CN111089965A - Antigen detection kit, preparation method thereof and antigen detection method - Google Patents
Antigen detection kit, preparation method thereof and antigen detection method Download PDFInfo
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Abstract
The invention belongs to the technical field of biochemistry, and particularly relates to an antigen detection kit, a preparation method thereof and an antigen detection method, wherein the antigen detection kit is composed of R1 liquid and R2 liquid, and the R1 liquid comprises: a monoclonal antibody containing a Fab fragment and an Fc fragment, another monoclonal antibody labeled with biotin and only containing the Fab fragment or a monoclonal antibody labeled with biotin and not homologous with the monoclonal antibody containing the Fab fragment and the Fc fragment; the R2 liquid comprises: streptavidin magnetic beads and secondary antibody marked by chemiluminescence marker. The embodiment of the invention provides an antigen detection kit, wherein the structures of two monoclonal antibodies in R1 liquid are not damaged, and the kit does not contain magnetic microspheres, so that the combination effect of an antigen and an antibody can be ensured, and the content of the antigen can be accurately measured by matching with R2 liquid.
Description
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to an antigen detection kit, a preparation method thereof and an antigen detection method.
Background
In the biochemical field, it is usually necessary to perform antigen detection using an antibody corresponding to an antigen, and for convenience of operation, the antibody corresponding to the antigen is first prepared into an antigen reagent detection kit, and in the detection process, the antigen can be detected only by adding the antigen reagent detection kit.
Most of the existing antigen reagent detection boxes are based on a double-antibody sandwich method, two monoclonal antibodies are usually modified by chemical substances, namely the two monoclonal antibodies are respectively coupled with a tracer substance and a magnetic microsphere, an antibody (coupled with the magnetic microsphere) -antigen-antibody (coupled with the tracer substance) compound is formed in the process of adding an antigen for detection, and then the antigen is detected by detecting the tracer substance on the antibody coupled with the tracer substance. However, in the above antigen reagent detecting kit, coupling with the magnetic microsphere or the tracer substance destroys the self-structure of the antibody, and affects the antibody binding of the antigen due to the steric hindrance effect, so that the final antigen detection result is not accurate enough.
Therefore, the existing antigen reagent detection kit also has the technical problem of influencing the combination of the antigen and the antibody, so that the result of the content of the antigen detected is not accurate enough.
Disclosure of Invention
The embodiment of the invention aims to provide an antigen detection kit, and aims to solve the technical problem that the existing antigen reagent detection kit has an inaccurate antigen detection result due to the influence on the combination of an antigen and an antibody.
The embodiment of the invention is realized by an antigen detection kit, which consists of R1 liquid and R2 liquid, wherein,
the R1 liquid comprises: a monoclonal antibody containing a Fab fragment and an Fc fragment, another monoclonal antibody labeled with biotin and containing only the Fab fragment or a monoclonal antibody labeled with biotin and being non-homologous with the monoclonal antibody containing the Fab fragment and the Fc fragment, and a buffer matrix;
the R2 liquid comprises: streptavidin magnetic beads, secondary antibody marked by chemiluminescence marker, and buffer matrix.
Another objective of the embodiments of the present invention is to provide a method for preparing an antigen detection kit, comprising the following steps:
adding biotin into a monoclonal antibody solution only containing Fab fragments for reaction;
adding phosphate buffer solution, and ultrafiltering to remove free biotin in the solution;
diluting with a buffer matrix after ultrafiltration concentration, and adding a monoclonal antibody containing a Fab fragment and an Fc fragment after dilution to obtain an R1 solution;
adding a chemiluminescent marker into a secondary antibody solution for reaction;
adding phosphate buffer solution, and ultrafiltering to remove free chemiluminescent marker;
and (3) diluting by using a buffer substrate after ultrafiltration concentration, and diluting and adding streptavidin magnetic beads to obtain an R2 solution.
Another objective of the embodiments of the present invention is to provide an antigen detection method, which includes the following steps: adding the R1 solution in the antigen detection kit into a sample to be detected containing the antigen for reaction; continuously adding the R2 solution in the antigen detection kit into a sample to be detected containing the antigen for reaction;
adding a buffer matrix for cleaning to remove unreacted substances in the R1 liquid and the R2 liquid;
and after adding a luminescence auxiliary reagent, receiving and calculating the number of light quanta by using a photomultiplier, wherein the luminescence auxiliary reagent is a reagent which can perform luminescence reaction with the chemiluminescent marker in a solution state to generate photons.
The antigen detection kit provided by the embodiment of the invention comprises R1 liquid and R2, wherein the R1 liquid comprises a monoclonal antibody containing a Fab fragment and an Fc fragment, another biotin-labeled monoclonal antibody only containing the Fab fragment or a biotin-labeled monoclonal antibody which is not homologous with the monoclonal antibody containing the Fab fragment and the Fc fragment, and a buffer matrix; r2 also includes streptavidin magnetic beads, chemiluminescent label labeled secondary antibody, and buffer matrix. In the antigen detection kit provided by the embodiment of the invention, one strain in the R1 solution is an unmarked monoclonal antibody, and the other strain is a biotin marked monoclonal antibody, the biotin is directly coupled to the amino group on the surface of the antibody, so the structures of the two monoclonal antibodies are not destroyed, the combination of the antigen and the antibody is not influenced, meanwhile, because the magnetic beads (magnetic microspheres) do not exist, the steric hindrance effect caused by the magnetic microspheres is avoided, the binding rate of the antigen and the antibody is ensured, furthermore, streptavidin magnetic beads in the R2 solution can be combined with biotin, and a secondary antibody labeled with a chemiluminescent marker can be specifically combined with a monoclonal antibody containing a Fab fragment and an Fc fragment, and at the moment, the introduction of the R2 solution also does not affect the combination of the antigen and the antibody, and finally, the content of the antigen can be accurately determined by detecting the chemiluminescent marker on the secondary antibody labeled with the chemiluminescent marker.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The embodiment of the invention provides an antigen detection kit, which consists of R1 liquid and R2 liquid, wherein,
the R1 liquid comprises: a monoclonal antibody containing a Fab fragment and an Fc fragment, another monoclonal antibody labeled with biotin and containing only the Fab fragment or a monoclonal antibody labeled with biotin and being non-homologous with the monoclonal antibody containing the Fab fragment and the Fc fragment, and a buffer matrix;
the R2 liquid comprises: streptavidin magnetic beads, secondary antibody marked by chemiluminescence marker, and buffer matrix.
