CN113358868A - Sperm acrosome enzyme chemiluminescence immunoassay kit and preparation method thereof - Google Patents
Sperm acrosome enzyme chemiluminescence immunoassay kit and preparation method thereof Download PDFInfo
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 114
- 238000003018 immunoassay Methods 0.000 title claims abstract description 54
- 238000002360 preparation method Methods 0.000 title claims abstract description 31
- 239000003550 marker Substances 0.000 claims abstract description 52
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- 108010090804 Streptavidin Proteins 0.000 claims abstract description 12
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- 238000002372 labelling Methods 0.000 claims description 17
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- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical group C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 claims description 7
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 7
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- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 claims description 6
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- 239000000427 antigen Substances 0.000 claims description 3
- 102000036639 antigens Human genes 0.000 claims description 3
- 108091007433 antigens Proteins 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 3
- 235000020958 biotin Nutrition 0.000 claims description 3
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- HUDPLKWXRLNSPC-UHFFFAOYSA-N 4-aminophthalhydrazide Chemical compound O=C1NNC(=O)C=2C1=CC(N)=CC=2 HUDPLKWXRLNSPC-UHFFFAOYSA-N 0.000 claims description 2
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 claims description 2
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims description 2
- 239000000047 product Substances 0.000 claims description 2
- 229910052707 ruthenium Inorganic materials 0.000 claims description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 2
- 229960001763 zinc sulfate Drugs 0.000 claims description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 2
- VIFYIFQGOLPNHA-UHFFFAOYSA-L zinc;dioxido-oxo-sulfanylidene-$l^{6}-sulfane Chemical compound [Zn+2].[O-]S([O-])(=O)=S VIFYIFQGOLPNHA-UHFFFAOYSA-L 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 23
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- DZBUGLKDJFMEHC-UHFFFAOYSA-O acridine;hydron Chemical compound C1=CC=CC2=CC3=CC=CC=C3[NH+]=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-O 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
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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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
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Abstract
The invention relates to a sperm acrosome enzyme chemiluminescence immunoassay kit and a preparation method thereof, belonging to the technical field of in vitro detection. The sperm acrosome enzyme chemiluminescence immunoassay kit comprises: the streptavidin is provided with a magnetic particle with a Tosyl functional group, a sperm acrosome enzyme antibody marked by a chemiluminescence marker and a sperm acrosome enzyme antibody marked by a coupling marker. The sperm acrosome enzyme chemiluminescence immunoassay kit can complete the detection of sperm acrosome enzyme by taking a full-automatic chemiluminescence immunoassay analyzer as a detection tool. The chemiluminescence immunoassay kit is matched with an instrument, so that the time required by clinical detection is shortened. The chemiluminescence immunoassay kit has high detection precision.
Description
Technical Field
The invention belongs to the technical field of in-vitro detection, and particularly relates to a sperm acrosome enzyme chemiluminescence immunoassay kit and a preparation method thereof.
Background
Male sterility refers to the childbearing age of couples, non-contraceptive sexual life, and the female is not naturally pregnant within one year due to male factors. Clinically, the fertility of men is generally evaluated by using semen conventional detection parameters. Although routine semen detection is meaningful, limitations exist. In order to make up for the deficiencies of the conventional semen detection, people search for some more valuable indexes, and sperm acrosome enzyme is one of the indexes. People find that the sperm acrosin is closely related to the concentration, the activity, the normal form rate and the like of the sperms, and has important application value for the diagnosis and the treatment of male infertility, and the low positive rate and the low activity of the sperm acrosin are main reasons for causing the male infertility. Therefore, the detection of sperm acrosin is of great significance.
