CN114609379A - Detection reagent and detection method for 3-methoxytyramine - Google Patents
Detection reagent and detection method for 3-methoxytyramine Download PDFInfo
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- CN114609379A CN114609379A CN202210447727.9A CN202210447727A CN114609379A CN 114609379 A CN114609379 A CN 114609379A CN 202210447727 A CN202210447727 A CN 202210447727A CN 114609379 A CN114609379 A CN 114609379A
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- reagent
- derivatization
- methoxytyramine
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
Abstract
The invention relates to the technical field of biochemical detection, in particular to a detection reagent and a detection method for 3-methoxytyramine. The pretreatment reagent provided by the invention can be used for rapidly treating a pretreatment sample, the first result detection only needs 30min, and then the result is obtained at each test interval of 18 seconds, so that the detection efficiency is greatly improved. The reagent has higher pretreatment quality on the sample, so that the detection result has higher sensitivity, specificity and repeatability, and the clinical compliance rate is greatly improved.
Description
Technical Field
The invention relates to the technical field of biochemical detection, in particular to a detection reagent and a detection method for 3-methoxytyramine.
Background
Pheochromocytoma and Paraganglioma (PPGL) are one of the causes of secondary hypertension, and the prevalence rates thereof have been on the rise in recent years. 3-methoxytyramine (3-methoxytyramine, 3-MT) can be used as a detection marker of PPGL, and provides certain help for diagnosis and identification of good and malignant PPGL. Furthermore, 3-MT is considered to be an important marker for identifying the presence or absence of metastasis of pheochromocytoma.
At present, the mainstream detection method in the market of 3-MT mainly comprises High Performance Liquid Chromatography (HPLC), high performance liquid electrochemical combination (HPLC-EDC), enzyme-linked immunosorbent assay (ELISA) and the like, but the HPLC detection time is long, the cost is high, the difference of different laboratory conditions is large, batch determination is difficult to realize, the accuracy and the reproducibility of the enzyme-linked immunosorbent assay are poor, the automation degree is low, the method is easily influenced by the environment, and the sensitivity is low.
The magnetic microsphere is a spherical composite material with the diameter of nanometer or micron. Has the characteristics of large specific surface area, high dispersion stability, superparamagnetism, functional groups, biocompatibility and the like, and is widely applied to the fields of cell separation and purification, immunodetection, nucleic acid analysis and immune engineering, serving as a carrier of targeted drug release and the like. Chemiluminescence immunoassay is characterized in that enzyme of immunoreaction acts on a luminous substrate to cause the luminous substrate to generate chemical reaction and release a large amount of energy to generate an excited intermediate. When the excited state intermediate returns to a stable ground state, photons can be emitted simultaneously. The light quantum yield can be measured by using a light-emitting signal detection instrument, and is in direct proportion to the amount of the substance to be measured in the sample, so that a standard curve can be established and the content of the substance to be measured in the sample can be calculated. The magnetic microsphere can be widely applied to a chemiluminescence immunoassay method as a solid phase support.
The magnetic particle chemiluminescence immunoassay method has the characteristics of high accuracy and high sensitivity, but the method has higher requirements for detecting samples, 3-MT in blood or urine samples cannot be directly detected by the magnetic particle chemiluminescence immunoassay method, and the detection time cost is undoubtedly increased by overlong pretreatment acylation process.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a detection reagent and a detection method for 3-methoxytyramine.
The invention provides a sample pretreatment reagent combination, which comprises: acidifying liquid, a derivatizing reagent and an acylation buffer, wherein:
the acidizing fluid is a hydrochloric acid solution or a sulfuric acid solution;
the derivatization reagent comprises acid anhydride or methyl-PEG 4-NHS ester;
the derivatization buffer is selected from any one of Tris buffer, PBS buffer and carbonic acid buffer.
The acidizing fluid is a hydrochloric acid solution with the concentration of 0.1-0.5 mol/L, and the pH value is less than 1.
In some embodiments, the hydrochloric acid solution has a concentration of 0.2mol/L hydrochloric acid solution and a pH < 1.
