CN116953225A - Preparation technology and detection method of homogeneous digital immunoassay sample under bright field - Google Patents
Preparation technology and detection method of homogeneous digital immunoassay sample under bright field Download PDFInfo
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- CN116953225A CN116953225A CN202310761064.2A CN202310761064A CN116953225A CN 116953225 A CN116953225 A CN 116953225A CN 202310761064 A CN202310761064 A CN 202310761064A CN 116953225 A CN116953225 A CN 116953225A
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
Abstract
The application belongs to the technical field of immunoassay, and discloses a homogeneous digital immunoassay sample under a bright field, a preparation technology and a detection method thereof. The preparation technology comprises the following steps: (1) Fixing the capture antibody on an activation site on the surface of a microsphere a, wherein the obtained microsphere a with the surface bound with the capture antibody is the capture microsphere; (2) Fixing the detection antibody on the activation site on the surface of the microsphere b, and obtaining the microsphere b with the surface bound with the detection antibody after sealing, namely the detection microsphere; (3) Adding the capture microsphere and the detection microsphere into the solution containing the marker, mixing and fully incubating to obtain a digital immunoassay sample containing the complex of the capture microsphere n-detection microsphere m. The method provided by the application has the advantages that the marker, the capture microsphere and the detection microsphere are mixed for incubation, steps such as cleaning, separation, enrichment and the like are not needed after incubation is finished, the method can be used for direct detection, the advantage of digital immunity is achieved, the steps of an immunoassay method are simplified, and the complexity and the cost are reduced.
Description
Technical Field
The application relates to the technical field of immunoassay, in particular to a digital immunoassay technology, and more particularly relates to a preparation technology and a detection method of a homogeneous digital immunoassay sample under a bright field.
Background
The immune analysis utilizes the specific reaction between antigen and antibody to identify the molecule to be detected, and detects the antibody, antigen, immune cell, etc. in serum or other sample to diagnose or determine the existence of biological molecule, so that it has wide application in clinical medicine, life science research, etc. This technique was first proposed by us scientists Yalow and Berson, and in 1977 Yalow was therefore awarded the nobel medical prize. Digital immunoassays are the more rapidly developing immunoassays in the last decade, and the main difference from the conventional immunoassays is the detection mode of signals. The signal detection of the traditional immunoassay method is to quantify the total signal intensity (such as light scattering intensity, light intensity of a specific wave band, etc.) of immune complex formed by immune reaction, and the signal intensity is continuously changed along with the content of the marker, so that the signal can be considered to be a continuous signal or an analog signal, while the digital immunoassay is to count immune complex formed by immune reaction one by one, and finally the obtained signal is a digital signal.
Rissin et al in 2010 proposed a digital ELISA method (Rissin et al, single-molecular enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentration, nature Biotechnology,28:595-599,2010) that dispersed immune complexes formed based on ELISA reactions into a 4.5 μm diameter, 3.25 μm deep array of microwells, and then detected "signaled" microwells using a fluorescence microscopy detection device. The Gai et al proposed a digital immunoassay protocol in the open field (Gai et al Nonstochastic protein counting analysis for precision biomarker detection: suppressing poisson noise at ultralow concentration. Analytical Chemistry,92:654-658, 2020) using 4.5 μm polystyrene microspheres (4.5 μm microspheres are clearly distinguishable under open field microscopy) as carriers for detection of antibodies, and after formation of a diabody sandwich immune complex, the polystyrene microspheres were individually counted by open field microscopy by washing, eluting, concentrating, enriching, etc.
The digital immunoassay method needs optical detection equipment (such as the method of Rissin et al) or more complex post-processing steps (such as the scheme of Gai et al) provided with a fluorescence detection module, so that the detection equipment has complex structure, higher cost and longer detection time, and is not beneficial to popularization of an automatic detection instrument.
Disclosure of Invention
The application aims to overcome the defects of the background technology and provide a preparation technology and a detection method of a homogeneous digital immunoassay sample in a bright field. The method can simplify the steps of the digital immunoassay method, and further reduce the complexity and cost of automatic detection equipment. Specifically, the method of the application mixes and incubates the marker, the capture microsphere and the detection microsphere together, and the steps of cleaning, separation, enrichment and the like are not needed after the incubation is finished, so that the method can directly detect the marker, belongs to a one-step immunoassay or a homogeneous immunoassay, and the immune complex formed by immune reaction is counted one by one, and finally the obtained signal is a digital signal, thus having the advantage of digital immunity.
