CN111551730A - Fluorescent microsphere sealing liquid and kit using same - Google Patents

Abstract

The invention provides fluorescent microsphere sealing liquid and a kit using the same. Specifically, the pH value of the fluorescent microsphere sealing liquid provided by the invention is 6.5-8.5; and the solvent is water, and the confining liquid comprises: 1 to 100mM buffer reagent, 5 to 30mM ethanolamine, 5 to 50g/L sodium caseinate, 0.1 to 5g/L nonionic surfactant, and 0.1 to 10g/L preservative.

Description

Fluorescent microsphere sealing liquid and kit using same

Technical Field

The invention belongs to the field of immunodetection, and particularly relates to fluorescent microsphere confining liquid and a kit using the same.

Background

Myoglobin (myoglobin, MYO) is a hemoglobin, which was first found in striated muscle cells (skeletal and cardiac), and binds oxygen in a reversible manner to enhance oxygen transport in mitochondria, playing an important role in the aerobic metabolism of cells. When myocardial cells die (e.g., a myocardial infarction occurs), enzymes and other proteins, such as myoglobin, are released into the peripheral blood circulation due to damage to the cell membrane and can be detected in the blood. After about 2-3 hours of symptoms, the myoglobin in blood can exceed the upper limit of normal, reach a peak value in 9-12 hours, and return to normal after 24-36 hours.

Myoglobin is a sensitive predictor of Acute Myocardial Infarction (AMI), but it is not specific to the myocardium. It is clear that myoglobin is a very good negative predictive marker, and any suspected AMI can be ruled out after two negative myoglobin tests are consecutively performed, and elevated myoglobin is also seen in the following syndromes: severe shock, open trauma, severe poisoning, end stage renal failure, myocarditis, acute infectious diseases, and the like.

The fluorescence immunochromatography technology is a novel membrane detection technology based on antigen-antibody specific immunoreaction. The technology takes strip fiber chromatography materials fixed with a detection line (coated antibody or coated antigen) and a quality control line as a stationary phase, a test solution as a mobile phase, a fluorescence labeled antibody or antigen (such as a conjugate formed by the antibody or the antigen and fluorescent microspheres) is fixed on a binding pad or a pre-sample cup, and an analyte moves on the chromatography strip through capillary action. The fluorescence immunochromatographic assay method has the advantages of high sensitivity, good stability, low interference of natural fluorescence and the like, and at present, the markers for fluorescence immunoassay mainly comprise fluorescein (fluorescent microspheres), quantum dots, up-conversion nanoparticles and the like.

However, when the sealing liquid is not suitable when the fluorescent microspheres mark the antibody, the non-specific adsorption is obviously higher in background signal or the signal is lower during detection, so that the detection sensitivity is lower; meanwhile, the marked fluorescent microspheres are easy to agglomerate and unstable, and the signal fluctuation is obvious during detection, so that the detection accuracy, stability and the like are influenced.

In summary, there is an urgent need in the art to develop a novel blocking solution with high blocking efficiency, improved detection sensitivity, and good stability-assisting effect.

Disclosure of Invention

The invention aims to provide a novel confining liquid which has high confining efficiency, can improve the detection sensitivity and has good stabilizing effect.

In a first aspect of the invention, a fluorescent microsphere sealing solution is provided,

the pH value of the sealing liquid is 6.5-8.5;

the solvent of the confining liquid is water, and

the confining liquid comprises: 1 to 100mM buffer reagent, 5 to 30mM ethanolamine, 5 to 50g/L sodium caseinate, 0.1 to 5g/L nonionic surfactant and 0.1 to 10g/L preservative.

In another preferred embodiment, the fluorescent microspheres refer to fluorescent microspheres with carboxylated surfaces.

In another preferred embodiment, the fluorescent microsphere is a fluorescent microsphere coupled with an antibody or an antigen through-COOH.

In another preferred example, the concentration of the buffer reagent in the confining liquid is 5-50 mM buffer reagent; preferably, it is 10 to 40 mM; more preferably, it is 10 to 20 mM.

In another preferred example, the concentration of ethanolamine in the confining liquid is 5-20 mM; preferably, it is 8 to 15 mM; more preferably, 8 to 12 mM; optimally, 10 ± 1 mM; and/or

In another preferred example, the concentration of the casein sodium in the sealing liquid is 5-30 g/L; preferably, the concentration is 5 to 20 g/L; more preferably, 7 to 15 g/L; most preferably, 10. + -.1 g/L.