Or the acidizing fluid is a sulfuric acid solution with the concentration of 0.1-0.5 mol/L, and the pH value is less than 1.
In some embodiments, the sulfuric acid solution has a concentration of 0.2mol/L hydrochloric acid solution and a pH < 1.
According to the invention, citric acid, nitric acid, sulfuric acid and hydrochloric acid are used for carrying out acidification treatment on the sample, and the result shows that the acidification effect of the citric acid and the nitric acid is not as good as that of the sulfuric acid and the hydrochloric acid. Among them, hydrochloric acid, particularly 0.2mol/L hydrochloric acid solution is most effective.
The derivatization reagent is liquid acid anhydride. In some embodiments, the anhydride is hexanoic anhydride or octanoic anhydride.
Or the derivatization reagent is methyl-PEG 4-NHS ester with the concentration of 40 mg/mL-200 mg/mL.
The invention tries on a plurality of derivatization reagents, and the result shows that the derivatization effect of the anhydride is better than that of methyl-PEG 4-NHS ester. The derivatization effect of the hexanoic anhydride or the caprylic anhydride in the anhydride is better, wherein the effect of the hexanoic anhydride is better than that of the caprylic anhydride.
In the invention, the pH value of the derivatization buffer solution is 8-9.
In the invention, the derivatization buffer solution also comprises a preservative with the mass fraction of 1%.
In some embodiments, the preservative is selected from ProClin300, Bronidox, NaN3At least one of them.
In the invention, the volume ratio of the acidification liquid, the derivatization reagent and the acylation buffer solution is 250: 25.
The invention also provides a urine pretreatment method, which treats urine by using the sample pretreatment liquid.
The pretreatment method comprises the steps of mixing urine with an acidizing fluid, acidizing for 20-40 min at 85-95 ℃, adding a derivatization buffer solution and a derivatization reagent, and carrying out oscillation reaction for 1-10 min.
In some embodiments, the pretreatment method comprises mixing urine with an acidification solution, acidifying at 90 ℃ for 30min, adding a derivatization buffer and a derivatization reagent, and oscillating for 5 min.
In the invention, the volume ratio of the urine to the acidizing fluid is 50 to (200-300). In some embodiments, the volume ratio of urine to acidified liquid is 50: 250.
In the invention, the volume ratio of the acidified solution to the derivatization buffer solution and the derivatization reagent is (250-350) to (200-300) to (20-30). In some embodiments, the volume ratio of the acidified solution to the derivatization buffer and the derivatization reagent is 300: 250: 25.
The invention also provides a detection reagent of 3-methoxytyramine, which comprises the pretreatment reagent combination, a magnetic particle suspension with the surface coated with 3-methoxytyramine derivatization antigen, and a horseradish peroxidase labeled derivatization 3-methoxytyramine antibody.
The invention also provides a detection method of the 3-methoxytyramine, which comprises the following steps: mixing the urine treated by the pretreatment method with the magnetic particle suspension and the enzyme-labeled antibody, and separating magnetic beads for detection;
the surface of the magnetic particle suspension is coated with 3-methoxytyramine derivative antigen;
the enzyme-labeled antibody is a derivatization 3-methoxytyramine antibody labeled by horseradish peroxidase.
The pretreatment reagent provided by the invention can be used for rapidly treating a pretreatment sample, the first result detection only needs 30min, and then the result is obtained at each test interval of 18 seconds, so that the detection efficiency is greatly improved. The reagent has higher pretreatment quality on the sample, so that the detection result has higher sensitivity, specificity and repeatability, and the clinical compliance rate is greatly improved.
Drawings
FIG. 1 shows the correlation of the kit of the present invention with LC-MS/MS.
Detailed Description
The invention provides a detection reagent and a detection method of 3-methoxytyramine, and a person skilled in the art can use the contents for reference and appropriately improve process parameters to realize the detection. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
example 1
Preparation of a pretreatment reagent:
acidifying liquid: the strong acid comprises 0.2mol/L hydrochloric acid solution, and the pH value is less than 1;
acylation buffer solution: tris buffer solution (with the concentration of 0.05M-0.5M) containing 0.1 wt% of preservative has the pH value of 8-9. The preservative is ProClin300, Bronidox or NaN3The result is not influenced;
derivatization reagent: 25 μ l of hexanoic anhydride.
