CN110346578B - Combined quantitative detection system for content of insulin-like growth factor binding protein-1 and fetal fibronectin - Google Patents
Combined quantitative detection system for content of insulin-like growth factor binding protein-1 and fetal fibronectin Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
- G01N33/558—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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
Abstract
The invention discloses a combined quantitative detection system for the contents of insulin-like growth factor binding protein-1 and fetal fibronectin, which comprises a kit and detection equipment; the kit is used with a matched instrument by using a double-antibody sandwich immunochromatography technology to realize quantitative detection of insulin-like growth factor binding protein-1 and fetal fibronectin in vaginal secretions or vaginal effluent of pregnant women, and provides a reference basis for diagnosing premature rupture of membranes and predicting premature delivery. The gold-labeled pad realizes the simultaneous detection of two proteins by adopting a method of labeling colloidal gold with two antibodies and mixing complex solution and spraying gold, realizes the simultaneous quantitative detection of two indexes by sampling once, and has the advantages of quick and simple operation and high detection accuracy.
Description
Technical Field
The invention relates to the field of medical detection, in particular to a combined quantitative detection system for contents of insulin-like growth factor binding protein-1 and fetal fibronectin.
Background
The rupture of the fetal membrane just before parturition is called premature rupture of the fetal membrane (PROM), and the premature rupture of the fetal membrane at gestational age less than 37 weeks is also called premature rupture of the fetal membrane (immature term). Premature rupture of the fetal membranes is the most common complication in the perinatal period, can cause the early yield to be increased, the mortality rate of the perinatal infants is increased, the intrauterine infection rate and the puerperium infection rate are increased, the incidence rate of the premature rupture of the fetal membranes after 37 weeks of gestation is about 10 percent, and the incidence rate of the premature rupture of the fetal membranes after 37 weeks of gestation is about 2.0 to 3.5 percent. PROM can be concurrently born prematurely, umbilical cord prolapse and mother-child infection, and once the PROM is born, the life of the mother-child can be threatened if the PROM is not properly treated, so that the accurate and timely diagnosis of the PROM is very important. Insulin-like growth factor binding protein-1 (IGFBP-1) mainly exists in amniotic fluid and is a marker protein in the amniotic fluid during pregnancy. When the fetal membrane is ruptured, amniotic fluid leaks into the cervix and vagina from the rupture of the fetal membrane, the IGFBP-1 contained in the amniotic fluid serves as a sign for diagnosing premature rupture of the fetal membrane, and the content of the IGFBP-1 in the amniotic fluid is extremely high and is 100-1000 times of that in maternal blood, so that the sensitivity is very high, trace amniotic fluid which cannot be detected by a conventional means can be detected, and the problems that the latent premature rupture of the fetal membrane and high-level rupture of the fetal membrane are difficult to detect are solved.
Preterm labor refers to labor between the full 28 and 37 gestational weeks, with domestic preterm labor accounting for 5% -15% of the total labor, with about 15% of preterm infants dying from the neonatal period. Preterm birth control is one of the major measures to reduce perinatal mortality and improve neonatal quality. Foreign scholars recommend advancing the upper limit of preterm birth definition to 20 weeks gestation. Fetal Fibronectin (fn), a matrix component outside uterine chorion cells, is present between the chorion and the decidua, and is mainly produced by trophoblast cells. Since fusion of chorion and decidua prevents the release of fn after 21 weeks of pregnancy, normal pregnant women have very low fn levels at 22-35 weeks of pregnancy, and fn is found in cervicovaginal secretions only when chorion is separated from decidua, extracellular matrix at the interface of chorion and decidua is mechanically damaged or degraded by proteolytic enzymes. Thus, the level of fn in cervicovaginal secretions between pregnancies of 22-35 weeks is strongly correlated with the occurrence or nonoccurrence of preterm labor. Preterm birth accounts for 5% -15% of the total labor in China, and about 15% of preterm infants die during neonatal life, so preterm birth control is one of the main measures to reduce perinatal mortality and improve neonatal quality.
