CN112326976B - Fluorescent quantitative detection kit for progesterone, estradiol and beta-human chorionic gonadotrophin - Google Patents
Fluorescent quantitative detection kit for progesterone, estradiol and beta-human chorionic gonadotrophin Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/577—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
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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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
- G01N33/74—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
- G01N33/743—Steroid hormones
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- Urology & Nephrology (AREA)
- Hematology (AREA)
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Abstract
The invention provides a fluorescent quantitative detection kit for progesterone, estradiol and beta-human chorionic gonadotrophin, and relates to the technical field of in-vitro diagnosis. The kit comprises a test strip, wherein the test strip comprises a substrate and a sample pad, a CP pad, an NC film and an absorption pad which are sequentially stuck on the substrate; the CP pad is coated with a fluorescein labeled conjugate of a murine antiprogestin monoclonal antibody, a murine antiestradiol monoclonal antibody, a murine anti-beta-human chorionic gonadotrophin monoclonal antibody and chicken IgY; the NC film is respectively provided with a detection line coated with progesterone antigen, estradiol antigen and beta-HCG monoclonal antibody and a quality control line coated with rabbit anti-chicken IgY monoclonal antibody. The kit can realize one-time sampling and simultaneously and rapidly, accurately and quantitatively detect three early pregnancy indexes, and provides more accurate reference basis for dynamically detecting the early hormone level change of pregnancy and predicting the pregnancy ending of early threatened abortion patients.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis, in particular to a fluorescent quantitative detection kit for progesterone, estradiol and beta-human chorionic gonadotrophin.
Background
Progesterone, β -human chorionic gonadotrophin and estradiol are extremely important in early pregnancy. Progesterone (progesterone, abbreviated Prog) is a steroid hormone produced by the corpus luteum in early gestation, β -human chorionic gonadotropin (human choronic gonadotrophin, abbreviated β -HCG) is a glycoprotein secreted by syntrophic cells, and estradiol (Estrodiol, abbreviated E2) is a natural steroid estrogen secreted by the ovarian maturation follicles.
In early gestation, progesterone is mainly produced by the corpus luteum of ovarian pregnancy and is replaced by placental syncytial trophoblasts after 8-10 weeks, so that progesterone levels reflect the functions of the corpus luteum and placenta of ovarian pregnancy. 7 weeks prior to gestation, progestin produced by the gestation of the corpus luteum plays a key role in whether pregnancy can continue. 8-10 weeks is the transition period of the function of the corpus luteum of ovary and placenta, the progestin level may be slightly reduced, and after 12 weeks, the placenta replaces the corpus luteum of ovary pregnancy, and the progestin level is also raised. The secretion of progesterone in early gestation mainly depends on the corpus luteum of the ovary, and when the corpus luteum is caused to secrete insufficient progestin level for some reason, the early spontaneous abortion can be possibly caused, and the appropriate progesterone level is beneficial to the maintenance of early gestation. The progesterone level influences the permeability of uterine smooth muscle cells, regulates the concentration of sodium and potassium ions in the cells, reduces the excitability of uterine smooth muscle, reduces the sensitivity of pregnant uterus to the outside, reduces the uterine contraction, and ensures the stable implantation growth and development of fertilized eggs. In early gestation, progesterone levels were relatively stable and had no correlation with the week of gestation. Numerous studies have shown that the boundary point for normal pregnancy and progesterone that can continue gestation after megastream th early days of appearance is 30nmol/L (10 ng/mL). Progesterone, while playing a vital role in early pregnancy and predicting early intrauterine pregnancy outcomes, in the latter, is not highly specific and progesterone is often used in combination with other blood-born hormones to predict intrauterine early pregnancy outcomes.
Estradiol, like progesterone, is also produced by the corpus luteum of the ovary in early gestation and is synthesized mainly by fetal-placenta units after 10 weeks gestation. By the end of gestation, estradiol and estrone values are 100 times those of non-pregnant women. Studies have shown that human corpus luteum produces large amounts of estradiol, but that successful implantation of embryos requires progesterone instead of estradiol. Proper estradiol levels are important for the maintenance of early pregnancy, difficult to maintain if the estradiol levels are too low, and rapid increases in estradiol levels reflect normal fetal survival and good placental function. Most scholars' clinical studies agree that the level of estradiol in patients who are normal intrauterine early pregnancy or who have early threatened abortion can continue should increase with increasing week of pregnancy, suggesting the idea that pregnancy may continue if a plateau or decline trend occurs. Estradiol is more significant and more specific than traditional progesterone, β -HCG in predicting early pregnancy outcome.