In another preferred example, the concentration of the nonionic surfactant in the sealing liquid is 0.3-3 g/L; preferably, it is 0.2-1 g/L; more preferably, 0.3 to 0.7 g/L; most preferably, 0.5. + -. 0.1 g/L.

In another preferred example, the concentration of the corrosion inhibitor in the sealing liquid is 0.5-5 g/L; preferably, it is 0.5-2 g/L; more preferably, 1. + -. 0.2 g/L.

In another preferred embodiment, the surfactant is selected from the group consisting of: tween 20.

In another preferred embodiment, the preservative is selected from the group consisting of: sodium azide, Proclin 300, or a combination thereof.

In another preferred example, the pH value of the sealing liquid is 7-8; preferably, the pH is 7.5-8; more preferably, the pH is 7.8. + -. 0.1.

In another preferred embodiment, the buffer reagent is used to form a buffer system.

In another preferred embodiment, the pH buffering agent is a PB buffering agent.

In another preferred embodiment, the PB buffering reagent consists of sodium dihydrogen phosphate and disodium hydrogen phosphate.

In another preferred embodiment, the blocking solution further optionally comprises a pH regulator.

In another preferred embodiment, the pH regulator is NaOH or HCl.

In a second aspect of the present invention, there is provided a method for preparing a sealing liquid as described in the first aspect, comprising the steps of:

(1) dissolving a buffer reagent in water to obtain a buffer solution;

(2) mixing ethanolamine, sodium caseinate, a non-ionic surfactant and a preservative with the buffer solution obtained in the step (1); and

(3) adding water to a constant volume to obtain the confining liquid as described in the first aspect.

In another preferred embodiment, step (3) optionally further comprises the step of adding a pH regulator to adjust pH before adding water.

In a third aspect of the invention, there is provided a blocked fluorescent microsphere conjugate,

the fluorescent microsphere conjugate subjected to blocking treatment is the fluorescent microsphere conjugate obtained by blocking treatment with the blocking solution according to the first aspect.

In another preferred embodiment, the fluorescent microsphere conjugate is a conjugate formed by a fluorescent microsphere and a first antibody or a first antigen.

In another preferred embodiment, the first antibody or first antigen refers to an antibody or antigen capable of specifically binding to the marker to be detected.

In another preferred embodiment, the marker is a protein; more preferably, the marker is myoglobin.

In another preferred embodiment, the fluorescent microsphere conjugate is a first antibody-fluorescent microsphere conjugate; more preferably, it refers to a myoglobin monoclonal antibody (preferably murine myoglobin monoclonal antibody) -fluorescent microsphere conjugate.

In a fourth aspect of the present invention, there is provided a method for preparing a blocked fluorescent microsphere conjugate according to the third aspect, comprising the steps of:

(i) providing a fluorescent microsphere conjugate without blocking treatment; and (ii) blocking with a blocking solution as described in the first aspect, thereby obtaining a blocked fluorescent microsphere conjugate as described in the third aspect.

In another preferred example, the treatment time of the sealing treatment is 0.1-5 h; preferably, 0.5 to 2 hours.

In another preferred example, the treatment temperature of the sealing treatment is 0-50 ℃; preferably, 10 to 40 ℃; more preferably, 15 to 30 ℃.

In another preferred example, the processing temperature of the sealing process is room temperature.

In a fifth aspect of the present invention, there is provided a kit for fluorescence immunoassay, wherein,

(i) the kit comprises: a confining liquid as described in the first aspect; alternatively, the first and second electrodes may be,

(ii) the kit comprises: a blocked fluorescent microsphere conjugate of the third aspect; alternatively, the first and second electrodes may be,

(iii) the kit comprises: a container carrying the blocked fluorescent microsphere conjugate of the third aspect; or

(iv) The kit comprises: a fluorescent immunochromatographic reagent strip comprising a conjugate pad; wherein the conjugate pad carries a blocked fluorescent microsphere conjugate as described in the third aspect; or

(v) The kit comprises: buffer reagent, ethanolamine, sodium caseinate, nonionic surfactant, preservative and instructions; wherein the buffer, ethanolamine, sodium caseinate, non-ionic surfactant and preservative are as defined in the first aspect and said instructions describe the formulation of the sealant fluid as described in the first aspect.

In another preferred embodiment, the description also describes the production method according to the second aspect.