In the embodiment of the present invention, it should be emphasized at first that the present invention is to improve and optimize the existing antigen detection kit based on the double-antibody sandwich method, and the main improvement point is to improve the marker of the antibody, and the improvement process is not affected by the specific selection of the antigen and the antibody, that is, the present invention is not to protect a specific antigen detection kit, but to protect all antigen detection kits based on the idea of improving the marker of the antibody disclosed in the present invention, the monoclonal antibodies used in the antigen detection kits are different for different antigens, but those skilled in the art can combine the parameters known in the art and determine the corresponding monoclonal antibodies and secondary antibodies used according to the kind of the antigen to be detected, for example, when the detection of procalcitonin (PCT antigen) is required, the first antibody and the second antibody can respectively select a mouse anti-human PCT monoclonal antibody and a biotin-labeled rabbit anti-human PCT monoclonal antibody, and then can obtain a detection kit for detecting procalcitonin, and when the cardiac troponin I (cTnI antigen) needs to be detected, the first antibody and the second antibody can respectively select a mouse anti-human cTnI monoclonal antibody and a biotin-labeled rabbit anti-human cTnI monoclonal antibody, and then can obtain a detection kit for detecting the cardiac troponin I.
In the embodiment of the invention, two monoclonal antibodies in the R1 liquid are used for being combined with an antigen in a sample to be detected to form a first antibody (biotin mark) -antigen-second antibody compound (no mark), and in the combination process, as biotin is directly coupled to an amino group on the surface of the antibody and the structure of the antibody is not affected, the two monoclonal antibodies in the R1 liquid can be well combined with the antigen, the steric hindrance effect caused by introducing a magnetic microsphere in the prior art is avoided, and the combination rate of the antibody and the antigen is further ensured.
In the embodiment of the invention, the streptavidin magnetic beads can be combined with biotin, namely can be combined with a biotin-labeled monoclonal antibody, however, since the biotin-labeled monoclonal antibody contains only a Fab fragment (antigen-binding fragment) and does not contain an Fc fragment (crystallizable fragment) or the biotin-labeled monoclonal antibody is not homologous to another unlabeled monoclonal antibody, the secondary antibody labeled with a chemiluminescent label is thus capable of specifically binding to another unlabeled monoclonal antibody, namely, after the solution R2 is added, a streptavidin magnetic bead-first antibody (biotin labeling) -antigen-second antibody (no labeling) -chemiluminescent marker-labeled secondary antibody complex can be formed, and at this time, the content of the antigen in the sample to be detected can be determined by measuring the content of the chemiluminescent marker on the chemiluminescent marker-labeled secondary antibody.
In the present embodiment, it is preferable that the concentration of the monoclonal antibody containing a Fab fragment and an Fc fragment in the R1 solution is 1 to 100. mu.g/mL, and the concentration of the biotin-labeled monoclonal antibody of another strain containing only a Fab fragment or the biotin-labeled monoclonal antibody that is not homologous to the monoclonal antibody containing a Fab fragment and an Fc fragment is 1 to 100. mu.g/mL.
In the embodiment of the present invention, preferably, in the R2 solution, the concentration of the chemiluminescent label-labeled secondary antibody is 1 to 50. mu.g/mL, and the concentration of the streptavidin-coated magnetic beads is 1 to 2000. mu.g/mL.
In the embodiment of the present invention, the chemiluminescent label is preferably acridinium ester compound, alkaline phosphatase or ruthenium terpyridyl, and specifically, the principle of luminescence of the chemiluminescent label is as follows: (1) under the alkaline condition, hydrogen peroxide can be added with the carbon atom at the 9 th position of acridine, superoxide anion formed by an addition product under the alkaline condition is used for nucleophilic attack on carbonyl carbon, a leaving group is separated, an unstable four-membered ring intermediate is further formed, an excited acridone is formed after ring opening, and photons are released in the process of returning to the ground state.
(2) The luminous substrate of the alkaline phosphatase is a 1, 2-dioxane derivative (adamantane derivative), namely the alkaline phosphatase can act on the 1, 2-dioxane derivative (adamantane derivative), and the substrate is catalyzed by the alkaline phosphatase to emit light under the action of a signal reagent.
(3) The terpyridyl ruthenium and the electron donor Tripropylamine (TPA) can simultaneously lose an electron on the surface of the anode to generate an oxidation reaction, divalent terpyridyl ruthenium is oxidized into trivalent terpyridyl ruthenium (strong oxidant), TPA is oxidized into an unstable cationic radical TPA + after losing the electron, the cationic radical TPA + can spontaneously lose a proton (H +) to form a radical TPA, thereby becoming a strong reducing agent, and an electron is delivered to the trivalent terpyridyl ruthenium to form excited terpyridyl ruthenium, the excited terpyridyl ruthenium is unstable, a photon with the wavelength of 620nm can be emitted, and the ground terpyridyl ruthenium is recovered.
In the present embodiment, when acridinium ester compounds or alkaline phosphatase or ruthenium terpyridyl are used as chemiluminescent markers, the released photons (which can be determined by the intensity of light) are directly proportional to the concentration of the antigen to be detected.
In the embodiment of the invention, the buffer matrix comprises 10-100 mM/L of phosphate buffer solution, 10-50 g/L of bovine serum albumin, 205-10 g/L of Tween-8, 8-9 g/L of sodium chloride and 0.2-0.5 g/L of sodium azide, and the addition of the buffer matrix is mainly used for providing a liquid phase environment for the direct combination of the antigen and the antibody and the combination of other corresponding substances, so that the reaction is facilitated. Furthermore, it is preferable that the buffer matrix in the R2 solution additionally includes a stability protection agent, because if the stability protection agent is added to the R1 solution, the binding between the antigen and the antibody will be interfered, and if the stability protection agent is added to the buffer matrix in the R2 solution, the binding between the antigen and the antibody will not be affected, but instead, the binding between the streptavidin-labeled magnetic beads and the biotin-labeled monoclonal antibody can be improved, thereby improving the sensitivity of the whole antigen detection cassette. Preferably, the stability protective agent is trehalose, and the concentration is controlled to be 10-20 g/L.