In order to achieve the purpose of the application, the application provides a homogeneous digital immunoassay sample under a bright field, and the preparation technology of the homogeneous digital immunoassay sample comprises the following steps:
(1) Preparing capture microspheres: fixing the capture antibody on an activation site on the surface of a microsphere a, wherein the obtained microsphere a with the surface bound with the capture antibody is the capture microsphere;
(2) Preparing detection microspheres: fixing the detection antibody on the activation site on the surface of the microsphere b, and obtaining the microsphere b with the surface bound with the detection antibody after sealing, namely the detection microsphere;
(3) Preparing a complex of a capturing microsphere n-detecting microsphere m: adding the capture microsphere and the detection microsphere into the solution containing the marker, mixing and fully incubating to obtain a digital immunoassay sample containing the complex of the capture microsphere n-detection microsphere m;
wherein the complex of the capture microsphere n-detection microsphere m is a complex formed by the capture microsphere, the marker and the detection microsphere through immune reaction, n refers to the number of the capture microsphere in the complex, and m refers to the number of the detection microsphere in the complex.
Further, the specific method for preparing the capture microsphere in the step (1) comprises the following steps: mixing the microsphere a with an activator for reacting for a period of time, and washing off unreacted redundant activator adhered to the microsphere a by using a washing buffer solution after the surface of the microsphere a is activated; adding a certain amount of sample of capture antibody into a container where the activated microsphere a is located, and after full incubation, binding part of capture antibody to the surface of the microsphere a; washing off the excessive unbound capture antibody by using a washing buffer solution, and collecting the microspheres a with the capture antibody bound on the surface, namely the capture microspheres.
Further, the specific method for preparing the detection microsphere in the step (2) comprises the following steps: mixing microsphere b (with diameter different from microsphere a and distinguishable under microscopic equipment) with activator for reacting for a period of time, and washing off unreacted excessive activator adhered on microsphere b by using washing buffer solution after the surface of microsphere b is activated; adding a certain amount of sample of detection antibody into a container where the activated microsphere b is located, and after full incubation, binding part of detection antibody to the surface of the microsphere b; washing off the excessive unbound detection antibody by using a washing buffer solution, and collecting the microspheres b with the surfaces bound with the detection antibody after sealing to obtain the detection microspheres.
Further, the specific method for preparing the complex of the capturing microsphere n-detecting microsphere m in the step (3) is as follows: preparing a marker solution containing a certain concentration or taking a certain volume of marker solution containing a marker sample with unknown concentration, sequentially or simultaneously adding a certain amount of capture microspheres and detection microspheres into the marker solution, mixing, fully incubating, and binding two binding sites of the marker with the capture microspheres and the detection microspheres respectively to obtain a digital immunoassay sample containing a complex of the capture microspheres and the detection microspheres.
Further, in some embodiments of the present application, the microspheres a, b may be magnetic microspheres or non-magnetic microspheres; when the microsphere a and the microsphere b are non-magnetic microspheres, the materials of the microsphere a and the microsphere b include, but are not limited to, polystyrene, polyethylene, polypropylene, silicon dioxide and the like.
Further, in some embodiments of the application, the diameter of the microspheres a, b is in the range of 0.2 μm to 50 μm, preferably 1 μm to 5 μm, the diameter of the microspheres b being different from the diameter of the microspheres a and distinguishable under the microscopy apparatus.
Further, in some embodiments of the present application, the microsphere a, microsphere b carries functional groups on the surface, including but not limited to carboxyl, amino, etc.; the activator in the step (1) and the step (2) is selected according to the functional groups carried on the surfaces of the microsphere a and the microsphere b, including but not limited to EDC/NHS, glutaraldehyde and the like.
Further, in some embodiments of the present application, the ratio of the capture antibody/detection antibody to the amount of the microspheres a/b in the step (1) and the step (2) ranges from 10 to 100000, preferably from 100 to 10000;
further, in some embodiments of the present application, the incubation in step (1) and step (2) is sufficient to allow the reaction to proceed, and no specific conditions are required, and the incubation may be performed at 37 ℃ for 30 minutes or at 25 ℃ for 2 hours; preferably, proper shaking is carried out in the incubation process, so that collision among reactants is accelerated, and the incubation time is further shortened.
Further, in some embodiments of the present application, the step (3) of adding a certain amount of the capturing microsphere and the detecting microsphere sequentially or simultaneously means that the capturing microsphere, the marker and the detecting microsphere are sequentially combined according to any one of (a) to (c):
(a) The capture microsphere is added into the marker solution to be combined with the marker, then the detection microsphere is added, and the detection microsphere is combined with the marker;
(b) The detection microsphere is added into the marker solution to be combined with the marker, then the capture microsphere is added, and the capture microsphere is combined with the marker;
(c) The capture microspheres and the detection microspheres are added into the marker solution together to be combined with the markers.
Further, in some embodiments of the application, the ratio of the number of captured microspheres to the number of detected microspheres in step (3) is in the range of 0.1 to 10, preferably 0.5 to 2.