Human chorionic gonadotrophin is a glycoprotein, the last 24 amino acid extension of the acid end of the beta-HCG subunit is unique, so that the specificity of the human chorionic gonadotrophin is clinically utilized to measure the beta-HCG of maternal serum. The trophoblast begins to secrete trace beta-HCG on the 6 th day after the fertilization of the mother, can be measured from the maternal serum on the 10 th day, and the concentration reaches a peak value of about 50-100KU/L from 8 to 10 weeks after the pregnancy, and is rapidly reduced for about 10 days. The main function of the beta-HCG is to maintain and enlarge the luteal phase of menstruation into the luteal phase of pregnancy, and to increase the secretion of steroid hormone to promote the development of villus and the formation of placenta, so that the activity of early pregnancy embryo development is in direct proportion to the level of the beta-HCG; meanwhile, beta-HCG can also prevent embryo trophoblast from being attacked by maternal lymphocytes. Because serum beta-HCG has larger fluctuation amplitude in the same pregnancy cycle, the early intrauterine development condition of pregnancy is generally predicted by observing the turnover rate of beta-HCG at present. The beta-HCG in pregnancy is multiplied, the smaller the pregnancy cycle number is, the larger the amplification is, when the pregnancy cycle is less than 6 weeks, the increment between the cycles is more than or equal to 4.8 times, the increment between the cycles is 3 times, the increment between the cycles is 1.3 times, the increment between the cycles is 0.5 times, the increment between the cycles is not obvious in 9-10 weeks, the average value is reduced by 32% from the 10 weeks to >12 weeks of pregnancy. Because the detection fluctuation range of the beta-HCG concentration in the same gestation period is larger, and the single detection accuracy is not high, the combined progestogen detection is mostly adopted clinically to improve the prediction accuracy.
The progesterone, the estradiol and the beta-human chorionic gonadotrophin are closely related to each other and complement each other. The effect of progesterone is equivalent to that of estradiol in early pregnancy, the effect of progesterone on reproductive organs is greatly weakened, and the effect of estradiol on the period of maintaining gestation of corpus luteum is also greatly influenced; beta-HCG promotes secretion of progestin by acting on the corpus luteum to maintain pregnancy, while estrogen is also produced by the corpus luteum of the ovary in early gestation. Therefore, the dynamic detection of the level changes of progesterone, estradiol and beta-human chorionic gonadotrophin in early gestation can early predict the gestation ending of early threatened abortion patients, and has great significance for clinical guidance and treatment.
In the prior art, only single detection kits of progesterone, estradiol and beta-human chorionic gonadotrophin are available, and the kit capable of simultaneously and efficiently detecting the progesterone, the estradiol and the beta-human chorionic gonadotrophin is lacking.
Disclosure of Invention
The invention aims to solve the technical problem of providing a fluorescent quantitative detection kit for progesterone, estradiol and beta-human chorionic gonadotrophin, which can realize one-time sampling and simultaneously and rapidly, accurately and quantitatively detect three early pregnancy indexes, namely progesterone, estradiol and beta-human chorionic gonadotrophin, and provides a more accurate reference basis for dynamically detecting early gestation hormone level change and predicting early threatened abortion patient pregnancy ending.
The fluorescent quantitative detection kit for the progesterone, the estradiol and the beta-human chorionic gonadotrophin comprises a test strip, wherein the test strip comprises a substrate and a sample pad, a CP pad, an NC membrane and an absorption pad which are sequentially adhered on the substrate; the CP pad is coated with a fluorescein labeled conjugate of a murine antiprogestin monoclonal antibody, a murine antiestradiol monoclonal antibody, a murine anti-beta-human chorionic gonadotrophin monoclonal antibody and chicken IgY; the NC film is respectively provided with a detection line coated with progesterone antigen, estradiol antigen and beta-HCG monoclonal antibody and a quality control line coated with rabbit anti-chicken IgY monoclonal antibody.