The antigen detection kit provided by the embodiment of the invention comprises R1 liquid and R2, wherein the R1 liquid comprises a monoclonal antibody containing a Fab fragment and an Fc fragment, another biotin-labeled monoclonal antibody only containing the Fab fragment or a biotin-labeled monoclonal antibody which is not homologous with the monoclonal antibody containing the Fab fragment and the Fc fragment, and a buffer matrix; r2 also includes streptavidin magnetic beads, chemiluminescent label labeled secondary antibody, and buffer matrix. In the antigen detection kit provided by the embodiment of the invention, one strain in the R1 solution is an unmarked monoclonal antibody, and the other strain is a biotin marked monoclonal antibody, the biotin is directly coupled to the amino group on the surface of the antibody, so the structures of the two monoclonal antibodies are not destroyed, the combination of the antigen and the antibody is not influenced, meanwhile, because the magnetic beads (magnetic microspheres) do not exist, the steric hindrance effect caused by the magnetic microspheres is avoided, the binding rate of the antigen and the antibody is ensured, furthermore, streptavidin magnetic beads in the R2 solution can be combined with biotin, and a secondary antibody labeled with a chemiluminescent marker can be specifically combined with a monoclonal antibody containing a Fab fragment and an Fc fragment, and at the moment, the introduction of the R2 solution also does not affect the combination of the antigen and the antibody, and finally, the content of the antigen can be accurately determined by detecting the chemiluminescent marker on the secondary antibody labeled with the chemiluminescent marker.
The embodiment of the invention also provides a preparation method of the antigen detection kit, which comprises the following steps: in step S102, biotin is added to a monoclonal antibody solution containing only Fab fragments for reaction.
Step S104, adding phosphate buffer solution, and performing ultrafiltration to remove free biotin in the solution.
And step S106, diluting the mixture by using a buffer matrix after ultrafiltration and concentration, and adding a monoclonal antibody containing a Fab fragment and an Fc fragment after dilution to obtain an R1 solution.
Step S108, adding the chemiluminescent label into the secondary antibody solution for reaction.
Step S110, adding phosphate buffer, and ultrafiltering to remove free chemiluminescent label from the solution.
And step S112, diluting by using a buffer matrix after ultrafiltration concentration, and diluting and adding streptavidin magnetic beads to obtain an R2 solution.
In the embodiment of the present invention, it should be noted that, step S102 to step S106 are used to prepare the components of the R1 liquid in the antigen detection kit, and step S108 to step S112 are used to prepare the R2 in the antigen detection kit, but it should be noted that, there is no strict sequence between step S102 to step S106 and step S108 to step S112, and it is not necessary to complete the steps in the same space, that is, step S102 to step S106 may be performed separately to prepare the R1 liquid, step S108 to step S112 may be performed separately to prepare the R2 liquid, and then the prepared R1 liquid and R2 liquid are combined together to obtain the antigen detection kit.
In the embodiments of the present invention, for the explanation of the monoclonal antibody, biotin, buffer matrix, secondary antibody, and chemiluminescent label, please refer to the explanation of the antigen detection kit, which is not repeated herein.
In the embodiment of the invention, preferably, in the process of adding biotin for reaction, the reaction temperature is controlled at 37 ℃, and the reaction time is controlled at 30-120 minutes.
In the embodiment of the invention, preferably, in the process of adding the chemiluminescent marker into the anti-antibody solution for reaction, the reaction temperature is controlled at 37 ℃, and the reaction time is controlled at 30-120 minutes.
The embodiment of the invention also provides a preparation method of another antigen detection kit, which comprises the following steps: step S202, adding biotin into the monoclonal antibody solution for reaction.
Step S204, adding phosphate buffer solution, and performing ultrafiltration to remove free biotin in the solution.
And step S206, diluting the mixture by using a buffer matrix after ultrafiltration concentration, and adding another monoclonal antibody which is not homologous with the monoclonal antibody after dilution to obtain an R1 solution.
In step S208, a chemiluminescent label is added to the secondary antibody solution for reaction.
Step S210, adding phosphate buffer, and ultrafiltering to remove free chemiluminescent label in the solution.
And step S212, diluting by using a buffer matrix after ultrafiltration concentration, and diluting and adding streptavidin magnetic beads to obtain an R2 solution.
In the present embodiment, the preparation method of the antigen detection kit is substantially similar to that of the antigen detection kit provided previously, and the difference is only that the selection of the monoclonal antibody labeled with biotin is different, and the details are not repeated here.
The embodiment of the invention also provides an antigen detection method, which comprises the following steps:
step S302, adding R1 liquid into the sample to be detected containing the antigen for reaction.
In the embodiment of the invention, the R1 solution comprises a monoclonal antibody containing a Fab fragment and an Fc fragment, another monoclonal antibody which is labeled by biotin and only contains the Fab fragment or a monoclonal antibody which is labeled by biotin and is not homologous with the monoclonal antibody containing the Fab fragment and the Fc fragment, and a buffer matrix.
In the present embodiment, it is preferable that the concentration of the monoclonal antibody containing the Fab fragment and the Fc fragment is 0.01 to 0.1mg/mL, and the concentration of the biotin-labeled monoclonal antibody containing only the Fab fragment or the biotin-labeled monoclonal antibody non-homologous to the monoclonal antibody containing the Fab fragment and the Fc fragment is 0.01 to 0.1 mg/mL.
And step S304, continuously adding the R2 solution into the sample to be detected containing the antigen for reaction.
In the embodiment of the invention, the streptavidin magnetic bead, the chemiluminescent marker labeled secondary antibody and the buffer matrix are arranged in a same way.
In the present embodiment, the concentration of the chemiluminescent label-labeled secondary antibody is preferably 0.01 to 0.05mg/mL, and the concentration of the streptavidin-labeled magnetic beads is preferably 0.1 to 0.5 mg/mL.
In the embodiment of the present invention, the chemiluminescent label is preferably acridinium ester compound or alkaline phosphatase or ruthenium terpyridyl.
And step S306, adding a buffer substrate for cleaning to remove unreacted substances in the R1 liquid and the R2 liquid.
