CN114487445B - Chorionic gonadotropin pre-coated ELISA plate, preparation method thereof, method for quantitatively detecting HCG in human urine and kit - Google Patents
Chorionic gonadotropin pre-coated ELISA plate, preparation method thereof, method for quantitatively detecting HCG in human urine and kit Download PDFInfo
- Publication number
- CN114487445B CN114487445B CN202011157485.7A CN202011157485A CN114487445B CN 114487445 B CN114487445 B CN 114487445B CN 202011157485 A CN202011157485 A CN 202011157485A CN 114487445 B CN114487445 B CN 114487445B
- Authority
- CN
- China
- Prior art keywords
- elisa plate
- hcg
- solution
- concentration
- chorionic gonadotrophin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Endocrinology (AREA)
- Reproductive Health (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明公开了一种绒毛膜促性腺激素预包被酶标板、其制备方法、定量检测人尿液中HCG的方法和试剂盒，其涉及酶联免疫分析技术领域。该制备方法包括：将加有HCG单克隆抗体的酶标板进行孵育、洗板、封闭和烘干；其中，HCG单克隆抗体中HCG抗体的浓度为2‑3μg/ml。该制备方法能够大批量的制备稳定性佳、保质期长的绒毛膜促性腺激素预包被酶标板，便于后续实验和检测使用，提升了分析效率。此外，本申请的方法可以对尿液中的HCG的含量进行大规模的检测，快速且成本低，灵敏度、准确度和重复性高。本发明的试剂盒，其包含了本申请的方法中涉及到的各种试剂，从而简化了人尿液中HCG的定量检测。The present invention discloses a chorionic gonadotropin pre-coated ELISA plate, a preparation method thereof, a method for quantitatively detecting HCG in human urine, and a kit, which relate to the technical field of enzyme-linked immunosorbent assay. The preparation method comprises: incubating, washing, sealing and drying an ELISA plate to which HCG monoclonal antibodies are added; wherein the concentration of HCG antibodies in the HCG monoclonal antibodies is 2-3 μg/ml. The preparation method can prepare chorionic gonadotropin pre-coated ELISA plates with good stability and long shelf life in large quantities, which is convenient for subsequent experiments and detections, and improves analysis efficiency. In addition, the method of the present application can perform large-scale detection of the content of HCG in urine, which is fast and low in cost, and has high sensitivity, accuracy and repeatability. The kit of the present invention comprises various reagents involved in the method of the present application, thereby simplifying the quantitative detection of HCG in human urine.
Description
Technical Field
The invention relates to the technical field of enzyme-linked immunoassay, in particular to a chorionic gonadotrophin pre-coated ELISA plate, a preparation method thereof, a method for quantitatively detecting HCG in human urine and a kit.
Background
The quantitative detection of HCG is an important link in the production of the medicine by a plurality of biological medicine enterprises, and the current technology for detecting HCG in urine comprises a colloidal gold method, an enzyme-linked immunoassay method, a radioimmunoassay method, a chemiluminescent immunoassay method, a fluorescent magnetic particle enzyme-linked immunoassay method, a time-resolved fluoroimmunoassay method and the like. However, the means commonly used for pharmaceutical enterprises include 3 kinds of methods, such as a colloidal gold method, an enzyme-linked immunoassay method and a fluorescent magnetic particle enzyme-linked immunoassay method (fully automatic fluorescent magnetic particle enzyme-linked immunoassay instrument AIA-360 of Tosoh Co., ltd., japan): the colloidal gold method can be used for large-scale and rapid detection, and has low cost, but can not be quantified, has low sensitivity, poor stability, limited detection range and low repeatability; the ELISA method can be used for rapid large-scale detection, and has low cost, but insufficient accuracy and deviation; the fluorescent magnetic particle ELISA method has high accuracy and repeatability, but the equipment and consumable materials have high cost, and large-scale detection cannot be performed.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a preparation method of a chorionic gonadotrophin precoating ELISA plate, which can prepare the chorionic gonadotrophin precoating ELISA plate with good stability and long quality guarantee period in a large scale, is convenient for subsequent experiments and detection and use, greatly saves time and operation cost, and improves analysis efficiency.
The invention aims to provide a method for quantitatively detecting HCG in human urine, which can detect the content of HCG in urine on a large scale, and has the advantages of rapidness, low cost, high sensitivity, high accuracy and high repeatability.
The application aims to provide a kit for quantitatively detecting HCG in human urine, which comprises various reagents involved in the method, so that the quantitative detection of HCG in human urine is simplified.
The invention is realized in the following way:
In a first aspect, an embodiment of the present invention provides a method for preparing a chorionic gonadotrophin precoated elisa plate, comprising:
Placing an ELISA plate added with an HCG monoclonal antibody at 2-8 ℃ for heat preservation for 8-12 hours, then placing the ELISA plate at 36-38 ℃ for incubation for 1-2 hours, washing the incubated ELISA plate with a washing solution, adding a sealing solution for sealing, incubating the ELISA plate at 36-38 ℃ for 1-2 hours, and then removing the sealing solution;
wherein the concentration of HCG antibody in the HCG monoclonal antibody is 2-3 mug/ml.
In an alternative embodiment, the HCG monoclonal antibody is obtained by diluting the HCG antibody with a coating buffer;
Preferably, the coating buffer is a mixed aqueous solution containing anhydrous disodium hydrogen phosphate, sodium chloride, potassium chloride, monopotassium phosphate and a preservative; wherein the concentration of anhydrous disodium hydrogen phosphate in the coating buffer solution is 1-2g/L, the concentration of sodium chloride is 7.5-8.5g/L, the concentration of potassium chloride is 0.1-0.3g/L, the concentration of monopotassium phosphate is 0.2-0.3g/L, and the concentration of preservative is 0.4-0.6ml/L.
In an alternative embodiment, the blocking solution is prepared by mixing sucrose, bovine serum albumin with a blocking buffer, wherein the concentration of sucrose is 8-12g/ml and the concentration of bovine serum albumin is 0.4-0.6g/ml;
The sealing buffer solution is a mixed aqueous solution of anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate and preservative, wherein the concentration of the anhydrous disodium hydrogen phosphate in the sealing buffer solution is 5-6g/L, the concentration of the sodium dihydrogen phosphate dihydrate is 1-2g/L, and the concentration of the preservative is 0.4-0.6ml/L.
In an alternative embodiment, removing the blocking fluid from the microplate comprises: firstly reversely buckling the ELISA plate to drain the sealing liquid, and then placing the ELISA plate at 36-38 ℃ for drying for 2-3h;
Preferably, after the elisa plate is dried, the method further comprises packaging and storing the elisa plate: and wrapping each dried ELISA plate and 2-3 bags of drying agent with a preservative film for 4-6 layers, and then storing at 2-8 ℃ for 1 month or below-10 ℃ for 6 months.
In an alternative embodiment, drying the elisa plate comprises: drying at 36-38deg.C for 2-3 hr.
In a second aspect, the embodiment of the invention provides a chorionic gonadotrophin precoated elisa plate prepared by the method for preparing a chorionic gonadotrophin precoated elisa plate according to any of the previous embodiments.