In the invention, the CP pad is coated with a fluorescein labeled conjugate of a murine anti-progesterone monoclonal antibody, a murine anti-estradiol monoclonal antibody, and a murine anti-beta-human chorionic gonadotrophin monoclonal antibody, which are the murine anti-Prog monoclonal antibody of product number E82321M from Meridian, the murine anti-E2 monoclonal antibody of product number 2E2 from Hytest, and the fluorescein labeled conjugate of the murine anti-beta-HCG monoclonal antibody of product number 27E8 from Hytest.
In the invention, the progesterone antigen coated on the NC film is from biosurfic company, and the product number is V56050; estradiol antigen is derived from Medix company, product number 710050; the beta-HCG monoclonal antibody is derived from Medix company, product number 100004.
In the invention, the chicken IgY is derived from WU-Han Yuan-Gu biotechnology-limited liability company and is numbered as BCB 005; the rabbit anti-chicken IgY monoclonal antibody is the antibody of the Wuhan Yuan Valley biotechnology company No. AB-038B.
In the invention, a clamping shell is arranged on the outer side of the test strip, the clamping shell comprises an upper clamping shell and a lower clamping shell which are mutually clamped, the lower clamping shell is provided with a clamping groove for placing the test strip, a sample adding port is arranged at the position, corresponding to a sample pad of the test strip, of the upper clamping shell, an observation port is arranged at the position, corresponding to an NC film of the test strip, of the upper clamping shell, and a detection line and a quality control line on the NC film are exposed at the observation port.
In the present invention, the kit further comprises a U disk with progesterone, estradiol and beta-human chorionic gonadotrophin standard curves.
The beneficial effects are that: the fluorescent quantitative detection kit for the progesterone, the estradiol and the beta-human chorionic gonadotrophin can realize one-time sampling and simultaneously, rapidly, accurately and quantitatively detect three early pregnancy indexes, namely the progesterone, the estradiol and the beta-human chorionic gonadotrophin, avoid repeated sampling of pregnant women, reduce the probability of stimulating the pregnant women to shrink or infect, and provide more accurate reference for dynamically detecting the level change of the early pregnancy hormones and predicting the pregnancy ending of early threatened abortion patients.
Drawings
The structure of the cartridge of FIG. 1 is schematically shown, wherein the cartridge comprises a 1-upper cartridge, a 2-lower cartridge, a 3-sample inlet, a 4-observation inlet and a 5-clamping groove.
FIG. 2 is a schematic diagram of the structure of the test strip, 6-substrate, 7-sample pad, 8-CP pad, 9-NC membrane, 10-absorbent pad.
Fig. 3 is a progesterone standard curve with progesterone concentration (ng/ml) on the abscissa and fluorescence signal intensity values on the ordinate.
FIG. 4 shows an estradiol standard curve, with estradiol concentration (pg/ml) on the abscissa and fluorescence signal intensity values on the ordinate.
FIG. 5 is a standard curve of beta-human chorionic gonadotrophin (hCG) with the abscissa representing beta-hCG concentration (mIU/ml) and the ordinate representing fluorescence signal intensity values.
Fig. 6 shows the correlation of progesterone detection, with the ordinate of the concentration of progesterone detected by the kit of the invention and the abscissa of the concentration of progesterone detected by the roche cobas e electrochemiluminescence fully automatic immunoassay system.
FIG. 7 shows the correlation of estradiol detection, the ordinate of which refers to the concentration of estradiol detected by the kit of the invention, and the abscissa of which refers to the concentration of estradiol detected by the full-automatic electrochemical luminescence immunoassay system of Roche cobas e.
FIG. 8 shows the correlation of the detection of beta-human chorionic gonadotrophin, the ordinate of which refers to the concentration of beta-human chorionic gonadotrophin detected by the kit of the present invention, and the abscissa of which refers to the concentration of beta-human chorionic gonadotrophin detected by the full-automatic electrochemical luminescence immunoassay system of Roche cobas e.