The chemiluminescence immunoassay has the advantages of high sensitivity, accurate quantification, no radioactivity risk, small sample requirement and the like. Compared with the traditional detection method, the chemiluminescence immunoassay kit has the advantages that the combination of the antigen and the antibody is carried out under the similar liquid condition, and the reaction is quicker and more sensitive; on the other hand, the chemiluminescence immunoassay kit and the chemiluminescence immunoassay instrument form a closed system, and the adding and detecting tasks of the reagent and the sample are automatically completed by the instrument, so that the error of manual operation is reduced, and the sensitivity and the accuracy of the whole system are improved. The invention provides a sperm acrosome enzyme chemiluminescence immunoassay kit and a preparation method thereof.
Disclosure of Invention
The invention aims to provide a sperm acrosome enzyme chemiluminescence immunoassay kit and a preparation method thereof.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the invention provides a sperm acrosome enzyme chemiluminescence immunoassay kit, which comprises: r1, R2, and R3 reagents;
the R1 reagent is streptavidin magnetic particles with Tosyl functional groups;
the R2 reagent is a sperm acrosome enzyme antibody marked by a chemiluminescent marker;
the R3 reagent is a sperm acrosome enzyme antibody marked by a coupling marker.
In the above technical solution, the mass ratio of the R1 reagent is 0.02% to 1%.
In the technical scheme, in the R2 reagent, the labeling ratio of the sperm acrosome enzyme antibody to the chemiluminescent label is 1: 3-20; the buffer solution is 100-500 mM Bistris, 0.3-1.0M zinc compound, and the final concentration of sperm acrosome enzyme antibody is 0.1-1 mu g/mL.
In the technical scheme, in the R3 reagent, the labeling ratio of the sperm acrosome enzyme antibody to the conjugate label is 1: 5-20; the buffer solution is 100-500 mM Bistris, 0.3-1.0M zinc compound, and the final concentration of sperm acrosome enzyme antibody is 0.2-3 mug/mL.
In the technical scheme, in the R1 reagent, the particle size of the magnetic particles is 4-6 μm.
In the technical scheme, in the R2 reagent, the chemiluminescent marker is acridinium ester, luminol, isoluminol or ruthenium terpyridyl.
In the above technical scheme, in the R3 reagent, the conjugated label is biotin or antigen.
In the technical scheme, the zinc compound is zinc sulfate, zinc chloride or zinc thiosulfate.
In the technical scheme, the chemiluminescence immunoassay kit further comprises a sperm acrosin calibrator, and the concentrations of the sperm acrosin calibrator are 0.0pg/mL, 2.0pg/mL, 5.0pg/mL, 10.0pg/mL, 20.0pg/mL, 50.0pg/mL and 100.0pg/mL of the sperm acrosin protein solution.
The invention also provides a preparation method of the sperm acrosome enzyme chemiluminescence immunoassay kit, which comprises the following steps:
step 1, preparation of R1 reagent
Taking 0.5 mL (50mg) of streptavidin magnetic particle solution with Tosyl functional groups with the concentration of 100mg/mL, placing the solution on a magnetic separator until the supernatant is not turbid, discarding the supernatant, and reserving magnetic particles; after repeated washing for 3 times, preparing an R1 reagent with the magnetic bead concentration of 0.072% in a buffer solution of 100-500 mMBistris and the pH of 6-8;
step 2, preparation of R2 reagent
Putting 500 mu g of sperm acrosome enzyme antibody into a centrifuge tube, adding carbonic acid buffer solution after ensuring that the antibody is positioned at the bottom of the centrifuge tube, fully and uniformly mixing, adding DMF solution of a chemiluminescent marker according to the labeling ratio of the sperm acrosome enzyme antibody to the chemiluminescent marker of 1: 3-20, and carrying out sealing and purification steps to obtain a sperm acrosome enzyme chemiluminescent marker antibody; preparing a R2 reagent with the final antibody concentration of 0.1-2 mug/mL by using a buffer solution of 100-500 mM Bistris, 0.3-1.0M zinc compound and pH 6-8 for a sperm acrosome enzyme chemiluminescence marker antibody concentrated solution;
step 3, preparation of R3 reagent
Taking 500 mu g of sperm acrosome enzyme antibody, dialyzing and purifying TRIS buffer solution with pH of 6.5, adding a conjugate marker according to the labeling ratio of the sperm acrosome enzyme antibody to the conjugate marker of 1: 5-20, and carrying out sealing and purification steps to obtain a sperm acrosome enzyme antibody labeled by the conjugate marker; preparing a sperm acrosome enzyme antibody concentrated solution marked by a coupling marker into an R3 reagent with the antibody final concentration of 0.2-3 mu g/mL by using a buffer solution of 100-500 mM Bistris, 0.3-1.0M zinc compound and pH 6-8;
and assembling the R1, R2 and R3 reagents into a finished product reagent, namely a sperm acrosome enzyme chemiluminescence immunoassay kit. When the sperm acrosome enzyme chemiluminescence immunoassay kit is used for detecting sperm acrosome enzyme, a full-automatic chemiluminescence immunoassay analyzer is utilized to detect a sperm acrosome enzyme calibrator, a standard curve is drawn, and the calibration curve is embedded in computer software; then testing the clinical sample according to the requirement, and calculating the concentration of the sperm acrosome enzyme according to the light quantum number of the sample.