In a third aspect, embodiments of the present invention provide a method for quantitatively detecting HCG in human urine, comprising: analysis using a chorionic gonadotrophin pre-coated elisa plate according to any of the previous embodiments;
Preferably, adding an HRP-labeled HCG antibody diluent into the chorionic gonadotrophin pre-coated ELISA plate prepared by the preparation method of the chorionic gonadotrophin pre-coated ELISA plate;
Respectively adding HCG standard substance solutions with different concentrations and urine sample diluents with different concentrations into the chorionic gonadotrophin pre-coated ELISA plate for incubation, plate washing, color development and termination, and then measuring absorbance values;
The read result is then processed: sorting the read results from small to large according to OD values, selecting 6 urine samples with gradient OD values, detecting the immune titer of the HCG by using a fluorescent magnetic particle ELISA analyzer, taking the OD value of the urine sample as an abscissa and the immune titer as an ordinate as a standard curve, and calculating a curve equation;
Wherein the HRP-labeled HCG antibody diluent is prepared by diluting an HRP-labeled HCG antibody solution with a labeled antibody diluent in a ratio of 1:1400-1600; the concentration of the urine sample in the urine sample diluent is 8-12 mu l/ml.
In an alternative embodiment, the HCG standard solution is obtained by diluting HCG standard to different gradient concentrations, wherein the HCG standard is diluted to a solution having an HCG titer of 9000-11000mIU/ml using a product of known titer.
In an alternative embodiment, the urine sample diluent is further added with a colorant with the concentration of 1-3 per mill;
preferably, the colorant is a blue food colorant.
In an alternative embodiment, the developing the elisa plate comprises: adding a color development liquid into the ELISA plate and incubating for 10-15min at 36-38 ℃;
Preferably, the color development liquid comprises 0.4-0.6ml of substrate solution A, 9.4-9.6ml of substrate solution B and 40-45 mu l of substrate solution C;
Wherein the substrate solution A is a mixed solution of 3, 5-tetramethyl benzidine and absolute ethyl alcohol, and the concentration of the 3, 5-tetramethyl benzidine is 1-3mg/ml;
The substrate solution B is a mixed aqueous solution of anhydrous disodium hydrogen phosphate and citric acid, the concentration of the anhydrous disodium hydrogen phosphate in the substrate solution B is 7-8g/L, and the concentration of the citric acid is 4-6g/L;
The volume concentration of the substrate solution C is 25-35% H 2O2 and water according to the following ratio of 1: 35-45% of diluted aqueous solution.
A fourth method, the present embodiment provides a kit for quantitatively detecting HCG in human urine, comprising a chorionic gonadotrophin pre-coated elisa plate according to any of the previous embodiments;
Preferably, the kit further comprises an HRP-labeled HCG antibody, a washing solution, a color development solution and a stop solution.
The invention has the following beneficial effects:
The preparation method of the chorionic gonadotrophin precoating ELISA plate provided by the application can prepare and obtain the chorionic gonadotrophin precoating ELISA plate with long shelf life and stable product, and a large number of chorionic gonadotrophin precoating ELISA plates are manufactured at one time, so that the subsequent experiments and detection use are facilitated, the time and the operation cost are greatly saved, and the analysis efficiency is improved. Because the titer of chorionic gonadotrophin in urine is low, the chorionic gonadotrophin pre-coated ELISA plate provided by the application can be detected under the condition of low concentration (2-3 mug/ml) of HCG antibody, and the chorionic gonadotrophin pre-coated ELISA plate provided by the application has high sensitivity. Furthermore, the method for quantitatively detecting HCG in human urine provided by the application combines the fluorescent magnetic particle ELISA method and the double-antibody sandwich ELISA method, and solves the problems of incapability of quantification, low sensitivity, poor stability, limited detection range and low repeatability compared with a colloidal gold method; compared with an independent ELISA method, the method solves the problems of insufficient accuracy and deviation; compared with a single fluorescent magnetic particle ELISA method and other tip detection technologies, the method solves the problems of high cost and incapability of large-scale detection; by combining the conditions, the method provided by the application can be used for detecting the HCG content in urine on a large scale, is quick and low in cost (96 plates are taken as an example, the average cost per hole is about 0.434 yuan), and has high sensitivity, accuracy and repeatability. The advantages are obvious. In addition, the method has important significance in diagnosing, monitoring and treating various diseases of human bodies and the like for a plurality of medical and health departments. The kit for quantitatively detecting the HCG in the human urine provided by the application comprises various reagents involved in the method, so that the quantitative detection of the HCG in the human urine is simplified.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The application provides a chorionic gonadotrophin pre-coated ELISA plate, a preparation method thereof, a method for quantitatively detecting HCG in human urine and a kit.
Wherein, the preparation method of the chorionic gonadotrophin precoating ELISA plate comprises the following steps: placing the ELISA plate added with the HCG monoclonal antibody at 2-8 ℃ for heat preservation for 8-12 hours, then placing the ELISA plate at 36-38 ℃ for incubation for 1-2 hours, washing the incubated ELISA plate with a washing solution, adding a sealing solution for sealing, incubating the ELISA plate at 36-38 ℃ for 1-2 hours, and then removing the sealing solution in the ELISA plate; wherein the concentration of HCG antibody in the HCG monoclonal antibody is 2-3 μg/ml.
Specifically, the preparation method of the chorionic gonadotrophin precoated ELISA plate comprises the following steps:
S101, preparation of reagent
HCG coated antibody split charging: the newly purchased HCG antibody (ABBEST; anti-h HCG McAb, product number 031047001 of Bowman biosciences, zhengzhou) is dissolved at room temperature, the packaging volume is calculated according to the concentration of 240 mug/count (coated 8 ELISA plates/count) according to the labeled concentration, packaging is carried out, the packaging is finished, the label is made, and the product is preserved below-10 ℃ and used in the effective period.
Coating buffer solution: the coating buffer solution is a mixed aqueous solution containing anhydrous disodium hydrogen phosphate, sodium chloride, potassium chloride, monopotassium phosphate and a preservative; wherein, the concentration of anhydrous disodium hydrogen phosphate in the coating buffer solution is 1-2g/L, the concentration of sodium chloride is 7.5-8.5g/L, the concentration of potassium chloride is 0.1-0.3g/L, the concentration of monopotassium phosphate is 0.2-0.3g/L, and the concentration of preservative is 0.4-0.6ml/L.
As a typical but non-limiting example, the preparation method of the coating buffer is: accurately weighing 1.44g of anhydrous disodium hydrogen phosphate, 7.9g of sodium chloride, 0.2g of potassium chloride and 0.24g of monopotassium phosphate, adding 800ml of purified water, stirring until the mixture is completely dissolved, adding 0.5ml (concentration of 0.05%) of proclin 300 preservative (ZB 116) into the mixture, fixing the volume to 1000ml, shaking the mixture uniformly, and storing the mixture at 2-8 ℃ for 1 month of effective period.
Blocking buffer: the buffer solution is a mixed aqueous solution of anhydrous disodium hydrogen phosphate, dihydrate sodium dihydrogen phosphate and preservative, wherein the concentration of the anhydrous disodium hydrogen phosphate in the sealing buffer solution is 5-6g/L, the concentration of the dihydrate sodium dihydrogen phosphate is 1-2g/L, and the concentration of the preservative is 0.4-0.6ml/L.
As a typical but non-limiting example, the blocking buffer is prepared by: accurately weighing 5.75g of anhydrous disodium hydrogen phosphate, 1.3g of sodium dihydrogen phosphate dihydrate, adding 800ml of purified water, stirring until the solution is completely dissolved, adding 0.5ml (concentration 0.05%) of proclin 300 preservative (flash organism, product number: ZB 116), fixing the volume to 1000ml, shaking uniformly, and preserving at 2-8 ℃ for 1 month of effective period.
