 (Std2), 21.56, 38.65 (Std3), 115.40, 145·60 (Std4), 350, 400.0 (Std5) ng / ml _;

3.2 Formation of sandwich composites and absorbance measurement

Take 1 1.8Nanorod-A1 and 4. lNanorod-G4 solution for each 15 μl, standard solution Stdl 270 μ1, Biotin-C9 and Biotin-C2 for 1 μl each, mix and react at 37 °C for 10 min to form a sandwich. Composite, scanning the absorption solution at 500-1000 nm absorption spectrum, recording the absorbance value A at the maximum absorption peak of 1.88 Nanorod-Al, 4. The absorbance value Al ₂ at the maximum absorption peak of Nanorod-G4 _;

Transfer the mixed solution to the well of the microplate, react at 37 ° C for 15 min, scan the absorption spectrum of the mixed solution at 500-1000 nm, and record the absorbance value A2 at the maximum absorption peak of 1.88 Nanorod-Al. 4. Absorbance at the maximum absorption peak of INanorocK Value A2 _2;

Find the value of A / kk /A2 ₂ respectively.

In the same operation, the corresponding ratios of other points of the CEA and AFP mixed antigen standard lines were obtained, and the results are shown in Table 3.

Table 3: CEA/AFP antigen hybrid standard system 1

Stdl Std2 Std3 Std4 Std5

CEA: k\J A2i 1 1.05 1.26 1.60 1.82

AFP: Al ₂ /A2 ₂ 1.02 1.12 1.33 1.55 1.76 The results of the above table are plotted, and the standard curves of CEA and AFP are obtained respectively (Figures 5 and 6).

4, sample testing

Replace the standard series of solutions with 7 unknown serum samples, repeat the steps of 3.2, and compare the AL/ Α2^Π Al ₂ /A2 ₂ with the standard curves of Figures 5 and 6, respectively, and measure the CEA of 9 samples. The AFP values are shown in Table 4.

Example 4: Simultaneous detection of carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and total prostate specific antigen (PSA) in serum

1, raw materials

The first antibody and the second antibody against PSA were purchased from Bi odes i gn Inc., US;

PSA antigen was purchased from China National Institute for the Control of Pharmaceutical and Biological Products;

Str printed tav i di n coated ELISA plate, commercially available;

The nanometer gold rod with a horizontal axis of 25 nm and a vertical axis of 47 nm has an aspect ratio of 1.88, a commercially available product, and a maximum absorption peak of -600 nm, hereinafter referred to as 1.88Nanorod;

The nanometer gold rod having a horizontal axis of 25 nm and a vertical axis of 73 nm, an aspect ratio of 2.92, a commercially available product, a maximum absorption peak of -700 nm, hereinafter referred to as 2.92 Nanorod _; The nanometer gold rod having a horizontal axis of 10 nm and a vertical axis of 41 nm has an aspect ratio of 4.1, and a commercially available product, a maximum absorption peak of -800, hereinafter referred to as 4. INanorod;

Detector: USB4000-FL (US Ocean Optics)

Tungsten halogen light source, wavelength 350~2000 let.

2, antibody labeling

Continue to use 1.88Nanorod-Al and 4. INanorod-G4 for CEA and AFP in "Example 3".

2. 1 2.92 Nanorod marker PSA secondary antibody

The marking method is the same as in Example 3. The absorbance at -730 nm was -1.0, and the Nanorod-labeled second antibody solution per ml was equivalent to about 10 g of G4 antibody, giving 2.92 Nanorod-PSA.

2.3 Biotin labeled primary antibody

Continue to use Biotin-C9 and Biotin-C2 for CEA and AFP in "Example 3". 2.3. 1 Biotin label PSA (primary antibody)

The labeling method was the same as in Example 3, and Biotin-PSA was obtained at a final concentration of 500 μg/ml.

3. Drawing of standard curve

3. 1 Preparation of CEA, AFP, PSA standard solutions

A mixed standard series of CEA, AFP, and PSA antigens was prepared in PBS (pH 7.4) at concentrations of 0, 0, 0 (Stdl), 3.0, 9.5, 1.1 (Std2), 24.6, 37.55, 4· 5 (Std3) , 110.60, 148.60, 18.5 (Std4), 350, 400.0, 50 (Std5) ng / ml _;