Example 2
Preparation of a pretreatment reagent:
acidifying liquid: the strong acid comprises 0.2mol/L hydrochloric acid solution, and the pH value is less than 1;
acylation buffer solution: PBS buffer solution (concentration is 0.05M-0.5M) containing 0.1 wt% preservative, and the pH value is 8-9. The preservative is ProClin300, Bronidox or NaN3The result is not influenced;
derivatization reagent: 25 μ l of caprylic anhydride.
Example 3
Preparation of a pretreatment reagent:
acidifying liquid: the strong acid comprises 0.2mol/L sulfuric acid solution, and the pH value is less than 1;
acylation buffer solution: a carbonic acid buffer solution (with the concentration of 0.05M-0.5M) containing 0.1 wt% of preservative, and the pH value is 8-9. The preservative is ProClin300, Bronidox or NaN3The result is not influenced;
derivatization reagent: methyl-PEG 4-NHS ester at a concentration of 40 mg/ml.
Comparative example 1
Preparation of a pretreatment reagent:
acidifying liquid: the strong acid comprises 0.2mol/L nitric acid solution, and the pH value is less than 1;
acylation buffer solution: tris buffer solution (with the concentration of 0.05M-0.5M) containing 0.1 wt% of preservative has the pH value of 8-9. The preservative is ProClin300, Bronidox or NaN3The result is not influenced;
derivatization reagent: 25 μ l of hexanoic anhydride.
Comparative example 2
Preparation of a pretreatment reagent:
acidifying liquid: the strong acid comprises 0.2mol/L citric acid solution, and the pH value is less than 1;
acylation buffer solution: tris buffer solution (with the concentration of 0.05M-0.5M) containing 0.1 wt% of preservative has the pH value of 8-9. The preservative is ProClin300, Bronidox or NaN3The result is not influenced;
derivatization reagent: 25 μ l of hexanoic anhydride.
Example 4
The 3-MT detection kit comprises the pretreatment reagent combination described in any one of embodiments 1-3.
And comprises: calibrator, magnetic particle suspension (coating 3-MT derived antigen), and horse radish peroxidase-labeled specific recognition derived 3-methoxytyramine antibody.
The preparation method comprises the following steps:
1. preparation of the calibrator
Prepared citric acid buffer solution (PH2-4) containing ProClin300, MIT (2-methyl-4-isothiazolin-3-one hydrochloride), and NaN as preservative3(sodium azide) orAnd two combinations are used as a calibrator diluent, the 3-MT antigen is diluted into 6 gradients, namely 0ng/mL, 30ng/mL, 90ng/mL, 270ng/mL, 810ng/mL and 2430ng/mL by the calibrator diluent, the 3-MT antigen is subpackaged into 1 mL/bottle, and the 1 mL/bottle is stored at the temperature of 2-8 ℃.
2. Preparation of magnetic particle suspensions
Preparation of solid-phase antigen: the immobilized competitive 3-MT antigen used in the invention includes, but is not limited to, derivatization with succinic anhydride, methyl-PEG 2-NHS ester, and methyl-PEG 8-NHS ester (the derivatized competitive antigen should be used in combination with a detection antigen, and the detection reagent selects an antibody with strong affinity for 3-MT derivatized with hexanoic anhydride, octanoic anhydride, and methyl-PEG 4-NHS ester, and relatively weak affinity for 3-MT derivatized with succinic anhydride, methyl-PEG 2-NHS ester, and methyl-PEG 8-NHS ester, so that detection of derivatized 3-MT can be achieved by using the solid competitive antigen derivatized with succinic anhydride, methyl-PEG 2-NHS ester, or methyl-PEG 8-NHS ester in combination with the selected antibody). Wherein the used dilution buffer is any one of Bis-Tris (Bis (2-hydroxymethyl) amino-Tris (hydroxymethyl) methane), Tris-NaCl (Tris hydroxymethyl aminomethane-sodium chloride) and PBS (phosphate buffer solution), and the pH value of the dilution buffer is 6.0-8.0; one or two of bovine serum albumin, bovine serum and casein are added into the diluent as protective proteins of the reagent. The preservative in the diluent is any one or two combinations of ProClin300, MIT (2-methyl-4-isothiazoline-3-ketone hydrochloride) and NaN3 (sodium azide).
