CN112630450A - Preparation method and application of chemiluminescence detection kit for quantitatively detecting concentration of ProAKAP4 - Google Patents
Preparation method and application of chemiluminescence detection kit for quantitatively detecting concentration of ProAKAP4 Download PDFInfo
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- CN112630450A CN112630450A CN202110094153.7A CN202110094153A CN112630450A CN 112630450 A CN112630450 A CN 112630450A CN 202110094153 A CN202110094153 A CN 202110094153A CN 112630450 A CN112630450 A CN 112630450A
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- 238000002038 chemiluminescence detection Methods 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 41
- 210000000582 semen Anatomy 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 16
- 238000001514 detection method Methods 0.000 claims description 15
- 239000000758 substrate Substances 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 12
- 230000005284 excitation Effects 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000011324 bead Substances 0.000 claims description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 6
- 229940098773 bovine serum albumin Drugs 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 6
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 claims description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 2
- 239000003755 preservative agent Substances 0.000 claims description 2
- 239000004094 surface-active agent Substances 0.000 claims description 2
- 238000003149 assay kit Methods 0.000 claims 4
- 208000007466 Male Infertility Diseases 0.000 abstract description 4
- 238000003018 immunoassay Methods 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 3
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 206010003883 azoospermia Diseases 0.000 description 20
- 230000000414 obstructive effect Effects 0.000 description 17
- 238000006243 chemical reaction Methods 0.000 description 11
- 238000005406 washing Methods 0.000 description 10
- 238000003908 quality control method Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 102100040079 A-kinase anchor protein 4 Human genes 0.000 description 2
- 101000890604 Homo sapiens A-kinase anchor protein 4 Proteins 0.000 description 2
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000035558 fertility Effects 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 239000003163 gonadal steroid hormone Substances 0.000 description 2
- 238000007885 magnetic separation Methods 0.000 description 2
- 230000004899 motility Effects 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000009612 semen analysis Methods 0.000 description 2
- ZMYKITJYWFYRFJ-UHFFFAOYSA-N 4-oxo-4-(2-phenylethylamino)butanoic acid Chemical compound OC(=O)CCC(=O)NCCC1=CC=CC=C1 ZMYKITJYWFYRFJ-UHFFFAOYSA-N 0.000 description 1
- 102100025905 C-Jun-amino-terminal kinase-interacting protein 4 Human genes 0.000 description 1
- 102000008130 Cyclic AMP-Dependent Protein Kinases Human genes 0.000 description 1
- 108010049894 Cyclic AMP-Dependent Protein Kinases Proteins 0.000 description 1
- 101001076862 Homo sapiens C-Jun-amino-terminal kinase-interacting protein 4 Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 108010067479 inhibin B Proteins 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000101 novel biomarker Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229960003604 testosterone Drugs 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a preparation method and application of a chemiluminescence detection kit for quantitatively detecting the concentration of ProAKAP4, belonging to the field of in-vitro diagnostic reagents. The ProAKAP4 chemiluminescence detection kit is prepared by introducing ProAKAP4, the lakeluminous substance base solution of the kit is colored, the luminous value is in negative correlation with the ProAKAP4 antibody content, the concentration of ProAKAP4 in semen can be rapidly and quantitatively detected by a full-automatic chemiluminescence immunoassay analyzer, the sensitivity and the specificity of diagnosis of male infertility are remarkably improved, and the kit has a good prediction value on the result of assisted reproduction.
Description
Technical Field
The invention relates to the field of in-vitro diagnostic reagents, in particular to a preparation method and application of a chemiluminescence detection kit for quantitatively detecting the concentration of ProAKAP 4.
Background
Azoospermia means that there is no sperm during ejaculation, which accounts for 10-15% of male infertility cases and 60% of all azoospermia cases, and the azoospermia is classified into obstructive azoospermia OA and non-obstructive azoospermia NOA, and the treatment of obstructive azoospermia OA and non-obstructive azoospermia NOA corresponds to different treatment methods.
Currently, the evaluation of sperm quality in clinic mainly adopts: semen analysis and sex hormone serology tests. Semen analysis is the most common detection method, but the analysis result is influenced by subjective factors, has low precision and poor repeatability, and has limited value for male fertility evaluation and clinical application; however, the result of male assisted reproduction PESA/TESE cannot be predicted by sex hormone serological detection such as FSH, testosterone, inhibin B and the like; in addition, the two detection methods cannot distinguish obstructive azoospermia OA and non-obstructive azoospermia NOA and cannot treat the obstructive azoospermia NOA symptomatically, so that a novel biomarker is urgently needed clinically and is used for objectively evaluating male fertility, identifying obstructive azoospermia OA and non-obstructive azoospermia NOA and predicting the outcome of assisted reproduction.