The invention has the beneficial effects that:
the sperm acrosome enzyme chemiluminescence immunoassay kit can complete the detection of sperm acrosome enzyme by taking a full-automatic chemiluminescence immunoassay analyzer as a detection tool. The chemiluminescence immunoassay kit is matched with an instrument, so that the time required by clinical detection is shortened. The chemiluminescence immunoassay kit has high detection precision.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a graph of the standard curve obtained in example 4, with the abscissa representing the concentration of the calibrator and the ordinate representing the number of photons of the reagent reaction, and the degree of fitting of the curve is 0.9900.
Detailed Description
In order to make the advantages, purposes and methods of the present invention more comprehensible, embodiments accompanying figures and specific examples are described in detail below. In the following description, numerous details are set forth to provide an understanding, but the present invention may be practiced otherwise than as described.
The invention provides a sperm acrosome enzyme chemiluminescence immunoassay kit, which comprises: r1, R2, and R3 reagents; the R1 reagent is streptavidin magnetic particles with Tosyl functional groups; the R2 reagent is a sperm acrosome enzyme antibody marked by a chemiluminescent marker; the R3 reagent is a sperm acrosome enzyme antibody marked by a coupling marker.
Preferably, the mass ratio of the R1 reagent-streptavidin magnetic particles with Tosyl functional groups is 0.02% to 1%, and more preferably, the streptavidin magnetic particles with Tosyl functional groups are prepared in a buffer solution to have a magnetic bead concentration of 0.072%.
Preferably, in the sperm acrosin antibody marked by the R2 reagent chemiluminescence marker, the marking ratio of the sperm acrosin antibody to the chemiluminescence marker is 1: 3-20, and more preferably the marking ratio is 1: 5; the buffer solution is 100-500 mM Bistris, 0.3-1.0M zinc compound, and the final concentration of sperm acrosome enzyme antibody is 0.1-1 mu g/mL; more preferably, the buffer solution is 100mM Bistris, 0.3-1.0M zinc chloride, and the final concentration of the antibody is 0.5 mu g/mL.
Preferably, in the sperm acrosome enzyme antibody marked by the R3 reagent coupling marker, the marking ratio of the sperm acrosome enzyme antibody to the coupling marker is 1: 5-20, and more preferably the marking ratio of the sperm acrosome enzyme antibody to the coupling marker is 1: 10; the buffer solution is 100-500 mM Bistris, 0.3-1.0M zinc compound, and the final concentration of sperm acrosome enzyme antibody is 0.2-3 mug/mL; more preferably, the buffer solution is 100mM Bistris, 0.3-1.0M zinc chloride, and the final concentration of the sperm acrosome enzyme antibody is 1.5 mu g/mL.
Preferably, the particle size of the R1 reagent-streptavidin magnetic particle is 4 to 6 μm, and more preferably 6 μm.