Detailed Description
Example 1 fluorescent quantitative detection kit for progesterone, estradiol and beta-human chorionic gonadotrophin
1. Composition of kit and preparation method thereof
A kit for the fluorescent quantitative detection of progesterone, estradiol and beta-human chorionic gonadotrophin comprising: test card, USB flash disk with standard curve.
(1) Structure of test card
The structure of the test card is described with reference to fig. 1 and 2 below.
The test card comprises a card shell and a test strip arranged in the card shell.
The test strip comprises a substrate 6 and a sample pad 7, a CP pad 8, an NC film 9 and an absorption pad 10 which are sequentially stuck on the substrate. The sample pad, CP pad, NC membrane and absorbent pad are overlapped with each other at their adjacent ends.
The clamping shell comprises an upper clamping shell 1 and a lower clamping shell 2 which are clamped with each other, a clamping groove 5 for placing a test strip is formed in the inner surface of the lower clamping shell 2, a sample adding port 3 is formed in the position, corresponding to a sample pad of the test strip, of the upper clamping shell, an observation port 4 is formed in the position, corresponding to an NC film of the test strip, of the upper clamping shell, and a T1 detection line, a T2 detection line, a T3 detection line and a quality control line C on the NC film are exposed at the observation port.
The clamping shell not only protects the test strip and prevents the test strip from being polluted by damage, but also plays a role in fixing, so that the test strip is not easy to slide and the measurement is affected. The upper clamping shell and the lower clamping shell can fix and compress the test strip, the labeled antibody and the sample on the CP pad can synchronously run, and the liquid flow rate is ensured to be uniform, so that the CV coefficient is further reduced, the precision and the accuracy are improved, the operation is convenient, the sample can be rapidly tested after being put flat and directly added, and no hard technical requirement is required for operators. In addition, the card shell and the detection equipment can be matched for use, a test card is inserted into a corresponding channel of the detection equipment (an immunofluorescence detector for Rayleigh biological production, TZ310 or TZ 320), then the sample is automatically and stably sent for detection, the accuracy of a detection result is further improved, and the measured value of the protein content is more accurate.
(2) Preparation of test card
① NC film preparation
From the sample pad to the absorption pad, a T1 detection line, a T2 detection line, a T3 detection line and a quality control line C are sequentially and equally arranged on an NC film (nitrocellulose film), prog antigen (Progesterone-11-Bovine Serum Albumin Conjugate, purchased from biosurfic company, product number V56050) is coated on the T1 detection line, and the coating concentration is 1mg/mL; e2 antigen (E2 (Estradiol) 6-HS ANTIGEN, available from Medix corporation, product number 710050) was coated on the T2 detection line at a concentration of 1mg/mL; t3 detection line coated with beta-HCG monoclonal antibody (purchased from Medix company, product number 100004) at a concentration of 1mg/mL; a rabbit anti-chicken IgY monoclonal antibody (purchased from Wohan Yuangu biotechnology Co., ltd., product number AB-038B) is coated on the quality control line C, and the coating concentration is 0.5mg/mL.
The distances between the T1 detection line and the T2 detection line, between the T2 detection line and the T3 detection line and between the T3 detection line and the quality control line C are the same, scribing is facilitated, and the detection effect is good. The T1 detection line is used for detecting Prog, the T2 detection line is used for detecting E2, and the T3 detection line is used for detecting beta-HCG. Scribing is carried out at a scribing concentration of 0.6 mu l/cm, and drying is carried out at 37 ℃ after scribing, so that NC films coated with T1, T2, T3 and C lines are obtained.
The concentration coating quality control lines C, T, T2 and T3 are adopted, the T, C lines are more uniform, the phenomenon of uneven or discontinuous depth is avoided, and meanwhile, the using amount of the used coating antibody is minimum, the cost is reduced, and the economic benefit is good.
② Preparation of CP pad
The mouse anti-Prog monoclonal antibody (purchased from meridian, product number E82321M), the mouse anti-E2 monoclonal antibody (purchased from hysest, product number 2E 2), the mouse anti- β -HCG monoclonal antibody (purchased from hysest, product number 27E 8) and chicken IgY (CHICKEN IGY Polyclonal antibody, purchased from wuhan former valley biotechnology, liability company, product number BCB 005) were labeled with biotin and then coupled with the dyight 800-labeled streptavidin, which was then sprayed on a glass fiber filter membrane, to obtain a CP pad.