Disclosure of Invention
The invention aims to provide a preparation method and application of a chemiluminescence detection kit capable of detecting the concentration of ProAKAP 4.
The technical scheme of the invention is as follows: a preparation method of a chemiluminescence detection kit for quantitatively detecting the concentration of ProAKAP4 comprises a reagent R1 and a reagent R2, wherein the reagent R1 is 0.01M PBS buffer solution containing a magnetic bead coating object of a magnetic bead coated anti-ProAKAP 4 antibody; reagent R2 was 0.01M PBS buffer containing acridinium ester labeled anti-ProAKAP 4 antibody.
In a further technical scheme, the reagent R1 and the reagent R2 are buffer solutions which are pH6.5-7.5 and contain surfactants, preservatives and proteins.
In a further technical scheme, the pH value of the reagent R1 is 7.2, and the reagent contains 0.1% Tween 20(v/v), 5% bovine serum albumin (w/v) and 0.5% Proclin300 (v/v).
In a further technical scheme, the pH value of the reagent R2 is 7.2, and the reagent contains 0.1% Tween 20(v/v), 5% bovine serum albumin (w/v) and 0.5% Proclin300 (v/v).
The technical scheme further comprises a chemiluminescent substrate solution A and a chemiluminescent substrate solution B, wherein the solution A is a pre-excitation solution, and the solution B is an excitation solution.
In a further technical scheme, the pre-excitation liquid is a hydrogen peroxide solution, and the excitation liquid is a sodium hydroxide solution.
The further technical scheme is that the kit prepared by the preparation method of the chemiluminescence detection kit for quantitatively detecting the concentration of ProAKAP4 is applied to the detection of the concentration of ProAKAP4 in semen.
The invention has the beneficial effects that:
ProAKAP4 is a protein precursor of the sperm-specific protein AKAP4 (a-kinase anchoring protein 4), maintains sperm structure, provides motility, gains sperm energy and fertilization, binds to the regulatory subunit of protein kinase a for activation, and is present only in the fibrous sheath of the major part of the sperm flagellum. ProAKAP4 was expressed in round sperm phase, and the concentration of ProAKAP4 varied from sperm to sperm, reflecting sperm functional motility and how sperm remain viable to the point of fertilization. ProAKAP4 is a good marker for assessing sperm quality. The concentration of ProAKAP4 negatively correlated with sperm DNA fragmentation rate. The ProAKAP4 chemiluminescence detection kit is prepared by introducing ProAKAP4, the lakeluminous substance base solution of the kit is colored, the luminous value is in negative correlation with the ProAKAP4 antibody content, the concentration of ProAKAP4 in semen can be rapidly and quantitatively detected by a full-automatic chemiluminescence immunoassay analyzer, the sensitivity and the specificity of diagnosis of male infertility are remarkably improved, and the kit has a good prediction value on the result of assisted reproduction.
Detailed Description
The invention will be further illustrated and understood by the following non-limiting examples.
The invention provides a preparation method of a chemiluminescence detection kit for quantitatively detecting the concentration of ProAKAP4, wherein the kit comprises a reagent R1, a reagent R2, a chemiluminescence substrate solution A and a chemiluminescence substrate solution B.
Wherein, the reagent R1 is 0.01M PBS buffer solution containing a magnetic bead coating material of a magnetic bead coated anti-ProAKAP 4 antibody; reagent R1 has a pH of 7.2 and contains 0.1% Tween 20(v/v), 5% bovine serum albumin (w/v) and 0.5% Proclin300 (v/v); reagent R2 is 0.01M PBS buffer solution containing acridinium ester labeled anti-ProAKAP 4 antibody; reagent R2 had a pH of 7.2 and contained 0.1% Tween 20(v/v), 5% bovine serum albumin (w/v) and 0.5% Proclin300 (v/v).
The liquid A adopts hydrogen peroxide solution as pre-excitation liquid, and the liquid B adopts sodium hydroxide solution as excitation liquid.