The chemiluminescence immunoassay kit also comprises a sperm acrosin calibrator, and the concentrations of the sperm acrosin calibrator are sperm acrosin protein solutions with the concentrations of 0.0pg/mL, 2.0pg/mL, 5.0pg/mL, 10.0pg/mL, 20.0pg/mL, 50.0pg/mL and 100.0 pg/mL.
When the sperm acrosome enzyme chemiluminescence immunoassay kit is used for detecting sperm acrosome enzyme, a full-automatic chemiluminescence immunoassay analyzer is utilized to detect a sperm acrosome enzyme calibrator, a standard curve is drawn, and the calibration curve is embedded in computer software; then testing the clinical sample according to the requirement, and calculating the concentration of the sperm acrosome enzyme according to the light quantum number of the sample.
The chemiluminescence immunoassay kit for detecting the sperm acrosin has the following advantages:
1. acridinium ester and the like are selected as the labeling materials of the chemiluminescence immunoassay system, the materials have the energy transition generated when the excited state returns to the ground state as direct chemiluminescence, the participation of enzyme is not needed, and the time and the cost are saved.
2. The streptavidin magnetic beads and the biotin-labeled antibody can be firmly combined together, so that nonspecific adsorption is reduced, the accuracy of a test sample is improved, and the anti-interference capability is strong.
3. The sperm acrosome enzyme chemiluminescence immunoassay kit is a full-automatic measurement sample, and directly gives a numerical value, reduces artificial operation errors, and realizes unattended operation.
4. The chemiluminescence immunoassay system of the acridinium ester has wide linear range.
5. The reagent and the instrument form a closed system, and the system error is small.
6. The sample is preserved for a longer time.
The chemiluminescence immunoassay kit can replace colloidal gold assay kits on the market in the aspect of accuracy, and provides faster and more accurate measurement data for customers.
Example 1 preparation of sperm acrosome enzyme chemiluminescence immunoassay kit:
preparation of R1 reagent: 0.5 mL (50mg) of a solution of streptavidin magnetic particles with Tosyl functional groups at a concentration of 100mg/mL was taken, placed on a magnetic separator until the supernatant was clear, discarded and the magnetic particles were retained. After 3 times of repeated washing, the reagent R1 with a magnetic bead concentration of 0.072% was prepared in 200mM Bistris, pH6.5 buffer.
Preparation of R2 reagent: putting 500 mu g of sperm acrosome enzyme antibody into a centrifuge tube, adding carbonic acid buffer solution after ensuring that the antibody is positioned at the bottom of the centrifuge tube, fully and uniformly mixing, adding a DMF (dimethyl formamide) solution of acridinium ester after uniformly mixing, and carrying out steps of sealing, purifying and the like to obtain the sperm acrosome enzyme chemiluminescent marker antibody. The sperm acrosome enzyme chemiluminescence marker antibody concentrated solution is prepared into an R2 reagent with the antibody final concentration of 0.1 mu g/mL by using 200mM Bistris, 0.3M zinc chloride and a buffer solution with the pH value of 6.5, wherein the labeling ratio of the sperm acrosome enzyme antibody to the chemiluminescence marker is 1: 3.
Preparation of R3 reagent: taking 500 mu g of sperm acrosin antibody, dialyzing and purifying TRIS buffer solution with pH of 6.5, adding activated long-chain sulfonated Biotin Sulfo-NHS-LC-Biotin, and carrying out steps such as blocking, purifying and the like to obtain the sperm acrosin coupled marker antibody. A sperm acrosome enzyme conjugate marker antibody concentrated solution is prepared into an R3 reagent with the antibody final concentration of 0.2 mu g/mL by using 200mM Bistris, 0.3M zinc chloride and a buffer solution with the pH value of 6.5, wherein the labeling ratio of the sperm acrosome enzyme antibody to the conjugate marker is 1: 20.