The murine anti-Prog monoclonal antibody (available from meridian company under product number E82321M) was coupled to biotin to give a biotin-Prog mAb conjugate solution as follows: the murine anti-Prog monoclonal antibody was added to DPBS (dulcitol phosphate buffer, available from HyClone, product number SH 30028.02) and biotin was then added to give a reaction system having a murine anti-Prog monoclonal antibody concentration of 2mg/ml and a biotin concentration of 120mg/ml. Mixing well with the added vortex, and quickly placing into a constant temperature culture oscillator to perform reaction after incubating for one hour at 25 ℃ and 600 rpm. After the reaction is finished, the reaction product is ultrafiltered by an ultrafiltration tube with the molecular weight cutoff of 30kDa, and the trapped fluid is taken out to obtain biotin-Prog mAb conjugate solution.
The murine anti-E2 monoclonal antibody (available from Hytest, product No. 2E 2) was conjugated to biotin to give a biotin-E2 mAb conjugate solution as follows: the murine anti-E2 monoclonal antibody is added into DPBS, and then biotin is added into the DPBS, so that the concentration of the murine anti-E2 monoclonal antibody in the reaction system is 2mg/ml, and the concentration of the biotin is 100mg/ml. Mixing well with the added vortex, and quickly placing into a constant temperature culture oscillator to perform reaction after incubating for one hour at 25 ℃ and 600 rpm. After the reaction is finished, the reaction product is ultrafiltered by an ultrafiltration tube with the molecular weight cutoff of 30kDa, and the trapped fluid is taken out to obtain biotin-E2 mAb conjugate solution.
The murine anti-beta-HCG monoclonal antibody (available from Hytest company, product number 27E 8) was coupled to biotin to give a biotin-beta-HCG mAb conjugate solution as follows: the murine anti-beta-HCG monoclonal antibody is added into DPBS, and then biotin is added into the DPBS, so that the concentration of the murine anti-beta-HCG monoclonal antibody in the reaction system is 2mg/ml, and the concentration of the biotin is 200mg/ml. Mixing well with the added vortex, and quickly placing into a constant temperature culture oscillator to perform reaction after incubating for one hour at 25 ℃ and 600 rpm. After the reaction is finished, the reaction product is ultrafiltered by an ultrafiltration tube with the molecular weight cutoff of 30kDa, and the trapped fluid is taken out to obtain biotin-beta-HCG mAb conjugate solution.
Chicken IgY (purchased from wuhan former cereal biotechnology liability company, number BCB 005) was coupled with biotin to obtain a biotin-chicken IgY conjugate solution according to the following method: chicken IgY and biotin are added into DPBS to make the concentration of chicken IgY in the reaction system be 2mg/ml and the concentration of biotin be 80mg/ml. Mixing well with the added vortex, and quickly placing into a constant temperature culture oscillator to perform reaction after incubating for one hour at 25 ℃ and 600 rpm. After the reaction is finished, the reaction product is ultrafiltered by an ultrafiltration tube with the molecular weight cut-off of 30kDa, and the cut-off liquid is taken to obtain biotin-chicken IgY conjugate solution.
The number of labelled biotin per antibody molecule was averaged using Thermo SCIENTIFIC PIERCE TM Biotin Quantitation Kit in biotin-Prog mAb conjugate solution, biotin-E2 mAb conjugate solution, biotin- β -HCG mAb conjugate solution and biotin-chicken IgY conjugate solution. The results were as follows: the concentration of biotin-Prog mAb conjugate solution was 1mg/ml, and the number of labeled biotin per mouse anti-Prog monoclonal antibody was 6 on average; the concentration of biotin-E2 mAb conjugate solution was 1mg/ml, and the number of labeled biotin per mouse anti-E2 monoclonal antibody was 7 on average; the concentration of biotin-beta-HCG mAb conjugate solution was 1mg/ml, and the number of labeled biotin per mouse anti-beta-HCG monoclonal antibody was 8 on average; the concentration of the biotin-chicken IgY conjugate solution was 1mg/ml, and the number of labeled biotin per chicken IgY was 7 on average.