In the embodiment of the invention, the buffer substrate comprises 10-100 mM/L of phosphate buffer solution, 10-50 g/L of bovine serum albumin, 205-10 g/L of Tween-205, 8-9 g/L of sodium chloride and 0.2-0.5 g/L of sodium azide.
In step S308, the light quantum number is received and calculated by a photomultiplier tube after adding the light-emitting auxiliary reagent.
In the embodiment of the present invention, the luminescence auxiliary reagent is a reagent capable of performing a luminescence reaction with a chemiluminescent label in a chemiluminescent label-labeled secondary antibody in a solution R2 to generate photons, and specifically, when the chemiluminescent label is an acridinium ester compound, the luminescence auxiliary reagent is solution a: 0.25mol/L sodium hydroxide solution and solution B: the concentration of nitric acid is 0.1mol/L, the mass fraction of hydrogen peroxide is 0.3%, when the chemiluminescence marker is alkaline phosphatase, the luminescence auxiliary reagent is 0.1 mol/L1, 2-dioxane derivative, when the chemiluminescence marker is terpyridyl ruthenium, the luminescence auxiliary reagent is Tripropylamine (TPA) buffer solution.
To further illustrate the beneficial effects of the present invention over the prior art, the following description will be made in conjunction with specific examples and relevant experimental data.
Example 1:
(1) preparing a procalcitonin (PCT, which is used for replacing procalcitonin in the following) detection kit:
adding 1mg of biotin-NHS into 20mg of rabbit anti-human PCT monoclonal antibody, and culturing for 30 minutes at 37 ℃;
ultrafiltering with 10mM/L phosphate buffer solution with pH of 7.0 to remove free biotin, and diluting with buffer matrix comprising 10mM/L phosphate buffer solution, 10g/L bovine serum albumin, 205 g/L Tween, 8g/L sodium chloride and 0.2g/L sodium azide until the concentration of the rabbit anti-human PCT monoclonal antibody labeled with biotin is 1 μ g/mL;
adding mouse anti-human PCT monoclonal antibody into the diluted buffer substrate until the concentration of the mouse anti-human PCT monoclonal antibody is 1 mu g/mL to prepare R1 liquid;
1mg of acridinium ester-NHS was added to 20mg of goat anti-mouse IgG secondary antibody and incubated at 37 ℃ for 30 minutes;
removing free acridinium ester by ultrafiltration with 10mM/L phosphate buffer solution with the pH value of 7.0, diluting the acridinium ester labeled goat anti-mouse IgG secondary antibody to the concentration of 1 mu g/mL after ultrafiltration concentration by using a buffer matrix, wherein the buffer matrix comprises 10mM/L phosphate buffer solution, 10g/L trehalose, 10g/L bovine serum albumin, 205 g/L tween-sodium, 8g/L sodium chloride and 0.2g/L sodium azide;
streptavidin magnetic beads were added to the diluted buffer substrate until the concentration of the streptavidin magnetic beads was 1. mu.g/mL, and R2 solution was obtained.
(2) Preparation of other auxiliary reagents
Preparation of auxiliary reagents and sample reagents:
solution A: 0.1mol/L nitric acid and 0.3% hydrogen peroxide mixed solution by mass fraction;
and B, liquid B: 0.25mol/L sodium hydroxide;
washing buffer solution: 10mM/L of phosphate buffer solution, 10g/L of bovine serum albumin, 205 g/L of tween-205, 8g/L of sodium chloride and 0.2g/L of sodium azide;
sample reagent: fifteen groups of sample reagents with PCT concentrations of 0.01ng/mL, 0.03ng/mL, 0.06ng/mL, 0.17ng/mL, 0.25ng/mL, 0.33ng/mL, 0.61ng/mL, 1.25ng/mL, 1.99ng/mL, 2.35ng/mL, 3.78ng/mL, 7.88ng/mL, 12.75ng/mL, 22.15ng/mL and 34.25ng/mL are prepared in sequence, wherein the sample reagent matrixes are selected from serum, the sample amount is 10ul, and the samples are sequentially marked as groups 1-15;
(3) procalcitonin detection process
Sequentially adding R1 solution in the procalcitonin detection kit into the 15 groups of samples, and reacting to form a biotin-labeled rabbit anti-human PCT monoclonal antibody-PCT antigen-mouse anti-human PCT monoclonal antibody compound;
continuously adding R2 solution in the procalcitonin detection kit, and reacting to form a rabbit anti-human PCT monoclonal antibody-PCT antigen-mouse anti-human PCT monoclonal antibody-acridinium ester labeled goat anti-mouse second antibody compound labeled by magnetic bead-streptavidin-biotin;
adding a washing buffer solution, and washing and removing unreacted solution;
adding solution A to dissociate acridinium ester label, adding solution B to make acridinium ester emit photon, and using photomultiplier to receive photon number.
In the embodiment of the present invention, the received light quantum number (RLU) is in a proportional relationship with the amount of procalcitonin, but usually, a calibration curve determination experiment performed in advance is required to determine the relationship between the received light quantum number and the procalcitonin, where the experimental procedure of the calibration curve determination experiment performed in advance is also the same as the above procedure, and the process of the calibration curve determination experiment in the present invention is not described in detail, and those skilled in the art should know the meaning of the calibration curve in the art.
In the 15 sets of samples provided in the embodiment of the present invention, a table of the back-extrapolated concentrations and the actual concentrations, which are inversely extrapolated based on the number of received photon counts and the calibration curve of the pre-determined procalcitonin, is shown in table 1.