3.2 Formation of sandwich composites and absorbance measurement

Take 1.18Nanorod-Al, 4. INanorod-G4 and 2.92 Nanorod- PSA solution for each 15 μl, standard solution Stdl 255 μ1, Biotin-C9, Biotin-C2 and Biotin-PSA for 1 μl each. The reaction was carried out at 37 ° C for 10 min to form a sandwich complex. The absorption spectrum of the mixed solution was measured at 500-1000 nm, and the absorbance value at the maximum absorption peak of 1.88 Nanorod-Al was recorded. Al^ 4. The absorbance value at the maximum absorption peak of Nanorod-G4 was Al. ₂ , 2.92 Nanorod-PSA absorbance value Al ₃ at the maximum absorption peak _;

Transfer the mixed solution to the well of the ELISA plate, react at 37 ° C for 15 min, scan the absorption spectrum of the mixed solution at 500-1000 nm, and record the absorbance value A2 at the maximum absorption peak of 1.88 Nanorod-Al. 4. Absorbance at the maximum absorption peak of INanorocK The absorbance value A2 ₃ at the maximum absorption peak of the value A2 ₂ , 2.92 Nanorod-PSA _{; the} values of A / 2 Al ₂ /A2 ₂ and Al ₃ / A2 ₃ were respectively obtained.

In the same operation, the corresponding ratios of other points of the CEA, AFP, and PSA mixed antigen standard lines were obtained, and the results are shown in Table 3. Table 3: CEA, AFP, PSA mixed antigen standard test results

The results of the above table are plotted to obtain the standard curves of CEA, AFP and PSA (Figures 7, 8, 9).

4, sample testing

Replace the standard series of solutions with 8 unknown serum samples, repeat the steps of 3.2, and substitute Al ASi , Al ₂ /A2 ₂ and Al ₃ /A2 ₃ into the standard curves of Figures 7, 8, and 9, respectively. The CEA, AFP, and PSA values of the samples are shown in Table 5.

Table 5: Simultaneous test results of CEA, AFP and PSA in the sample

The results of Examples 3 and 4 show that the method of the present invention not only has a uniform and rapid reaction, requires no washing in the whole process, is easy to operate, and is very suitable for multi-indicator joint inspection, and can obtain accurate results, while greatly reducing cost and saving time. Example 5

Examples 2 and 3 were repeated except that the concentration of AFP in the sample was measured by the single index (AFP) assay of Example 2 for 4 serum samples of unknown concentration, and the double index (CEA of Example 3) was used. /AFP) Detection method to detect the concentration of AFP in the sample. Each sample was repeated 3 times and averaged. The results showed that: compared with the concentration of AFP measured by single-index detection method, the concentration of AFP measured by double-index detection method was basically the same, and the ratio (AFP concentration/double index detection method measured by single-index detection method) The measured AFP concentration is between 0.90 and 1.11.

This indicates that the multi-index joint detection method of the present invention has high accuracy. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

Rights request

An immunoassay method for detecting the presence or absence and/or quantity of a test object, comprising the steps of:

(c) irradiating the mixed solution of the step (b) with light, allowing the light to pass through the mixed solution, and measuring the transmitted light to obtain the absorbance of the mixed solution, which is recorded as the first absorbance A1;

(f) comparing the first absorbance A1 with the second absorbance A2 to determine the presence or absence of the analyte and

/ or quantity.

2. The method according to claim 1, wherein the analyte comprises: a protein, a nucleic acid.

3. The method according to claim 1, wherein the light absorbing substance refers to a substance having a molar absorption coefficient ε > 10 ⁸ L · mol - ¹ · cm ^-1 .

4. The method of claim 1 wherein the light absorbing material is selected from the group consisting of colloidal gold, nanogold rods, nanosilver rods, or combinations thereof.

5. The method of claim 1 wherein: said first conjugate is labeled with a capture agent; more preferably, said capture agent comprises: biotin, magnetic microspheres, or a combination.

6. The method according to claim 1, further comprising the step of: (f) further comprising: comparing a ratio of the first absorbance A1 to the second absorbance A2 to a standard curve or a comparison to determine the object to be tested Quantity.

7. The method according to claim 1, wherein the analyte is an antigen, and the first binder and the second binder are antibodies that bind to the antigen simultaneously; or

The test substance is an antibody, and the first conjugate and the second conjugate are antibodies (anti-antibodies) that can bind to the antigen of the antibody or the antibody at the same time.

8. The method according to claim 1, wherein in the step (e), the light path transmitted through the mixed solution has an optical path of 0.1 to 200 cm.

9. The method of claim 1 wherein said analyte comprises 2-10 different substances, and said detection mixture contains a first combination of said different analytes and Second conjugate.

An immunoassay detecting apparatus for detecting the presence or absence and/or quantity of a test object according to the method of claim 1, wherein the apparatus comprises:

(b) a detector for detecting absorbance; and

(c) A description of the use of the method of claim 1.