CN111504959A - C-peptide rare earth fluorescent microsphere kit, detection card and preparation method thereof - Google Patents
C-peptide rare earth fluorescent microsphere kit, detection card and preparation method thereof Download PDFInfo
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- CN111504959A CN111504959A CN202010193464.4A CN202010193464A CN111504959A CN 111504959 A CN111504959 A CN 111504959A CN 202010193464 A CN202010193464 A CN 202010193464A CN 111504959 A CN111504959 A CN 111504959A
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Abstract
The invention discloses a C peptide rare earth fluorescent microsphere kit, a detection card and a preparation method thereof, wherein the detection card comprises a card shell and a detection test strip arranged in the card shell, the detection test strip comprises a bottom lining, a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper, the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are sequentially overlapped and stuck on the bottom lining along the length direction, a microsphere line formed by a C peptide monoclonal antibody marked by the C peptide rare earth fluorescent microsphere is sprayed on the combination pad, the rare earth fluorescent microsphere is a rare earth ion complex doped fluorescent microsphere, a detection line and a quality control line are arranged on the nitrocellulose membrane in parallel, the detection line is coated by the C peptide monoclonal antibody, and the quality control line is coated by a goat anti-mouse IgG antibody.
Description
Technical Field
The invention relates to a C peptide rare earth fluorescent microsphere kit, a detection card and a preparation method thereof.
Background
In vivo, a secretion product proinsulin of an islet β cell is taken as a precursor substance and is cleaved into two parts, namely a molecule of C peptide after enzyme digestion, and a molecule of insulin, wherein the molecule of C peptide exists with insulin, but compared with insulin, the concentration of the C peptide in blood is far higher than that of the insulin, the half-life period is longer, and the interference caused by the insulin used for treatment can be better eliminated by measuring the concentration level of the C peptide in the blood, so that the functional state of the islet β cell can be accurately reflected.
The fluorescent polymer microsphere generally refers to a polymer microsphere which has a particle size of between nanometer and micrometer, is loaded with a fluorescent substance and generates fluorescence by external energy excitation. The preparation method of the fluorescent microsphere and the fluorescent substance applied to the fluorescent microsphere are many and are generally limited to inorganic quantum dots and organic dyes. The excitation spectrum of the inorganic quantum dots is wide, and the emission wavelength is adjustable, so that the inorganic quantum dots have fluorescence of various colors; the organic dye comprises a plurality of dyes with different excitation wavelengths, and can meet the requirements of various applications. However, the sensitivity and accuracy of biological detection are limited by the problems of large interference of background fluorescence and stray fluorescence, short fluorescence lifetime, and the like of the two types of fluorescent substances. Meanwhile, the lanthanide rare earth nano material has excellent chemical and optical properties: high luminous efficiency, long fluorescence lifetime, large Stokes shift (the Stokes shift is the difference between the strongest wavelengths in the absorption spectrum and the emission spectrum of the same electron transition), narrow emission spectrum, and the like. Therefore, the rare earth complex can effectively replace inorganic quantum dots and organic dyes, so that the rare earth fluorescent microspheres are obtained, the interference of background fluorescence and stray light is eliminated, and the biological detection sensitivity and accuracy are improved.
The current detection means of C peptide level mainly comprises a chemical method and an immunological method. The chemical method has the advantages of easy operation, fast detection and the like, but has poor selectivity, is easy to be interfered by external environment, has strong dependence of luminous intensity on the environment and is easy to generate false positive. The immunization method is based on antigen-antibody specific binding, and has high specificity and sensitivity. Therefore, the C peptide rare earth fluorescent microsphere immunochromatography detection card is prepared by using the rare earth fluorescent microsphere as a labeled antibody material, so that the interference of non-specific fluorescence can be effectively eliminated, and the analysis sensitivity is greatly improved.
Disclosure of Invention
The invention aims to provide a C peptide rare earth fluorescent microsphere kit, a detection card and a preparation method thereof.