Example 2:
the same as other steps of example 1, except that (1) the preparation of the procalcitonin detection kit specifically comprises the following steps:
adding 1mg of biotin-NHS into 20mg of rabbit anti-human PCT monoclonal antibody, and culturing for 120 minutes at 37 ℃;
ultrafiltering with 10mM/L phosphate buffer solution with pH of 7.0 to remove free biotin, and diluting with buffer matrix comprising 10mM/L phosphate buffer solution, 10g/L bovine serum albumin, 205 g/L Tween, 8g/L sodium chloride and 0.2g/L sodium azide until the concentration of the rabbit anti-human PCT monoclonal antibody labeled with biotin is 100 μ g/mL;
adding mouse anti-human PCT monoclonal antibody into the diluted buffer substrate until the concentration of the mouse anti-human PCT monoclonal antibody is 100 mu g/mL to prepare R1 liquid;
1mg of acridinium ester-NHS was added to 20mg of goat anti-mouse IgG secondary antibody and incubated at 37 ℃ for 120 minutes;
removing free acridinium ester by ultrafiltration with 10mM/L phosphate buffer solution with the pH value of 7.0, diluting the acridinium ester labeled goat anti-mouse IgG secondary antibody to 50 mu g/mL after ultrafiltration concentration by using a buffer matrix, wherein the buffer matrix comprises 10mM/L phosphate buffer solution, 10g/L trehalose, 10g/L bovine serum albumin, 205 g/L tween-sodium, 8g/L sodium chloride and 0.2g/L sodium azide;
streptavidin magnetic beads were added to the diluted buffer substrate until the concentration of the streptavidin magnetic beads was 2000. mu.g/mL, and R2 solution was obtained.
In the embodiment of the invention, the same calibration curve of procalcitonin is needed, and the back-inferred concentration of procalcitonin can be reversely deduced based on the received light quantum number, and the specific experimental data are shown in table 2. It should be noted that, in order to control variables, the PCT concentration of each sample in the sample reagent prepared in example 2 should be the same as that of each sample reagent in example 1, but in actual preparation, solutions with exactly the same concentration cannot be prepared at 100%, and certain errors exist, but all errors are within the range allowed by experiments, and for convenience of calculation, the actual concentration in example 2 is assumed to be the same as that in example 1, and the same procedure is adopted in the following examples and comparative examples.
Example 3:
the same as other steps of example 1, except that (1) the preparation of the procalcitonin detection kit specifically comprises the following steps:
adding 1mg of biotin-NHS into 20mg of rabbit anti-human PCT monoclonal antibody, and culturing for 60 minutes at 37 ℃;
ultrafiltering with 10mM/L phosphate buffer solution with pH of 7.0 to remove free biotin, and diluting with buffer matrix comprising 10mM/L phosphate buffer solution, 10g/L bovine serum albumin, 205 g/L Tween, 8g/L sodium chloride and 0.2g/L sodium azide until the concentration of the rabbit anti-human PCT monoclonal antibody labeled with biotin is 50 μ g/mL;
adding mouse anti-human PCT monoclonal antibody into the diluted buffer substrate until the concentration of the mouse anti-human PCT monoclonal antibody is 50 mug/mL to prepare R1 liquid;
1mg of acridinium ester-NHS was added to 20mg of goat anti-mouse IgG secondary antibody and incubated at 37 ℃ for 60 minutes;
removing free acridinium ester by ultrafiltration with 10mM/L phosphate buffer solution with the pH value of 7.0, diluting the acridinium ester labeled goat anti-mouse IgG secondary antibody to 25 mu g/mL by using a buffer matrix after ultrafiltration concentration, wherein the buffer matrix comprises 10mM/L phosphate buffer solution, 10g/L trehalose, 10g/L bovine serum albumin, 205 g/L tween-sodium, 8g/L sodium chloride and 0.2g/L sodium azide;
streptavidin magnetic beads were added to the diluted buffer substrate until the concentration of the streptavidin magnetic beads was 1000. mu.g/mL, and R2 solution was obtained.
Similarly, the actual concentration of each sample and the back-extrapolated concentration based on the number of received photons and the PCT standard curve are shown in table 3.
Example 4:
(2) preparation of a human cardiac troponin (cTn I, which is used for replacing human cardiac troponin) detection kit:
adding 1mg of biotin-NHS into 20mg of rabbit anti-human cTn I monoclonal antibody, and culturing for 60 minutes at 37 ℃;
ultrafiltering with 10mM/L phosphate buffer solution with pH value of 7.0 to remove free biotin, and diluting with buffer matrix to obtain biotin-labeled rabbit anti-human cTnI monoclonal antibody with concentration of 50 μ g/mL, wherein the buffer matrix comprises 10mM/L phosphate buffer solution, 10g/L bovine serum albumin, tween-205 g/L, 8g/L sodium chloride and 0.2g/L sodium azide;
adding a mouse anti-human cTn I monoclonal antibody into the diluted buffer substrate until the concentration of the mouse anti-human cTn I monoclonal antibody is 50 mug/mL to prepare an R1 solution;
1mg of acridinium ester-NHS was added to 20mg of goat anti-mouse IgG secondary antibody and incubated at 37 ℃ for 60 minutes;
removing free acridinium ester by ultrafiltration with 10mM/L phosphate buffer solution with the pH value of 7.0, diluting the acridinium ester labeled goat anti-mouse IgG secondary antibody to 25 mu g/mL by using a buffer matrix after ultrafiltration concentration, wherein the buffer matrix comprises 10mM/L phosphate buffer solution, 10g/L trehalose, 10g/L bovine serum albumin, 205 g/L tween-sodium, 8g/L sodium chloride and 0.2g/L sodium azide;
streptavidin magnetic beads were added to the diluted buffer substrate until the concentration of the streptavidin magnetic beads was 1000. mu.g/mL, and R2 solution was obtained.
(2) Preparation of other auxiliary reagents
Preparation of auxiliary reagents and sample reagents:
solution A: 0.1mol/L nitric acid and 0.3% hydrogen peroxide mixed solution by mass fraction;
and B, liquid B: 0.25mol/L sodium hydroxide;
washing buffer solution: 10mM/L of phosphate buffer solution, 10g/L of bovine serum albumin, 205 g/L of tween-205, 8g/L of sodium chloride and 0.2g/L of sodium azide;
sample reagent: fifteen groups of sample reagents with cTn I concentration of 3pg/mL, 10pg/mL, 55pg/mL, 121pg/mL, 247pg/mL, 477pg/mL, 687pg/mL, 1017pg/mL, 1487pg/mL, 2188pg/mL, 4644pg/mL, 7558pg/mL, 10147pg/mL, 25795pg/mL and 46857pg/mL are prepared in sequence, wherein the sample reagent matrix is serum, the sample amount is 10ul, and the groups are marked as 1-15 groups in sequence;
(3) process for detecting human cardiac troponin
Sequentially adding R1 solution in the human cardiac troponin detection kit into the 15 groups of samples, and reacting to form a biotin-labeled rabbit anti-human cTn I monoclonal antibody-cTn I antigen-mouse anti-human cTn I monoclonal antibody compound;
continuously adding R2 solution in the human cardiac troponin detection kit, reacting to form a rabbit anti-human cTnI monoclonal antibody-cTnI antigen-mouse anti-human cTnI monoclonal antibody-acridinium ester labeled goat anti-mouse second antibody compound labeled by magnetic beads-streptavidin-biotin;
adding a washing buffer solution, and washing and removing unreacted solution;
adding solution A to dissociate acridinium ester label, adding solution B to make acridinium ester emit photon, and using photomultiplier to receive photon number.