Sealing liquid: is prepared by mixing sucrose, bovine serum albumin and a blocking buffer solution, wherein the concentration of sucrose is 8-12g/ml, and the concentration of bovine serum albumin is 0.4-0.6g/ml.
As a typical but non-limiting example, the method of preparing the sealing liquid is: 30g (concentration 10%) of sucrose, 1.5g (concentration 0.5%) of Bovine Serum Albumin (BSA) are accurately weighed, 300ml of blocking buffer is added, and the mixture is stirred until the mixture is completely dissolved, thus obtaining the finished product which is ready to be used.
Phosphate buffer stock (10×pbs): as a typical but non-limiting example, 14.38g of anhydrous disodium hydrogen phosphate, 79g of sodium chloride, 2g of potassium chloride and 2.4g of monopotassium phosphate are accurately weighed, 800ml of purified water is added, and the mixture is stirred until the mixture is completely dissolved, is stirred to 1000ml in volume, is uniformly shaken, and is stored at room temperature for 3 days in the effective period.
Washing solution (PBST): as a typical but non-limiting example, 400ml of phosphate buffer mother liquor, 3600ml of purified water and 4ml (0.1%) of tween-20 are measured, and the mixture is uniformly mixed and stirred to obtain the product which is prepared and used at present.
It will be appreciated that the amounts of the above agents may be varied or adjusted within certain limits in accordance with the proportions described above.
S102, preparation of pre-coated ELISA plates
Preparation: taking 16 new ELISA plates (high adsorption transparent ELISA plates, CIH-F8T, of Severe Biotechnology Co., ltd.) and marking each plate (category, date and number) for later use;
Dilution of HCG monoclonal antibodies: the coating buffer (160 ml was required for 16 pieces) was taken at 10ml per ELISA plate and added to the beaker. Taking out 2 pieces of HCG antibody (coating), melting at room temperature, adding into coating buffer solution, washing with coating buffer solution for 2 times, ensuring complete transfer, no residue, and stirring. The diluted antibody is HCG monoclonal antibody, and the concentration of HCG antibody in the coated HCG monoclonal antibody is 2-3 mug/ml (preferably 3 mug/ml).
Adding HCG monoclonal antibody: taking diluted HCG antibody solution, sequentially adding the diluted HCG antibody solution into 16 prepared blank ELISA plates according to 100 μl/hole by using an 8-channel pipette, wherein 8 blocks are divided into 2 groups; after each addition, checking whether the addition is missed or excessive, packaging with fresh-keeping film, and placing into refrigerator at 2-8deg.C overnight (at least 8 hr, preferably 8-12 hr).
Incubation: the next day, the ELISA plate which is at 2-8 ℃ overnight is taken out and put into a constant temperature incubator which is constant temperature to 36-38 ℃ for incubation for 1-2h. Every 8 blocks are in a group, 2 groups are added, the 1 st group is firstly put in, and the 2 nd group is put in after 30-50min interval.
Washing the plate: after the incubation time, the ELISA plate was removed, washed with 400. Mu.l wash solution/well and 2 times/plate with a plate washer. After washing, the ELISA plate is reversely buckled on clean water-absorbing filter paper, the ELISA plate is firmly beaten on the filter paper by holding the ELISA plate by hand for 2-3 times, and no residual liquid is observed in the ELISA plate.
Closing: taking the prepared sealing liquid, sequentially adding the sealing liquid into the washed ELISA plates by using an 8-channel pipetting gun according to 180 mu l/hole, wherein 8 blocks are divided into 2 groups, checking once every adding one group, packaging by using a preservative film after confirming no errors, and placing the sealing liquid into a constant-temperature incubator at the temperature of 36-38 ℃ for incubation for 2-3 hours.
And (3) drying: and taking out the ELISA plate after the incubation time is up, throwing off the sealing liquid therein by force, reversely buckling the ELISA plate on clean water-absorbing filter paper, holding the ELISA plate by hand, beating the ELISA plate on the filter paper by force, and observing that no residual liquid exists in the ELISA plate. And then inversely buckling the ELISA plate in a constant-temperature incubator, swinging the ELISA plate out, drying at 36-38 ℃ for about 2 hours, and observing that no visible liquid exists in the holes of the ELISA plate.
S103, packaging and storing
Taking the dried ELISA plates, wrapping 5 layers of the ELISA plates together with 2 bags of drying agents by using a preservative film, and ensuring the wrapping to be tight and airtight. Storing for a short period (after the use in 1 month), and placing at 2-8 ℃; long-term storage below-10deg.C, and effective period of 6 months.
The chorionic gonadotrophin pre-coated ELISA plate prepared by the preparation method can be prepared in advance, so that the analysis time for quantitatively detecting HCG in human urine is reduced, resources are greatly saved, and the chorionic gonadotrophin pre-coated ELISA plate can be effectively and stably stored for a long time and is convenient to take and use.
In addition, the application also provides a kit for quantitatively detecting HCG in human urine, which comprises the chorionic gonadotrophin pre-coated ELISA plate, an HRP-labeled HCG antibody, a washing solution, a chromogenic solution and a stop solution.
The raw materials and the preparation methods of the various reagents involved in the kit are specifically described below.
HRP-labeled HCG antibody split charging: newly purchased horseradish peroxidase (HRP) -labeled HCG antibody (ABBEST; anti-h beta HCG McAb, labling HRP, product number 03104700201 of Baiman Biotechnology Co., zheng, state), split-packed at 56 μl each after thawing at room temperature, labeled after split-packing, and stored below-10deg.C for use in the validity period.
Washing solution (PBST): 400ml of phosphate buffer mother liquor, 3600ml of purified water and 4ml (0.1%) of tween-20 are measured, and the mixture is evenly mixed and stirred, thus obtaining the compound preparation. Wherein, phosphate buffer mother liquor (10×pbs): 14.38g of anhydrous disodium hydrogen phosphate, 79g of sodium chloride, 2g of potassium chloride and 2.4g of monopotassium phosphate are accurately weighed, 800ml of purified water is added, and the mixture is stirred until the mixture is completely dissolved, the volume is fixed to 1000ml, and the mixture is uniformly shaken and stored at room temperature, and the effective period is 1 month.
HCG standard preparation: taking Shanghai Lizhu biological technology limited company coke as a product with known titer in a company division workshop, diluting the product into a solution with HCG titer of 9000-11000mIU/ml (preferably 10000 mIU/ml) by using a sample diluent, subpackaging according to each 0.5ml, marking after subpackaging, and preserving at the temperature below-10 ℃ for 2 years. Wherein, sample diluent: 10ml of phosphate buffer mother solution is measured, 90ml of purified water is added, 0.2g (concentration is 0.2%) of BSA is weighed and dissolved, and after being mixed uniformly, the mixture is placed in a refrigerator for 2-8 ℃ for preservation, and the validity period is 2 days.
Color development liquid: the color development liquid comprises 0.4-0.6ml of substrate solution A, 9.4-9.6ml of substrate solution B and 40-45 mu l of substrate solution C. As a typical but non-limiting example, accurately measuring and mixing 0.5ml of substrate solution A, 9.5ml of substrate solution B and 42 μl of substrate solution C, and preparing the product. (wherein, chromogenic substrate A: the chromogenic substrate A is a mixed solution of 3, 5-tetramethyl benzidine and absolute ethyl alcohol, wherein, the concentration of 3, 5-tetramethylbenzidine was 1-3mg/ml specifically, 50mg TMB powder (3, 5-tetramethylbenzidine, ai Ke reagent, CAS: 54827-17-7) is dissolved in 25ml absolute ethyl alcohol, after being mixed evenly, the mixture is placed in a refrigerator for preservation at 2-8 ℃ to prepare a chromogenic substrate solution A, and the effective period is 3 months, wherein the chromogenic substrate solution B is a mixed aqueous solution of anhydrous disodium hydrogen phosphate and citric acid, the concentration of the anhydrous disodium hydrogen phosphate in the chromogenic substrate solution B is 7-8g/L, the concentration of the citric acid is 4-6g/L, as a typical non-limiting example, 3.73g of anhydrous disodium hydrogen phosphate and 2.35g of citric acid are taken, purified water is added to 500ml, after being mixed evenly, the refrigerator is placed at 2-8 ℃ for preservation to prepare a chromogenic substrate solution B, and the effective period is 1 month.