The technical scheme for realizing the purpose of the invention is as follows: the C peptide rare earth fluorescent microsphere detection card comprises a card shell and a detection test strip arranged in the card shell; the detection test strip comprises a bottom lining, and a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially lapped and stuck on the bottom lining along the length direction; a microsphere line formed by the C peptide monoclonal antibody marked by the C peptide rare earth fluorescent microsphere is sprayed on the combination pad; the rare earth fluorescent microspheres are rare earth ion complex doped fluorescent microspheres; the nitrocellulose membrane is provided with a detection line and a quality control line in parallel, wherein the detection line is close to the combination pad, and the quality control line is far away from the combination pad; the detection line is coated with a C peptide monoclonal antibody, and the quality control line is coated with a goat anti-mouse IgG antibody.
The particle size of the rare earth fluorescent microsphere is 221.65 +/-9.48 nm.
The content of the C peptide monoclonal antibody marked by the C peptide rare earth fluorescent microspheres sprayed on the bonding pad is 50-200 mug of antibody/200 mug L of fluorescent microspheres, and the coating concentration of the C peptide monoclonal antibody on the detection line is 0.3-2 mg/m L.
The sample pad adopts the blood filtering membrane, preferably the whole blood filtering membrane, can realize whole blood direct detection, can filter the erythrocyte completely to need not to handle, directly use.
The clamping shell comprises an upper plastic shell and a lower plastic shell which are clamped with each other; the plastic upper shell is provided with a sample adding hole, an observation window and a one-dimensional code information area; the position of the sample adding hole corresponds to the position of the sample pad; the position of the observation window corresponds to the position of the nitrocellulose membrane; and the one-dimensional code information area is sprayed with one-dimensional codes which are used for the identification of the instrument and correspond to the detection items.
The plastic upper shell is provided with a sample adding groove, and the groove wall of the sample adding groove is an inward concave arc surface; the sample adding hole is arranged on the bottom surface of the sample adding groove; a gap is arranged between the bottom surface of the sample adding groove and the detection test strip.
The plastic upper shell is also provided with an anti-skid area; the anti-skidding area is an inwards concave arc surface, and a plurality of salient points are uniformly distributed in the anti-skidding area.
The present invention also provides: the C peptide rare earth fluorescent microsphere kit comprises a detection card and an ID card containing a calibration curve; the detection card adopts the C peptide rare earth fluorescent microsphere detection card.
The kit is used for quantitatively detecting the content of C Peptide (CP) in human serum, blood plasma and whole blood by utilizing an immunochromatography technology according to the principle of a double-antibody sandwich method. The C-peptide (CP) in the sample is bound to a fluorescently labeled anti-C-peptide (CP) antibody to form an immune complex. The immune complex flows upwards under the action of capillary and reacts with the anti-CP antibody in the test area, and a fluorescence signal is generated in the reaction area after the immune complex is excited by an excitation light source with a specific wavelength. The intensity of the signal is proportional to the content of C-peptide (CP) in the sample.
The present invention also provides: the preparation method of the C peptide rare earth fluorescent microsphere kit comprises the following steps:
preparing rare earth fluorescent microspheres;
step two, activating the rare earth fluorescent microspheres;
step three, preparing a C peptide monoclonal antibody marked by rare earth fluorescent microspheres;
step four, preparing a sample pad and a combination pad;
step five, processing the nitrocellulose membrane;
and step six, sequentially and mutually overlapping and sticking the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper on the bottom lining to obtain a test paper board, and then cutting the test paper board into test paper strips.
Assembling the test strip in a card shell;
and step eight, preparing the ID card containing the standard curve.