In the embodiment of the present invention, it should be noted that, at this time, a calibration curve of human cardiac troponin is used, and the calibration curve is also determined by a calibration curve determination experiment performed in advance, wherein the experimental procedure of the calibration curve determination experiment performed in advance is the same as the above procedure, and is not repeated herein, and wherein a table of the back-pushed concentration and the actual concentration based on the number of received light quanta and the back-pushed of the calibration curve of human cardiac troponin determined in advance is shown in table 4.
Example 5:
the same as other steps of example 3 except that, in the preparation of R2 solution in which the dilution step was carried out using a buffer substrate after ultrafiltration concentration, 10mM/L phosphate buffer, 10g/L bovine serum albumin, 205 g/L Tween, 8g/L sodium chloride and 0.2g/L sodium azide were used as the buffer substrate, and trehalose was not contained as compared with example 3.
See table 5 for the actual concentration of each sample and the back-extrapolated concentration based on the number of received photons and PCT standard curve.
Comparative example 1:
the same as other steps in example 3, except that (1) the steps for preparing the procalcitonin detection kit are specifically as follows:
adding 1mg of acridinium ester into 20mg of rabbit anti-human PCT monoclonal antibody, and culturing for 60 minutes at 37 ℃; removing free acridine ester by ultrafiltration with 10mM/L phosphate buffer solution with the pH value of 7.0, diluting the acridine ester labeled rabbit anti-human PCT monoclonal antibody to the concentration of 50 mu g/mL by using a buffer matrix after ultrafiltration concentration to obtain R1 liquid, wherein the buffer matrix comprises 10mM/L phosphate buffer solution, 10g/L bovine serum albumin, 205 g/L tween-sodium, 8g/L sodium chloride and 0.2g/L sodium azide;
adding 1mg of magnetic microspheres into 20mg of mouse anti-human PCT monoclonal antibody, and culturing at 37 ℃ for 60 minutes;
removing free magnetic microspheres by ultrafiltration with 10mM/L phosphate buffer solution with the pH value of 7.0, diluting the solution with a buffer matrix after ultrafiltration concentration until the concentration of the acridinium ester labeled goat anti-mouse IgG secondary antibody is 50 mu g/mL to obtain R2 solution, wherein the buffer matrix comprises 10mM/L phosphate buffer solution, 10g/L trehalose, 10g/L bovine serum albumin, 205 g/L tween-sodium, 8g/L sodium chloride and 0.2g/L sodium azide.
The procalcitonin detection kit obtained at this time is composed of one PCT monoclonal antibody marked by a chemiluminescence reagent and the other PCT monoclonal antibody marked by a magnetic microsphere, namely the conventional procalcitonin detection kit.
In the 15 sets of samples provided in the embodiment of the present invention, a table of the back-extrapolated concentrations and the actual concentrations, which are inversely extrapolated based on the number of received photon counts and the calibration curve of the pre-determined procalcitonin, is shown in table 6.
Comparative example 2:
the same as other steps in example 3, except that (1) the steps for preparing the procalcitonin detection kit are specifically as follows:
adding 1mg of biotin-NHS into 20mg of mouse anti-human PCT monoclonal antibody, and culturing for 60 minutes at 37 ℃;
ultrafiltering with 10mM/L phosphate buffer solution with pH of 7.0 to remove free biotin, and diluting with buffer matrix comprising 10mM/L phosphate buffer solution, 10g/L bovine serum albumin, 205 g/L Tween, 8g/L sodium chloride and 0.2g/L sodium azide until the concentration of biotin-labeled mouse anti-human PCT antibody is 50 μ g/mL;
adding mouse anti-human PCT monoclonal antibody into the diluted buffer substrate until the concentration of the mouse anti-human PCT monoclonal antibody is 50 mug/mL to prepare R1 liquid;
1mg of acridinium ester-NHS was added to 20mg of goat anti-mouse IgG secondary antibody and incubated at 37 ℃ for 60 minutes;
removing free acridinium ester by ultrafiltration with 10mM/L phosphate buffer solution with the pH value of 7.0, diluting the acridinium ester labeled goat anti-mouse IgG secondary antibody to 25 mu g/mL by using a buffer matrix after ultrafiltration concentration, wherein the buffer matrix comprises 10mM/L phosphate buffer solution, 10g/L trehalose, 10g/L bovine serum albumin, 205 g/L tween-sodium, 8g/L sodium chloride and 0.2g/L sodium azide;
streptavidin magnetic beads were added to the diluted buffer substrate until the concentration of the streptavidin magnetic beads was 1000. mu.g/mL, and R2 solution was obtained.
In the 15 sets of samples provided in the embodiment of the present invention, a table of the back-extrapolated concentrations and the actual concentrations, which are inversely extrapolated based on the number of received photon counts and the calibration curve of the pre-determined procalcitonin, is shown in table 7.
Table 1: example 1 Experimental data
Table 2: example 2 Experimental data
Table 3: example 3 Experimental data
Table 4: example 4 Experimental data
Table 5: example 5 Experimental data
Table 6: comparative example 1 experimental data
Table 7: comparative example 2 experimental data
The experimental data in tables 1, 2 and 3 show that the procalcitonin detection kit prepared based on the principle of the antigen reagent detection kit disclosed by the invention has a good detection effect on calcitonin, the error is concentrated within 5%, and the sensitivity is about 0.03ng/ml, and the experimental data in table 4 further shows that the core invention point of the invention is not in the specific selection of the antigen and the antibody, so that the selection of the antigen and the antibody is not specially limited, and various antigens can be applied to the invention. The experimental data in tables 3 and 5 show that when the stability protective agent is added into the buffer matrix of R2, the sensitivity of procalcitonin is improved to a certain extent, and the sensitivity is improved from about 0.2ng/ml to 0.03 ng/ml. As can be seen from the experimental data in tables 3 and 6, the antigen detection kit disclosed by the present invention has higher accuracy and smaller error in measurement compared to the conventional antigen detection kit based on the double-antibody sandwich method, and as can be seen from the experimental data in tables 3 and 7, the non-homology of the two monoclonal antibodies in the R1 solution is not limited in table 7, the secondary antibody labeled with the chemiluminescent label cannot be specifically identified, and can be simultaneously bound to the two monoclonal antibodies, so that the ratio of the amount of the chemiluminescent label to the amount of the antigen is higher than 1: 1, and the measurement result is overall higher, the error is larger, and the amount of the antigen cannot be accurately measured.
In addition, it can be seen by combining the experimental processes of example 3 and example 4 that the components of the R2 solutions in the prepared antigen detection kit are the same for different antigen detections, that is, in the actual operation process, only the corresponding R1 solution needs to be prepared for the antigen to be detected and the prepared R2 solution is matched to obtain the antigen detection kit, which greatly simplifies the detection process and effectively improves the detection efficiency.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. An antigen detection kit, which is characterized by consisting of an R1 liquid and an R2 liquid, wherein,
the R1 liquid comprises: a monoclonal antibody containing a Fab fragment and an Fc fragment, another monoclonal antibody labeled with biotin and containing only the Fab fragment or a monoclonal antibody labeled with biotin and being non-homologous with the monoclonal antibody containing the Fab fragment and the Fc fragment, and a buffer matrix;
the R2 liquid comprises: streptavidin magnetic beads, secondary antibody marked by chemiluminescence marker, and buffer matrix.
2. The antigen detection kit according to claim 1, wherein the concentration of the monoclonal antibody containing the Fab fragment and the Fc fragment in the R1 solution is 1 to 100. mu.g/mL, and the concentration of the biotin-labeled another monoclonal antibody containing only the Fab fragment or the biotin-labeled monoclonal antibody that is not homologous to the monoclonal antibody containing the Fab fragment and the Fc fragment is 1 to 100. mu.g/mL.
3. The antigen detection kit according to claim 1, wherein the concentration of the chemiluminescent-label-labeled secondary antibody in the R2 solution is 1 to 50 μ g/mL, and the concentration of the streptavidin-coated magnetic beads is 1 to 2000 μ g/mL.
4. The antigen detection kit according to claim 1, wherein the chemiluminescent label is an acridinium ester compound or alkaline phosphatase or ruthenium terpyridyl.
5. The antigen detection kit of claim 1, wherein the buffer matrix comprises: 10-100 mM/L of phosphate buffer solution, 10-50 g/L of bovine serum albumin, 205-10 g/L of tween-205, 8-9 g/L of sodium chloride and 0.2-0.5 g/L of sodium azide.
6. The preparation method of the antigen detection kit is characterized by comprising the following steps:
adding biotin into a monoclonal antibody solution only containing Fab fragments for reaction;
adding phosphate buffer solution, and ultrafiltering to remove free biotin in the solution;
diluting with a buffer matrix after ultrafiltration concentration, and adding a monoclonal antibody containing a Fab fragment and an Fc fragment after dilution to obtain an R1 solution;
adding a chemiluminescent marker into a secondary antibody solution for reaction;
adding phosphate buffer solution, and ultrafiltering to remove free chemiluminescent marker;
and (3) diluting by using a buffer substrate after ultrafiltration concentration, and diluting and adding streptavidin magnetic beads to obtain an R2 solution.
7. The preparation method of the antigen detection kit is characterized by comprising the following steps:
adding biotin into a monoclonal antibody solution for reaction;
adding phosphate buffer solution, and ultrafiltering to remove free biotin in the solution;
diluting by using a buffer matrix after ultrafiltration concentration, and adding another monoclonal antibody which is not homologous with the monoclonal antibody after dilution to obtain an R1 solution;
adding a chemiluminescent marker into a secondary antibody solution for reaction;
adding phosphate buffer solution, and ultrafiltering to remove free chemiluminescent marker;
and (3) diluting by using a buffer substrate after ultrafiltration concentration, and adding streptavidin magnetic beads after dilution to obtain an R2 solution.
8. The method for preparing the antigen detection kit according to claim 6 or 7, wherein the reaction temperature is controlled at 37 ℃ and the reaction time is controlled at 30-120 minutes in the process of adding biotin for reaction.
9. The method for preparing the antigen detection kit according to claim 6 or 7, wherein the reaction temperature is controlled at 37 ℃ and the reaction time is controlled at 30-120 minutes in the process of adding the chemiluminescent label into the secondary antibody solution for reaction.