In another preferred embodiment, the kit is a kit for quantitatively determining a marker based on an immunofluorescence double antibody sandwich method.

In another preferred embodiment, the marker is myoglobin.

In a sixth aspect of the present invention, there is provided a method of detecting a marker in a sample, comprising the steps of:

(1) contacting the sample with the conjugate subjected to blocking treatment as described in the third aspect, or contacting the sample with a fluorescent microsphere conjugate obtained by blocking treatment with a blocking solution as described in the first aspect, wherein the fluorescent microsphere conjugate is as defined in the third aspect; thereby obtaining a sample possibly containing the compound of the marker and the fluorescent microsphere conjugate; and

(2) and (2) detecting the compound of the marker and the fluorescent microsphere conjugate in the sample obtained in the step (1).

In another preferred embodiment, the sample possibly containing the conjugate complexes of the marker and the fluorescent microspheres is passed through a fibrous membrane comprising a detection line and a quality control line, and the fluorescence signals on the detection line and the quality control line are obtained by an instrument, so as to detect (preferably quantitatively detect) the conjugate complexes of the marker and the fluorescent microspheres in the sample obtained in step (1).

In a seventh aspect of the present invention, there is provided a fluorescence immunochromatographic kit for detecting a marker, the kit comprising:

(a) a pre-loading cup loaded with the first antibody-fluorescent microsphere conjugate subjected to the blocking treatment or the first antigen-fluorescent microsphere conjugate subjected to the blocking treatment; wherein the first antibody fluorescent microsphere conjugate subjected to blocking treatment or the first antigen-fluorescent microsphere conjugate subjected to blocking treatment is the first antibody-fluorescent microsphere conjugate or the first antigen-fluorescent microsphere conjugate obtained by blocking treatment with the blocking solution of the first aspect; and

(b) a reagent strip, wherein the reagent strip comprises:

(b1) a fibrous membrane comprising a detection line and a quality control line; wherein the detection line is coated with a second antibody or a second antigen capable of specifically binding to the marker, and the quality control line is coated with a third antibody or a third antigen capable of capturing the first antibody-fluorescent microsphere conjugate or the first antigen-fluorescent microsphere conjugate;

(b2) a sample pad; and

(b3) an absorbent pad.

In another preferred embodiment, the first antibody and the second antibody are the same or different.

In another preferred embodiment, the marker is myoglobin.

In another preferred embodiment, the first antibody is a myoglobin monoclonal antibody; preferably, the monoclonal antibody is a myoglobin antibody of murine origin.

In another preferred embodiment, the second antibody is a myoglobin monoclonal antibody; preferably, the monoclonal antibody is a myoglobin antibody of murine origin.

In another preferred embodiment, the third antibody is a polyclonal antibody; preferably, the antibody is goat anti-mouse IgG polyclonal antibody.

In another preferred embodiment, the reagent strip further comprises a bottom plate.

In another preferred example, the bottom plate is provided with a sample pad, a nitrocellulose membrane and an absorbent pad in sequence along the chromatographic direction.

In another preferred example, the bottom plate is a PVC bottom plate.

In another preferred example, the fiber membrane is a nitrocellulose membrane.

In another preferred embodiment, the kit further comprises a reagent card.

In another preferred embodiment, the reagent card is used for fixing the pre-sample cup and the reagent strip.

In another preferred embodiment, the fixing is a detachable fixing or a non-detachable fixing.

In an eighth aspect of the present invention, there is provided a method of detecting a marker in a sample, comprising the steps of:

(1) providing a fluorescent immunochromatographic kit for detecting a marker as described in the seventh aspect;

(2) adding a sample to be detected into the pre-sample adding cup, and reacting a marker possibly contained in the sample with the first antibody-fluorescent microsphere conjugate or the first antigen-fluorescent microsphere conjugate carried by the pre-sample adding cup, so as to obtain a sample possibly containing a marker and fluorescent microsphere conjugate compound;

(3) adding the sample obtained in the step (2) to a sample pad in the reagent strip; and

(4) and reading the fluorescence signals of the detection line and the quality control line of the fiber membrane in the reagent strip so as to obtain the quantitative structure of the marker in the sample.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the structure of ethanolamine.

FIG. 2 shows a comparison of stability monitoring data for labeled particles made with preferred and control confining liquids over 60 days; wherein the dotted line represents the threshold limit for day zero signal values (1 ± 15%), the days on the abscissa represents the days of storage (days) of the prepared fluorescent microsphere conjugates, and the signal values refer to TAP.