Preparation of solid-phase competitive antigen: coating with magnetic particles, fully and uniformly mixing the magnetic particle stock solution, taking the magnetic particle stock solution according to 0.1-0.3 mu l of each part, coating by 100 parts, adding 200-400 mu l of phosphate buffer solution for cleaning for 3-5 times, and then adsorbing and fixing the magnetic particles by using a magnet and removing the supernatant; after washing, adding 20mg/ml EDC (carbodiimide) and NHS (succinimide ester) activating agents, adding 20 ul of 5mg/ml BSA, uniformly mixing, shaking and activating for 1-3 h; and after activation, washing for 2 times by using a sodium acetate buffer solution with the pH value of 4.5, washing for 5min each time, then adding a derivatized 3-MT antigen according to 0.02-0.2 mu g/T for coating, uniformly mixing, shaking and coating for 2h, then adsorbing and fixing magnetic particles by using a magnet, removing supernatant, adding 200-400 mu l of phosphate buffer solution containing 2% protective protein, sealing for 4 times, finally fixing the volume to 2ml by using the phosphate buffer solution containing 2% protective protein, and storing at the temperature of 2-8 ℃ for later use.
3. Preparation of enzyme-labeled antibody solution: adding 2% bovine serum albumin into one of prepared Tris-NaCl buffer solution, PBS and Bis-Tris, mixing to obtain a horse radish peroxidase labeled specific recognition derivatization 3-methoxytyramine antibody solution diluent, uniformly mixing specific antibodies of 3-MT molecules according to the concentration of 0.05-1 mu g/ml, and storing at 2-8 ℃.
Example 5
The urine sample is pretreated by the reagents described in examples 1-3 and comparative examples 1-2: the specific pretreatment method comprises the following steps:
and (3) putting 50 mu l of urine specimen into a container with a cover (the volume is 2-10 ml), adding 250 mu l of acidizing fluid, acidizing for 30min in a water bath at 90 ℃, taking out, naturally cooling, adding 250 mu 1 of acylation buffer solution and 25 mu 1 of derivatization reagent, and performing oscillation reaction for 5min to obtain the derivatized antigen to be detected. And detecting the treated antigen.
The detection method comprises the following steps:
performing oscillation reaction on derivatized antigen to be detected 50 mu l and magnetic particle suspension (coating 3-MT derivatized antigen) 20 mu l and horseradish peroxidase-labeled specific recognition derivatized 3-methoxytyramine antibody for 30min, fixing magnetic particles by using a magnet, removing supernatant, cleaning for 5 times, adding a substrate, catalyzing the substrate to emit light, matching with a chemiluminescence apparatus, detecting a signal value, detecting the first result only in30 min, and then outputting the result at intervals of 18 seconds. And (4) calculating the concentration of the urine sample by using the four parameters through the calibrator.
TABLE 1 test results
The results show that: the detection results obtained by the detection methods of the embodiments 1 to 3 are basically consistent with the known concentration, the deviation is less than or equal to 5%, and the detection results of the comparative examples 1 to 2 are lower than the known concentration, so that the sample cannot be completely processed.
Effect verification
1. Assay sensitivity detection
The pretreatment reagent provided in example 1 was used to conduct assay sensitivity assessment according to the protocol for determining detection limit and quantitative limit described in example 5 in EP17A, 5 clinical urine samples with a value close to 0 were prepared, each sample was repeated 3 times for a total of 4 days to obtain 60 non-negative data, and if less than 60, the required test and the total of 60 were required, and the assay sensitivity was calculated by recording the data.