The two substances, namely insulin-like growth factor binding protein-1 and fetal fibronectin, are released into the vagina of a pregnant woman when uterus contracts and placenta and decidua are stripped, and when the two substances in the vagina of the pregnant woman abnormally rise, the premature rupture can be predicted. The method for detecting the premature rupture of the fetal membranes at present has the highest accuracy by detecting whether the insulin-like growth factor binding protein-1 appears or not, and the accuracy can reach 90 percent; the negative predictive value of fetal fibronectin detection in biomarkers is extremely high and therefore of great value, and the american society of obstetricians and gynecologists (ACOG) recommend a routine item for preterm birth diagnosis.
As mentioned above, there are currently clinical methods for diagnosing premature birth and premature rupture of membranes, but premature rupture of membranes and premature birth are closely related and can be relatively independent of common obstetrical symptoms, because premature rupture of membranes is a part of the cause of premature birth, and the occurrence of premature rupture of membranes does not mean premature birth, so that both premature birth and premature rupture of membranes need to be clinically evaluated for risk. However, the current clinical diagnosis methods are performed by sampling for multiple times and performing the sampling separately, so that the complexity of the operation is increased, and the multiple sampling of the pregnant women can stimulate the pregnant women to contract uterus and increase the probability of infection, thereby increasing the risks of premature birth and premature rupture of membranes.
At present, the IGFBP-1 has high specificity for diagnosing premature rupture of the fetal membrane, but is only limited to detecting the integrity degree of the fetal membrane, and even if the fetal membrane is intact, the risk of whether the pregnant woman has premature delivery is still unknown; the negative predictive value of fFN is high, but if fFN is positive, it cannot be judged whether fFN is from amniotic fluid or chorionic decidua space, and whether fFN is at risk of premature delivery due to premature rupture of fetal membranes. Although IGFBP-1 and fFN are recommended indexes in clinical guidelines, a technology for combining diagnosis of two substances and realizing simultaneous quantitative detection of protein content by sampling once has not been developed.
Patent application No. 201020565061.X discloses a combined detection kit for preterm delivery and premature rupture of membranes of pregnant women, which realizes that the preterm delivery and the premature rupture of membranes can be diagnosed simultaneously by one-time sampling, but the kit has the following defects:
(1) the scheme is qualitative detection, namely only negative and positive results can be given, the accurate content of the index protein cannot be known, and the qualitative detection has discrimination errors depending on naked eye judgment, so that wrong result analysis is easy to cause, and even doctor judgment and clinical application are seriously influenced;
(2) although the scheme only provides the steps of respectively preparing two gold-labeled pads for assembly, two detection strips are actually prepared, the two detection strips are arranged in a card or are pasted together back to back, and a test strip is not really made;
(3) the scheme is a naked test strip, and the test strip is easy to be polluted by visual observation, so that the judgment of a measurement result is influenced.
Therefore, it is highly desirable to provide a novel detection system for simultaneously determining the content of insulin-like growth factor binding protein-1 and fetal fibronectin to solve the above problems.
Disclosure of Invention
The invention aims to provide a combined quantitative detection system for the contents of insulin-like growth factor binding protein-1 and fetal fibronectin, which can realize the simultaneous and rapid quantitative detection of the contents of two proteins by one-time sampling and provide a reference basis for diagnosing premature rupture of membranes and predicting premature delivery.
In order to solve the technical problems, the invention adopts a technical scheme that: provides a combined quantitative detection system for the content of insulin-like growth factor binding protein-1 and fetal fibronectin, which comprises a kit and detection equipment;
the kit mainly comprises a detection card, a standard curve comparison table and sample diluent, wherein the detection card comprises a card shell and a test strip arranged in the card shell, the test strip comprises a substrate, a sample pad, a gold label pad, an NC membrane and an absorption pad, the sample pad, the gold label pad and the absorption pad are sequentially adhered to the substrate, and the gold spraying method of two antibodies for labeling colloidal gold and mixing complex solution is adopted for realizing the simultaneous detection of two proteins;
the detection equipment is used for quantitatively detecting the two proteins in the kit and provides a reference basis for diagnosing premature rupture of membranes and predicting premature delivery.
In a preferred embodiment of the present invention, the card housing comprises an upper card housing and a lower card housing which are clamped with each other, the upper card housing is provided with a sample adding hole located at the upper part of the sample pad and an observation port located at the upper part of the NC film, and the inner surface of the lower card housing is provided with a slot for placing the test strip.