Example 2: preparation of sperm acrosome enzyme chemiluminescence immunoassay kit
Preparation of R1 reagent: 0.5 mL (50mg) of a solution of streptavidin magnetic particles with Tosyl functional groups at a concentration of 100mg/mL was taken, placed on a magnetic separator until the supernatant was clear, discarded and the magnetic particles were retained. After 3 times of repeated washing, R1 reagent with a bead concentration of 0.072% was prepared in 100mM Bistris, pH7 buffer.
Preparation of R2 reagent: putting 500 mu g of sperm acrosome enzyme antibody into a centrifuge tube, adding carbonic acid buffer solution after ensuring that the antibody is positioned at the bottom of the centrifuge tube, fully and uniformly mixing, adding a DMF (dimethyl formamide) solution of acridinium ester after uniformly mixing, and carrying out steps of sealing, purifying and the like to obtain the sperm acrosome enzyme chemiluminescent marker antibody. A sperm acrosome enzyme chemiluminescence marker antibody concentrated solution is prepared into an R2 reagent with the antibody final concentration of 1 mu g/mL by using a buffer solution of 100mM Bistris, 1.0M zinc chloride and pH7, wherein the labeling ratio of the sperm acrosome enzyme antibody to the chemiluminescence marker is 1: 5.
Preparation of R3 reagent: taking 500 mu g of sperm acrosin antibody, dialyzing and purifying TRIS buffer solution with pH of 6.5, adding activated long-chain sulfonated Biotin Sulfo-NHS-LC-Biotin, and carrying out steps such as blocking, purifying and the like to obtain the sperm acrosin coupled marker antibody. A sperm acrosome enzyme conjugated marker antibody concentrated solution is prepared into an R3 reagent with the antibody final concentration of 2 mu g/mL by using a buffer solution of 100mM Bistris, 1.0M zinc chloride and pH7, wherein the labeling ratio of the sperm acrosome enzyme antibody to the conjugated marker is 1: 10.
Example 3: preparation of sperm acrosome enzyme chemiluminescence immunoassay kit
Preparation of R1 reagent: 0.5 mL (50mg) of a solution of streptavidin magnetic particles with Tosyl functional groups at a concentration of 100mg/mL was taken, placed on a magnetic separator until the supernatant was clear, discarded and the magnetic particles were retained. After repeated washing for 3 times, R1 reagent with a magnetic bead concentration of 0.072% was prepared in a buffer solution of 500mM Bistris, 0.05% Tween-20, 0.1% Proclin300, pH 7.5.
Preparation of R2 reagent: putting 500 mu g of sperm acrosome enzyme antibody into a centrifuge tube, adding carbonic acid buffer solution after ensuring that the antibody is positioned at the bottom of the centrifuge tube, fully and uniformly mixing, adding a DMF (dimethyl formamide) solution of acridinium ester after uniformly mixing, and carrying out steps of sealing, purifying and the like to obtain the sperm acrosome enzyme chemiluminescent marker antibody. The sperm acrosome enzyme chemiluminescence label antibody concentrated solution is prepared into an R2 reagent with the antibody final concentration of 0.5 mu g/mL by using a buffer solution of 500mM Bistris, 0.5M zinc chloride and pH8, wherein the labeling ratio of the sperm acrosome enzyme antibody to the chemiluminescence label is 1: 10.
Preparation of R3 reagent: taking 500 mu g of sperm acrosin antibody, dialyzing and purifying TRIS buffer solution with pH of 6.5, adding activated long-chain sulfonated Biotin Sulfo-NHS-LC-Biotin, and carrying out steps such as blocking, purifying and the like to obtain the sperm acrosin coupled marker antibody. The sperm acrosome enzyme conjugated marker antibody concentrated solution is prepared into an R3 reagent with the antibody final concentration of 1.5 mu g/mL by using 500mM Bistris, 0.5M zinc chloride and a buffer solution with the pH of 8, wherein the labeling ratio of the sperm acrosome enzyme antibody to the conjugated marker is 1: 5.