Stop solution: slowly adding 10ml of concentrated sulfuric acid into 80ml of purified water, mixing, and preserving at room temperature for 3 months.
The application also provides a method for quantitatively detecting HCG in human urine, which combines a fluorescent magnetic particle ELISA method and a double-antibody sandwich ELISA method to quantitatively detect HCG in human urine.
Specifically, it comprises the following steps:
Adding an HRP-labeled HCG antibody diluent into the chorionic gonadotrophin pre-coated ELISA plate;
Respectively adding HCG standard substance solutions with different concentrations and urine sample diluents with different concentrations into a chorionic gonadotrophin pre-coated ELISA plate for incubation, plate washing, color development and termination, and then measuring absorbance values at two wavelengths of 450nm/630nm by using an ELISA reader;
The read result is then processed: sorting the read results from small to large according to OD values, selecting 6 urine samples with gradient OD values, detecting the immune titer of the HCG by using a fluorescent magnetic particle ELISA analyzer, taking the OD value of the sample as an abscissa and the immune titer as an ordinate as a standard curve, and calculating a curve equation;
Wherein, the HRP-labeled HCG antibody diluent is prepared by diluting an HRP-labeled HCG antibody solution with enzyme-labeled antibody diluent in a ratio of 1:1400-1600; wherein the concentration of the urine sample in the urine sample dilution is 8-12. Mu.l/ml.
More specifically, the method comprises the following steps:
S201, preparation
And taking the HCG ELISA plate, enzyme-labeled antibody diluent, chromogenic substrate A solution, chromogenic substrate B solution, chromogenic substrate C solution and stop solution required to be used for detection out of a refrigerator refrigerating chamber at least 30min in advance, and balancing in a room temperature environment.
S202, preparing according to the reagent in the kit.
S203, preparing HCG standard solution
Taking one HCG standard product (10000 mIU/ml) after split charging, and melting at room temperature. Taking a 10ml centrifuge tube, accurately measuring 9.9ml of sample diluent, adding 0.1ml of HCG standard substance, shaking for 10-30 s, and uniformly mixing to obtain the HCG solution with the titer of 100 mIU/ml. 5 centrifuge tubes (1.5 ml) were separately prepared, and HCG solutions having a titer of 100mIU/ml were added according to the following table, and HCG standard solutions having a titer of 0mIU/ml, 8-12mIU/ml, 18-22mIU/ml, 38-42mIU/ml, 58-62mIU/ml, and 78-82mIU/ml were prepared from the sample dilutions.
| Concentration of standard solution | 100MIU/ml HCG solution addition | Sample diluent addition |
| 0mIU/ml | 0μl | 1000μl |
| 8-12mIU/ml | 80-120μl | 880-920μl |
| 18-22mIU/ml | 180-220μl | 780-820μl |
| 38-42mIU/ml | 380-420μl | 580-620μl |
| 58-62mIU/ml | 580-620μl | 380-420μl |
| 78-82mIU/ml | 780-820μl | 180-220μl |
The HCG standard solution is used as an external standard (simultaneously used as a negative control and a positive control) of the ELISA, after the ELISA is finished, the OD value of the standard solution is used as an abscissa, the concentration of the standard solution (namely the gradient titer of the standard solution) is used as an ordinate to be used as a standard curve, a curve equation is calculated, and the whole ELISA is monitored according to the OD value of the standard solution and the R 2 value of the standard curve equation.
It should be understood that other options for the concentration gradient of the HCG standard solution are possible.
S204, diluting a urine sample:
taking 1.5ml centrifuge tube, adding 990 μl sample diluent (0.2%o of top-mounted compound food colorant, blue, water-oil dual-purpose pigment, jin Jiahe food Co., dongguan city, inc.) in advance, adding 10 μl sample, and mixing by vortex to obtain urine sample diluent.
S205, diluted HRP-labeled HCG antibody
5.5Ml of enzyme-labeled antibody dilution and 3.5 μl of HRP-labeled HCG antibody solution were diluted at a ratio of about 1:1500 and mixed. The preparation is carried out on the same day, wherein the enzyme-labeled antibody diluent is commercially available under the trade name: HRP conjugate stabilizer/diluent II, manufacturer: huzhou english-division biotechnology limited, product number: HRP-SD-002.
S206, adding HRP labeled HCG antibody
Taking the ELISA plate which is balanced at the room temperature in advance, and adding 50 mu l of diluted HRP-labeled HCG antibody into each hole by using an 8-channel row gun.
S207, diluting the sample and the standard solution with urine
The HCG standard solution with the concentration of 0mIU/ml, 8-12mIU/ml, 18-22mIU/ml, 38-42mIU/ml, 58-62mIU/ml and 78-82mIU/ml is added, 50 mu l of each well, 2-3 wells are repeated for each gradient, and then diluted multiple urine samples (more than 6 urine samples) are added, and 50 mu l of each well is added. Each urine sample was replicated in 2-3 wells. And (3) after the sample addition is finished, placing the ELISA plate into a constant temperature incubator at 36-38 ℃ for incubation for 30min.
S208, washing plate
And (3) washing the plate by using a plate washing machine for 3 times according to the using rules of the plate washing machine, and finally, drying the ELISA plate on the absorbent paper.
S209, developing
100 Μl of color development solution (ready-to-use) is added to each well, and incubated in a constant temperature incubator at 36-38deg.C for 10-15min.
S210, terminate
The reaction was stopped by adding 50. Mu.l of stop solution to each well.
S211, reading
And (3) slightly shaking and uniformly mixing the ELISA plate added with the stop solution on a tabletop, then placing the plate into an ELISA reader, operating according to the operation rule of the ELISA reader, and measuring the absorbance value by the ELISA reader at the dual wavelength of 450/630 nm.
S212, result conversion
And sequencing the read results from small to large according to OD values, selecting 6 different urine samples with gradient OD values (referring to the positions of 6 HCG standard solution points), and detecting the HCG immune titer by using an AIA-360 (fully automatic fluorescent magnetic particle enzyme-linked immunosorbent assay) of Tosoh Co. And taking the OD value of the urine sample as an abscissa and the immune titer detected by the AIA-360 full-automatic analyzer as an ordinate as a standard curve, and calculating a curve equation.
When the immune titer of the urine sample is detected, the immune titer of each urine sample can be calculated by substituting the OD value of each urine sample measured by the method into an equation.