In a preferred embodiment of the invention, the NC membrane is provided with T2 detection lines, T1 detection lines and quality control lines at equal intervals in sequence, the T2 detection line is coated with IGFBP-1 monoclonal antibody 2, the T1 detection line is coated with fn monoclonal antibody 2, and the quality control line is coated with goat anti-mouse antibody.
In a preferred embodiment of the present invention, the sample pad and the gold label pad are glass fiber films.
In a preferred embodiment of the present invention, the standard curve comparison table is disposed on a bar code or an SD card, and the bar code is printed on the surface of the card shell.
In order to solve the technical problem, the invention adopts another technical scheme that: the preparation method of the gold-labeled pad for simultaneously determining the content of insulin-like growth factor binding protein-1 and fetal fibronectin comprises the following steps:
(1) respectively marking the fetal fibronectin antibody 1 and the insulin-like growth factor binding protein-1 antibody 1 in the colloidal gold solution with the adjusted pH value;
(2) mixing the two marker precipitates, adding a gold-labeled complex solution with the pH value of 7.0-7.4 for redissolving, and uniformly mixing to obtain a gold-labeled solution for preparing a gold-labeled pad;
(3) and (3) spraying the gold marking solution prepared in the step (2) on a hydrophilic untreated gold marking pad by using a gold spraying instrument, and drying at 37 ℃ to obtain a marked gold marking pad.
In a preferred embodiment of the present invention, in step (1), the method for labeling fetal fibronectin antibody 1 comprises the following steps:
(101) adding 0.1mol/L potassium carbonate solution into 1ml of colloidal gold to adjust the pH value to the optimum labeling pH value of fFN, wherein the pH value is 6.9-7.4;
(102) after uniformly mixing, dropwise adding 6 mu g of fetal fibronectin antibody 1 with the concentration of 0.1mg/ml while rapidly stirring, and reacting for 30 min;
(103) then, 50. mu.l of 10% BSA was added thereto, and the mixture was stirred uniformly to react for 15min, and then, 20. mu.l of 5% PEG20000 was added thereto to react for 15 min.
In a preferred embodiment of the present invention, in step (1), the method for labeling insulin-like growth factor binding protein-1 antibody 1 comprises the steps of:
(111) adding 0.1mol/L potassium carbonate solution into 1ml of colloidal gold to adjust the pH value to the optimal labeling pH value of IGFBP-1, wherein the pH value is 7.0-7.7;
(112) after mixing, dropwise adding 6 mug of insulin-like growth factor binding protein-1 antibody 1 with the concentration of 0.1mg/ml while stirring rapidly, and reacting for 30 min;
(113) then, 50. mu.l of 10% BSA was added thereto, and the mixture was stirred uniformly to react for 15min, and then, 20. mu.l of 5% PEG20000 was added thereto to react for 15 min.
In a preferred embodiment of the present invention, in the step (2), the composition of the gold-labeled solution includes:
PB buffer: a matrix component having a pH of 7.0 to 7.4;
10% sucrose and 5% trehalose;
1％BSA；
0.5％PVP；
0.5％Tween20。
the invention also provides a gold-labeled pad prepared by the preparation method for simultaneously determining the contents of insulin-like growth factor binding protein-1 and fetal fibronectin.
The invention has the beneficial effects that:
(1) the combined detection system of the kit and the matched equipment can accurately determine the content of the insulin-like growth factor binding protein-1 and the fetal fibronectin, avoid the error result caused by visual judgment, has wider application range of the IGFBP-1 and the fFN combined application to the diagnosis of premature rupture of fetal membranes and the prediction of premature delivery, can determine whether the risk is simple premature birth or premature birth combined with premature rupture of the fetal membrane, is convenient for doctors to carry out targeted treatment on patients, simultaneously realizes the simultaneous quantitative detection of two indexes by one-time sampling, can detect the two indexes by using the same vaginal secretion specimen, the method judges the severity of premature rupture of the fetal membranes and the risk probability of premature delivery according to the protein content value, avoids sampling the pregnant woman for many times, reduces the probability of stimulating the pregnant woman to contract uterine or infect, is convenient for a doctor to operate, improves the compliance of a patient, and has the advantages of quick and simple operation;
(2) the detection card is combined with the test strip for testing by the card shell, thereby protecting the test strip from being polluted, playing a role of fixing, ensuring that the test strip is not easy to slide, influencing the determination, and ensuring that the flow rate of the sample adding quantity is uniform, thereby reducing the CV value, improving the precision and further improving the accuracy of the detection; in addition, the card shell can be matched with detection equipment for use, and the card shell is inserted into a corresponding channel of the detection equipment, so that automatic and stable sample feeding detection is realized, the accuracy of a detection result is further improved, and the measured value of the protein content is more accurate;
(3) the gold label pad adopts a method of marking colloidal gold by two antibodies and mixing complex solution to spray gold to realize simultaneous detection of two proteins, really realizes that markers of two marked pH values stably exist on one complex solution and the gold label pad, can achieve the purpose of no mutual interference between two detection items, can drive the quantity of marked gold to the maximum extent, improves the sensitivity, ensures that the final detection line is more uniform, does not have the phenomenon of different depths or discontinuity, further reduces the CV value and improves the precision;
(4) the invention can rapidly and sensitively detect the content of the specific protein at the same time, avoids complex operation, can adapt to various detection environments, has a printing function, can store the detection result for a long time, is convenient for contrast analysis, and ensures that the detection has universality.