Example 4: preparation of sperm acrosome enzyme chemiluminescence immunoassay kit
Preparation of R1 reagent: 0.5 mL (50mg) of a solution of streptavidin magnetic particles with Tosyl functional groups at a concentration of 100mg/mL was taken, placed on a magnetic separator until the supernatant was clear, discarded and the magnetic particles were retained. After 3 times of washing, R1 reagent with a magnetic bead concentration of 0.072% was prepared in 200mM Tris, pH6.5 buffer.
Preparation of R2 reagent: putting 500 mu g of sperm acrosome enzyme antibody into a centrifuge tube, adding carbonic acid buffer solution after ensuring that the antibody is positioned at the bottom of the centrifuge tube, fully and uniformly mixing, adding a DMF (dimethyl formamide) solution of acridinium ester after uniformly mixing, and carrying out steps of sealing, purifying and the like to obtain the sperm acrosome enzyme chemiluminescent marker antibody. A concentrated solution of the sperm acrosome enzyme chemiluminescent marker antibody is prepared into an R2 reagent with the antibody final concentration of 0.1 mu g/mL by using a buffer solution of 200mM Bistris and pH6.5, wherein the labeling ratio of the sperm acrosome enzyme antibody to the chemiluminescent marker is 1: 20.
Preparation of R3 reagent: taking 500 mu g of sperm acrosin antibody, dialyzing and purifying TRIS buffer solution with pH of 6.5, adding activated long-chain sulfonated Biotin Sulfo-NHS-LC-Biotin, and carrying out steps such as blocking, purifying and the like to obtain the sperm acrosin coupled marker antibody. A concentrated solution of the antibody of the sperm acrosome enzyme conjugate marker is prepared into an R3 reagent with the final antibody concentration of 0.2 mu g/mL by using a buffer solution of 200mM Bistris and pH6.5, wherein the labeling ratio of the sperm acrosome enzyme antibody to the conjugate marker is 1: 5.
Example 5: sperm acrosome enzyme chemiluminescence immune detection kit performance evaluation
The linear detection was performed on the kit assembled in example 1: the concentrations of the calibrators were 0.0pg/mL, 2.0pg/mL, 5.0pg/mL, 10.0pg/mL, 20.0pg/mL, 50.0pg/mL, and 100.0pg/mL, and the light quantum number were plotted as a standard curve, and the linear correlation coefficient r was 0.9999.
| Concentration of | Quantum number of light |
| 0 | 110 |
| 2.0 | 779 |
| 5.0 | 2824 |
| 10.0 | 9208 |
| 20.0 | 26908 |
| 50.0 | 114457 |
| 100.0 | 289183 |
The definition of the analysis sensitivity is that the light quantum number is measured for 20 times on a zero value calibrator, the average value of the light quantum number is taken and two times of standard deviation is added, and the light quantum number is taken into a straight line formed by the zero value calibrator and adjacent calibration to obtain the sensitivity; and calculating the sensitivity of the sperm acrosome enzyme chemiluminescence immunoassay kit to be 0.80 pg/mL.
Example 6: sperm acrosome enzyme chemiluminescence immune detection kit performance evaluation
The linear detection was performed on the kit assembled in example 2: and (3) making a standard curve of the concentrations of the calibrators of 0.0pg/mL, 2.0pg/mL, 5.0pg/mL, 10.0pg/mL, 20.0pg/mL, 50.0pg/mL and 100.0pg/mL and the light quantum number to obtain a linear correlation coefficient r of 0.9997, wherein the linear range is 0.5-100 pg/mL.
| Concentration of | Quantum number of light |
| 0 | 102 |
| 2.0 | 805 |
| 5.0 | 2652 |
| 10.0 | 9366 |
| 20.0 | 26327 |
| 50.0 | 115569 |
| 100.0 | 288564 |
The definition of the analysis sensitivity is that the light quantum number is measured for 20 times on a zero value calibrator, the average value of the light quantum number is taken and two times of standard deviation is added, and the light quantum number is taken into a straight line formed by the zero value calibrator and adjacent calibration to obtain the sensitivity; the sensitivity of the sperm acrosome enzyme chemiluminescence immunoassay kit is calculated to be 0.51 pg/mL.
Example 7: sperm acrosome enzyme chemiluminescence immune detection kit performance evaluation
The linear detection was performed on the kit assembled in example 3: and (3) making standard curves of the concentrations of the calibrators of 0.0pg/mL, 2.0pg/mL, 5.0pg/mL, 10.0pg/mL, 20.0pg/mL, 50.0pg/mL and 100.0pg/mL and the light quantum number to obtain a linear correlation coefficient r of 0.9993, wherein the linear range is 0.5-100 pg/mL.
The definition of the analysis sensitivity is that the light quantum number is measured for 20 times on a zero value calibrator, the average value of the light quantum number is taken and two times of standard deviation is added, and the light quantum number is taken into a straight line formed by the zero value calibrator and adjacent calibration to obtain the sensitivity; and calculating the sensitivity of the sperm acrosome enzyme chemiluminescence immunoassay kit to be 0.20 pg/mL.
Example 8: sperm acrosome enzyme chemiluminescence immune detection kit performance evaluation
The linear detection was performed on the kit assembled in example 4: and (3) making a standard curve of the concentrations of the calibrators of 0.0pg/mL, 2.0pg/mL, 5.0pg/mL, 10.0pg/mL, 20.0pg/mL, 50.0pg/mL and 100.0pg/mL and the light quantum number (see the figure 1) to obtain a linear correlation coefficient r which is 0.9983 and the linear range of 0.5-100 pg/mL.
| Concentration of | Quantum number of light |
| 0 | 236 |
| 2.0 | 1036 |
| 5.0 | 2949 |
| 10.0 | 9987 |
| 20.0 | 27521 |
| 50.0 | 122697 |
| 100.0 | 277456 |
The definition of the analysis sensitivity is that the light quantum number is measured for 20 times on a zero value calibrator, the average value of the light quantum number is taken and two times of standard deviation is added, and the light quantum number is taken into a straight line formed by the zero value calibrator and adjacent calibration to obtain the sensitivity; and calculating the sensitivity of the sperm acrosome enzyme chemiluminescence immunoassay kit to be 0.20 pg/mL.
Example 9: validity period real-time stability verification of sperm acrosome enzyme chemiluminescence immunoassay kit
The kit assembled in the above examples 1 to 4 was used to test three identical lyophilized samples every four months to verify the real-time stability.
As can be seen from the above examples 1-3, the test calibrator of the example added with zinc chloride has better linear correlation and sensitivity; the real-time stability at 12 months of life is also better than example 4 without the addition of zinc chloride: the deviations were within. + -. 10%, while those of example 4 without zinc chloride were within. + -. 15%. Therefore, zinc chloride is an important component of the present invention, and may affect the shelf life stability of the kit of the present invention.
The chemiluminescent label, the conjugated label and the zinc compound in the above embodiments may be replaced by other substances defined above, and the above technical effects may also be achieved, which are not illustrated here.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A sperm acrosome enzyme chemiluminescence immunoassay kit, comprising: r1, R2, and R3 reagents; it is characterized in that the preparation method is characterized in that,
the R1 reagent is streptavidin magnetic particles with Tosyl functional groups;
the R2 reagent is a sperm acrosome enzyme antibody marked by a chemiluminescent marker;
the R3 reagent is a sperm acrosome enzyme antibody marked by a coupling marker.
2. The sperm acrosome enzyme chemiluminescence immunoassay kit according to claim 1, wherein the mass ratio of the R1 reagent is 0.02% to 1%.