The method comprises the following specific steps of adding 50m L ultrapure water into a conical flask, vacuumizing for 30min to remove oxygen in water, weighing 0.015g of an emulsifier SDBS (sodium dodecyl benzene sulfonate) into a 50m L three-neck flask, adding 23m L of the ultrapure water treated by the previous step, magnetically stirring to dissolve the SDBS to obtain a water phase, respectively putting 0.1m L of MAA (methacrylic acid) and 0.9m L of styrene into a 1.5m L centrifugal tube, simultaneously weighing 0.03g of the europium complex into the mixed solution, ultrasonically dispersing uniformly to obtain an oil phase, sucking the oil phase in the previous step by a dropper, dropwise adding the oil phase into the water phase while stirring to uniformly mix, subjecting the emulsified mixed solution to ultrasonic fine precipitation for 30min, preparing a KPS (potassium persulfate) initiator with a certain concentration (30 mg/m), dropwise adding the potassium persulfate) into the water phase for dropwise adding, stirring while stirring, performing ultrasonic fine precipitation for 70 min, adding the potassium persulfate solution for 10 min, performing ultrasonic precipitation for 70 min, performing ultrasonic precipitation at a time, adding the supernatant fluid for 70 min, performing ultrasonic precipitation for 10 rpm, and obtaining a centrifugal precipitation reaction for a final time, and adding the supernatant fluid, and performing ultrasonic precipitation for 10 rpm.
By adopting the technical scheme, the invention has the following beneficial effects that (1) a microsphere line formed by the C peptide monoclonal antibody marked by the C peptide rare earth fluorescent microsphere is sprayed on the C peptide rare earth fluorescent microsphere detection card combination pad, the rare earth fluorescent microsphere can be synthesized in a batch control mode, has the advantages of uniform size and appearance, good water solubility, easiness in surface modification, high single-particle luminous intensity, long fluorescence life and the like, has the advantages of low background, strong fluorescent signal, high signal-to-noise ratio and the like compared with the imported fluorescent microsphere in the application of an in-vitro detection kit, and obviously improves the sensitivity of C peptide detection.
(2) The sample pad of the C peptide rare earth fluorescent microsphere detection card adopts a blood filtering membrane, preferably a whole blood filtering membrane, can realize direct detection of whole blood, can completely filter red blood cells, does not need treatment, and can be directly used.
(3) The shell of the C peptide rare earth fluorescent microsphere detection card comprises an upper plastic shell and a lower plastic shell which are mutually clamped, and the detection test strip can be replaced according to the requirement due to the structure, so that the shell can be recycled.
(4) The sample adding groove is formed in the card shell plastic upper shell of the C peptide rare earth fluorescent microsphere detection card, the groove wall of the sample adding groove is an inwards concave arc surface, and the bottom surface of the sample adding groove is provided with the sample adding hole, so that a sample can be prevented from remaining in the sample adding hole; a gap is arranged between the bottom surface of the sample adding groove and the detection test strip, so that the sample can be prevented from overflowing, and the accuracy and the sensitivity can be improved.
(5) The anti-skid region is arranged on the card shell plastic of the C peptide rare earth fluorescent microsphere detection card, and a plurality of salient points are uniformly distributed in the anti-skid region, so that the detection card is convenient to operate by hands.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the present disclosure taken in conjunction with the accompanying drawings, in which
FIG. 1 is a schematic structural diagram of a C-peptide rare earth fluorescent microsphere detection card.
FIG. 2 is a schematic structural diagram of a detection test strip of the C-peptide rare earth fluorescent microsphere detection card of the present invention.
FIG. 3 is a transmission electron microscope image of the rare earth europium complex doped fluorescent microsphere in example 1 of the present invention.
FIG. 4 shows the fluorescence emission spectrum and excitation spectrum of the rare earth europium complex doped fluorescent microsphere in example 1 of the present invention.
FIG. 5 is a standard curve diagram of the C peptide rare earth fluorescent microsphere kit of example 1.
The reference numbers in the drawings are:
the device comprises a bottom lining 1, a sample pad 2, a combination pad 3, a nitrocellulose membrane 4, absorbent paper 5, a detection line 6, a quality control line 7, a plastic upper shell 8, a plastic lower shell 9, a sample adding hole 10, an observation window 11, a one-dimensional code information area 12, a sample adding groove 13 and an anti-skid area 14.
Detailed Description
(example 1)
The C peptide rare earth fluorescent microsphere kit comprises a detection card and an ID card containing a calibration curve; the detection card comprises a card shell and a detection test strip arranged in the card shell.