Drawings
FIG. 1 is a schematic view of the structure of the test card;
FIG. 2 is a schematic structural diagram of the test strip;
FIG. 3 is a schematic structural view of the detection apparatus;
FIG. 4 is a functional block diagram of the detection device;
the parts in the drawings are numbered as follows: 1. the detection device comprises a detection card 11, a card shell 111, a sample adding hole 112, an observation port 12, a test strip 121, a substrate 122, a sample pad 123, a gold mark pad 124, an NC membrane 125, an absorption pad 2, a cold light source 3, a photoelectric sensor 4, a sliding block 5 and a guide rail.
Detailed Description
The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention.
Referring to fig. 1 and 2, an embodiment of the present invention includes:
a combined quantitative detection system for the contents of insulin-like growth factor binding protein-1 (IGFBP-1) and fetal fibronectin (fFN) comprises a kit and detection equipment. The detection equipment is used for quantitatively detecting the two proteins in the kit and provides a reference basis for diagnosing premature rupture of membranes and predicting premature delivery. Preferably, the detection equipment adopts an HR201 colloidal gold test paper analyzer.
The kit mainly comprises a detection card 1, a standard curve comparison table and a sample diluent, wherein the sample diluent mainly comprises a Tris buffer solution, and the pH value of the sample diluent is 8.5. Referring to fig. 1, the test card 1 includes a card case 11, and a test strip 12 disposed in the card case 11. Referring to fig. 2, the test strip 12 includes a substrate 121, a sample pad 122, a gold label pad 123, an NC film 124, and an absorbent pad 125, which are sequentially attached to the substrate 121. The sample pad 122 and the gold pad 123 are glass fiber films. The absorbent pad 125 provides suction to absorb the reacted liquid, thereby smoothing the chromatography. The NC membrane 124 is sequentially provided with T2 detection lines, T1 detection lines and a quality control line C at equal intervals, an IGFBP-1 monoclonal antibody 2 is coated on a T2 detection line, an fFN monoclonal antibody 2 is coated on a T1 detection line, and a goat anti-mouse antibody is coated on the quality control line. The distances between the T2 detection line and the T1 detection line and between the T1 detection line and the quality control line C are the same, the detection effect is good, the scribing is convenient, fFN is detected by the T1, and IGFBP-1 is detected by the T2.
The card shell 11 comprises an upper card shell and a lower card shell which are mutually clamped, a sample adding hole 111 positioned at the upper part of the sample pad 122 and an observation port 112 positioned at the upper part of the NC membrane 124 are formed in the upper card shell, a T2 detection line, a T1 detection line and a quality control line C on the NC membrane 124 are positioned in the middle of the observation port 112, and a clamping groove for placing the test strip 12 is formed in the inner surface of the lower card shell. The clamping shell 11 not only protects the test strip and prevents the test strip from being damaged and polluted, but also plays a role in fixing, so that the test strip is not easy to slide and the measurement is influenced. Card shell can be with examination strip 12 fixed and slight pressure testing from top to bottom, and the mark antibody and the sample diluent on the gold mark pad 123 can the synchronous operation, guarantee the liquid velocity of flow homogeneous, further reduce the CV coefficient, improve precision, and convenient operation puts flat direct application of sample and can test fast simultaneously, does not have hard technical requirement to operating personnel. In addition, the card shell 11 can be matched with detection equipment for use, and the card shell 11 is inserted into a corresponding channel of the detection equipment, so that automatic and stable sample feeding detection is realized, the accuracy of a detection result is further improved, and the measured value of the protein content is more accurate.