The analytical sensitivity was calculated according to the method of EP17 using EXCEL data templates with a 5% tolerance for type I errors of 2.425 ng/ml.
2. Detection of precision
The precision evaluation was carried out according to EP05-A3, 3-MT reagent was used, and high/medium/low value quality control was used in combination, and the detection was repeated 20 times, and the data was recorded, and the calculation results are shown in Table 2.
TABLE 2 detection of the precision of the kit of the invention
As can be seen from Table 2, the established detection method has a variation of less than 3% in all three concentration levels, and has good precision.
3. Clinical alignment
45 clinical urine samples of known concentration (LC-MS/MS fixed value) were tested by the kit of the present invention, and the correlation is shown in FIG. 1: y is 0.8925x +33.45, r2 is 0.9961, which shows that the kit can accurately detect 3-MT in human urine.
4. Stability evaluation
And (3) examining the stability of the detection reagent, taking the reagent, placing the reagent in a 37 ℃ incubator for acceleration for 10 days, taking out the reagent, taking another 2-8 ℃ preservation reagent, and simultaneously detecting the same clinical urine specimen, wherein the detection results are shown in Table 3.
TABLE 4 stability test of the kits of the invention
As can be seen from Table 4, the established detection method has better stability, and the reduction of 10 percent is less than 10 percent when the detection method is stored at 37 ℃ for 10 days than at 2-8 ℃.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (10)
1. The reagent combination for sample pretreatment is characterized by comprising: acidifying liquid, a derivatization reagent and an acylation buffer, wherein:
the acidizing fluid is a hydrochloric acid solution or a sulfuric acid solution;
the derivatization reagent comprises acid anhydride or methyl-PEG 4-NHS ester;
the derivatization buffer is selected from any one of Tris buffer, PBS buffer and carbonic acid buffer.
2. The reagent combination of claim 1, wherein the acidifying solution is a hydrochloric acid solution with a concentration of 0.1-0.5 mol/L or a sulfuric acid solution with a concentration of 0.1-0.5 mol/L, and the pH value is less than 1.
3. The reagent combination of claim 1, wherein the derivatizing reagent is a liquid anhydride or methyl-PEG 4-NHS ester at a concentration of 40mg/ml to 200 mg/ml.
4. The reagent combination according to claim 1 or 3, characterized in that the anhydride is hexanoic anhydride or octanoic anhydride.
5. The reagent combination of claim 1, wherein the derivatization buffer has a pH of 8 to 9; the derivatization buffer solution also comprises a preservative with the mass fraction of 1%, wherein the preservative is selected from ProClin300, BRONIDOX and NaN3At least one of them.
6. A method for pretreating urine, characterized in that the urine is treated with the reagent combination according to any one of claims 1 to 5.
7. The pretreatment method according to claim 6, comprising mixing urine with an acidification solution, acidifying at 85-95 ℃ for 20-40 min, adding a derivatization buffer solution and a derivatization reagent, and carrying out a shaking reaction for 1-10 min.
8. The pretreatment method according to claim 7,
the volume ratio of the urine to the acidizing fluid is 50: 200-300;
the volume ratio of the acidified solution to the derivatization buffer solution to the derivatization reagent is (250-350) to (200-300) to (20-30).
A detection reagent for 3-methoxytyramine, which comprises the reagent combination of any one of claims 1 to 5, a magnetic particle suspension coated with a 3-methoxytyramine-derived antigen, and a horse radish peroxidase-labeled derivatized 3-methoxytyramine antibody.
A method for detecting 3-methoxytyramine, which is characterized by comprising the following steps: mixing the urine treated by the pretreatment method of any one of claims 6 to 8 with a magnetic particle suspension and an enzyme-labeled antibody, and separating magnetic beads for detection;
the surface of the magnetic particle suspension is coated with 3-methoxytyramine derivative antigen;
the enzyme-labeled antibody is a derivatization 3-methoxytyramine antibody labeled by horseradish peroxidase.
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