FIG. 3 shows a comparison of the release and dispersibility of the marking particles produced with the preferred and control confining liquids. In FIG. 3, the ordinate represents the fluorescence intensity and the abscissa represents 180 points collected in the detection section. The abscissa 30-70 is the control line, and the abscissa 110-160 is the detection line.

Detailed Description

The inventors have conducted extensive and intensive studies. It has surprisingly been found that a blocking solution with appropriate concentrations of ethanolamine and sodium caseinate and a specific pH is particularly suitable for blocking labeled fluorescent microspheres or fluorescent latex microspheres (e.g. labeled with antibodies). The sealing liquid has high sealing efficiency, and the fluorescent microspheres sealed by the sealing liquid are not easy to agglomerate stably, the detection signals are stable, and the detection sensitivity is high. Based on this, the inventors have completed the present invention.

Term(s) for

As used herein, a "marker", which may also be referred to as a "biomarker", refers to a biochemical marker characteristic of a common physiological or pathological or therapeutic process that can be objectively measured and evaluated, and from which the progress of the biological process in which the body is currently located can be determined.

As used herein, the term "reagent strip" refers to a chromatographic reagent strip based on, for example, a double antibody sandwich assay for the detection of a marker. Generally, the reagent strip comprises: a sample pad for adding a sample or a specimen (the sample or specimen may be reacted or complexed with a fluorescent microsphere conjugate (such as an antibody-fluorescent microsphere conjugate) or not) sequentially arranged along a chromatography direction (i.e., a flow direction of the sample or a sample liquid in detection), and a fiber membrane (such as a nitrocellulose membrane) including a detection line and a quality control line. Wherein, the detection line can specifically capture the fluorescent microsphere conjugate compounded with the marker, and the quality control line can capture the fluorescent microsphere conjugate not compounded with the marker. When the sample is added or the sample is not complexed or reacted with the fluorescent microsphere conjugates, a conjugate pad carrying or immobilized the fluorescent microsphere conjugates (e.g., antibody-fluorescent microsphere conjugates) is also included between the sample pad and the fibrous membrane.

The terms "fluorescent microspheres" and "fluorescent latex microspheres" are used interchangeably herein.

As used herein, Tween 20(Tween 20) is a nonionic surfactant, has antigen repairing effect, can be adsorbed with fluorescent microsphere in hydrophobic manner, has strong elution effect on nonspecific adsorption, and can reduce the hydrophobic effect between proteins, thereby improving the recognition capability of specific antibody.

As used herein, the sample to be tested can be a treated sample or an untreated sample, so long as the sample meets the detection requirements when contacted with the blocked fluorescent microspheres. Preferably, the sample may be treated (e.g., filtered, diluted, etc.) or untreated blood, plasma, body fluid, etc.

As used herein, ethanolamine (the structure of which is shown below) and sodium caseinate can be bound to the binding sites (-COOH) of the antibody that is not cross-linked on the surface of the fluorescent microsphere, and also bound to the surface of the fluorescent microsphere in an adsorption-coated manner, thereby preventing non-specific binding of the murine myoglobin monoclonal antibody.

As used herein, BSA (bovine serum albumin) is a common blocker ingredient.

Fluorescent microsphere sealing liquid

In order to solve the problems existing in the prior art that (i) the detection sensitivity is reduced due to higher background signal or lower detection signal after the fluorescent latex microspheres are sealed in the fluorescence immunochromatography because of the sealing liquid; (ii) the invention provides a confining liquid for fluorescent microspheres, which is the same as the confining liquid in the first aspect.

In one embodiment, the present invention provides a sealant fluid having a formulation as shown below: the molar concentration of PB (namely the total concentration of sodium dihydrogen phosphate and disodium hydrogen phosphate) is 10-40 mM, the molar concentration of ethanolamine is 5-30 mM, the mass fraction of sodium caseinate is 0.5-5% (based on the total mass of water), the mass fraction of Tween 20 is 0.03-0.3% (based on the total mass of water), the mass fraction of Proclin 300 or sodium azide is 0.05-1% (based on the total mass of water), and the balance is water, and the pH is 6.5-8.5. Preferably, the blocking solution is used in labeling of the murine myoglobin monoclonal antibody-fluorescent microsphere conjugate.

The invention relates to a fluorescent microsphere conjugate and a preparation method thereof

The invention also provides a fluorescent microsphere conjugate and a preparation method thereof.

The fluorescent microsphere conjugate provided by the invention is subjected to sealing treatment by the sealing liquid in the first aspect.

In a specific embodiment, taking the murine myoglobin monoclonal antibody-fluorescent microsphere conjugate as an example, the preparation method of the fluorescent microsphere conjugate subjected to blocking treatment provided by the invention comprises the following steps:

replacing a fluorescent microsphere stock solution with a labeling buffer solution with the pH value of 5.5-8 (preferably, the labeling buffer solution is a PB buffer solution (phosphate buffer solution));

secondly, activating the fluorescent latex microspheres (for example, activating the fluorescent latex microspheres by adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS)), and separating the activated fluorescent latex microspheres (for example, separating by centrifuging and removing supernatant) after the reaction is finished;

thirdly, adding the mouse myoglobin monoclonal antibody and the activated fluorescent latex microspheres for reaction, and separating the fluorescent latex microspheres after the reaction (such as a method for removing supernatant through centrifugation);

adding the fluorescent microsphere sealing solution of the invention and the reacted murine myoglobin monoclonal antibody-fluorescent microsphere conjugate, sealing (preferably sealing for 1 hour at room temperature), and separating the sealed fluorescent microsphere conjugate (such as by centrifuging and removing supernatant);

fifthly, storing the obtained rat myoglobin monoclonal antibody-fluorescent microsphere conjugate in a fluorescent storage solution, preferably, the storage concentration is 0.2-2 mg/ml.

Myoglobin fluorescence immunochromatography kit

The invention also provides a fluorescence immunochromatography kit capable of rapidly detecting a marker (such as myoglobin), wherein the kit comprises:

a pre-sample cup, an optional reagent card, a reagent strip (the reagent strip comprises an optional PVC bottom plate, a sample pad, a nitrocellulose membrane and an absorption pad);

wherein, the pre-loading cup is dried with (or loaded with) a first blocking-treated antibody (or antigen) -fluorescent microsphere conjugate (for example, when the marker is myoglobin, a blocking-treated murine myoglobin monoclonal antibody-fluorescent microsphere conjugate is dried), and the first antibody can be specifically bound with the marker;

the nitrocellulose membrane is provided with a detection line and a quality control line;

wherein the detection line is coated with a second antibody (or antigen) capable of specifically binding to the marker (for example, when the marker is myoglobin, the second antibody (or antigen) may be a monoclonal antibody against myoglobin of murine origin); the quality control line is coated with a third antibody (or antigen) (e.g., when the marker is myoglobin, the third antibody (or antigen) is goat anti-mouse IgG polyclonal antibody);

preferably, the sample pad, the nitrocellulose membrane and the absorbent pad are lapped and carried at one time on a PVC base plate; preferably, the pre-sample cup and the PVC base plate are fixed in the reagent card;

the formula of the sealing liquid used for sealing the first antibody (or antigen) -fluorescent microsphere conjugate (such as the murine myoglobin monoclonal antibody-fluorescent microsphere conjugate) is as follows: the molar concentration of PB is 10-40 mM, the molar concentration of ethanolamine is 5-30 mM, the mass fraction of casein sodium is 0.5-5% (based on the total mass of water), the percentage concentration of Tween 20 is 0.03-0.3% (based on the total mass of water), the mass fraction of Proclin 300 or sodium azide is 0.05-1% (based on the total mass of water), the balance is water, and the pH value is 6.5-8.5.

The detection principle of the kit is as follows: the kit of the invention adopts an immunofluorescence double antibody sandwich method to quantitatively detect the concentration of a marker (such as the concentration of myoglobin in human plasma) in a sample. Typically, the method comprises: adding a sample to be tested (e.g., 100 + -10 microliters; preferably, the sample to be tested is obtained after a biological sample (e.g., blood, plasma, body fluid, etc.) is subjected to a treatment (e.g., dilution and/or filtration treatment)) to a pre-sample cup (reaction tube container) containing a first antibody (or antigen) -fluorescent microsphere conjugate (e.g., murine myoglobin monoclonal antibody-fluorescent microsphere conjugate), taking a certain amount (e.g., 60 + -6 microliters of sample) of the pre-sample cup after a reaction for a certain time, and adding the sample after the reaction to the pre-sample cup from a sample pad (e.g., dropping the sample onto the sample pad through a test hole of a reagent card); preferably, the sample to be tested is a sample obtained by diluting (e.g. diluting with a diluent) a biological sample (e.g. plasma) 20-60 times (preferably 30-50 times, e.g. 40 times);