The biotin-Prog mAb conjugate was coupled to the dlight 800-labeled streptavidin (available from Thermo scientific, product number 46421) as follows: biotin-Prog mAb conjugate was added to DPBS, shaken at 25 ℃, 500rpm, and then the dyight 800-labeled streptavidin was rapidly added to give Prog mAb-labeled conjugate. The concentration of biotin-Prog mAb conjugate in the reaction system was 0.56mg/ml, and that of Dyight 800-labeled streptavidin was 0.3mg/ml.
The biotin-E2 mAb conjugate, biotin-beta-HCG mAb conjugate and biotin-chicken IgY conjugate are respectively coupled with Dyight 800 labeled streptavidin by the same method to respectively obtain E2 mAb labeled conjugate, beta-HCG mAb labeled conjugate and chicken IgY labeled conjugate. The concentration of biotin-antibody conjugate in the reaction system is 0.56mg/ml, and the concentration of Dyight 800 labeled streptavidin is 0.3mg/ml.
Preparing a conjugate containing 0.1mg/ml Prog mAb label, 0.1mg/ml E2 mAb label, 0.15mg/ml beta-HCG mAb label and 0.05mg/ml chicken IgY label by taking QC-buffer as a solvent to obtain a CP coating liquid. And coating the CP coating liquid on a glass fiber pad (the size is 10mm multiplied by 280 mm) by using a BIODOT coating machine, spraying the CP coating liquid with the spraying amount of 2.2ul/cm, and putting the glass fiber pad into a vacuum drying oven for treatment for at least 24 hours to obtain the CP pad. Wherein QC-buffer is an aqueous solution containing 5% (mass percentage concentration) of human serum albumin, 0.1% (mass percentage concentration) of PVP90, 2% (mass percentage concentration) of trehalose and 0.5% (mass percentage concentration) of sodium azide.
③ Sample pad
The treatment solution was an aqueous solution containing 6.055mg/ml Tris base (Tris-hydroxymethyl aminomethane), 1.2mg/ml EDTA-Na 2, 2mg/ml Casein, 8% Tween-20 and 5% trehalose by volume, pH 7.2.
And soaking the polyester fiber membrane by adopting a treatment liquid, and drying to obtain the sample pad.
The sample pad prepared by the method has the advantages of good use effect, good positive and negative control effects, and positive samples of Prog, E2 and beta-HCG can be well distinguished from negative samples by using detection equipment for detection.
④ Assembly
And sequentially adhering the sample pad, the CP pad, the NC film and the absorption pad on the substrate, cutting into strips to prepare test strips, and assembling with a matched clamping shell. Then filling the aluminum foil bags, filling a bag of drying agent into the aluminum foil bags, and sealing the aluminum foil bags by using a heat sealing machine. And finally, preparing the test card.
Wherein the absorbent pad is made of a mixture of glass fibers and cotton wool (available from Ahlstrom company, cat# 320), and the substrate is made of polyvinyl chloride.
(3) USB flash disk with standard curve
The U disk is provided with a progesterone standard curve, an estradiol standard curve and a beta-human chorionic gonadotrophin standard curve.
The preparation method of each material standard curve is as follows:
Manufacturing a progesterone standard curve: the national standard of progesterone (57-83-0) was formulated with PBS (10 mM, pH 7.2) at a concentration of 0.25ng/ml, 0.5ng/ml, 2.5ng/ml, 10ng/ml, 40ng/ml, 160ng/ml, 300ng/ml. The progesterone standard with each concentration is respectively dripped into the sample-adding hole of the test card, and then put into an immunofluorescence detector (TZ 310 or TZ 320) produced by Rayleigh biology, and reacted for 8min at 18 ℃. After the reaction is finished, scanning T1 and a quality control line C by the immunofluorescence detector (TZ 310 or TZ 320) under the conditions of 777nm of excitation light wavelength and 794nm of receiving light wavelength to obtain a fluorescence signal intensity value, and preparing a standard curve by taking the concentration of progesterone as an abscissa and the fluorescence signal intensity value as an ordinate, wherein the equation is y=1.0922x+0.9297, R 2 =0.9995, x is the concentration of progesterone, and y is the fluorescence signal intensity value, as shown in figure 3. The linear range of the test card for progesterone detection is 0.25ng/ml to 300ng/ml.