Referring to fig. 2, the test strip comprises a bottom lining, and a sample pad 2, a binding pad 3, a nitrocellulose membrane 4 and a absorbent paper 5 which are sequentially overlapped and stuck on the bottom lining 1 along the length direction.
The binding pad 3 is sprayed with a microsphere line formed by C peptide monoclonal antibodies (the content is 50-200 mug of antibodies/200 mug L of fluorescent microspheres) marked by C peptide rare earth fluorescent microspheres, the rare earth fluorescent microspheres are fluorescent microspheres doped with rare earth ion complexes, the particle size is 221.65 +/-9.48 nm, preferably the rare earth fluorescent microspheres are doped with rare earth europium elements (the solid content is 1%, w/w), the particle size is 221.65 +/-9.48 nm, and the rare earth fluorescent microspheres are stable under the ground state and emit fluorescence with the wavelength range of 600-620 nm under the action of an excitation light source of 320-380 nm.
The nitrocellulose membrane 4 is provided with a detection line 6 and a quality control line 7 in parallel, wherein the detection line 6 is close to the combination pad 3, the quality control line 7 is far away from the combination pad 3, the distance between the detection line 6 and the quality control line 7 is 3-5 mm, the detection line 6 is coated with a C peptide monoclonal antibody, the quality control line 7 is coated with a goat anti-mouse IgG antibody, the coating concentration of the C peptide monoclonal antibody on the detection line 6 is 0.3-2 mg/m L, the coating amount is 0.5-1.5 mu L coating liquid amount/cm membrane, and the coating concentration of the goat anti-mouse IgG antibody in the quality control line 7 is 0.3-2 mg/m L, and the coating amount is 0.5-1.5 mu L coating liquid amount/cm membrane.
The sample pad 2 adopts a blood filtering membrane, preferably a whole blood filtering membrane, can realize the direct detection of whole blood, can completely filter red blood cells, and does not need to be processed and directly used.
Referring to fig. 1, the card shell comprises an upper plastic shell 8 and a lower plastic shell 9 which are clamped with each other, so that the test strip can be replaced as required, and the card shell can be reused; the plastic upper shell 8 is provided with a sample adding hole 10, an observation window 11 and a one-dimensional code information area 12; the position of the sample adding hole 10 corresponds to the position of the sample pad 2; the position of the observation window 11 corresponds to the position of the nitrocellulose membrane 4; the one-dimensional code information area 12 is sprayed with a one-dimensional code corresponding to the detection item for recognition by the instrument.
Still referring to fig. 1, a sample adding slot 13 is arranged on the plastic upper shell 8, and the slot wall of the sample adding slot 13 is an inward concave arc surface; the sample adding hole 10 is arranged on the bottom surface of the sample adding groove 13, so that a sample can be prevented from remaining in the sample adding hole 10; a gap is arranged between the bottom surface of the sample adding groove 13 and the detection test strip, so that the sample can be prevented from overflowing, and the accuracy and the sensitivity can be improved.
The plastic upper shell 8 is also provided with an anti-skid area 14; the anti-skid area 14 is an inwards concave arc surface, and a plurality of salient points are uniformly distributed in the anti-skid area 14.