The standard curve comparison table is arranged on a bar code or an SD card, the bar code is printed on the surface of the card shell 11, the bar code or the SD card is matched with detection equipment for use, and the concentration of the labeled protein can be calculated according to the standard curve of the measured signal value for introducing the function of the standard curve of the batch of kits.
The kit in the detection system is used with matched detection equipment by using a double-antibody sandwich immunochromatography technology to realize quantitative detection of insulin-like growth factor binding protein-1 (IGFBP-1) and fetal fibronectin (fFN) in vaginal secretions or vaginal effluent of a pregnant woman.
When in test, the sample is dripped into the sample adding hole 111 of the test card 1 of the kit, the fFN in the sample is combined with the colloidal gold-labeled fFN monoclonal antibody 1 coated on the gold-labeled pad 123 in advance, the IGFBP-1 in the sample is combined with the colloidal gold-labeled IGFBP-1 monoclonal antibody 1 coated on the gold-labeled pad in advance, the combination is chromatographed upwards under the capillary effect, then the fFN combination is combined and captured by the fFN monoclonal antibody 2 fixed on the corresponding test area (T1 detection line) on the NC membrane 124, the IGFBP-1 combination is combined and captured by the IGFBP-1 monoclonal antibody 2 fixed on the corresponding test area (T2 detection line) on the NC membrane 124, the more fFNs in the sample, the more combinations accumulated on the T1 detection line, and the signal intensity reflects the number of the captured fFNs; the more IGFBP-1 in the sample, the more conjugate accumulated on the T2 detection line, and the signal intensity reflects the amount of IGFBP-1 that was captured. The signal intensity of the T1 test area and the T2 test area is detected by a detection device, and then the detection results of the fFN and the IGFBP-1 are calculated through a calibration curve stored on a bar code or an SD card.
The measuring system of the detection device scans C, T1 and T2 areas by using a specific light source to obtain a light absorption electric signal, then analyzes the light absorption electric signal quantitatively to analyze a target object to be detected, and provides a rapid quantitative detection result for samples (insulin-like growth factor binding protein-1 (IGFBP-1) and fetal fibronectin (fFN) in vaginal secretion or vaginal effluent of a pregnant woman) by using only one step.
With reference to fig. 3 and 4, the detection device includes a measurement card, a light source system, a photoelectric detection system, a thermal printer with a main circuit control board, a display screen, etc., the light source system includes a cold light source 2 and a photoelectric sensor 3, and the main circuit control board includes a DC-DC circuit, an amplifying circuit, and a control circuit. The detection device transmits a detection card 1 carrying a gold immunochromatographic test paper principle with complete reaction to the inside of the device through a mechanical motion part (a guide rail 5, a slide block 4 and the like), a bar code carrying sample information and item information passes through single-sided glass with an angle of 45 degrees and is read by a bar code scanning head, so that the device can interpret the IGFBP-1 and fFN items, the interpretation process comprises irradiating a detection area C, T1 and a T2 line by a specific wavelength LED light source, a photoelectric detection system receives an optical signal of the wavelength light source through the sample chromatography test paper, the conversion from the optical signal to an electric signal is completed, the signal amplification is carried out, an analog signal is converted into a digital signal through an AD conversion board, the digital signal finally enters a main board algorithm system, the final detection value is obtained through conversion of a standard curve preset in a bar code or a card, and the result display is carried out through a display screen and a printer, the gold immunochromatographic quantitative analysis process of the sample is completed through the operation principle in the device.