Making an estradiol standard curve: the high concentration sample of the Estradiol is diluted to 5pg/ml, 20pg/ml, 80pg/ml, 320pg/ml, 640pg/ml, 1280pg/ml, 2560pg/ml and 3000pg/ml of standard Estradiol by using a fixed value of a Roche Estradiol II detection kit (assigned by a Roche cobas e electrochemiluminescence full-automatic immunoassay system). Estradiol standards of various concentrations are respectively dripped into the sample-adding holes of the test card, and then placed into an immunofluorescence detector (TZ 310 or TZ 320) produced by Rayleigh biology, and reacted for 8min at 18 ℃. After the reaction is finished, the immunofluorescence detector (TZ 310 or TZ 320) scans T2 and a quality control line C under the conditions of an excitation light wavelength of 777nm and a receiving light wavelength of 794nm to obtain fluorescence signal intensity values, and a standard curve is manufactured by taking the concentration of estradiol as an abscissa and the fluorescence signal intensity value as an ordinate, wherein the equation is y= 1.1208x-5.4193, R 2 =0.9978, x is the concentration of estradiol, and y is the fluorescence signal intensity value, as shown in fig. 4. The test card has a linear range of 5-3000pg/ml for estradiol detection.
Preparation of beta-human chorionic gonadotrophin standard curve: beta-human chorionic gonadotrophin standard (75/551) was diluted with PBS (10 mM, pH 7.2) to a concentration of 2mIU/ml, 20mIU/ml, 200mIU/ml, 20000mIU/ml. Beta-human chorionic gonadotrophin standard substances with various concentrations are respectively dripped into sample adding holes of a test card, and then placed into an immunofluorescence detector (TZ 310 or TZ 320) produced by Rayleigh biology, and reacted for 8min at 18 ℃. After the reaction is finished, the immunofluorescence detector (TZ 310 or TZ 320) scans T3 and a quality control line C respectively under the conditions of an excitation light wavelength of 777nm and a receiving light wavelength of 794nm to obtain fluorescence signal intensity values, and a standard curve is manufactured by taking the concentration of beta-human chorionic gonadotrophin as an abscissa and the fluorescence signal intensity value as an ordinate, wherein the equation is y=1.0192x+80.128, R 2 =0.9978, x is the concentration of beta-human chorionic gonadotrophin, and y is the fluorescence signal intensity value, as shown in figure 5. The linear range of detection is 2-20000mIU/ml.
2. Method for using kit
During testing, a sample to be detected is dripped into a sample inlet of a test card, the test card is placed into an immunofluorescence detector (TZ 310 or TZ 320) produced by Rayleigh biology, the reaction is carried out for 8min at 18 ℃, then fluorescent signal intensity values of T1, T2 and T3 detection lines are detected by the immunofluorescence detector (TZ 310 or TZ 320) under the conditions of 777nm of excitation light wavelength and 794nm of receiving light wavelength, and then the concentration of progesterone, estradiol and beta-human chorionic gonadotrophin in the sample to be detected is obtained by calculation according to a standard curve stored on a U disk. The detection result is calculated to be reliable only when the fluorescence signal intensity value at the quality control line C is larger than 0.2.
According to the conventional method, the detection limit of the kit provided by the invention on progesterone is 0.1ng/mL, the detection limit of the kit on estradiol is 3pg/mL, and the detection limit of the kit on beta-human chorionic gonadotropin is 1mIU/mL.
The kit is adopted to detect high, medium, low concentration and negative samples of Prog, E2 and HCG respectively, each level is repeatedly measured for 10 times, and the Coefficient of Variation (CV) is calculated, and the result is as follows: the coefficient of variation was found to be less than 4%.
Example 2 comparison of the effects of the test strip of the invention with other test strips
Control strips 1-3 were prepared.
According to the preparation method of the test strip of the present invention, a control test strip 1 was prepared, except that the Prog antibody (purchased from Meridian, product number E82321M) in the Prog mAb-labeled conjugate on the CP pad was replaced with the Prog antibody of product number HPRO-2 from hysest company.