under chromatography, the sample passes from the sample pad to the nitrocellulose membrane. A marker (e.g., myoglobin) if present in the sample, which forms a complex with a first antibody (or antigen) -fluorescent microsphere conjugate (e.g., a murine myoglobin monoclonal antibody-fluorescent microsphere conjugate); after the reacted sample is added to the sample pad, and passes through the cellulose membrane under the action of chromatography, the reacted sample is combined with a second antibody (or antigen) (such as a mouse myoglobin monoclonal antibody) on the detection line (if a marker exists in the sample), so that a fluorescent immune complex is formed on the detection line, and the rest fluorescent microsphere conjugates which do not form the fluorescent immune complex on the detection line in the reacted sample are combined with a third antibody (or antigen) on the detection line. The concentration of the myoglobin in the sample is in direct proportion to the fluorescence signal of the detection line, and the fluorescence signal intensity of the detection line is corrected by the fluorescence signal intensity of the quality control line, so that the quantitative detection of the marker (such as the myoglobin) is realized.

The main advantages of the invention include:

(a) the sealing liquid has high sealing efficiency and short sealing time (only about 1 hour), and the fluorescent microsphere conjugate sealed by the sealing liquid has less non-specific adsorption.

(b) The fluorescent microspheres sealed by the sealing liquid have good stability.

(c) The kit or reagent strip prepared by the fluorescent microsphere conjugate sealed by the sealing liquid has the advantages of low background signal, high sensitivity and good stability.

(d) The kit has the advantages of simple operation, high sensitivity, economy, practicability and the like.

(e) The immunofluorescence chromatography kit such as the myoglobin immunofluorescence chromatography kit has short reaction time (only 12min, wherein the reaction time with the closed fluorescent microspheres is about 2min, and the reaction time of the reagent strip is about 10min), excellent accuracy and high sensitivity.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

EXAMPLE 1 preferred sealant formulations and methods for their preparation

One preferred formulation of the sealant fluid of the present invention is: the molar concentration of PB is 10mM, the mass fraction of sodium caseinate is 1%, the molar concentration of ethanolamine is 10mM, the mass fraction of Tween 20 is 0.05%, the mass fraction of sodium azide is 0.1%, and the pH is adjusted to 7.8.

The preparation method of the confining liquid comprises the following steps: 0.0992g of disodium hydrogen phosphate and 0.0358g of sodium dihydrogen phosphate are weighed and dissolved in 80mL of water, the mixture is shaken and mixed evenly, after the disodium hydrogen phosphate and the sodium dihydrogen phosphate are dissolved completely, 1g of casein sodium (purchased from Sigma), 0.0611g of ethanolamine, 0.05g of Tween 20 and 0.1g of sodium azide are added, and the mixture is shaken and mixed evenly. The pH was adjusted to 7.8 with hydrochloric acid and sodium hydroxide and finally made to 100mL at room temperature.

Example 2

Preparation of mouse myoglobin monoclonal antibody-fluorescent microsphere conjugate (labeled particle):

firstly, replacing a fluorescent microsphere original solution with a labeling buffer solution (namely a PB buffer solution) with the pH value of 5.5-7.0 through centrifugation and washing;

adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to activate the fluorescent latex microspheres, centrifuging to remove supernatant after the reaction is finished, and washing once again;

thirdly, adding the mouse myoglobin monoclonal antibody to react with the activated fluorescent latex microspheres, centrifuging to remove supernatant after the reaction is finished, and washing once again;

adding the fluorescent confining liquid and the reacted murine myoglobin monoclonal antibody-fluorescent microsphere conjugate, sealing at room temperature for 1 hour, and centrifuging to remove supernatant;

fifthly, storing the obtained rat myoglobin monoclonal antibody-fluorescent microsphere conjugate in a fluorescent storage solution, wherein the storage concentration is 0.5 mg/ml.

EXAMPLE 3 preparation of hemoglobin immunofluorescent chromatography kit

Preparation of myoglobin immunofluorescence chromatography reagent strip:

ultrasonically dispersing the mouse myoglobin monoclonal antibody-fluorescent microsphere conjugate obtained in the embodiment 2, adding Tris buffer solution and a protective agent, uniformly mixing by oscillation, dropwise adding 5 microliters of mixed solution into each pre-sampling cup, and drying in vacuum;

coating 2mg/ml goat anti-mouse IgG polyclonal antibody and 1mg/ml mouse myoglobin monoclonal antibody with coating liquid, fixing the strips of 1.0mm on a nitrocellulose membrane by a gold spraying and membrane scribing instrument, attaching the nitrocellulose membrane, an absorption pad and a sample pad on a PVC (polyvinyl chloride) bottom plate in sequence, cutting the nitrocellulose membrane into a width of 3.2mm, putting the nitrocellulose membrane, the absorption pad and the sample pad into a reagent card, putting a sample cup with a dried mouse myoglobin monoclonal antibody-fluorescent microsphere conjugate in the reagent card, and clamping the reagent and tabletting. The structures of the reagent card and the pre-sample cup are described in CN 205038217U.

Comparative example 1 preparation of fluorescent microsphere conjugates (labeled particles)

The preparation method was the same as in example 2 except that the blocking solution used in the step (iv) was replaced with 0.1% BSA.

Comparative example 2 preparation of hemoglobin immunofluorescence chromatography kit

The preparation method is the same as that of example 3, except that: the conjugate prepared in example 2 used in step (r) was replaced with the conjugate prepared in comparative example 1.

Test example 1 comparison of blocking Effect

In test example 1, the effect control of the preferred blocking solution of the present invention and the control blocking solution (0.1% BSA) is compared, including stability and lot-to-lot difference of the murine myoglobin monoclonal antibody-fluorescent microsphere conjugate (labeled particles for short).

Sample standard substance: myoglobin (purchased from hai peptide) was formulated with sample diluent FBSS (ereaded) to a standard solution of the desired (specified) concentration.

The test method comprises the following steps:

adding sample amount of a pre-adding sample cup: 100 microliter

Sample loading amount of the sample pad: 60 microliter

Detection (reaction) time: for 10 minutes.

Reading the information of the test strip window by using a matched detection instrument, and calculating the area of the T peak and the area of the C peak by using fluorescence analysis software. The signal value is TAP, TAP 5000 × TA/(TA + CA). Wherein TA is the fluorescence peak area of a T line of the detection line, and CA is the fluorescence peak area of a C line of the quality control line.

Test example 1.1 confining liquid stability test

The kits prepared in example 3, respectively, tested 0ng/ml, 100ng/ml and 400ng/ml of sample standards and read and software analyzed to obtain signal value (TAP) results (shown in FIG. 2). Wherein, the mouse myoglobin monoclonal antibody-fluorescent microsphere conjugate used in the preparation of the kit is prepared by the method of the embodiment 2 and is stored for 0, 7, 14, 30, 45 and 60 days respectively.

As can be seen from FIG. 2, the labeling particles prepared using the blocking solution of the present invention (fluorescent conjugates prepared by the method of example 2) had excellent stability within 60 days, fluctuation within. + -. 15% and low background signal (signal value < 10). Whereas the labeled particles (fluorescent conjugates prepared by the method of comparative example 1) prepared with the control blocking solution (0.1% BSA) showed a continuous decrease in signal within 60 days, which was finally over 15% with a higher background signal (signal value > 40). As can be seen from a comparison of FIG. 2, the labeled particles prepared by the blocking solution of the present invention have significantly more excellent stability, and the accuracy of the labeled particles prepared by the blocking solution of the present invention is better than that of the labeled particles prepared by the control blocking solution (0.1% BSA) as seen by the length of the error bar in the figure.

Test example 1.2

The standard solutions were tested with different batches of the kit and read and analyzed by software to obtain the signal value (TAP), with the test results shown in Table 1. The kits of different batches are different batches of kits obtained by respectively preparing different batches of the blocked fluorescent microsphere conjugates (namely, the labeled particles) by adopting the blocking solution prepared by the same batch according to the methods in the examples and the comparative examples.

Table 1 compares the difference data between the batches of labeled particles prepared for the preferred blocking solutions and the control blocking solution.

It can be seen from table 1 that the lot-to-lot differences of the labeled particles of different lots produced from the same lot of the preferred blocking solution are better than those of the control blocking solution (0.1% BSA), and that the lot-to-lot CVs of the preferred blocking solution of different concentrations are all smaller than those of the control blocking solution (0.1% BSA), and it can be seen that the blocking solution of the present application has excellent storage stability itself. It can also be seen that the blocking solution preferably produces labeled particles with a background signal significantly lower than the control blocking solution (0.1% BSA).

Test example 1.3

Samples with a myoglobin concentration of 400ng/ml were tested using the kits of example 3 and comparative example 2, respectively, and fluorescence signals of the test line and the quality control line were obtained by an instrument, and the results are shown in FIG. 3.

From FIG. 3, it can be seen that the labeled particles prepared from the control blocking solution (0.1% BSA) have poor release effect on the membrane, and the labeled particles are clearly remained at the front end (150-180) of the NC membrane. The marked particles prepared by the optimized confining liquid have good release effect and stable integral baseline. Meanwhile, the fluorescence intensity of the detection baseline of the marking particles of the preferred confining liquid and the control confining liquid is compared, and the fluorescence intensity of the control confining liquid baseline is about 100, while the fluorescence intensity of the preferred confining liquid baseline is about 50. It is shown that the marking particles obtained from the preferred blocking solution have better dispersibility and flowability, and less residue on the film.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (10)

1. A fluorescent microsphere sealing liquid is characterized in that,

the pH value of the sealing liquid is 6.5-8.5;

the solvent of the confining liquid is water, and

the confining liquid comprises: 1 to 100mM buffer reagent, 5 to 30mM ethanolamine, 5 to 50g/L sodium caseinate, 0.1 to 5g/L nonionic surfactant, and 0.1 to 10g/L preservative.

2. The confining liquid as recited in claim 1 wherein in said confining liquid,

the concentration of the buffer reagent is 5-50 mM; preferably, it is 10 to 40 mM; and/or

The concentration of the ethanolamine is 5-20 mM; and/or

The concentration of the casein sodium is 5-30 g/L; and/or

The concentration of the nonionic surfactant is 0.3-3 g/L; and/or

The concentration of the corrosion inhibitor is 0.5-5 g/L.

3. The sealant fluid of claim 1 wherein said surfactant is selected from the group consisting of: tween 20; and/or the preservative is selected from the group consisting of: sodium azide, Proclin 300, or a combination thereof.

4. The sealing fluid according to claim 1, wherein the pH of the sealing fluid is 7 to 8; preferably, the pH is 7.5-8; more preferably, the pH is 7.8. + -. 0.1.

5. The sealant fluid of claim 1 wherein the pH buffering agent is a PB buffering agent.

6. A method of preparing the sealant fluid of claim 1, comprising the steps of:

(1) dissolving a buffer reagent in water to obtain a buffer solution;

(2) mixing ethanolamine, sodium caseinate, a non-ionic surfactant and a preservative with the buffer solution obtained in the step (1); and

(3) adding water to a constant volume to obtain the confining liquid as claimed in claim 1.

7. A blocked fluorescent microsphere conjugate, which is obtained by blocking the fluorescent microsphere conjugate with the blocking solution according to claim 1.

8. The method of preparing the blocked fluorescent microsphere conjugate of claim 7, comprising the steps of:

(i) providing a fluorescent microsphere conjugate without blocking treatment; and (ii) blocking with the blocking solution of claim 1 to obtain the blocked fluorescent microsphere conjugate of claim 7.

9. A kit for fluorescence immunoassay, characterized in that,

(i) the kit comprises: the sealant fluid of claim 1; alternatively, the first and second electrodes may be,

(ii) the kit comprises: the blocked fluorescent microsphere conjugate of claim 7; alternatively, the first and second electrodes may be,

(iii) the kit comprises: a container carrying the blocked fluorescent microsphere conjugate of claim 7; or

(iv) The kit comprises: a fluorescent immunochromatographic reagent strip comprising a conjugate pad; wherein the conjugate pad carries the blocked fluorescent microsphere conjugate of claim 7; or

(v) The kit comprises: buffer reagent, ethanolamine, sodium caseinate, nonionic surfactant, preservative and instructions; wherein the buffer, ethanolamine, sodium caseinate, nonionic surfactant and preservative are as defined in claim 1 and said instructions load the sealant formulation and formulation method.

10. A method of detecting a marker in a sample comprising the steps of:

(1) contacting a sample with a blocking-treated conjugate according to claim 7, or with a fluorescent microsphere conjugate blocked with a blocking solution according to claim 1, wherein the fluorescent microsphere conjugate is as defined in claim 7; thereby obtaining a sample possibly containing the compound of the marker and the fluorescent microsphere conjugate; and

(2) and (2) detecting the compound of the marker and the fluorescent microsphere conjugate in the sample obtained in the step (1).

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