The method for quantitatively detecting HCG in human urine provided by the application combines the fluorescent magnetic particle ELISA method and the double-antibody sandwich ELISA method, and solves the problems of incapability of quantification, low sensitivity, poor stability, limited detection range and low repeatability compared with a colloidal gold method; compared with an independent ELISA method, the method solves the problems of insufficient accuracy and deviation; compared with a single fluorescent magnetic particle ELISA method and other tip detection technologies, the method solves the problems of high cost and incapability of large-scale detection; by combining the conditions, the method provided by the application can be used for detecting the HCG content in urine on a large scale, is quick and low in cost (96 plates are taken as an example, the average cost per hole is about 0.434 yuan), and has high sensitivity, accuracy and repeatability. The advantages are obvious. In addition, the method has important significance in the aspects of diagnosis, monitoring and treatment of various diseases of human bodies and the like in a plurality of medical and health departments.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
S1, preparation of pre-coated ELISA plate for determination of HCG immune titer in pregnant urine by ELISA method
S101, preparation of reagent
HCG coated antibody split charging: the newly purchased HCG antibody (ABBEST; anti-h HCG McAb, product number 031047001 of Bowman biosciences, zhengzhou) is dissolved at room temperature, the packaging volume is calculated according to the concentration of 240 mug/count (coated 8 ELISA plates/count) according to the labeled concentration, packaging is carried out, the packaging is finished, the label is made, and the product is preserved below-10 ℃ and used in the effective period.
Coating buffer solution: accurately weighing 1.44g of anhydrous disodium hydrogen phosphate, 7.9g of sodium chloride, 0.2g of potassium chloride and 0.24g of monopotassium phosphate, adding 800ml of purified water, stirring until the mixture is completely dissolved, adding 0.5ml (concentration of 0.05%) of proclin 300 preservative (ZB 116) into the mixture, fixing the volume to 1000ml, shaking the mixture uniformly, and storing the mixture at 2-8 ℃ for 1 month of effective period.
Blocking buffer: accurately weighing 5.75g of anhydrous disodium hydrogen phosphate, 1.3g of sodium dihydrogen phosphate dihydrate, adding 800ml of purified water, stirring until the solution is completely dissolved, adding 0.5ml (concentration 0.05%) of proclin 300 preservative (flash organism, product number: ZB 116), fixing the volume to 1000ml, shaking uniformly, and preserving at 2-8 ℃ for 1 month of effective period.
Sealing liquid: 30g (concentration 10%) of sucrose and 1.5g (concentration 0.5%) of BSA are accurately weighed, 300ml of blocking buffer is added, and the mixture is stirred until complete dissolution, thus obtaining the finished product.
Phosphate buffer stock (10×pbs): 14.38g of anhydrous disodium hydrogen phosphate, 79g of sodium chloride, 2g of potassium chloride and 2.4g of monopotassium phosphate are accurately weighed, 800ml of purified water is added, the mixture is stirred until the mixture is completely dissolved, the volume is fixed to 1000ml, the mixture is uniformly shaken, and the mixture is stored at room temperature and has a valid period of 3 days.
Washing solution (PBST): 400ml of phosphate buffer mother liquor, 3600ml of purified water and 4ml (0.1%) of tween-20 are measured, and the mixture is evenly mixed and stirred, thus obtaining the compound preparation.
S102, preparation of pre-coated ELISA plates
Preparation: taking 16 new ELISA plates (high adsorption transparent ELISA plates, CIH-F8T, of Severe Biotechnology Co., ltd.) and marking each plate (category, date and number) for later use;
Dilution of HCG monoclonal antibodies: the coating buffer (160 ml was required for 16 pieces) was taken at 10ml per ELISA plate and added to the beaker. Taking out 2 pieces of HCG antibody (coating), melting at room temperature, adding into coating buffer solution, washing with coating buffer solution for 2 times, ensuring complete transfer, no residue, and stirring. The final concentration of the diluted antibody was 3. Mu.g/ml.
Adding HCG monoclonal antibody: the diluted HCG antibody solution was sequentially added to the prepared 16 blank ELISA plates at 100. Mu.l/well using an 8-channel pipette. Wherein 8 blocks are divided into 2 groups; after each addition, checking whether the addition is missed or excessive, and after the addition is confirmed, packaging the fresh-keeping film, and placing the fresh-keeping film into a refrigerator at the temperature of 2-8 ℃ overnight (at least 8 hours).
Incubation: the next day, the ELISA plate which is at 2-8deg.C overnight is taken out and put into a constant temperature incubator which is constant temperature to 36-38deg.C, and incubated for 1h. Every 8 blocks are in a group, 2 groups are added, the 1 st group is firstly put in, and the 2 nd group is put in after 40min interval.
Washing the plate: after the incubation time, the ELISA plate was removed, washed with 400. Mu.l wash solution/well and 2 times/plate with a plate washer. After washing, the ELISA plate is reversely buckled on clean water-absorbing filter paper, the ELISA plate is strongly beaten on the filter paper by the right hand, the ELISA plate is beaten for 2-3 times, and no residual liquid is observed in the ELISA plate.
Closing: the prepared sealing liquid is taken and sequentially added into the washed ELISA plate by an 8-channel pipette according to 180 mu l/hole. Wherein 8 blocks are divided into 2 groups, each time one group is added, the fresh-keeping film is used for packaging after the confirmation, and the fresh-keeping film is put into a constant temperature incubator for incubation for 2 hours at the temperature of 36-38 ℃.
And (3) drying: taking out the ELISA plate after the incubation time, forcibly throwing off the sealing liquid therein, reversely buckling the ELISA plate on clean water-absorbing filter paper, forcibly beating the ELISA plate on the filter paper by holding the ELISA plate, beating for 2-3 times, and observing that no residual liquid exists in the ELISA plate. And then inversely buckling the ELISA plate in a constant-temperature incubator, swinging the ELISA plate out, drying at 36-38 ℃ for about 2 hours, and observing that no visible liquid exists in the holes of the ELISA plate.
S103, packaging and storing
Taking the dried ELISA plates, wrapping 5 layers of the ELISA plates together with 2 bags of drying agents by using a preservative film, and ensuring the wrapping to be tight and airtight. Storing for a short period (after the use in 1 month), and placing at 2-8 ℃; long-term storage below-10deg.C, and effective period of 6 months.
S2, HCG immune titer detection in pregnant urine
S201, preparation
And taking the HCG ELISA plate, enzyme-labeled antibody diluent, chromogenic substrate A solution, chromogenic substrate B solution, chromogenic substrate C solution and stop solution required to be used for detection out of a refrigerator refrigerating chamber at least 30min in advance, and balancing in a room temperature environment.
S202, preparation of reagent
HRP-labeled HCG antibody split charging: newly purchased HRP-labeled HCG antibody (ABBEST; anti-h beta HCG McAb, labling HRP, product number 03104700201 of Baiman Biotechnology Co., zheng), thawed at room temperature, packaged, labeled, stored below-10 ℃, and used during the effective period.
Phosphate buffer stock (10×pbs): 14.38g of anhydrous disodium hydrogen phosphate, 79g of sodium chloride, 2g of potassium chloride and 2.4g of monopotassium phosphate are accurately weighed, 800ml of purified water is added, and the mixture is stirred until the mixture is completely dissolved, the volume is fixed to 1000ml, and the mixture is uniformly shaken and stored at room temperature, and the effective period is 1 month.
Washing solution (PBST): 400ml of phosphate buffer mother liquor, 3600ml of purified water and 4ml (0.1%) of tween-20 are measured, and the mixture is evenly mixed and stirred, thus obtaining the compound preparation.
Sample dilution: 10ml of phosphate buffer mother solution is measured, 90ml of purified water is added, 0.2g (concentration is 0.2%) of BSA is weighed and dissolved, and after being mixed uniformly, the mixture is placed in a refrigerator for preservation at 2-8 ℃ and the validity period is 2 days.