The control strip 2 was prepared according to the preparation method of the test strip of the present invention, except that E2 antigen of Medix company product number 710050 on NC membrane was replaced with E2 antigen of biosurfic company product number V56110, and E2 antibody (Hytest, 2E 2) in E2 mAb-labeled conjugate on CP pad was replaced with E2 antibody of Holmes company product number HM 177A-1177A.
The control strip 3 was prepared according to the preparation method of the test strip of the present invention except that the β -HCG antibody having a product number 100004 of Medix company on the NC film was replaced with the β -HCG antibody having a product number 28A4 of Hytest company.
See in particular table 1.
The test strips and the control test strips of the invention can detect the linear detection range and the detection limit of the Prog, E2 and HCG. From Table 2, it can be seen that the test strips of the present invention have significantly better linear detection range and detection limit than the control strips for the detection of Prog, E2 and HCG.
Table 1 composition of each test strip
The materials in the note form are given in the form of "purchase Source, product number".
TABLE 2 detection limits and Linear detection ranges for each test strip
Example 3 detection of specific serum samples Using the kit of the invention
According to the preparation method of the test strip, a batch of detection kits are prepared, and correlation comparison is carried out on the detection kits and serum samples assigned by the Roche cobas e electrochemiluminescence full-automatic immunoassay system. 50 samples of progesterone, estradiol and beta-human chorionic gonadotrophin are detected, the detection result of the full-automatic electrochemical luminescence immunoassay system of the Rogowski cobas e is taken as a control group, and the correlation of the detection result of the invention is compared, and the results are shown in figures 6-8. It can be seen that the detection result of the kit is very accurate.
Claims (5)
1. The fluorescent quantitative detection kit for the progesterone, the estradiol and the beta-human chorionic gonadotrophin is characterized by comprising a test strip, wherein the test strip comprises a substrate and a sample pad, a CP pad, an NC membrane and an absorption pad which are sequentially adhered on the substrate; the CP pad is coated with a fluorescein labeled conjugate of a murine antiprogestin monoclonal antibody, a murine antiestradiol monoclonal antibody, a murine anti-beta-human chorionic gonadotrophin monoclonal antibody and chicken IgY; the NC film is respectively provided with a detection line coated with progesterone antigen, estradiol antigen and beta-human chorionic gonadotrophin monoclonal antibody and a quality control line coated with rabbit anti-chicken IgY monoclonal antibody; the CP pad is coated with a murine antiprogestin monoclonal antibody, a murine antiestradiol monoclonal antibody, and a fluorescein-labeled conjugate of a murine anti-beta-human chorionic gonadotrophin monoclonal antibody, which are respectively a murine antiprogestin monoclonal antibody with the product number E82321M of the company Meridian, a murine antiestradiol monoclonal antibody with the product number 2E2 of the company Hytest, and a fluorescein-labeled conjugate of a murine anti-beta-human chorionic gonadotrophin monoclonal antibody with the product number 27E8 of the company Hytest.
2. Kit according to claim 1, characterized in that the N C membrane-coated progesterone antigen is derived from biosurfic company, product number V56050; estradiol antigen is derived from Medix company, product number 710050; the beta-human chorionic gonadotrophin monoclonal antibody is derived from Medix company under product number 100004.
3. The kit according to claim 2, wherein the chicken IgY is derived from the wuhan former cereal biotechnology liability company, numbered BCB005; the rabbit anti-chicken IgY monoclonal antibody is the antibody of the Wuhan Yuan Valley biotechnology company No. AB-038B.
4. The kit of claim 3, wherein a clamping shell is arranged on the outer side of the test strip, the clamping shell comprises an upper clamping shell and a lower clamping shell which are mutually clamped, the lower clamping shell is provided with a clamping groove for placing the test strip, a sample adding port is arranged at a position, corresponding to a sample pad of the test strip, of the upper clamping shell, an observation port is arranged at a position, corresponding to an NC film of the test strip, of the upper clamping shell, and both a detection line and a quality control line on the NC film are exposed at the observation port.
5. The kit of claim 4, further comprising a U disk with progesterone, estradiol, and beta-human chorionic gonadotrophin standard curves.
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