The preparation method of the kit comprises the following steps:
1. the gold-labeled pad 123 realizes the simultaneous detection of two proteins by adopting a method of labeling colloidal gold with two antibodies and spraying gold with a mixed complex solution. Under alkaline conditions, the surface of the colloidal gold particles is negatively charged, and can form non-covalent electrostatic attraction and firm combination with positively charged groups of target protein, and when the markers are aggregated at the antigen-antibody reaction position to reach a certain density, red spots visible to naked eyes appear. The success of the binding of the gold colloid to the protein depends on the pH value, the optimum pH value is generally close to or slightly larger than the isoelectric point of the labeled substance, so that the gold colloid can be firmly bound, and the dosage of the labeled substance depends on the size of the gold colloid particles. The preparation method of the gold mark pad 123 comprises the following steps:
(1) respectively marking the fetal fibronectin antibody 1 and the insulin-like growth factor binding protein-1 antibody 1 in the colloidal gold solution with the adjusted pH value; the method specifically comprises the following steps:
the method for marking the fetal fibronectin antibody 1 comprises the following steps:
(101) adding 0.1mol/L potassium carbonate solution into 1ml of colloidal gold to adjust the pH value to the optimum labeling pH value of fFN, wherein the pH value is 6.9-7.4;
(102) after uniformly mixing, dropwise adding 6 mu g of fetal fibronectin antibody 1 with the concentration of 0.1mg/ml while rapidly stirring, and reacting for 30 min;
(103) then, 50. mu.l of 10% BSA was added thereto, and the mixture was stirred uniformly to react for 15min, and then, 20. mu.l of 5% PEG20000 was added thereto to react for 15 min.
The method for labeling insulin-like growth factor binding protein-1 antibody 1 comprises the following steps:
(111) adding 0.1mol/L potassium carbonate solution into 1ml of colloidal gold to adjust the pH value to the optimal labeling pH value of IGFBP-1, wherein the pH value is 7.0-7.7;
(112) after mixing, dropwise adding 6 mug of insulin-like growth factor binding protein-1 antibody 1 with the concentration of 0.1mg/ml while stirring rapidly, and reacting for 30 min;
(113) then, 50. mu.l of 10% BSA was added thereto, and the mixture was stirred uniformly to react for 15min, and then, 20. mu.l of 5% PEG20000 was added thereto to react for 15 min.
BSA and PEG20000 are used as stabilizers to protect the stability of the colloidal gold markers, so that the colloidal gold can be used for blocking redundant sites on the colloidal gold particles, the colloidal gold can be conveniently stored for a long time, the nonspecific adsorption reaction of the immune gold complex can be prevented or reduced, and the BSA and the PEG20000 are used for blocking and then mixing so that the two colloidal gold markers cannot generate cross adsorption after mixing.
(2) Adopting low-temperature ultracentrifugation to remove unmarked protein/antibody and insufficiently marked colloidal gold, and the specific method comprises the following steps: centrifuging at 8000rpm for 30min, removing supernatant, mixing the two marker precipitates, adding a gold-labeled solution with the pH value of 7.0-7.4 for redissolving, wherein the pH ensures that the two antibody markers can exist stably, mixing the two antibody-labeled colloidal gold precipitates for redissolving to 40 mu l, and uniformly mixing after complete redissolution to obtain a gold-labeled solution for preparing a gold-labeled pad;
wherein the gold-labeled complex solution comprises the following components:
PB buffer: a matrix component having a pH of 7.0 to 7.4 to provide an optimum miscible environment;
10% sucrose and 5% trehalose: providing protection for the immunogold complex;
1% BSA: further eliminating the nonspecific adsorption reaction of the immune gold compound during mixing and dissolving;
0.5% PVP: promoting the rewetting and releasing of the immune gold compound during detection;
0.5% Tween 20: in order to reduce CV, the gold-labeled pad was not pretreated, and Tween20 was added to compensate for the loss of hydrophilicity due to the non-treatment of the gold-labeled pad.
(3) And (3) spraying the gold standard solution prepared in the step (2) on a hydrophilic untreated gold standard pad by using a gold spraying instrument, setting the gold spraying concentration to be 3 mu l/cm for spraying, and then drying at 37 ℃ to obtain the marked gold standard pad. The two antibodies are mixed to redissolve and spray gold, so that the quantity of the labeled gold can be driven to the maximum extent, the sensitivity of the antibody is improved, C, T1 and T2 are more uniform, the phenomenon that the depth is not uniform or discontinuous is avoided, the CV value is further reduced, the precision of the CV value is improved, and the accuracy of the result is improved.