HCG standard preparation: taking Shanghai Lizhu biological technology limited company coke as a product with known titer in a company division workshop, diluting the product into a solution with the HCG titer of 10000mIU/ml by using a sample diluent, subpackaging according to each 0.5ml, marking after subpackaging, and preserving at the temperature below-10 ℃ for 2 years.
Chromogenic substrate A solution: 50mg of TMB powder (3, 5-tetramethylbenzidine, ai Ke reagent, CAS: 54827-17-7) is taken and dissolved in 25ml of absolute ethyl alcohol, and the mixture is placed in a refrigerator for preservation at 2-8 ℃ after uniform mixing, and the effective period is 3 months.
Chromogenic substrate solution B: taking 3.73g of anhydrous disodium hydrogen phosphate and 2.35g of citric acid, adding purified water to 500ml, uniformly mixing, and placing in a refrigerator for preservation at 2-8 ℃ for 1 month of validity period.
Color development substrate C solution: taking 1.25ml of 30% H 2O2, adding purified water to 50ml, uniformly mixing, and placing in a refrigerator for preservation at 2-8 ℃ for 3 months.
Color development liquid: accurately measuring and evenly mixing 0.5ml of substrate solution A, 9.5ml of substrate solution B and 42 mu l of substrate solution C to obtain the product.
Stop solution: slowly adding 10ml of concentrated sulfuric acid into 80ml of purified water, mixing, and preserving at room temperature for 3 months.
S203, preparing HCG standard solution
Taking one HCG standard product (10000 mIU/ml) after split charging, and melting at room temperature. 9.9ml of sample diluent is accurately measured by a 10ml centrifuge tube, 0.1ml of HCG standard substance is added, and shaking is carried out for 10-30 s to mix evenly, thus obtaining 100mIU/ml HCG solution. Another 1.5ml centrifuge tube was used for 5 branches, and 100mIU/ml of HCG solution and sample dilution were added to prepare 0mIU/ml, 10mIU/ml, 20mIU/ml, 40mIU/ml, 60mIU/ml, 80mIU/ml of HCG standard solution according to the following table.
| Concentration of standard solution | 100MIU/ml HCG solution addition | Sample diluent addition |
| 0mIU/ml | 0μl | 1000μl |
| 10mIU/ml | 100μl | 900μl |
| 20mIU/ml | 200μl | 800μl |
| 40mIU/ml | 400μl | 600μl |
| 60mIU/ml | 600μl | 400μl |
| 80mIU/ml | 800μl | 200μl |
The HCG standard solution is used as an external standard of the ELISA (simultaneously used as a negative control and a positive control), after the ELISA is finished, OD values of the standard solution are respectively used as an abscissa, 0mIU/ml, 10mIU/ml, 20mIU/ml, 40mIU/ml, 60mIU/ml and 80mIU/ml are respectively used as an ordinate as a standard curve, a curve equation is calculated, and the whole ELISA is monitored according to the OD values of the standard solution and the R 2 values of the standard curve equation.
S204, diluting a urine sample:
taking 1.5ml centrifuge tube, adding 990 μl sample diluent (0.2%o of top-up compound food colorant, blue, water-oil pigment, jin Jiahe food Co., dongguan City, inc. is added in advance), adding 10 μl sample, and mixing by vortex.
S205, diluted HRP-labeled HCG antibody
Sample dilutions were diluted 5.5ml, and HRP-labeled HCG antibody solution 3.5 μl, i.e., at a ratio of about 1:1500, and mixed well. Is prepared on the same day of use.
S206, adding HRP labeled HCG antibody
Taking the ELISA plate which is balanced at the room temperature in advance, and adding 50 mu l of diluted HRP-labeled HCG antibody into each hole by using an 8-channel row gun.
S207, diluting the sample and the standard solution by adding urine (one-step incubation, simultaneously adding the HRP-labeled HCG antibody and the sample into the ELISA plate for incubation)
HCG standard solutions with concentrations of 0mIU/ml, 10mIU/ml, 20mIU/ml, 40mIU/ml, 60mIU/ml and 80mIU/ml are added to the ELISA plate, 50 μl per well is repeated for 2 wells per concentration, then diluted different urine samples are added, 50 μl per well is repeated for 2 wells per urine sample. And (3) after the sample addition is finished, placing the ELISA plate into a constant temperature incubator at 36-38 ℃ for incubation for 30min.
S208, washing plate
And (3) washing the plate by using a plate washing machine for 3 times according to the using rules of the plate washing machine, and finally, drying the ELISA plate on the absorbent paper.
S209, developing
100 Μl of color development solution (ready-to-use) is added to each well, and incubated in a constant temperature incubator at 36-38deg.C for 10-15min.
S210, terminate
The reaction was stopped by adding 50. Mu.l of stop solution to each well.
S211, reading
And (3) slightly shaking and uniformly mixing the enzyme label plate added with the terminator on a table top, then placing the enzyme label plate into an enzyme label instrument, operating according to the operation rule of the enzyme label instrument, and measuring the absorbance value by the enzyme label instrument at the dual wavelength of 450/630 nm.
S212, result conversion
And sequencing the read results from small to large according to OD values, selecting 6 urine samples with gradient OD values (referring to the positions of 6 HCG standard solution points), and detecting the HCG immune titer by using an AIA-360 (fully automatic fluorescent magnetic particle enzyme-linked immunosorbent assay) of Tosoh Co. And taking the OD value of the sample as an abscissa and the immune titer detected by the AIA-360 full-automatic analyzer as an ordinate as a standard curve, and calculating a curve equation.
When the immune titer of each urine sample is detected, the immune titer of each urine sample can be calculated only by bringing the OD value of each urine sample measured according to the method into an equation.
Example 2
This example is essentially the same as example 1, except that in example 1, an HRP-labeled HCG antibody is used and the sample is added to the microplate simultaneously and then incubated in one step.
Whereas in this example a two-step incubation was performed: firstly adding a sample, placing the ELISA plate into a constant temperature incubator at 36-38 ℃ for incubation for 30min, then adding an HRP-marked HCG antibody into the ELISA plate, and then placing the ELISA plate into the constant temperature incubator at 36-38 ℃ for incubation for 30min.
Example 3
This example is basically the same as example 1 except that the amounts of the chromogenic substrate A, chromogenic substrate B and chromogenic substrate C in the chromogenic solution in this example are different from those in example 1.
In this example, the color-developing solution includes 0.4ml of color-developing substrate A solution, 9.6ml of color-developing substrate B solution, and 45. Mu.l of color-developing substrate C solution;
Wherein, chromogenic substrate A liquid: dissolving 50mg of TMB powder in 50ml of absolute ethyl alcohol, uniformly mixing, and placing in a refrigerator for preservation at 2-8 ℃ for 3 months.
Chromogenic substrate solution B: taking 4g of anhydrous disodium hydrogen phosphate and 6g of citric acid, adding purified water to 500ml, uniformly mixing, and placing in a refrigerator for preservation at 2-8 ℃ for 1 month of effective period.
Color development substrate C solution: taking 1.35ml of 30% H 2O2, adding purified water to 50ml, uniformly mixing, and placing in a refrigerator for preservation at 2-8 ℃ for 3 months.
Comparative example 1
Comparative example 1 was analyzed using only fluorescent magnetic particle enzyme-linked immunoassay.
Specifically, the selected urine sample is directly placed in an AIA-360 (full-automatic fluorescent magnetic particle enzyme-linked immunoassay) of Tosoh corporation, and the HCG immune titer is directly detected.
Comparative example 2
Comparative example 2 was analyzed using only a double antibody sandwich enzyme-linked immunoassay.