The method converts the marker into a double-antibody sandwich immune complex through immune reaction, and the double-antibody sandwich immune complex connects the capture microsphere and the detection microsphere. The capture microspheres and detection microspheres were chosen to be of different diameters and both exceeded 0.2 μm. And directly observing a sample after immune reaction through bright field microscopy equipment, recording a double-sphere compound formed by tightly adhering the capture microspheres and the detection microspheres as a signal value, and recording 0 as the rest of the separately dispersed capture microspheres, the detection microspheres, the compound formed by non-specific combination of the capture microspheres and the capture microspheres, the compound formed by non-specific combination of the detection microspheres and the like.
In another aspect, the present application also provides a method for digitally detecting a homogeneous digital immunoassay sample in the above-mentioned bright field, the method comprising: preparing a plurality of groups of digital immunoassay samples with different marker concentrations, wherein the digital immunoassay samples to be detected have unknown marker concentrations; transferring the sample to a detection area of microscopic equipment, scanning and imaging the sample in a bright field after the microspheres are precipitated to the bottom of the detection area, and storing picture data; recording the accumulated signal values of all the 'capture microsphere n-detection microsphere m' complexes in the picture data of each sample; establishing a standard curve according to the marker concentration and the recorded signal value; and determining the marker concentration according to the signal value of the digital immunoassay sample to be detected.
The capture microsphere and the detection microsphere can distinguish differences under microscopic equipment due to different diameters. n=0, i.e. the detection microsphere not bound to the capture microsphere, a single detection microsphere or a plurality of detection microspheres bound together by non-specificity are observed in the microscopy device; m=0, i.e., capture microspheres that are not bound to the detection microsphere, a single capture microsphere or a plurality of capture microspheres that are non-specifically bound together are observed in the microscopy device; none of the above is a target signal, and no counting is required. In the complex 'capture microsphere n-detection microsphere m' which needs to be counted, n is an integer more than or equal to 1, and m is also an integer more than or equal to 1.
Because the number of the capture antibodies coated on the surfaces of the capture microspheres is more than or equal to 1, 1 capture microsphere can be combined with more than 1 marker (the number of the markers is m, and m is more than or equal to 1), and the marker is further combined with the detection microsphere to form a capture microsphere-detection microsphere m complex, and the complex count is m.
Since the number of detection antibodies coated on the surface of the detection microsphere is not less than 1, 1 detection microsphere may be combined with more than 1 marker (the number of the markers is n, n is not less than 1), and the marker is further combined with the capture microsphere to form a complex of the capture microsphere n-detection microsphere, and the complex count is n.
And (3) observing and counting the number of the complex 'capture microsphere n-detection microsphere m' which needs to be counted in the single digital immunoassay sample, and summing the number of the complex 'capture microsphere n-detection microsphere m' which needs to be counted, so as to obtain a signal value of the sample, and S.
It should be noted that, due to the reaction kinetics between molecules, the reaction efficiency of the active groups is less than 100%, and the dead volume effect of transferring the sample to the detection area, etc., may result in a certain degree of loss of molecules and/or microspheres, and a standard curve needs to be established for calibration.
Advantages of the present application over conventional methods include, but are not limited to:
(1) The preparation of the homogeneous digital immunoassay sample reduces the dead volume effect caused by liquid transfer, and avoids the negative effects of molecular loss, microsphere loss and the like to a certain extent.
(2) According to the application, the target signal and the interfering substance are distinguished through the diameter of the microsphere, a separation step is not needed, the nonmagnetic microsphere can be used for capturing the microsphere and detecting the microsphere, the cost of the nonmagnetic microsphere made of polymer materials is lower, the density is low, and the immune complex can be formed by fully mixing and incubating.
(3) The application directly detects the signal of the digital immunoassay sample in the open field, the light path of the required microscopic equipment is simple, and a fluorescence detection module is not required to be added.
(4) The homogeneous digital immunoassay sample can be prepared by a one-step method (capturing microsphere and preparing immune microsphere in advance), so that the automatic process is simplified, the volume and cost of an automatic instrument are reduced, and the popularization is facilitated.
Drawings
FIG. 1 is a schematic illustration of a sample preparation and detection method of the present application;
FIG. 2 is a graph of sample concentration versus measured signal and linear fit results for example 1 of the present application;
FIG. 3 is a graph of sample concentration versus measured signal and linear fit results for example 2 of the present application.
Detailed Description
In order to make the objects, technical schemes and advantages of the present application more apparent, the present application will be further described in detail with reference to the accompanying drawings and examples, wherein the examples are respectively different systems, and homogeneous digital immunoassay samples are prepared and tested to verify that the present application has universality. Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application. It is to be understood that the following description is intended to be illustrative of the application and not restrictive.
The raw materials and equipment used in the present application are common raw materials and equipment used in the art and are commercially available products unless otherwise specified. The methods used in the present application are conventional in the art unless otherwise specified.
The terms "comprising," "including," "having," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.
The conjunction "consisting of …" excludes any unspecified element, step or component. If used in a claim, such phrase will cause the claim to be closed, such that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of …" appears in a clause of the claim body, rather than immediately following the subject, it is limited to only the elements described in that clause; other elements are not excluded from the stated claims as a whole.
When an equivalent, concentration, or other value or parameter is expressed as a range, preferred range, or a range bounded by a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when ranges of "1 to 5" are disclosed, the described ranges should be construed to include ranges of "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a numerical range is described herein, unless otherwise indicated, the range is intended to include its endpoints and all integers and fractions within the range.
The singular forms include plural referents unless the context clearly dictates otherwise. "optional" or "any" means that the subsequently described event or event may or may not occur, and that the description includes both cases where the event occurs and cases where the event does not.
Approximating language, in the specification and claims, may be applied to modify an amount that would not limit the application to the specific amount, but would include an acceptable portion that would be close to the amount without resulting in a change in the basic function involved. Accordingly, the modification of a numerical value with "about", "about" or the like means that the present application is not limited to the precise numerical value. In some examples, the approximating language may correspond to the precision of an instrument for measuring the value. In the description and claims of the application, the range limitations may be combined and/or interchanged, if not otherwise specified, including all the sub-ranges subsumed therein.
The indefinite articles "a" and "an" preceding an element or component of the application are not limited to the requirement (i.e. the number of occurrences) of the element or component. Thus, the use of "a" or "an" should be interpreted as including one or at least one, and the singular reference of an element or component includes the plural reference unless the amount clearly dictates otherwise.
Furthermore, the descriptions of the terms "one embodiment," "some embodiments," "examples," "particular examples," or "some examples," etc., described below mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms are not necessarily for the same embodiment or example. The technical features of the respective embodiments of the present application may be combined with each other as long as they do not collide with each other.
Example 1
The embodiment provides a preparation technology and a detection method of a homogeneous digital immunoassay sample using magnetic capture microspheres, nonmagnetic detection microspheres and novel crown N protein markers as main raw materials, and the operation flow is shown in figure 1, and is specifically as follows.
Step one, preparing capture microspheres:
(1) Cleaning: 60 mu L of JSR magnetic microsphere stock solution with diameter of 1.5 mu m and carboxyl connected to the surface is taken, wherein the solution contains about 5.76 multiplied by 10 8 Placing the magnetic microspheres in a 2mL centrifuge tube, adding 600 μl of PBST buffer (10 mM, pH=7.4, containing 0.05% Tween-20), repeatedly blowing for 10 times, removing supernatant with the aid of a magnetic rack, and repeatedly washing for 2 times according to the above operation;
(2) Activating: to the washed magnetic microspheres, 100. Mu.L of EDC solution (concentration: 1 mg/mL), 150. Mu.L of NHS solution (concentration: 1 mg/mL), 400. Mu.L of MES buffer (50 mM, pH=5.5) were added, and the mixture was homogenized by a vortex shaker for 30 seconds, and then the centrifuge tube was fixed on a rotary mixer, and the mixture was placed in an incubator at 37℃for 30 minutes; removing the supernatant with the aid of a magnetic frame, adding 600 mu L of PBST buffer solution (10 mM, pH=7.4, containing 0.05% Tween-20), repeatedly blowing for 10 times, removing the supernatant with the aid of the magnetic frame, and repeatedly washing for 2 times according to the operation to obtain the magnetic microsphere with the activated surface; activated magnetic microspheres were resuspended in 600 μl of PBS buffer (10 mm, ph=7.4);
(3) Coating: adding 7 mu L of a capture antibody of new crown N protein (Cov 19-MAb-15 antibody of Phpeng organism with the concentration of 4.38 mg/mL) into the activated magnetic microsphere, uniformly mixing for 30 seconds by using a vortex oscillator, fixing a centrifuge tube on a rotary mixer, and placing the mixture in a constant temperature oven at 37 ℃ for rotary incubation for 2 hours; removing the supernatant with the aid of a magnetic frame, adding 600 mu L of PBST buffer solution (10 mM, pH=7.4, containing 0.05% Tween-20), repeatedly blowing for 10 times, removing the supernatant with the aid of the magnetic frame, and repeatedly washing for 2 times according to the operation to obtain capture microspheres of the capture antibody coated on the surface; the capture microspheres were resuspended in 500 μl of PBS buffer (10 mm, ph=7.4) for use.