The method for mixing the two antibodies together by the marking scheme can sample and add the sample at one time, rapidly measure the contents of the insulin-like growth factor binding protein-1 and the fetal fibronectin at the same time, further improve the precision and reduce the CV value, thereby improving the accuracy of the detection result, and has the advantages of less used antibodies and raw materials, cost saving, higher result accuracy, rapid and simple economic applicability and the like.
2. NC film:
both the T1 and T2 test lines were coated at a concentration of 1.2mg/mL, and the C line was coated at a concentration of 0.7 mg/mL. Streaking is generally carried out at a streaking concentration of 1. mu.l/cm, and the coated antibody/antigen is streaked and then dried at 37 ℃ to obtain a coated streaking rubber plate (i.e., coating the NC membrane with lines T1, T2 and C). By adopting the concentration coating quality control line C, T1 and the T2 detection line, the using effect is better after coating, the C, T line is more uniform, the phenomenon that the depth is not uniform or discontinuous is avoided, the used coating antibody is the least, the cost is reduced, and the economic benefit is good.
3. Sample pad:
setting the pH of the sample pad treatment solution to 8.5, drying and storing, and preparing the sample pad according to the method. The sample pad 122 prepared by the pH has the best use effect and better positive and negative control results, the detection equipment in the detection system is used for detection, the cutoff values of the IGFBP-1 and the fFN have obvious detection signal values, the negative signal value is 0, the test results of the positive standard sample signal values with different concentration gradients have obvious gradients, and the signal value is higher when the concentration is higher.
4. The treated sample pad 122, gold label pad 123, coated NC film 124 and absorbent pad 125 are combined and cut into strips to form test strips 12, which are assembled with the mating cartridge case 11.
5. Taking an aluminum foil bag and a drying agent; opening a heat sealing machine and preheating; the test strip 12 and the card shell 11 are assembled firstly, then the test strip is put into an aluminum foil bag, a bag of drying agent is put into the aluminum foil bag, and the aluminum foil bag is sealed by a heat sealing machine. Finally, the preparation of the kit is completed.
The test strip and the matched detection equipment thereof are prepared under the same production environment condition according to the method, the high, medium and low 3 concentrations of the fFN and the IGFBP-1 are respectively taken, the measurement is repeatedly carried out for 10 times at each level, the Coefficient of Variation (CV) is calculated, the high, medium and low 3 concentrations of the fFN are respectively 500ng/ml, 200ng/ml and 50ng/ml, and the high, medium and low 3 concentrations of the IGFBP-1 are respectively 200ng/ml, 80ng/ml and 20 ng/ml.
Test bars produced from the same batch of reagents were tested for intra-batch CV and the results are given in the following table:
three reagent test strips were prepared according to the above method under the same production environment conditions, taking high, medium and low 3 concentrations of fmn and IGFBP-1, testing with a kit, 3 times for each batch, calculating the mean of 3 determinations for each batch, testing the batch-to-batch CV, and the results are as follows:
the above results are in accordance with national regulations, the CV in batch and between batches is within 15%, and the standard qualified indexes of CV in batch and between batches (CV < 15%) are also in accordance with the regulations of the invention.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (9)
1. A preparation method of a gold-labeled pad for simultaneously determining the content of insulin-like growth factor binding protein-1 and fetal fibronectin comprises the following steps:
(1) respectively marking the fetal fibronectin antibody 1 and the insulin-like growth factor binding protein-1 antibody 1 in the colloidal gold solution with the adjusted pH value;
(2) removing unmarked protein/antibody and insufficiently marked colloidal gold by low-temperature ultracentrifugation, mixing the two marker precipitates, adding a gold-labeled complex solution with the pH value of 7.0-7.4 for redissolving, and uniformly mixing to obtain a gold-labeled solution for preparing a gold-labeled pad;
(3) and (3) spraying the gold marking solution prepared in the step (2) on a hydrophilic untreated gold marking pad by using a gold spraying instrument, and drying at 37 ℃ to obtain a marked gold marking pad.
2. The method for preparing a gold-labeled pad for simultaneously determining the contents of insulin-like growth factor-binding protein-1 and fetal fibronectin according to claim 1, wherein in the step (1), the method for labeling the fetal fibronectin antibody 1 comprises the following steps:
(101) adding 0.1mol/L potassium carbonate solution into 1ml of colloidal gold to adjust the pH value to the optimum labeling pH value of the fetal fibronectin antibody 1, wherein the pH value is 6.9-7.4;
(102) after uniformly mixing, dropwise adding 6 mug of fetal fibronectin antibody 1 with the concentration of 0.1mg/ml while rapidly stirring, and reacting for 30 min;
(103) and adding 50 mul 10% BSA, stirring uniformly, reacting for 15min, adding 20 mul 5% PEG20000, and reacting for 15 min.