The preparation method of the C peptide rare earth fluorescent microsphere kit comprises the following steps:
the preparation method comprises the steps of firstly, preparing rare earth fluorescent microspheres, namely, adding 50m L ultrapure water into a conical flask, vacuumizing for 30min to remove oxygen in water, weighing 0.015g of emulsifier SDBS (sodium dodecyl benzene sulfonate) into a 50m L three-neck flask, adding 23m L of the ultrapure water treated in the steps, magnetically stirring to dissolve the SDBS to obtain a water phase, respectively putting 0.1m L of MAA (methacrylic acid) and 0.9m L of styrene into a 1.5m 5 centrifugal tube, simultaneously weighing 0.03g of europium complex into the mixed solution, ultrasonically dispersing uniformly to obtain an oil phase, sucking the oil phase in the steps by a dropper, dropwise adding the oil phase into the water phase while stirring to uniformly mix, ultrasonically emulsifying the oil-water mixed solution for 30min through a water bath, preparing an initiator KPS (potassium persulfate) aqueous solution with a certain concentration (30mg/m L), pouring the initiator KPS (potassium persulfate) aqueous solution into the ultrasonically-water mixed solution, ultrasonically stirring for uniformly mixing, adding the ultrasonically emulsifying the ultrasonically, adding the ultrasonically-water mixed solution into the three-phase ultrasonically emulsified mixed solution, adding the three-ultrasonically emulsified solution for 30min, carrying out a three-neck flask at a temperature of 8665 min, carrying out a 25 min, carrying out a centrifugal reaction at a temperature of 70 min, carrying out a centrifugal precipitation at a centrifugal reaction, carrying a centrifugal reaction at a centrifugal precipitation speed of 30.
The fluorescent microspheres are characterized by a Transmission Electron Microscope (TEM), and the transmission electron microscope images show the morphology and the nanometer size of the rare earth fluorescent microspheres synthesized in the embodiment. As shown in FIG. 3, the rare earth fluorescent microsphere synthesized by the embodiment has good monodispersity and complete morphology. The fluorescence spectrum shown in FIG. 4 shows the wavelength ranges of the excitation spectrum and the emission spectrum of the fluorescent microspheres.
And step two, activating the rare earth fluorescent microspheres, namely ultrasonically treating the fluorescent microspheres for 2min, centrifuging the fluorescent microspheres 200 mu L at 14000rpm for 15min, washing precipitates to 1m L by using a MES solution with the pH value of 6.0, ultrasonically treating the precipitates for 2min, adding 50 mu l of 100mg/m L carbodiimide, uniformly mixing the precipitates for 5min, adding 100mg/m L N-hydroxy thiosuccinimide with the pH value of 100 mu L, centrifuging the precipitates at 14000rpm for 15min after uniformly mixing the precipitates for 15min, and washing the precipitates to 1m L by using the MES solution with the pH value of 6.0.
And step three, preparing the C peptide monoclonal antibody marked by the rare earth fluorescent microspheres, namely adding the C peptide monoclonal antibody into the activated fluorescent microspheres after carrying out ultrasonic treatment for 2min according to 100 mu g/200 mu L, mixing the mixture evenly for 2 h, sealing the mixture by 50mM containing 0.5% BSA and 0.1% glycine and Tris-HCl sealing solution with the pH value of 8.0 for 1 h, then carrying out high-speed centrifugation at 14000rpm for 15min, washing the mixture twice by buffer solution in Tris-HCl preservation solution with the pH values of 1% NaCl, 0.5% BSA, 0.5% sucrose, 0.1% Tween20 and 8.0, carrying out ultrasonic treatment to resuspend the mixture to 200 mu L, and storing the mixture in a dark place at 4 ℃.
Step four, preparing a sample pad 2 and a bonding pad 3: the sample pad 2 selects a whole blood filter membrane, so that the whole blood can be directly detected, red blood cells can be completely filtered, and the whole blood filter membrane is not required to be processed and can be directly used; on the binding pad 3, the C peptide monoclonal antibody labeled with the rare earth fluorescent microspheres was diluted 15 times with a microsphere diluent (20 mm tris-Hcl buffer containing 0.5% BSA, 25% sucrose) and uniformly sprayed one line (i.e., microsphere line) at a dose of 4 μ l liquid/cm of the sample pad 2. The mixture was placed in an oven and dried overnight at 37 ℃.
And step five, treating the nitrocellulose membrane 4, namely respectively adjusting the concentration of the C peptide monoclonal antibody and the goat anti-mouse IgG antibody to 0.5mg/m L and 1mg/m L by using coating buffer solution (10 mMPBS buffer solution containing 2.5 percent of cane sugar), coating the membranes by using coating solution amount of 1 mu L per cm, respectively serving as a detection line 6 and a quality control line 7, parallel scribing on the nitrocellulose membrane 4 for coating, wherein the interval between the quality control line 7 and the detection line 6 is 4mm, airing in an oven with the humidity of less than 30 percent and the temperature of 37 ℃ for 10 hours, and sealing the bags for later use.