The action process of the kit is as follows: the ProAKAP4 chemiluminescence quantitative detection kit is used for detecting the concentration of ProAKAP4, a two-step double-antibody sandwich immunoassay method is adopted in the ProAKAP4 chemiluminescence quantitative detection kit, ProAKAP4 in serum to be detected is combined with an anti-ProAKAP 4 antibody coated on a magnetic bead and then is combined with an anti-ProAKAP 4 antibody marked by acridinium ester to form a magnetic bead coating-ProAKAP 4-reagent R2 sandwich type immune compound, a substrate is added to detect a luminescence value, and the concentration of ProAKAP4 in a sample is calculated through a standard curve.
The kit is used for detecting the semen of 5 obstructive azoospermia patients and the semen of 5 non-obstructive azoospermia patients, and concretely explains the operation process of the kit and the application of the diagnostic effect.
The specific operation process of the kit detection is as follows:
1) the kit is loaded into a full-automatic chemiluminescence immunoassay analyzer, and before the kit is loaded, the reagent R1 needs to be turned over slightly for about 30 times to ensure that magnetic bead particles are dispersed uniformly, and the reagent R1 does not need to be mixed uniformly after the reagent R1 is loaded for the first time;
2) installing the reagent R1 and the reagent R2 on a reagent rack, selecting a reagent position on an instrument operation interface, scanning a two-dimensional code on the reagent rack for information recording, and then placing the reagent rack into a reagent bin.
3) Preparing a ProAKAP4 calibrator and a ProAKAP4 quality control product according to the ProAKAP4 calibrator specification and the ProAKAP4 quality control product specification;
4) preparing a sample diluent according to a sample diluent specification;
5) preparing washing liquid according to the instruction of concentrated washing liquid;
6) preparing a substrate solution A and a substrate solution B according to a substrate solution specification;
7) calibration: and clicking the curve, selecting a ProAKAP4 project, scanning two-dimensional code information attached to the kit, and automatically generating the curve. Putting the ProAKAP4 calibrator on a sample rack, pushing the sample rack in, editing sample information on an operation interface, selecting ProAKAP4 items, making 2 multiple holes for each calibrator, setting the sample type as 'CA', and clicking 'operation' after determining.
8) And (3) detection: the sample is balanced to room temperature before use and is fully and uniformly mixed, the ProAKAP4 quality control products and the sample are placed on a sample rack and pushed into the sample rack, the sample information is edited on an operation interface, a ProAKAP4 project is selected, and the 'operation' is clicked after the determination. The system will perform the following operations: the total incubation time was controlled for 15 minutes,
(1) 20 samples (calibrator, quality control and sample) to be measured are transmitted to a liquid absorption point,
(2) the reaction cup is loaded into the operation channel,
(3) respectively sucking 30yL of the substance to be detected into the reaction cups,
(4) the reaction cup is transported to a reagent bin, 50yL reagent Rl is added,
(5) after shaking and mixing, the reaction cup is transported to an incubation bin and incubated for 10 minutes at 37 ℃,
(6) transporting the reaction cup to a washing channel for magnetic separation, washing the reaction mixture with a washing solution, repeating the magnetic separation-washing for 4 times,
(7) the cuvette was transported to the reagent station again, 50yL of reagent R2 was added,
(8) after shaking and mixing, the reaction cup is transported to an incubation bin and incubated for 5 minutes at 37 ℃,
(9) transporting the reaction cup to a washing channel for magnetic separation, washing the reaction mixture with a washing solution, repeating the magnetic separation-washing for 4 times,
(10) conveying the reaction cup to a substrate channel, adding 1OOyL substrate liquid A, shaking and mixing,
(11) conveying the reaction cup to a detection channel, grabbing the reaction cup to a detection bin, adding 1OOyL substrate solution B, immediately detecting a luminescent signal, and calculating the concentration of ProAKAP 4;
(12) and (5) grabbing the reaction cup to a waste bin.
9) And (4) judging a result:
and (3) generating a standard curve by using a four-parameter Logistic curve fitting data reduction method (4PLC, Y weighting), so as to obtain the corresponding ProAKAP4 concentration result range of the obstructive azoospermia OA and the non-obstructive azoospermia, and comparing the result range with the detection result of the chemiluminescence detection kit for quantitatively detecting the concentration of ProAKAP4, so that the obstructive azoospermia OA and the non-obstructive azoospermia can be distinguished and diagnosed. In conclusion, the proAKAP4 chemiluminescence detection kit obtained by the preparation method provided by the invention has good test stability, can be used for identifying obstructive azoospermia OA and non-obstructive azoospermia NOA in male infertility, and is a detection kit with excellent performance.