3. The method for preparing a gold pad for simultaneously determining the contents of insulin-like growth factor-binding protein-1 and fetal fibronectin as claimed in claim 1, wherein the method for labeling insulin-like growth factor-binding protein-1 antibody 1 in step (1) comprises the following steps:
(111) adding 0.1mol/L potassium carbonate solution into 1ml of colloidal gold to adjust the pH value to the optimum labeling pH value of the insulin-like growth factor binding protein-1 antibody 1, wherein the pH value is 7.0-7.7;
(112) after uniformly mixing, dropwise adding 6 mug of insulin-like growth factor binding protein-1 antibody 1 with the concentration of 0.1mg/ml while rapidly stirring, and reacting for 30 min;
(113) and adding 50 mul 10% BSA, stirring uniformly, reacting for 15min, adding 20 mul 5% PEG20000, and reacting for 15 min.
4. The method for preparing a gold-labeled pad for simultaneously determining the content of insulin-like growth factor-binding protein-1 and fetal fibronectin as claimed in claim 1, wherein in the step (2), the composition of the gold-labeled complex solution comprises:
PB buffer: a matrix component having a pH of 7.0 to 7.4;
10% sucrose and 5% trehalose;
1%BSA；
0.5% PVP; and
0.5%Tween20。
5. a gold-labeled pad prepared by the method for preparing a gold-labeled pad for simultaneous determination of insulin-like growth factor-binding protein-1 and fetal fibronectin contents according to any one of claims 1 to 4.
6. A combined quantitative detection system for the contents of insulin-like growth factor binding protein-1 and fetal fibronectin is characterized by comprising a kit and detection equipment;
the kit mainly comprises a detection card, a standard curve comparison table and a sample diluent, wherein the detection card comprises a card shell and a test strip arranged in the card shell, the test strip comprises a substrate, a sample pad, a gold-labeled pad, an NC membrane and an absorption pad, which are sequentially adhered on the substrate, and the gold-labeled pad adopts the gold-labeled pad of claim 5 to realize the simultaneous detection of two proteins; the standard curve comparison table is arranged on a bar code or an SD card, and the bar code is printed on the surface of the card shell;
the detection equipment comprises a light source system, a photoelectric detection system, a main circuit control board, a thermal printer and a display screen, wherein the light source system comprises a cold light source and a photoelectric sensor, and the main circuit control board comprises a DC-DC circuit, an amplifying circuit and a control circuit; the detection device transmits a detection card of a gold immunochromatographic test paper principle which completely reacts to the inside of the device through a mechanical movement part, a bar code carrying sample information and project information passes through single-sided glass with an angle of 45 degrees, information reading is carried out through a bar code scanning head, the device judges IGFBP-1 and fFN projects, a specific light source is used for scanning an NC membrane to obtain a light absorption electric signal, then the light absorption electric signal is analyzed, a target object to be detected is quantitatively analyzed, quantitative detection of two proteins in the kit is realized, and a reference basis is provided for diagnosing premature rupture of a fetal membrane and predicting premature birth.
7. The system of claim 6, wherein the cartridge comprises an upper cartridge and a lower cartridge which are clamped with each other, the upper cartridge is provided with a sample hole on the upper part of the sample pad and an observation port on the upper part of the NC membrane, and the inner surface of the lower cartridge is provided with a slot for placing a test strip.
8. The system of claim 6, wherein T2 detection lines, T1 detection lines and quality control lines are sequentially arranged on the NC membrane at equal intervals, the IGFBP-1 monoclonal antibody 2 is coated on the T2 detection line, the fFN monoclonal antibody 2 is coated on the T1 detection line, and the goat anti-mouse antibody is coated on the quality control line.
9. The system of claim 6, wherein the sample pad and gold-labeled pad are glass fiber membranes.
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Inventor after: Yu Juanping Inventor after: Hu Rong Inventor after: Wang Zhongliang Inventor before: Hu Rong Inventor before: Wang Zhongliang |