Step two, preparing detection microspheres:
(1) Cleaning: 400. Mu.L of a latex microsphere stock solution with a diameter of 3 μm and carboxyl groups attached to the surface was taken, wherein the solution contained about 3X 10 8 Placing the latex microspheres in a 2mL centrifuge tube, centrifuging at 12000g for 5 min, removing the supernatant, adding 600 μl of PBST buffer (10 mM, pH=7.4, containing 0.05% Tween-20), repeatedly blowing for 10 times, centrifuging at 12000g for 5 min, removing the supernatant, and repeatedly washing for 2 times according to the above operation;
(2) Activating: to the above washed latex microspheres, 100. Mu.L of EDC solution (concentration: 1 mg/mL), 150. Mu.L of NHS solution (concentration: 1 mg/mL), 400. Mu.L of MES buffer (50 mM, pH=5.5) were added, and the mixture was homogenized by a vortex shaker for 30 seconds, and then the centrifuge tube was fixed on a rotary mixer, and the mixture was placed in an incubator at 37℃for 30 minutes by rotary incubation; centrifuging at 12000g for 5 min, removing supernatant, adding 600 μl of PBST buffer (10 mM, pH=7.4, containing 0.05% Tween-20), repeatedly blowing for 10 times, centrifuging at 12000g for 5 min, removing supernatant, and repeatedly washing for 2 times to obtain surface activated latex microsphere; the activated latex microspheres were resuspended in 1000 μl of PBS buffer (10 mm, ph=7.4);
(3) Coating: adding 22 mu L of detection antibody of new crown N protein (Cov 19-MAb-17 antibody of Phpeng organism, concentration of 7.03 mg/mL) into the activated latex microsphere, uniformly mixing for 30 seconds by using a vortex oscillator, fixing a centrifuge tube on a rotary mixer, and placing the mixture in a constant temperature oven at 37 ℃ for rotary incubation for 2 hours; centrifuging at 6000g for 5 minutes, and removing the supernatant; 600 μl of PBST buffer (10 mM, pH=7.4, containing 0.05% Tween-20) was added, the mixture was repeatedly blown 10 times, centrifuged at 6000g for 5 minutes, and the supernatant was removed; repeating the washing for 2 times according to the operation;
(4) Closing: to the above latex microspheres coated with the detection antibody, 850 μl of PBS buffer (10 mm, ph=7.4) and 15 μl of 20% BSA were added, mixed with a vortex shaker for 30 seconds, then the centrifuge tube was fixed on a rotary mixer, and placed in an incubator at 37 ℃ for rotary incubation for 1 hour; centrifuging at 6000g for 5 minutes, and removing the supernatant; 600 μl of PBST buffer (10 mM, pH=7.4, containing 0.05% Tween-20) was added, the mixture was repeatedly blown 10 times, centrifuged at 6000g for 5 minutes, and the supernatant was removed; repeating the washing for 2 times according to the operation to obtain the detection microsphere with the surface coated with the detection antibody; the detection microspheres were resuspended in 860 μl of PBS buffer (10 mm, ph=7.4) for use.
Step three, preparing a digital immunoassay sample containing the gradient concentration marker:
(1) Gradient concentration marker configuration: the novel crown N protein stock with the concentration of 3.6mg/mL is diluted with PBS buffer (10 mM, pH=7.4) to a solution with the concentration of 100ng/mL, and the label is marked as S0; s is diluted into solutions of 10ng/mL, 1ng/mL, 100pg/mL, 10pg/mL and 0pg/mL in a gradient manner, the volumes of the solutions are 200 mu L, the labels are respectively marked as S1, S2, S3, S4 and S5, and 3 parts of each concentration solution is prepared;
(2) Incubation of immune microspheres: and (3) respectively taking 5 mu L of the capture microsphere prepared in the first step and 10 mu L of the detection microsphere prepared in the second step, adding into S1-S5, uniformly mixing for 30 seconds by using a vortex oscillator, fixing a centrifuge tube on a rotary mixer, and placing in a constant temperature oven at 37 ℃ for rotary incubation for 1 hour to obtain a digital immunoassay sample.
Fourth, bright field optical detection:
(1) And (3) tabletting: uniformly mixing the digital immunoassay sample prepared in the step three by using a vortex oscillator for 30 seconds, taking 10 mu L of the sample to be dripped on a glass slide, and covering the glass slide above the dripped sample;
(2) Data acquisition and statistics: transferring the sample subjected to film making to a slide placing area of an optical microscope, adjusting the focal length to clear images in the bright field, storing the pictures of the sample in the bright field by utilizing the photographing function of a CMOS camera, and recording the captured microspheres n -detecting microspheres m "number of complexes;
step five, establishing a standard curve:
the above S1 to S5 samples were measured and recorded, and the average value of 3 measurements was used as the final signal response value of the sample, and the Standard Deviation (SD) was calculated. Taking the concentration of the sample as the abscissa and the final signal response value of the sample as the ordinate, respectively taking Log values, drawing a scatter diagram, linearly fitting the scatter diagram to establish a standard curve, and calculating the fitting degree R 2 As shown in fig. 2. The limit of detection (LOD) was calculated by extrapolation and equals 3 times SD of the background signal.
Example 2
The embodiment provides a preparation and detection method of a homogeneous digital immunoassay sample using nonmagnetic capture microspheres, nonmagnetic detection microspheres and novel crown N protein markers as main raw materials, and the operation flow is shown in figure 1, and is specifically as follows.
Step one, preparing capture microspheres:
(1) Cleaning: 50 mu L of a stock solution of nano-micro latex microspheres with the diameter of 2 mu m and carboxyl groups connected to the surface is taken, wherein the stock solution approximately contains 6.05X10 8 Placing the magnetic microspheres in a 2mL centrifuge tube, adding 600 μl of PBST buffer (10 mM, pH=7.4, containing 0.05% Tween-20), repeatedly blowing for 10 times, centrifuging at 12000g for 5 min, removing supernatant, and repeatedly washing for 2 times according to the above operation;
(2) Activating: to the washed 2 μm latex microspheres, 100. Mu.L of EDC solution (1 mg/mL concentration), 150. Mu.L of NHS solution (1 mg/mL concentration), 400. Mu.L of MES buffer (50 mM, pH=5.5) were added, and the mixture was homogenized by a vortex mixer for 30 seconds, and then the centrifuge tube was fixed on a rotary mixer and placed in an incubator at 37℃for 30 minutes of rotary incubation; centrifuging at 12000g for 5 min, removing supernatant, adding 600 μl of PBST buffer (10 mM, pH=7.4, containing 0.05% Tween-20), repeatedly blowing for 10 times, centrifuging at 12000g for 5 min, removing supernatant, and repeatedly washing for 2 times to obtain 2 μm latex microsphere with activated surface; the activated 2 μm latex microspheres were resuspended in 600 μl of PBS buffer (10 mm, ph=7.4);
(3) Coating: adding 7 mu L of a capture antibody of new crown N protein (Cov 19-MAb-15 antibody of Phpeng organism, concentration of 4.38 mg/mL) into the activated 2 mu m latex microsphere, uniformly mixing for 30 seconds by using a vortex oscillator, fixing a centrifuge tube on a rotary mixer, and placing the mixture in a constant temperature oven at 37 ℃ for rotary incubation for 2 hours; centrifuging at 6000g for 5 min, removing supernatant, adding 600 μl of PBST buffer (10 mM, pH=7.4, containing 0.05% Tween-20), repeatedly blowing for 10 times, centrifuging at 6000g for 5 min, removing supernatant, and repeatedly washing for 2 times to obtain surface coated capture antibody capture microsphere; the capture microspheres were resuspended in 500 μl of PBS buffer (10 mm, ph=7.4) for use.
Step two, preparing detection microspheres:
(1) Cleaning: 400 mu L of emulsion microsphere stock solution with diameter of 3 mu m and carboxyl connected to the surface is taken, wherein the emulsion microsphere stock solution contains about 3 multiplied by 10 8 Placing the latex microspheres in a 2mL centrifuge tube, centrifuging at 12000g for 5 min, removing the supernatant, adding 600 μl of PBST buffer (10 mM, pH=7.4, containing 0.05% Tween-20), repeatedly blowing for 10 times, centrifuging at 12000g for 5 min, removing the supernatant, and repeatedly washing for 2 times according to the above operation;
(2) Activating: to the washed 3 μm latex microspheres, 100. Mu.L of EDC solution (1 mg/mL concentration), 150. Mu.L of NHS solution (1 mg/mL concentration), 400. Mu.L of MES buffer (50 mM, pH=5.5) were added, and the mixture was homogenized by a vortex mixer for 30 seconds, and then the centrifuge tube was fixed on a rotary mixer and placed in an incubator at 37℃for 30 minutes of rotary incubation; centrifuging at 12000g for 5 min, removing supernatant, adding 600 μl of PBST buffer (10 mM, pH=7.4, containing 0.05% Tween-20), repeatedly blowing for 10 times, centrifuging at 12000g for 5 min, removing supernatant, and repeatedly washing for 2 times to obtain 3 μm latex microsphere with activated surface; the activated latex microspheres were resuspended in 1000 μl of PBS buffer (10 mm, ph=7.4);
(3) Coating: adding 22 mu L of detection antibody of new crown N protein (Cov 19-MAb-17 antibody of Phpeng organism, concentration of 7.03 mg/mL) into the activated 3 mu m latex microsphere, uniformly mixing for 30 seconds by using a vortex oscillator, fixing a centrifuge tube on a rotary mixer, and placing in a constant temperature oven at 37 ℃ for rotary incubation for 2 hours; centrifuging at 6000g for 5 minutes, and removing the supernatant; 600 μl of PBST buffer (10 mM, pH=7.4, containing 0.05% Tween-20) was added, the mixture was repeatedly blown 10 times, centrifuged at 6000g for 5 minutes, and the supernatant was removed; repeating the washing for 2 times according to the operation;
(4) Closing: to the above 3 μm latex microspheres coated with the detection antibody, 850. Mu.L of PBS buffer (10 mM, pH=7.4) and 15. Mu.L of 20% BSA were added, mixed with a vortex shaker for 30 seconds, and then the centrifuge tube was fixed on a rotary mixer, and placed in an incubator at 37℃for 1 hour under rotary incubation; centrifuging at 6000g for 5 minutes, and removing the supernatant; 600 μl of PBST buffer (10 mM, pH=7.4, containing 0.05% Tween-20) was added, the mixture was repeatedly blown 10 times, centrifuged at 6000g for 5 minutes, and the supernatant was removed; repeating the washing for 2 times according to the operation to obtain the detection microsphere with the surface coated with the detection antibody; the detection microspheres were resuspended in 860 μl of PBS buffer (10 mm, ph=7.4) for use.