Specifically: the selected urine sample was subjected to the operation under the operation conditions of example 1 (steps S201 to S211), the absorbance value of the urine sample was detected, the absorbance value of the urine sample was substituted into the standard curve obtained in step S203 (the standard solution has an immunological titer gradient of 0mIU/ml, 10mIU/ml, 20mIU/ml, 40mIU/ml, 60mIU/ml, 80mIU/ml as the ordinate), and the immunological titer of the urine sample was calculated.
Comparative example 3
Which is substantially the same as in example 1, except that: in comparative example 3, the step of "taking out an ELISA plate at 2 to 8℃overnight and placing it in a constant temperature incubator which has been kept at 36 to 38℃for incubation for 1 hour" was omitted, and only the operation at 2 to 8℃overnight was performed.
Comparative example 4
Comparative example 4 is substantially the same as example 1 except that the blocking liquid in comparative example 4 is different from example 1:
The blocking solution in comparative example 4 was: 20% fresh bovine serum, 0.01mol/L PBS solution.
Comparative example 5
Comparative example 5 is substantially the same as example 1 except that the coating buffer in comparative example 5 is different from example 1:
The coating buffer in comparative example 5 was: the carbonate salt having a pH of 9.6 was dissolved in deionized water by weighing 2.93g of Na 2CO3 1.59g、NaHCO3, and then adding water to 1L.
1. Comparative experimental analysis
The analytical methods provided in examples 1-3 and comparative examples 1-5 above were performed on 3 urine samples, respectively, in 5 parallel experiments to determine the immunotiter of the 3 urine samples, and the results are shown in Table 1; standard Deviation (SD), average (M), coefficient of variation (C.V) of the parallel results for each experiment are shown in table 2, where C.V =sd/m×100%:
Table 1: examples 1-3, comparative examples 1-5 determination of the immunotitres of the obtained samples 1-3
Table 2: analysis of results of examples 1 to 3 and comparative examples 1 to 5
Analysis of results for examples 1-3 and comparative examples 1-5: the values of C.V% in examples 1-3 and comparative examples 1,2 and 4 are lower than 5%, which means that the repeatability of the method is better, C.V values in comparative examples 3 and 5 are higher, which means that the repeatability is worse, and as can be seen from the results shown by the average value (M) in examples 1-3 and comparative example 1, the immunotiter obtained in examples 1-3 is almost identical to that in comparative example 1, which means that the method has accurate results, since the method provided by the application only needs to use the full-automatic fluorescent magnetic particle ELISA analyzer AIA-360 of Tosoh corporation to detect the immunotiter of 6 urine samples, the subsequent urine samples can directly measure the absorbance by using the standard curve of step S121, and compared with the full-automatic fluorescent magnetic particle ELISA analyzer AIA-360 of Tosoh, the method has about 42 times lower cost than that in comparative example 1 (specifically, 96 plates are used as the average enzyme-linked immunosorbent assay results of about 0.434), and the comparative example 1 has a relatively stable double enzyme-linked immunosorbent assay; the results of comparative example 3 and comparative example 1 have larger errors, which indicates that the operation at 2-8 ℃ over night alone can result in incomplete incubation, and the quality of the prepared chorionic gonadotrophin pre-coated elisa plate product is poor, the subsequent analysis is affected, and the repeatability is poor. The results of comparative example 4 are almost the same as those of comparative example 1, and the C.V value is low, but the cost is about 0.26 yuan/96 hole when bovine serum albumin is selected as the sealing liquid in example 1, and the cost is about 7 yuan/96 hole when new born bovine serum is selected as the sealing liquid in comparative example 4, so that the detection cost is increased by the new born bovine serum coated in comparative example 4, and bacteria are easy to grow after the liquid new born bovine serum is unsealed, and the new born bovine serum is not easy to store as bovine albumin; the experimental results of comparative example 5 have a large difference from comparative example 1, and C.V has a high value and poor reproducibility, indicating that the coating buffer selected in example 1 of the present application can improve the accuracy of the results.
2. Stability test analysis
The chorionic gonadotrophin precoated ELISA plate obtained in example 1 was stored in a refrigerator at-10deg.C, and stability of the ELISA plate was measured at 7 days, 14 days, 30 days, 60 days, 120 days, and 180 days, and the measurement results are shown in Table 3.
Table 3: stability test results
Stability test results analysis: after the chorionic gonadotrophin precoating ELISA plate is stored for 180 days at the temperature of minus 10 ℃, the OD value of each standard substance is not obviously reduced, and the R 2 value is more than or equal to 0.99, which indicates that the chorionic gonadotrophin precoating ELISA plate prepared by the preparation method of the chorionic gonadotrophin precoating ELISA plate provided by the application has better stability.
In summary, the preparation method of the chorionic gonadotrophin precoating ELISA plate provided by the application can prepare and obtain the chorionic gonadotrophin precoating ELISA plate with long quality guarantee period and stable product, and a large number of chorionic gonadotrophin precoating ELISA plates are manufactured at one time, so that the subsequent experiments and detection use are facilitated, the time and the operation cost are greatly saved, and the analysis efficiency is improved. Furthermore, the method for quantitatively detecting HCG in human urine provided by the application combines the fluorescent magnetic particle ELISA method and the double-antibody sandwich ELISA method, and solves the problems of incapability of quantification, low sensitivity, poor stability, limited detection range and low repeatability compared with a colloidal gold method; compared with an independent ELISA method, the method solves the problems of insufficient accuracy and deviation; compared with a single fluorescent magnetic particle ELISA method and other tip detection technologies, the method solves the problems of high cost and incapability of large-scale detection; by combining the conditions, the method provided by the application can be used for detecting the HCG content in urine on a large scale, is quick and low in cost (96 plates are taken as an example, the average cost per hole is about 0.434 yuan), and has high sensitivity, accuracy and repeatability. The advantages are obvious. In addition, the method has important significance in diagnosing, monitoring and treating various diseases of human bodies and the like for a plurality of medical and health departments. The kit for quantitatively detecting the HCG in the human urine provided by the application comprises various reagents involved in the method, so that the quantitative detection of the HCG in the human urine is simplified.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A method for preparing a chorionic gonadotrophin pre-coated elisa plate, comprising the steps of:
Placing an ELISA plate added with an HCG monoclonal antibody at 2-8 ℃ for heat preservation for 8-12 hours, then placing the ELISA plate at 36-38 ℃ for incubation for 1-2 hours, washing the incubated ELISA plate with a washing solution, adding a sealing solution for sealing, incubating the ELISA plate at 36-38 ℃ for 1-2 hours, and then removing the sealing solution;
Wherein the concentration of HCG antibody in the HCG monoclonal antibody is 2-3 mug/ml; the HCG monoclonal antibody is obtained by diluting the HCG antibody with a coating buffer solution; the coating buffer solution is a mixed aqueous solution containing anhydrous disodium hydrogen phosphate, sodium chloride, potassium chloride, monopotassium phosphate and a preservative; the blocking solution is prepared by mixing sucrose, bovine serum albumin and a blocking buffer solution.
2. The method for preparing chorionic gonadotrophin precoating elisa plate according to claim 1, wherein the concentration of anhydrous disodium hydrogen phosphate in the coating buffer is 1-2g/L, the concentration of sodium chloride is 7.5-8.5 g/L, the concentration of potassium chloride is 0.1-0.3g/L, the concentration of potassium dihydrogen phosphate is 0.2-0.3g/L and the concentration of preservative is 0.4-0.6ml/L.