10. An antigen detection method, comprising the steps of:
adding the R1 solution in the antigen detection kit according to any one of claims 1 to 5 into a sample to be detected containing an antigen for reaction;
continuously adding the R2 solution in the antigen detection kit according to any one of claims 1 to 5 into a sample to be detected containing the antigen for reaction;
adding a buffer matrix for cleaning to remove unreacted substances in the R1 liquid and the R2 liquid;
and after adding a luminescence auxiliary reagent, receiving and calculating the number of light quanta by using a photomultiplier, wherein the luminescence auxiliary reagent is a reagent which can perform luminescence reaction with the chemiluminescent marker in a solution state to generate photons.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112326953A (en) * | 2020-11-03 | 2021-02-05 | 广东海洋大学深圳研究院 | Method for directionally labeling polybiotin by using antibody |
| CN113030461A (en) * | 2021-03-12 | 2021-06-25 | 北京华科泰生物技术股份有限公司 | IgG antibody marked by amino group or carboxyl group-containing substance, human IgG magnetic particle chemical detection kit and preparation method thereof |
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| CN113640511A (en) * | 2021-08-04 | 2021-11-12 | 宁波瑞源生物科技有限公司 | Magnetic particle electrochemiluminescence kit |
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Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4659678A (en) * | 1982-09-29 | 1987-04-21 | Serono Diagnostics Limited | Immunoassay of antigens |
| CN1719256A (en) * | 2005-07-15 | 2006-01-11 | 北京倍爱康生物技术股份有限公司 | Magnetic separation enzymatic chemical luminous immune detection method of human thyroglobulin antibody |
| CN103293310A (en) * | 2013-05-08 | 2013-09-11 | 成都创宜生物科技有限公司 | Kit for detecting premature rupture of fetal membranes |
| CN104614522A (en) * | 2015-02-10 | 2015-05-13 | 深圳市新产业生物医学工程股份有限公司 | Helicobacter pylori antibody detection kit, detection method and application |
| CN104698172A (en) * | 2015-02-10 | 2015-06-10 | 深圳市新产业生物医学工程股份有限公司 | Kit for detecting hepatitis B surface antigen and detection method and application of kit |
| US20150376722A1 (en) * | 2014-06-27 | 2015-12-31 | Abbott Laboratories | COMPOSITIONS AND METHODS FOR DETECTING HUMAN PEGIVIRUS 2 (HPgV-2) |
| WO2016127301A1 (en) * | 2015-02-10 | 2016-08-18 | 深圳市新产业生物医学工程股份有限公司 | Rt3 chemiluminescent immunological detection reagent kit, and detection method and application therefor |
| JP2016194437A (en) * | 2015-03-31 | 2016-11-17 | 株式会社Ｌｓｉメディエンス | Measurement method of prostate specific antigen and measuring kit |
| WO2017107974A1 (en) * | 2015-12-23 | 2017-06-29 | 中国人民解放军第二军医大学 | Detection test kit for serum psmd4 proteins and detection method and application thereof |
| CN108445214A (en) * | 2018-02-02 | 2018-08-24 | 浙江艾明德生物科技有限公司 | A kind of kit and preparation method quantitatively detecting d-dimer |
| CN109187972A (en) * | 2018-08-17 | 2019-01-11 | 迪瑞医疗科技股份有限公司 | A kind of magnetic microparticle chemiluminescence kit of quantitative detection plasma renin content and preparation method thereof |
| CN109239356A (en) * | 2018-09-12 | 2019-01-18 | 迪瑞医疗科技股份有限公司 | C peptide chemiluminescence immunity detection reagent and preparation method thereof and detection method |
-
2019
- 2019-11-18 CN CN201911130561.2A patent/CN111089965B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4659678A (en) * | 1982-09-29 | 1987-04-21 | Serono Diagnostics Limited | Immunoassay of antigens |
| CN1719256A (en) * | 2005-07-15 | 2006-01-11 | 北京倍爱康生物技术股份有限公司 | Magnetic separation enzymatic chemical luminous immune detection method of human thyroglobulin antibody |
| CN103293310A (en) * | 2013-05-08 | 2013-09-11 | 成都创宜生物科技有限公司 | Kit for detecting premature rupture of fetal membranes |
| US20150376722A1 (en) * | 2014-06-27 | 2015-12-31 | Abbott Laboratories | COMPOSITIONS AND METHODS FOR DETECTING HUMAN PEGIVIRUS 2 (HPgV-2) |
| CN104614522A (en) * | 2015-02-10 | 2015-05-13 | 深圳市新产业生物医学工程股份有限公司 | Helicobacter pylori antibody detection kit, detection method and application |
| CN104698172A (en) * | 2015-02-10 | 2015-06-10 | 深圳市新产业生物医学工程股份有限公司 | Kit for detecting hepatitis B surface antigen and detection method and application of kit |
| WO2016127301A1 (en) * | 2015-02-10 | 2016-08-18 | 深圳市新产业生物医学工程股份有限公司 | Rt3 chemiluminescent immunological detection reagent kit, and detection method and application therefor |
| JP2016194437A (en) * | 2015-03-31 | 2016-11-17 | 株式会社Ｌｓｉメディエンス | Measurement method of prostate specific antigen and measuring kit |
| WO2017107974A1 (en) * | 2015-12-23 | 2017-06-29 | 中国人民解放军第二军医大学 | Detection test kit for serum psmd4 proteins and detection method and application thereof |
| CN108445214A (en) * | 2018-02-02 | 2018-08-24 | 浙江艾明德生物科技有限公司 | A kind of kit and preparation method quantitatively detecting d-dimer |
| CN109187972A (en) * | 2018-08-17 | 2019-01-11 | 迪瑞医疗科技股份有限公司 | A kind of magnetic microparticle chemiluminescence kit of quantitative detection plasma renin content and preparation method thereof |
| CN109239356A (en) * | 2018-09-12 | 2019-01-18 | 迪瑞医疗科技股份有限公司 | C peptide chemiluminescence immunity detection reagent and preparation method thereof and detection method |
Non-Patent Citations (1)
| Title |
|---|
| 周齐洋;金晶;江波;先小龙;颜彬;吕华;苏恩本;: "化学发光磁微粒免疫分析法定量检测HBsAg", 临床检验杂志, no. 11 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112326953A (en) * | 2020-11-03 | 2021-02-05 | 广东海洋大学深圳研究院 | Method for directionally labeling polybiotin by using antibody |
| CN113030461A (en) * | 2021-03-12 | 2021-06-25 | 北京华科泰生物技术股份有限公司 | IgG antibody marked by amino group or carboxyl group-containing substance, human IgG magnetic particle chemical detection kit and preparation method thereof |
| CN113030461B (en) * | 2021-03-12 | 2022-04-22 | 北京华科泰生物技术股份有限公司 | IgG antibody marked by amino group or carboxyl group-containing substance, human IgG magnetic particle chemical detection kit and preparation method thereof |
| CN113267631A (en) * | 2021-05-17 | 2021-08-17 | 江苏普若维生物技术有限责任公司 | Preparation method of electrochemical kit for detecting TNF-alpha |
| CN113433318A (en) * | 2021-06-29 | 2021-09-24 | 山东中鸿特检生物科技有限公司 | Kit for detecting alpha-fetoprotein heteroplasmon AFP-L3 content and detection method and application thereof |
| CN113640511A (en) * | 2021-08-04 | 2021-11-12 | 宁波瑞源生物科技有限公司 | Magnetic particle electrochemiluminescence kit |
| CN113640511B (en) * | 2021-08-04 | 2024-01-30 | 宁波瑞源生物科技有限公司 | Magnetic particle electrochemiluminescence kit |
| CN116047087A (en) * | 2022-12-29 | 2023-05-02 | 宁波瑞源生物科技有限公司 | Method for judging free biotin in sample |
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