Step three, preparing a digital immunoassay sample containing the gradient concentration marker:
(1) Gradient concentration marker configuration: the novel crown N protein stock with the concentration of 3.6mg/mL is diluted with PBS buffer (10 mM, pH=7.4) to a solution with the concentration of 100ng/mL, and the label is marked as S0; s is diluted into solutions of 10ng/mL, 1ng/mL, 100pg/mL, 10pg/mL and 0pg/mL in a gradient manner, the volumes of the solutions are 200 mu L, the labels are respectively marked as S1, S2, S3, S4 and S5, and 3 parts of each concentration solution is prepared;
(2) Incubation of immune microspheres: and (3) respectively taking 5 mu L of the capture microsphere prepared in the first step and 10 mu L of the detection microsphere prepared in the second step, adding into S1-S5, uniformly mixing for 30 seconds by using a vortex oscillator, fixing a centrifuge tube on a rotary mixer, and placing in a constant temperature oven at 37 ℃ for rotary incubation for 1 hour to obtain a digital immunoassay sample.
Fourth, bright field optical detection:
(1) And (3) tabletting: uniformly mixing the digital immunoassay sample prepared in the step three by using a vortex oscillator for 30 seconds, taking 10 mu L of the sample to be dripped on a glass slide, and covering the glass slide above the dripped sample;
(2) Data acquisition and statistics: transferring the sample to slide placing region of optical microscope, adjusting focal length to clear image, storing image of sample under bright field by photographing function of CMOS camera, and recording (capturing microsphere) n -detecting microspheres m ) The number of complexes;
step five, establishing a standard curve:
the above S1 to S5 samples were measured and recorded, and the average value of 3 measurements was used as the final signal response value of the sample, and the Standard Deviation (SD) was calculated. Taking the concentration of the sample as the abscissa and the final signal response value of the sample as the ordinate, respectively taking Log values, drawing a scatter diagram, linearly fitting the scatter diagram to establish a standard curve, and calculating the fitting degree R 2 As shown in fig. 3. The limit of detection (LOD) was calculated by extrapolation and equals 3 times SD of the background signal.
It will be readily appreciated by those skilled in the art that the foregoing is merely illustrative of the present application and is not intended to limit the application, but any modifications, equivalents, improvements or the like which fall within the spirit and principles of the present application are intended to be included within the scope of the present application.
Claims (10)
1. The preparation technology of the homogeneous digital immunoassay sample in the bright field is characterized by comprising the following steps of:
(1) Preparing capture microspheres: fixing the capture antibody on an activation site on the surface of a microsphere a, wherein the obtained microsphere a with the surface bound with the capture antibody is the capture microsphere;
(2) Preparing detection microspheres: fixing the detection antibody on the activation site on the surface of the microsphere b, and obtaining the microsphere b with the surface bound with the detection antibody after sealing, namely the detection microsphere;
(3) Preparing a complex of a capturing microsphere n-detecting microsphere m: adding the capture microsphere and the detection microsphere into the solution containing the marker, mixing and fully incubating to obtain a digital immunoassay sample containing the complex of the capture microsphere n-detection microsphere m;
wherein the complex of the capturing microsphere n-detecting microsphere m is a complex formed by capturing microsphere, a marker and detecting microsphere through immune reaction, n refers to the number of capturing microsphere in the complex, m refers to the number of detecting microsphere in the complex, and m and n are integers more than or equal to 0.