And step six, sequentially and mutually overlapping and adhering a sample pad 2 (with the size of 17 × 300mm and made of a blood filtering membrane material), a bonding pad 3 (with the size of 16 × 300mm and made of glass fiber cotton material), a nitrocellulose membrane 4 (with the size of 25 × 300mm) and absorbent paper 5 (with the size of 28 × 300mm) on a bottom liner 1 (with the size of 80 × 300mm) to obtain a test paper board, and cutting the test paper board into test paper strips.
And step seven, assembling the test strip in the card shell.
Step eight, preparing an ID card containing a standard curve: each box contains an ID card (same batch of standard curves) of a standard curve, quality control products with different concentrations are measured through a detection card, the concentration of the quality control products is taken as an abscissa, a fluorescence signal ratio is taken as an ordinate, the standard curve is drawn, the ID card is written into the standard curve to generate a one-dimensional code, and a dry fluorescence immunoassay analyzer is used for reading corresponding one-dimensional code information on a reagent card and measuring the corresponding concentration.
The quantitative detection of the C peptide rare earth fluorescent microsphere kit of the embodiment is as follows:
①, drawing of Standard Curve
The time-resolved fluorescence detection card prepared in example 1 was added with C-peptide antigen quality control samples of different concentrations (7 concentrations of 0.3, 1, 2, 4, 8, 15, 20ng/m L, three replicates were set for each concentration, C-peptide antigen was diluted with calf serum to obtain 20 ng/L mother liquor, and then diluted with calf serum to different quality control sample concentrations), after chromatography for 15min, C, T-line fluorescence signals and C/T values were read by AFS-1000 dry fluorescence immunoassay analyzer manufactured by tibo bio-science and technology limited, the experimental results and analyses are shown in table 1:
and (3) drawing a standard curve by using the C peptide quality control substance concentration and the sample signal T/C average value, wherein the curve data are shown in the table 1, and the standard curve is shown in the figure 5. Wherein the R value is 0.9974, and the concentration of C peptide contained in the sample is quantitatively determined through the line.
② testing the performance of time-resolved fluorescent test paper card
The lowest detection limit, namely, the repeated measurement is carried out for 20 times by using a zero-value sample, the mean M and the standard deviation ID of 20 times of results are calculated, the detection limit (M +2ID) of the method is reported by adding two times of standard deviation to a blank mean, the result is 0.24ng/M L, and the sensitivity is 0.3ng/M L.
Linear range takes 7 concentration values between 0.2 ~ 10ng/m L, and every concentration repeated measurement is three times, will determine concentration average and carry out linear analysis with theoretical concentration, obtains linear equation y 0.996x +0.0912, r 0.9995, shows that the utility model discloses the kit is fine at 0.3 ~ 20ng/m L linear range within the relevance.
The precision is that three batches of the C peptide rare earth fluorescent microsphere kit are used for respectively detecting 15 and 4ng/m L repetitive quality control products, each batch of the kit is parallelly detected for 10 times by the repetitive quality control products, the CV in the three batches with the concentration of 15ng/m L is respectively 7.01 percent, 5.72 percent and 6.14 percent, the CV in the three batches with the concentration of 15ng/m L is 6.21 percent, the CV in the three batches with the concentration of 4ng/m L is respectively 6.86 percent, 8.77 percent and 7.81 percent, and the CV in the three batches is 7.63 percent and is within 10 percent.