3. The sperm acrosome enzyme chemiluminescence immunoassay kit of claim 1, wherein in the R2 reagent, the labeling ratio of sperm acrosome enzyme antibody to chemiluminescence label is 1: 3-20; the buffer solution is 100-500 mM Bistris, 0.3-1.0M zinc compound, and the final concentration of sperm acrosome enzyme antibody is 0.1-1 mu g/mL.
4. The sperm acrosome enzyme chemiluminescence immunoassay kit according to claim 1, wherein in the R3 reagent, the labeling ratio of the sperm acrosome enzyme antibody to the conjugate label is 1: 5-20; the buffer solution is 100-500 mM Bistris, 0.3-1.0M zinc compound, and the final concentration of sperm acrosome enzyme antibody is 0.2-3 mug/mL.
5. The sperm acrosome enzyme chemiluminescence immunoassay kit of claim 1, wherein in the R1 reagent, the magnetic particles have a particle size of 4-6 μm.
6. The sperm acrosome enzyme chemiluminescent immunoassay kit of claim 1, wherein the chemiluminescent label of the R2 reagent is acridinium ester, luminol, isoluminol or ruthenium terpyridyl.
7. The sperm acrosome enzyme chemiluminescent immunoassay kit of claim 1, wherein the conjugate label of the R3 reagent is biotin or an antigen.
8. The sperm acrosome enzyme chemiluminescence immunoassay kit of claim 3 or 4, wherein the zinc compound is zinc sulfate, zinc chloride or zinc thiosulfate.
9. The sperm acrosome enzyme chemiluminescent immunoassay kit of claim 1 further comprising a sperm acrosome enzyme calibrator having a concentration of a sperm acrosome enzyme protein solution of 0.0pg/mL, 2.0pg/mL, 5.0pg/mL, 10.0pg/mL, 20.0pg/mL, 50.0pg/mL and 100.0 pg/mL.
10. A preparation method of a sperm acrosome enzyme chemiluminescence immunoassay kit is characterized by comprising the following steps:
step 1, preparation of R1 reagent
Taking 0.5 mL (50mg) of streptavidin magnetic particle solution with Tosyl functional groups with the concentration of 100mg/mL, placing the solution on a magnetic separator until the supernatant is not turbid, discarding the supernatant, and reserving magnetic particles; after repeated washing for 3 times, preparing an R1 reagent with the magnetic bead concentration of 0.072% in a buffer solution with the pH of 6-8 and the concentration of 100-500 mM Bistris;
step 2, preparation of R2 reagent
Putting 500 mu g of sperm acrosome enzyme antibody into a centrifuge tube, adding carbonic acid buffer solution after ensuring that the antibody is positioned at the bottom of the centrifuge tube, fully and uniformly mixing, adding DMF solution of a chemiluminescent marker according to the labeling ratio of the sperm acrosome enzyme antibody to the chemiluminescent marker of 1: 3-20, and carrying out sealing and purification steps to obtain a sperm acrosome enzyme chemiluminescent marker antibody; preparing a R2 reagent with the final antibody concentration of 0.1-2 mug/mL by using a buffer solution of 100-500 mM Bistris, 0.3-1.0M zinc compound and pH 6-8 for a sperm acrosome enzyme chemiluminescence marker antibody concentrated solution;
step 3, preparation of R3 reagent
Taking 500 mu g of sperm acrosome enzyme antibody, dialyzing and purifying TRIS buffer solution with pH of 6.5, adding a conjugate marker according to the labeling ratio of the sperm acrosome enzyme antibody to the conjugate marker of 1: 5-20, and carrying out sealing and purification steps to obtain a sperm acrosome enzyme antibody labeled by the conjugate marker; preparing a sperm acrosome enzyme antibody concentrated solution marked by a coupling marker into an R3 reagent with the antibody final concentration of 0.2-3 mu g/mL by using a buffer solution of 100-500 mM Bistris, 0.3-1.0M zinc compound and pH 6-8;
and assembling the R1, R2 and R3 reagents into a finished product reagent, namely a sperm acrosome enzyme chemiluminescence immunoassay kit.
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