2. The preparation technology of homogeneous digital immunoassay samples in the open field according to claim 1, wherein the specific method for preparing the capture microspheres in step (1) is as follows: mixing the microsphere a with an activator for reacting for a period of time, and washing off unreacted redundant activator adhered to the microsphere a by using a washing buffer solution after the surface of the microsphere a is activated; adding a certain amount of sample of capture antibody into a container where the activated microsphere a is located, and after full incubation, binding part of capture antibody to the surface of the microsphere a; washing off the excessive unbound capture antibody by using a washing buffer solution, and collecting the microspheres a with the capture antibody bound on the surface, namely the capture microspheres.
3. The preparation technology of homogeneous digital immunoassay samples in the open field according to claim 1, wherein the specific method for preparing the detection microsphere in the step (2) is as follows: mixing the microsphere b with an activator for reacting for a period of time, and washing off unreacted redundant activator adhered to the microsphere b by using a washing buffer solution after the surface of the microsphere b is activated; adding a certain amount of sample of detection antibody into a container where the activated microsphere b is located, and after full incubation, binding part of detection antibody to the surface of the microsphere b; washing off the excessive unbound detection antibody by using a washing buffer solution, and collecting the microspheres b with the surfaces bound with the detection antibody after sealing to obtain the detection microspheres.
4. The preparation technology of homogeneous digital immunoassay samples in the open field according to claim 1, wherein the specific method for preparing the "capture microsphere n-detection microsphere m" complex in step (3) is as follows: preparing a marker solution containing a certain concentration or taking a certain volume of marker solution containing a marker sample with unknown concentration, sequentially or simultaneously adding a certain amount of capture microspheres and detection microspheres into the marker solution, mixing, fully incubating, and binding two binding sites of the marker with the capture microspheres and the detection microspheres respectively to obtain a digital immunoassay sample containing a complex of the capture microspheres and the detection microspheres.
5. The technique for preparing a homogeneous digital immunoassay sample under an open field according to claim 1, wherein the microspheres a, b are magnetic or non-magnetic microspheres; when the microsphere a and the microsphere b are nonmagnetic microspheres, the microsphere a and the microsphere b are made of polystyrene, polyethylene, polypropylene or silicon dioxide; preferably, the diameter of the microspheres a, b is in the range of 0.2 μm to 50 μm, preferably 1 μm to 5 μm, the diameter of the microspheres b being different from the diameter of the microspheres a and distinguishable under the microscopy apparatus.
6. The preparation technology of homogeneous digital immunoassay samples in the open field according to claim 1, wherein the surfaces of the microspheres a and b carry functional groups, and the activating agents in the step (1) and the step (2) are selected according to the functional groups carried on the surfaces of the microspheres a and b; preferably, the functional group is carboxyl or amino.
7. The technique for preparing a homogeneous digital immunoassay sample under an open field according to claim 1, wherein the ratio of the capture antibody/detection antibody to the amount of microspheres a/b in the step (1) and the step (2) is in the range of 10 to 100000, preferably 100 to 10000; preferably, the incubation in the step (1) and the step (2) is sufficient for the reaction to be sufficient, and no specific condition is required to be referred to; preferably, appropriate shaking is performed in the incubation process, so that collision between reactants is accelerated, and incubation time is further shortened.
8. The technique for preparing a homogeneous digital immunoassay sample in the open field according to claim 1, wherein, in the step (3), a certain amount of capture microspheres and detection microspheres are added sequentially or simultaneously, wherein the capture microspheres, the markers and the detection microspheres are combined in any order of (a) - (c):
(a) The capture microsphere is added into the marker solution to be combined with the marker, then the detection microsphere is added, and the detection microsphere is combined with the marker;
(b) The detection microsphere is added into the marker solution to be combined with the marker, then the capture microsphere is added, and the capture microsphere is combined with the marker;
(c) The capture microspheres and the detection microspheres are added into the marker solution together to be combined with the markers.
9. The method for preparing a homogeneous digital immunoassay sample in the open field according to claim 1, wherein the ratio of the number of captured microspheres to the number of detected microspheres in step (3) is in the range of 0.1 to 10, preferably 0.5 to 2.
10. A method for the digital detection of a homogeneous digital immunoassay sample in the presence of a bright field obtained by a preparation technique according to any one of claims 1 to 9, characterized in that it comprises: preparing a plurality of groups of digital immunoassay samples with different marker concentrations, wherein the digital immunoassay samples to be detected have unknown marker concentrations; transferring the sample to a detection area of microscopic equipment, scanning and imaging the sample in a bright field after the microspheres are precipitated to the bottom of the detection area, and storing picture data; recording the accumulated signal values of all the 'capture microsphere n-detection microsphere m' complexes in the picture data of each sample; establishing a standard curve according to the marker concentration and the recorded signal value; and determining the marker concentration according to the signal value of the digital immunoassay sample to be detected.
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