The accuracy is that a quality control substance with the concentration of 1ng/m L is selected as a detection sample and is divided into 3 parts with the same volume, 15ng/m L and 4ng/m L accuracy quality control substances are respectively added into 2 parts of the samples to prepare 2 recovered samples with different adding concentrations, the concentration of the added substance to be detected is calculated, the solution without the detected substance with the same amount is added into the other part of the sample to prepare a basic sample, the samples are repeatedly analyzed for 3 times, the average value is taken to calculate, the recovery rate (the concentration of the analyzed sample-the concentration of the basic sample)/the adding concentration × 100% is calculated, the recovery rate of the recovered sample 15ng/m L is 97.78%, the recovery rate of the recovered sample 4ng/m L is 95.53%, the average recovery rate is 96.56%, and the deviation is within 10%.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
- C peptide rare earth fluorescent microsphere detection card, its characterized in that: comprises a card shell and a detection test strip arranged in the card shell; the detection test strip comprises a bottom lining, and a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially lapped and stuck on the bottom lining along the length direction; a microsphere line formed by the C peptide monoclonal antibody marked by the C peptide rare earth fluorescent microsphere is sprayed on the combination pad; the rare earth fluorescent microspheres are rare earth ion complex doped fluorescent microspheres; the nitrocellulose membrane is provided with a detection line and a quality control line in parallel, wherein the detection line is close to the combination pad, and the quality control line is far away from the combination pad; the detection line is coated with a C peptide monoclonal antibody, and the quality control line is coated with a goat anti-mouse IgG antibody.
- 2. The C peptide rare earth fluorescent microsphere detection card according to claim 1, characterized in that: the particle size of the rare earth fluorescent microsphere is 221.65 +/-9.48 nm.
- 3. The detection card of the C peptide rare earth fluorescent microspheres of claim 1, wherein the content of C peptide monoclonal antibodies marked by the C peptide rare earth fluorescent microspheres sprayed on the bonding pad is 50-200 μ g of antibodies/200 μ L of fluorescent microspheres, and the coating concentration of the C peptide monoclonal antibodies on the detection line is 0.3-2 mg/m L.
- 4. The C peptide rare earth fluorescent microsphere detection card according to claim 1, characterized in that: the sample pad adopts a blood filtering membrane.
- 5. The C peptide rare earth fluorescent microsphere detection card according to claim 1, characterized in that: the clamping shell comprises an upper plastic shell and a lower plastic shell which are clamped with each other; the plastic upper shell is provided with a sample adding hole, an observation window and a one-dimensional code information area; the position of the sample adding hole corresponds to the position of the sample pad; the position of the observation window corresponds to the position of the nitrocellulose membrane; and the one-dimensional code information area is sprayed with one-dimensional codes which are used for the identification of the instrument and correspond to the detection items.
- 6. The C peptide rare earth fluorescent microsphere detection card according to claim 5, characterized in that: the plastic upper shell is provided with a sample adding groove, and the groove wall of the sample adding groove is an inward concave arc surface; the sample adding hole is arranged on the bottom surface of the sample adding groove; a gap is arranged between the bottom surface of the sample adding groove and the detection test strip.
- 7. The C peptide rare earth fluorescent microsphere detection card according to claim 5, characterized in that: the plastic upper shell is also provided with an anti-skid area; the anti-skidding area is an inwards concave arc surface, and a plurality of salient points are uniformly distributed in the anti-skidding area.
- 8. A C peptide rare earth fluorescent microsphere kit is characterized in that: the method comprises the steps of detecting a card and an ID card containing a calibration curve; the detection card adopts the C peptide rare earth fluorescent microsphere detection card as claimed in one of claims 1 to 5.
- 9. The method for preparing a kit for detecting atrophic gastritis of claim 8, which comprises the following steps:preparing rare earth fluorescent microspheres;step two, activating the rare earth fluorescent microspheres;step three, preparing a C peptide monoclonal antibody marked by rare earth fluorescent microspheres;step four, preparing a sample pad and a combination pad;step five, processing the nitrocellulose membrane;and step six, sequentially and mutually overlapping and sticking the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper on the bottom lining to obtain a test paper board, and then cutting the test paper board into test paper strips.Assembling the test strip in a card shell;and step eight, preparing the ID card containing the standard curve.
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| CN202010193464.4A CN111504959B (en) | 2020-03-18 | 2020-03-18 | C peptide rare earth fluorescent microsphere kit, detection card and preparation method thereof |
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