Claims (7)
1. A preparation method of a chemiluminescence detection kit for quantitatively detecting the concentration of ProAKAP4 is characterized in that the kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 is 0.01M PBS buffer solution containing a magnetic bead coating object of an anti-ProAKAP 4 antibody coated by magnetic beads; reagent R2 was 0.01M PBS buffer containing acridinium ester labeled anti-ProAKAP 4 antibody.
2. The method for preparing a chemiluminescent assay kit for quantitatively detecting the concentration of ProAKAP4 according to claim 1, wherein the method comprises the following steps: the reagent R1 and the reagent R2 are buffer solutions with pH6.5-7.5 and containing surfactants, preservatives and proteins.
3. The method for preparing a chemiluminescent assay kit for quantitatively detecting the concentration of ProAKAP4 according to claim 2, wherein the method comprises the following steps: the reagent R1 has a pH of 7.2 and contains 0.1% Tween 20(v/v), 5% bovine serum albumin (w/v) and 0.5% Proclin300 (v/v).
4. The method for preparing a chemiluminescent assay kit for quantitatively detecting the concentration of ProAKAP4 according to claim 2, wherein the method comprises the following steps: reagent R2 had a pH of 7.2 and contained 0.1% Tween 20(v/v), 5% bovine serum albumin (w/v) and 0.5% Proclin300 (v/v).
5. The method for preparing a chemiluminescent assay kit for quantitatively detecting the concentration of ProAKAP4 according to claim 1, wherein the method comprises the following steps: the chemiluminescent substrate solution also comprises a chemiluminescent substrate solution A and a chemiluminescent substrate solution B, wherein the solution A is a pre-excitation solution, and the solution B is an excitation solution.
6. The method for preparing the chemiluminescence detection kit for quantitatively detecting the concentration of ProAKAP4 according to claim 5, wherein the method comprises the following steps: the pre-excitation liquid is hydrogen peroxide solution, and the excitation liquid is sodium hydroxide solution.
7. Use of the kit prepared by the method for preparing the chemiluminescent detection kit for the quantitative detection of the concentration of proAKAP4 according to any one of claims 1 to 6 in the detection of the concentration of proAKAP4 in semen.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110094153.7A CN112630450A (en) | 2021-01-25 | 2021-01-25 | Preparation method and application of chemiluminescence detection kit for quantitatively detecting concentration of ProAKAP4 |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110094153.7A CN112630450A (en) | 2021-01-25 | 2021-01-25 | Preparation method and application of chemiluminescence detection kit for quantitatively detecting concentration of ProAKAP4 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160356785A1 (en) * | 2013-09-05 | 2016-12-08 | INSERM (Institut National de la Santé et de la Recherche Médicale | Methods for determining human sperm quality |
| CN109085363A (en) * | 2018-07-26 | 2018-12-25 | 苏州呼呼健康科技有限公司 | Detection reagent, detection kit and the application and detection method of AKAP4 antigen detection |
| CN110146692A (en) * | 2019-05-28 | 2019-08-20 | 迪瑞医疗科技股份有限公司 | One kind being based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system and detection kit |
| WO2020008403A1 (en) * | 2018-07-05 | 2020-01-09 | Spqi | Peptide suitable as standard for assaying proakap4, and corresponding assay kit and use |
-
2021
- 2021-01-25 CN CN202110094153.7A patent/CN112630450A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160356785A1 (en) * | 2013-09-05 | 2016-12-08 | INSERM (Institut National de la Santé et de la Recherche Médicale | Methods for determining human sperm quality |
| WO2020008403A1 (en) * | 2018-07-05 | 2020-01-09 | Spqi | Peptide suitable as standard for assaying proakap4, and corresponding assay kit and use |
| CN109085363A (en) * | 2018-07-26 | 2018-12-25 | 苏州呼呼健康科技有限公司 | Detection reagent, detection kit and the application and detection method of AKAP4 antigen detection |
| CN110146692A (en) * | 2019-05-28 | 2019-08-20 | 迪瑞医疗科技股份有限公司 | One kind being based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system and detection kit |
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