3. The method for preparing chorionic gonadotrophin precoated elisa plate according to claim 1, wherein the concentration of sucrose in the blocking solution is 8-12g/ml and the concentration of bovine serum albumin is 0.4-0.6g/ml; the sealing buffer solution is a mixed aqueous solution of anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate and preservative, wherein the concentration of the anhydrous disodium hydrogen phosphate in the sealing buffer solution is 5-6g/L, the concentration of the sodium dihydrogen phosphate dihydrate is 1-2 g/L, and the concentration of the preservative is 0.4-0.6ml/L.
4. The method of preparing a chorionic gonadotrophin precoated elisa plate according to claim 1, wherein removing the blocking solution from the elisa plate comprises: and reversely buckling the ELISA plate to drain the sealing liquid, and then placing the ELISA plate at 36-38 ℃ for drying for 2-3h.
5. The method for preparing a chorionic gonadotrophin precoated elisa plate according to claim 4, further comprising packaging and storing the elisa plate after drying the elisa plate: and wrapping each dried ELISA plate and 2-3 bags of drying agent together with a preservative film for 4-6 layers, and then placing the ELISA plate at 2-8 ℃ for 1 month or below-10 ℃ for 6 months.
6. A chorionic gonadotrophin pre-coated ELISA plate is characterized in that, which is prepared by the method for preparing the chorionic gonadotrophin precoated enzyme label plate according to any one of claims 1 to 5.
7. Use of the chorionic gonadotrophin pre-coated elisa plate according to claim 6 for preparing a kit for quantitative detection of HCG in human urine.
8. A kit for quantitatively detecting HCG in human urine, comprising: the chorionic gonadotrophin precoated elisa plate of claim 6.
9. The kit for quantitatively detecting HCG in human urine according to claim 8, further comprising an HRP-labeled HCG antibody, an HCG standard, a wash solution, a color developing solution, and a stop solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011157485.7A CN114487445B (en) | 2020-10-26 | 2020-10-26 | Chorionic gonadotropin pre-coated ELISA plate, preparation method thereof, method for quantitatively detecting HCG in human urine and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011157485.7A CN114487445B (en) | 2020-10-26 | 2020-10-26 | Chorionic gonadotropin pre-coated ELISA plate, preparation method thereof, method for quantitatively detecting HCG in human urine and kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114487445A CN114487445A (en) | 2022-05-13 |
| CN114487445B true CN114487445B (en) | 2024-11-22 |
Family
ID=81471120
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011157485.7A Active CN114487445B (en) | 2020-10-26 | 2020-10-26 | Chorionic gonadotropin pre-coated ELISA plate, preparation method thereof, method for quantitatively detecting HCG in human urine and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114487445B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103048475A (en) * | 2012-12-18 | 2013-04-17 | 苏州浩欧博生物医药有限公司 | Nano magnetic particle chemiluminiscence assay kit for free HCG (human chorionic gonadotropin) beta subunit, preparation method for Nano magnetic particle chemiluminiscence assay kit and detection method adopting Nano magnetic particle chemiluminiscence assay kit |
| CN109975558A (en) * | 2019-05-16 | 2019-07-05 | 杭州普鲁米生物科技有限公司 | The method of flash type homogeneous chemistry luminescence technology detection human chorion gonadotrophic hormone beta |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4722889A (en) * | 1985-04-02 | 1988-02-02 | Leeco Diagnostics, Inc. | Immunoassays using multiple monoclonal antibodies and scavenger antibodies |
| CN102866254B (en) * | 2012-09-21 | 2015-01-21 | 潍坊市康华生物技术有限公司 | High-sensitivity antenatal screening kit of Down's Syndrome in pregnant women at second trimester as well as preparation and detection methods of kit |
| CN104764877A (en) * | 2014-05-14 | 2015-07-08 | 陈岩松 | Immunity chromatography test method and test paper |
| CN109320599A (en) * | 2018-09-11 | 2019-02-12 | 天津市湖滨盘古基因科学发展有限公司 | A kind of 1994151 mutain of human chorionic gonadotrophin and application |
-
2020
- 2020-10-26 CN CN202011157485.7A patent/CN114487445B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103048475A (en) * | 2012-12-18 | 2013-04-17 | 苏州浩欧博生物医药有限公司 | Nano magnetic particle chemiluminiscence assay kit for free HCG (human chorionic gonadotropin) beta subunit, preparation method for Nano magnetic particle chemiluminiscence assay kit and detection method adopting Nano magnetic particle chemiluminiscence assay kit |
| CN109975558A (en) * | 2019-05-16 | 2019-07-05 | 杭州普鲁米生物科技有限公司 | The method of flash type homogeneous chemistry luminescence technology detection human chorion gonadotrophic hormone beta |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114487445A (en) | 2022-05-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104749367B (en) | A kind of Procalcitonin. light-induced chemiluminescent immunoassay kit and preparation method thereof | |
| CN108398550B (en) | Composition, chip, preparation method of chip and detection device comprising chip | |
| CN110646620A (en) | Method for simultaneously detecting four inflammation markers and kit thereof | |
| CN112051403A (en) | C-reactive protein chemiluminescence immunoassay kit and preparation method and application thereof | |
| CN108398564B (en) | anti-TORCH-IgG type antibody spectrum chip, preparation method thereof and TORCH detection kit | |
| CN114216897A (en) | sST2 chemiluminescence detection kit and detection method thereof | |
| CN113514449A (en) | Application and detection method of the kit for detection of serum amyloid A by spatial proximity chemiluminescence | |
| CN111289758B (en) | Kit for quantitative detection of H-FABP and method for quantitative detection of H-FABP | |
| CN108398554B (en) | anti-TORCH-IgM antibody spectrum chip, preparation method thereof and TORCH detection kit | |
| CN115389761B (en) | Insulin fluorescence immunochromatography assay kit and preparation method thereof | |
| CN114594251A (en) | IL-6 fluorescence immunochromatographic kit and preparation method thereof | |
| CN114487445B (en) | Chorionic gonadotropin pre-coated ELISA plate, preparation method thereof, method for quantitatively detecting HCG in human urine and kit | |
| CN113238055A (en) | Kit for detecting procalcitonin by using space proximity chemiluminescence method, and detection method and application thereof | |
| CN113655227A (en) | Multi-connected detection kit for screening neonatal diseases, and preparation method and use method thereof | |
| CN104749355B (en) | Detection method and detection kit of sulfonamides (SAs) | |
| CN111007248A (en) | Chemiluminescence immunoassay kit for detecting rubella virus IgM antibody | |
| CN114544973B (en) | Kit, and preparation method and application thereof | |
| CN116430032A (en) | Chemiluminiscence detection kit for procalcitonin and detection method thereof | |
| JP3228791B2 (en) | Measuring method of antigen or antibody in sample | |
| CN112147341A (en) | A kind of detection gastrin G-17 time-resolved fluorescence immunoassay kit and preparation method and detection method thereof | |
| CN107219216A (en) | A kind of method of aflatoxin B1 in chemiluminescence immunoassay detection baby rice powder | |
| CN116466072A (en) | Dry-type fluorescence immunochromatography detection card, kit and detection method for quantitatively detecting procalcitonin and heparin binding protein | |
| CN117347626B (en) | Human insulin-like growth factor binding protein-1 chemiluminescence immunoassay kit and preparation method thereof | |
| CN101368970A (en) | Chemical luminescence immune assay determination reagent kit for detecting Beta-2 microglobulin | |
| NL2026488B1 (en) | Method for preparing a protein chip plate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |