CN113848330B - Detection method of homocysteine and vitamin B12, detection test strip and application thereof - Google Patents
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Abstract
The invention provides a detection method of homocysteine and vitamin B12, a detection test strip and application thereof, and belongs to the technical field of analytical chemistry and clinical detection. The detection method comprises the step of performing the detection in a solid-phase immunochromatography mode by adopting a competition method. The immunochromatographic test strip based on the quantum dots comprises a bottom plate, wherein a sample pad, a combination pad, a detection pad and a water absorption pad are sequentially arranged on the bottom plate, and adjacent pads are connected at the joint in an overlapping manner. The detection pad takes a nitrocellulose membrane as a base pad, and a detection line 1, a detection line 2 and a quality control line are sequentially arranged on the nitrocellulose membrane. The invention optimizes the dissociation and detection processes of homocysteine and vitamin B12, thereby realizing the rapid, sensitive and specific detection of the substances.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry and clinical detection, and particularly relates to a detection method of homocysteine and vitamin B12, a detection test strip and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Homocysteine (Hcy) is a sulfhydryl-containing amino acid and an important intermediate metabolite in the metabolic process of methionine in a human body. It was found that, under disease conditions, enzyme metabolism is impaired and Hcy concentration in blood is increased. Hcy has been considered by the international medical community as an independent risk factor for the development of atherosclerosis and cardiovascular and cerebrovascular diseases such as thrombosis and coronary heart disease, and the clinical significance of Hcy is even considered to exceed that of cholesterol. Furthermore, elevated Hcy is a sensitive biomarker reflecting a deficiency in human vitamin B12.
Vitamin B12 (Vitamin B12, VB 12) is a polycyclic compound containing 3-valent cobalt, 4 reduced pyrrole rings are connected together to become 1 corrin macrocycle (similar to porphyrin), and the compound is the only essential water-soluble Vitamin containing metal elements, plays an important role in the substance metabolism process in the body, can cause megaloblastic anemia in the body in the absence of the essential water-soluble Vitamin, and can induce various neurological diseases. Therefore, the homocysteine and VB12 detection method with high selectivity and sensitivity has important value.
The existing detection methods aiming at homocysteine and VB12 mainly comprise high performance liquid chromatography-ultraviolet spectrum, Atomic Absorption Spectrum (AAS), enzyme-linked immunosorbent assay (ELISA), spectrophotometry, microbiological analysis and the like. However, these detection methods generally have the problems of high detection limit, long analysis time, high price, need of professional technicians and the like.
Quantum Dots (QDs) are a novel luminescent nanomaterial with unique photoluminescence, chemical stability and excellent biocompatibility in the detection field. However, the current fluorescent detection system based on Quantum Dots (QDs) has strict requirements on precursor substances and detection environment, long analysis time and high cost, cannot meet the requirement of rapid and mass detection, and has complex detection flow; it is not very specific for homocysteine and VB 12. Meanwhile, homocysteine and VB12 exist in vivo in association with proteins, and therefore, when a biological sample such as serum or plasma is detected, the sample needs to be dissociated so that homocysteine and VB12 are released from the associated proteins for detection. However, the pretreatment process is often too complex and the dissociation is not complete, resulting in inaccurate detection results.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a detection method of homocysteine and vitamin B12, a detection test strip and application thereof. The invention optimizes the dissociation and detection processes of homocysteine and vitamin B12, thereby realizing the rapid, sensitive and specific detection of the substances.
The invention is realized by the following technical scheme:
in a first aspect of the invention, there is provided a method for detecting homocysteine and vitamin B12, said method comprising performing the assay as a solid phase immunochromatographic assay using a competition method.
Specifically, when the analyte is homocysteine, the detection method comprises the following steps:
s1, adding a sample to be tested into the diluent for dilution and dissociation, and converting the sample to be tested into a sample to be tested of S-adenosyl homocysteine (SAH) through an enzymatic reaction;
s2, adding the substance to be detected to an immunochromatography test strip based on quantum dots, and carrying out fluorescence detection on the test strip after color development is completed.
The diluent comprises a dissociator R1 and a converting agent R2, wherein the R1 and the R2 are mixed according to the volume ratio of 7-10: 1 (preferably 9: 1), the dissociator R1 is a Tris-HCl solution containing 1-10 mM (preferably 5 mM) DTT, and the R2 is a Tris-HCl solution containing 1-10U/ml (preferably 5U/ml) S-adenosylhomocysteine hydrolase, and the sample to be detected is catalyzed to be converted into SAH.
Specifically, when the analyte is vitamin B12, the detection method includes:
s1, adding a sample to be tested into the diluent for dilution and dissociation, and converting the sample to be tested into a substance to be tested of cyanocobalamin under the cyanide condition after reduction;
s2, adding the substance to be detected to an immunochromatography test strip based on quantum dots, and carrying out fluorescence detection on the test strip after color development is completed.
Wherein the diluent is a strong alkali solution containing thioglycerol, DTT and potassium cyanide; more specifically, the strong alkali solution is 0.01-0.1M (preferably 0.05M) NaOH solution, and the final concentration of the thioglycerol is 0.5-2 mM, preferably 1 mM); the final concentration of DTT is 1-10 mM, preferably 5 mM; the final concentration of potassium cyanide is 0.001 to 0.01%, preferably 0.005%.
The sample to be tested may be a blood product including, but not limited to, whole blood, serum, and plasma.
The immunochromatographic test strip comprises a base plate, wherein a sample pad, a combination pad, a detection pad and a water absorption pad are sequentially arranged on the base plate, and the adjacent pads are connected at the joint in an overlapping manner.
Wherein, the sample pad is sprayed with a slow release film.
Specifically, the sample pad may be made of a glass cellulose membrane material, which has excellent hydrophilicity and is rapidly re-wetted, so that the conjugate is sufficiently released.
The slow release film is obtained by adding gelatin, hydroxypropyl methylcellulose (HPMC) and glycerol into water and uniformly stirring, the film forming property of the slow release film is controlled by adjusting the proportion of the gelatin, the HPMC and the glycerol and the water content, the aim of controlling the slow release film to be dissolved at regular time 5min after the sample liquid is added is fulfilled, and the whole process has no influence on the detection result.
The mass ratio of the gelatin to the hydroxypropyl methyl cellulose to the glycerol is 1-3: 1:1, preferably 2:1:1, and the mass volume ratio of the gelatin to the water is 1-3 g: 50 ml; preferably 2 g: 50 ml;
in a specific embodiment of the invention, 2g of gelatin is added into 50ml of purified water, soaked for 2 hours, then kept at a constant temperature of about 65 ℃ and stirred at a constant speed to be dissolved; then adding 1g of hydroxypropyl methylcellulose (HPMC) and 1g of glycerol, and fully stirring and uniformly mixing to obtain the hydroxypropyl methyl cellulose (HPMC) and the glycerol.
The specific process is as follows: mixing a blood sample and a dissociating agent to prepare a sample solution, dripping a certain amount of sample solution at a sample adding hole, wherein a film at the position plays a role in physical isolation, so that a substance to be detected in the sample is fully dissociated and converted by the dissociating agent, dissolving the film after 5min, releasing the dissociated substance to be detected into a sample pad, and then starting to display the plate.
The detection pad takes a nitrocellulose membrane (NC membrane) as a base pad, and a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane.
When the analyte is homocysteine, the analyte is,
the binding pad is sprayed with a quantum dot labeled SAH antibody and a quantum dot labeled chicken IgY antibody; furthermore, the mass ratio of the two components can be 0.5-2: 1, and preferably 1: 1.
The quantum dots are CdSe/ZnS quantum dots, and have the advantages of small half-peak width of an emission spectrum, high luminous efficiency, high sensitivity, stable performance and the like compared with common fluorescent substances.
The SAH antibody is an SAH monoclonal antibody; specifically, the quantum dot labeled SAH antibody is obtained by adopting the following preparation method:
adding CdSe/ZnS quantum dots into a buffer solution to activate the carboxyl of the quantum dots, and connecting the CdSe/ZnS quantum dots with the amino group of the SAH monoclonal antibody.
Specifically, the preparation method comprises the following steps:
adding CdSe/ZnS quantum dots into MES buffer solution for activation, adding EDC and NHS, adding SAH monoclonal antibody, mixing, reacting, performing centrifugal elution with borate buffer solution, redissolving, and diluting.
The MES buffer solution is neutral or weakly acidic, and is further preferably 5-7 in pH, for example 6.5.
The borate buffer is neutral or weakly alkaline, and is further preferably pH 7-9, such as pH 8.4.
The detection line is coated with SAH-BSA antigen, namely the SAH antigen coupled with BSA;
the quality control line is coated with goat anti-chicken IgY antibody.
The water absorption pad can be a water absorption plate or water absorption paper;
the bottom plate can be made of PVC plates.
When the test substance is vitamin B12,
the binding pad is sprayed with a quantum dot labeled vitamin B12 antibody and a quantum dot labeled chicken IgY antibody; furthermore, the mass ratio of the two components can be 0.5-2: 1, and preferably 1: 1.
The quantum dots are CdSe/ZnS quantum dots, and have the advantages of small half-peak width of an emission spectrum, high luminous efficiency, high sensitivity, stable performance and the like compared with common fluorescent substances.
The vitamin B12 antibody is a vitamin B12 monoclonal antibody; specifically, the quantum dot labeled vitamin B12 antibody is obtained by the following preparation method:
adding CdSe/ZnS quantum dots into a buffer solution to activate the carboxyl of the quantum dots, and then connecting the quantum dots with the amino group of the vitamin B12 monoclonal antibody.
Specifically, the preparation method comprises the following steps:
adding CdSe/ZnS quantum dots into MES buffer solution for activation, adding EDC and NHS, adding vitamin B12 monoclonal antibody, mixing, reacting, performing centrifugal elution with borate buffer solution, redissolving, and diluting.
The MES buffer solution is neutral or weakly acidic, and is further preferably 5-7 in pH, for example 6.5.
The borate buffer is neutral or weakly alkaline, and is further preferably pH 7-9, such as pH 8.4.
The detection line is coated with a vitamin B12 coupled antigen;
the quality control line is coated with goat anti-chicken IgY antibody.
In a third aspect of the invention, there is provided the use of the above-mentioned detection method or test strip in any one or more of:
1) detecting homocysteine;
2) vitamin B12 assay.
The detection may be a qualitative or quantitative detection, preferably a quantitative detection.
The beneficial technical effects of one or more technical schemes are as follows:
according to the technical scheme, the dissociation and detection processes of homocysteine and vitamin B12 are optimized, so that the substances can be quickly, sensitively and specifically detected, and the method has good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic view of the test strip of the present invention;
wherein, the test paper comprises 1-a sample pad, 2-a combination pad, 3-NC membrane, 4-absorbent paper, 5-PVC bottom plate, 6-slow release membrane, 7-homocysteine test line, 8-VB12 test line and 9-quality control line;
FIG. 2 is a schematic diagram of the external structure of the detection card of the present invention; the detection card is obtained by packaging a detection card shell with a test strip;
wherein, 10 is a sample adding hole, and 11 is a detection window;
FIG. 3 is a standard curve of homocysteine according to the invention;
FIG. 4 is a graph showing the standard curve of vitamin B12 according to the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is intended to include the plural unless the context clearly dictates otherwise, and it should be further understood that when the term "comprises" or "comprising" is used in this specification, it indicates the presence of the feature, step, operation, device, component or group thereof.
The principle of the homocysteine detection method based on the quantum dot fluorescence technology is as follows:
an SAH-BSA antigen is marked on an NC membrane to serve as a detection line, and a quantum dot marked SAH antibody (Q-Ab) and a quantum dot marked chicken IgY antibody are sprayed on a binding pad to serve as the binding pad. After sample addition, the antigen to be detected (SAH) in the sample is respectively combined with the quantum dot labeled antibody to form a quantum dot labeled antibody-antigen complex. And then, the sample passes through a detection line area solidified with the SAH-BSA antigen, the labeled antibody which is not combined with the antigen to be detected is combined with the detection line antigen to form a quantum dot labeled antibody-antigen complex, the uncombined quantum dot labeled chicken IgY antibody continuously moves forwards and is combined with the goat anti-chicken IgY antibody in the quality control line area, and the quality control calibration reaction is completed. The quantum dots are excited by a matched instrument to generate fluorescence signals, so that a qualitative result can be obtained, the content of the antigen to be detected (SAH) in the sample and the intensity of the fluorescence signals are in negative correlation within a certain range, and the concentration of the sample to be detected can be obtained through calculation of a standard curve, so that a quantitative detection result is obtained.
The principle of the detection method based on the quantum dot fluorescence technology VB12 is as follows:
the kit adopts a competition method and carries out determination in a solid-phase immunochromatography mode. Coating vitamin B12-BSA antigen on a nitrocellulose membrane (NC membrane) as a detection line, and coating goat-anti-chicken IgY on the NC membrane as a quality control line; the quantum dot marked VB12 monoclonal antibody and the quantum dot marked chicken IgY are uniformly mixed and sprayed on a nitrocellulose membrane to prepare the binding pad. During testing, a sample is dripped into a sample adding hole, and the vitamin B12 in the sample is combined with the vitamin B12 monoclonal antibody of the quantum dot mark pre-coated on the nitrocellulose membrane, so that the combination amount of the folic acid monoclonal antibody of the quantum dot mark and the vitamin B12 coupling antigen pre-coated on a detection line is reduced, and the fluorescence band of the detection line (T line) is weakened or disappears. Fluorescence bands appear in the control line region regardless of the presence of vitamin B12 in the sample. The quantum dots are excited by a matched instrument to generate fluorescent signals, so that a qualitative result can be obtained, the ratio (T/C) of the light signal intensity on the detection line to the light signal intensity of the quality control line is in negative correlation with the sample concentration, the concentration of a sample to be detected can be obtained through standard curve calculation, and a quantitative detection result can be obtained.
According to the application, after a serum, plasma or whole blood sample is treated by a sample diluent, the sample is directly sampled and dripped into a sample adding hole by a liquid transferring device, and the sample is added, and after 15min of a display board, the sample is taken out for detection.
1) The accuracy of the obtained experimental result is good, and the relative deviation with the national standard is within +/-10%;
2) the obtained experimental result has good repeatability, and CV does not exceed 10%;
3) the sample does not need special treatment, and serum, plasma and whole blood samples can be used for detection;
4) the kit and the dissociation liquid do not need to be refrigerated, and can be transported and stored at normal temperature.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
The embodiment provides homocysteine, vitamin B12 joint detection kit, including the test paper strip, the test paper strip includes the PVC board, sets up nitrocellulose membrane (NC membrane) on the PVC board, and NC membrane upper end is provided with the combination pad of polyester film material, and the combination pad upper end is provided with the sample pad of cellophane membrane material, and the other end of the NC membrane that corresponds with this two is equipped with water absorption pad (paper). The bonding pad is coated with a coating with the mass ratio of 1: 1:1, quantum dot labeled homocysteine antibody, quantum dot labeled vitamin B12 antibody and quantum dot labeled chicken IgY antibody; the nitrocellulose membrane (NC membrane) is provided with a detection line 1, a detection line 2 and a quality control line, wherein the detection line 1 is coated with homocysteine antigen, the detection line 2 is coated with vitamin B12 antigen, and the quality control line is coated with goat anti-chicken IgY; the quantum dots are cadmium selenide quantum dots.
The preparation method of the kit for combined detection of homocysteine and vitamin B12 in the embodiment comprises the following specific operation steps:
1) preparation of a coating film: taking an NC membrane and a PVC plate, pasting a non-spotting surface of the NC membrane on a set position of the PVC plate, taking homocysteine antigen with the concentration of 1.0mg/ml, vitamin B12 antigen and goat anti-chicken IgY, coating the NC membrane on a detection line 1, a detection line 2 and a quality control line position of the NC membrane by cutting the membrane by a membrane cutting and gold spraying instrument according to 1 mul/cm, and drying for 8h at 37 ℃ to obtain the coating membrane.
2) Preparing a bonding pad:
a: adding 50 μ L of cadmium selenide quantum dots into 4ml of 10mM MES buffer (pH 6.5), activating for 15min, and storing at 4 deg.C in dark place;
b: taking the quantum dot marking solution prepared in the step a, sequentially adding 15 mul of 20mg/ml EDC and 15 mul of 20mg/ml NHS respectively, adding 200 mul of 5mg/ml SAH antibody, and reacting for 2h in a dark place;
c: b, transferring the marked mixed solution in the step b to a 100KD ultrafiltration tube for centrifugation, and centrifuging for 15min at 3500g/min to ensure that the volume of the centrifuged liquid is 1/10 less than the original volume; eluting 5 times with 50mM borate buffer (pH 8.4) to ensure that the liquid volume is less than 1/10 of the original volume after each centrifugation;
d: c, diluting the marked antibody obtained in the step c by using a compound solution, and uniformly mixing to obtain a quantum dot marked homocysteine antibody; the double solution is 20mM Tris-HCl buffer solution containing 1% BSA, 1% PEG-200, 5% trehalose and 5% mannitol.
e: preparing a quantum dot labeled vitamin B12 antibody and a quantum dot labeled chicken IgY antibody according to the same method of the steps a-d; the three labeled antibodies were then mixed at a volume ratio of 1: 1:1, preparing a bonding pad coating solution after uniformly mixing, spraying the coating solution on a polyester pad by using a film-scratching gold spraying instrument according to the concentration of 3 mu l/cm, and drying the polyester pad for 4 hours at 37 ℃ to obtain the bonding pad;
3) assembling:
tearing off the sticky paper at the corresponding position on the bottom plate, and sticking the absorbent paper above the NC film of the coating film to cover the NC film by 1.5 mm; pasting the bonding pad below the NC membrane of the coating membrane, and covering the NC membrane by 1.5 mm; and (3) sticking the sample pad below the bonding pad, and covering the bonding pad for 3mm to obtain the assembled large plate.
4) Film covering: covering the prepared slow release film on the sample adding hole of the assembled large-plate sample pad, and fixing the upper part and the lower part of the slow release film by using invisible adhesive tapes respectively.
The preparation method of the sustained-release membrane comprises the following steps: adding 2g of gelatin into 50ml of purified water, soaking for 2 hours, keeping the temperature to about 65 ℃, and stirring at a constant speed to dissolve the gelatin; then adding 1g of hydroxypropyl methylcellulose (HPMC) and 1g of glycerol, and fully stirring and uniformly mixing to obtain the hydroxypropyl methyl cellulose (HPMC) and the glycerol.
5) Slitting and casing: a microcomputer cutting machine is used, the width of the test strip is set, and then the test strip is cut to obtain the prepared test strip; and (3) putting the test strip into a detection card shell in the forward direction, pressing the shell and packaging well to obtain the single-person homocysteine and vitamin B12 combined detection card. The detection card shell is formed by buckling an upper part and a lower part, the upper part of the detection card shell is provided with a sample adding hole and a detection window, and the sample adding hole corresponds to the position of the slow release membrane and is used for adding samples to the detection test strip; the inspection line and the quality control line can be observed through the inspection window.
6) The kit also contains a sample diluent, and when the reagent strip is used for detecting homocysteine, the sample diluent specifically comprises a dissociating agent R1 and a transforming agent R2 according to an optimal formula, wherein the dissociating agent R1 is a Tris-HCl solution containing dithiothreitol (DTT, the concentration is 5 mM), R2 is a Tris-HCl solution containing S-adenosyl homocysteine hydrolase (SAHH, the concentration is 5U/ml), and R1 and R2 are mixed according to a volume ratio of 9:1, mixing.
When used for the detection of vitamin B12, samples of dilutions were made according to the optimal formulation, specifically a strong alkaline solution containing thioglycerol, DTT and potassium cyanide, adding thioglycerol, DTT and potassium cyanide to a 0.05M NaOH solution to final concentrations of 1mM, 5mM and 0.005 mM, respectively.
The kit for combined detection of homocysteine and vitamin B12 comprises a test strip for combined detection of homocysteine and vitamin B12 and a corresponding sample diluent. According to the technical scheme, the same homocysteine and vitamin B12 are combined with the test strip for detection, so that homocysteine and vitamin B12 can be detected respectively.
Of course, the test strip for separately detecting homocysteine and vitamin B12 can also be prepared, and the difference is only that when the test strip for separately detecting homocysteine is prepared, the coating on the bonding pad has a mass ratio of 1:1 quantum dot-labeled homocysteine antibody and quantum dot-labeled chicken IgY antibody; the nitrocellulose membrane (NC membrane) is only provided with a detection line and a quality control line, the detection line is coated with homocysteine antigen, and the quality control line is coated with goat anti-chicken IgY; when the test strip for separately detecting vitamin B12 is prepared, the coating on the bonding pad has a mass ratio of 1:1, a mixture of a quantum dot labeled vitamin B12 antibody and a quantum dot labeled chicken IgY antibody; the nitrocellulose membrane (NC membrane) is only provided with a detection line and a quality control line, the detection line is coated with vitamin B12 antigen, and the quality control line is coated with goat anti-chicken IgY. The specific preparation method is adjusted correspondingly.
Comparative example 1
The preparation method was the same as example 1 except that 2g of Hydroxypropylmethylcellulose (HPMC) and 0.5g of glycerin were added at the time of preparing the sustained-release film.
Comparative example 2
The preparation method is the same as that of example 1, and the difference is only that when the SAH antibody and the VB12 antibody are marked by the quantum dots, the buffer system is phosphate buffer solution with pH 7.0.
Comparative example 3
The preparation method is the same as that of example 1, and is only different in that when the SAH antibody is marked by the quantum dots, the volume of the added SAH antibody with the concentration of 5mg/ml is 100 mul; when VB12 antibody was quantum dot-labeled, VB12 antibody was added at a concentration of 5mg/ml in a volume of 100. mu.l.
Comparative example 4
The preparation method is the same as that of example 1, except that when the analyte is homocysteine, the volume ratio of R1 to R2 in the sample diluent is 8: 1 to prepare a diluent.
Comparative example 5
The preparation was identical to example 1, except that when the test substance was vitamin B12, the final concentration of thioglycerol in the sample dilution was 2 mM.
Effect verification
The samples to be tested are diluted by diluent and dripped into the sample adding holes, the timing is started, the time of the slow release pad for obstructing the sample liquid (the time from the sample liquid adding to the sample pad) and the detection results of the 20 samples are recorded.
The blocking times are shown in table 1 below:
TABLE 1
The results are shown in table 2 below:
TABLE 2
The data in the table 2 show that the time for the sample liquid to enter the sample pad can be accurately controlled to be about 5min (300 s) by using the prepared slow release pad in the kit embodiment of the invention, so that the dissociation effect of the sample is ensured, the sensitivity and the accuracy are higher, and the correlation coefficients of the detection result and the assignment result of the sample are both more than 0.975, which is far superior to the detection result of the comparative example 1.
And (3) parallelly comparing and testing 40 clinical samples in the example 1 with the comparative examples 2 and 3, diluting the sample to be tested with a diluent, dripping the diluted sample into a sample adding hole, and recording the detection results of 40 samples after 15 min.
The results are shown in table 3 below:
TABLE 3
The data in the table 3 show that the buffer system of the labeled antibody and the addition amount of the antibody used in the kit embodiment of the present invention are the most suitable label system after optimization, the sensitivity and the accuracy of the reaction after sample addition are ensured, and the correlation coefficients of the detection result of the sample and the assignment result are both above 0.975, which is far superior to the detection results of comparative example 2 and comparative example 3.
And (3) comparing and testing 40 clinical samples in parallel with comparative examples 4 and 5 in the example 1, diluting the sample to be tested with a diluent, dripping the diluted sample into a sample adding hole, and recording the detection results of 40 samples after 15 min.
The results are shown in table 4 below:
TABLE 4
The data in the table 4 show that the component proportion in the sample diluent used in the kit embodiment of the present invention is already optimal, the target detection substance can be sufficiently dissociated after being mixed with the sample, the accuracy of the sample detection result after sample addition is ensured, and the correlation coefficients of the sample detection result and the assignment result are both above 0.975, which is far superior to the detection results of comparative example 4 and comparative example 5.
Test example:
diluting homocysteine to 1. mu.M, 2. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 50. mu.M; vitamin B12 was diluted to 100pg/ml, 250pg/ml, 500pg/ml, 1000pg/ml, 2000pg/ml, 3000pg/ml as standard solutions, and blank solutions were prepared at the same time.
50 mul of standard solutions with different concentrations are respectively added into the sample adding holes of the detection card prepared in the embodiment 1, after 15min, the detection card finished by the display plate is put into a fluorescence immunoassay analyzer (Shandong Zifeng Biotechnology Co., Ltd.) for interpretation, the fluorescence values of detection lines and quality control lines with different concentrations are obtained, and the corresponding relation between the fluorescence value of the standard with each concentration and the concentration of the standard is recorded. And (4) establishing a standard curve equation according to the fluorescence signal-calibration concentration value of the measurement result. As shown in FIGS. 2-3, the regression equation of the standard curve of homocysteine is: y = 1/(0.1627 x + 1.2465); the standard curve equation for vitamin B12 is: y = 1/(0.0161 x + 4.3667). And burning the standard curve into an ID card to prepare a homocysteine and vitamin B12 calibration card.
In addition, a blank solution was added to the test card prepared in example 1, and the concentration of homocysteine in the reverse generation was 0. mu. mol/L, and the concentration of biotin B12 in the reverse generation was 0 pg/ml.
100 random clinical samples are taken, the kit prepared in the embodiment 1 of the invention is adopted to detect the concentrations of homocysteine and vitamin B12, and the detection method comprises the following steps: inserting the calibration card into a fluorescence immunoassay analyzer (Shandong Zifeng Biotechnology Co., Ltd.) to calibrate the apparatus; adding 10 μ l sample into 90 μ l sample diluent, mixing, adding 50 μ l sample solution dropwise onto the sample pad, placing the display panel into a fluorescence immunoassay analyzer (Shandong Zifeng Biotechnology Co., Ltd.) for 15min, and measuring and recording the detection results of homocysteine and vitamin B12.
When the reagent is used for detecting homocysteine, the sample diluent specifically comprises a dissociating agent R1 and a converting agent R2 according to an optimal formula, wherein the dissociating agent R1 is a Tris-HCl solution containing dithiothreitol (DTT, the concentration is 5 mM), R2 is a Tris-HCl solution containing S-adenosine homocysteine hydrolase (SAHH, the concentration is 5U/ml), and the volume ratio of R1 to R2 is 9:1, mixing.
When used for the detection of vitamin B12, samples of dilutions were made according to the optimal formulation, specifically a strong alkaline solution containing thioglycerol, DTT and potassium cyanide, adding thioglycerol, DTT and potassium cyanide to a 0.05M NaOH solution to final concentrations of 1mM, 5mM and 0.005 mM, respectively.
The results obtained are shown in Table 5 below:
TABLE 5
Therefore, the kit can directly and quantitatively detect the concentrations of homocysteine and vitamin B12, is convenient and quick, can obtain results in 15min, and is suitable for popularization and use in primary hospitals and physical examination centers.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A method for detecting homocysteine and vitamin B12, wherein said method comprises using a competition method to perform the detection in the form of solid-phase immunochromatography;
the detection method is carried out by adopting an immunochromatographic test strip based on quantum dots, the immunochromatographic test strip based on the quantum dots comprises a bottom plate, a sample pad, a combination pad, a detection pad and a water absorption pad are sequentially arranged on the bottom plate, and the adjacent pads are connected in an overlapping way at the joint;
a slow release film is sprayed on the sample pad;
the sample pad is made of a glass cellulose membrane material;
the slow release film is obtained by adding gelatin, hydroxypropyl methylcellulose and glycerol into water and uniformly stirring;
the mass ratio of the gelatin to the hydroxypropyl methylcellulose to the glycerol is 1-3: 1:1, and the mass volume ratio of the gelatin to the water is 1-3 g: 50 ml;
the detection pad takes a nitrocellulose membrane as a base pad, and a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane.
2. The method of detecting homocysteine and vitamin B12 according to claim 1 wherein said method of detection comprises:
when the analyte is homocysteine, the analyte is,
s1, adding the sample to be tested into the diluent for dilution and dissociation, and adding enzyme to convert the sample to be tested into the object to be tested of S-adenosyl homocysteine;
s2, adding the substance to be detected to an immunochromatography test strip based on quantum dots, and carrying out fluorescence detection on the test strip after color development is finished;
the diluent comprises a dissociating agent R1 and a converting agent R2; wherein the dissociating agent R1 contains a Tris-HCl solution of 1-10 mM DTT, R2 is a Tris-HCl solution of 1-10U/ml S-adenosylhomocysteine hydrolase, and the sample to be detected is catalyzed to convert into SAH;
wherein, R1 and R2 are mixed according to the volume ratio of 7-10: 1.
3. The method of detecting homocysteine and vitamin B12 according to claim 1 wherein when the test substance is vitamin B12, said method comprises:
s1, adding a sample to be tested into the diluent for dilution and dissociation, and converting the sample to be tested into cyanocobalamin under the cyanide condition after reduction to obtain a sample to be tested;
s2, adding the substance to be detected to an immunochromatography test strip based on quantum dots, and carrying out fluorescence detection on the test strip after color development is finished;
the diluent is a strong alkali solution containing thioglycerol, DTT and potassium cyanide; wherein the strong alkali solution is 0.01-0.1M NaOH solution, the final concentration of the thioglycerol is 0.5-2 mM, and the final concentration of the DTT is 1-10 mM; the final concentration of potassium cyanide is 0.001-0.01%.
4. The test method according to claim 2 or 3, wherein the sample to be tested is a blood product, and the blood product includes whole blood, serum, and plasma.
5. The immunochromatographic test strip based on the quantum dots is characterized by comprising a bottom plate, wherein a sample pad, a combination pad, a detection pad and a water absorption pad are sequentially arranged on the bottom plate, and the adjacent pads are connected at the joint in an overlapping manner;
a slow release film is sprayed on the sample pad;
the sample pad is made of a glass cellulose membrane material;
the slow release film is obtained by adding gelatin, hydroxypropyl methylcellulose and glycerol into water and uniformly stirring;
the mass ratio of the gelatin to the hydroxypropyl methylcellulose to the glycerol is 1-3: 1:1, and the mass volume ratio of the gelatin to the water is 1-3 g: 50 ml;
the detection pad takes a nitrocellulose membrane as a base pad, and a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane.
6. The immunochromatographic test strip of claim 5, wherein when the analyte is homocysteine, the binding pad is sprayed with a quantum dot labeled SAH antibody and a quantum dot labeled chicken IgY antibody; the mass ratio of the two is 0.5-2: 1;
the SAH antibody is an SAH monoclonal antibody;
the quantum dot labeled SAH antibody is obtained by adopting the following preparation method:
adding CdSe/ZnS quantum dots into MES buffer solution for activation, adding EDC and NHS, adding SAH monoclonal antibody, mixing for full reaction, performing centrifugal elution by using borate buffer solution, and diluting after redissolution to obtain the CdSe/ZnS quantum dots;
the MES buffer solution is neutral or weakly acidic;
the borate buffer solution is neutral or weakly alkaline;
the detection line is coated with SAH-BSA antigen, namely the SAH antigen coupled with BSA;
the quality control line is coated with goat anti-chicken IgY antibody;
the water absorption pad is a water absorption plate or water absorption paper;
the bottom plate is made of a PVC plate.
7. The immunochromatographic test strip of claim 5, wherein when the test substance is vitamin B12, the binding pad is sprayed with quantum dot labeled vitamin B12 antibody and quantum dot labeled chicken IgY antibody; the mass ratio of the two is 0.5-2: 1;
the vitamin B12 antibody is a vitamin B12 monoclonal antibody;
the quantum dot labeled vitamin B12 antibody is obtained by the following preparation method:
adding CdSe/ZnS quantum dots into MES buffer solution for activation, adding EDC and NHS, adding vitamin B12 monoclonal antibody, mixing for full reaction, performing centrifugal elution by using borate buffer solution, and diluting after redissolution to obtain the CdSe/ZnS quantum dots;
the MES buffer solution is neutral or weakly acidic;
the borate buffer solution is neutral or weakly alkaline;
the detection line is coated with a vitamin B12 coupled antigen;
the quality control line is coated with goat anti-chicken IgY antibody;
the water absorption pad is a water absorption plate or water absorption paper;
the bottom plate is made of a PVC plate.
8. Use of the test method according to any one of claims 1 to 4 or the immunochromatographic test strip according to any one of claims 5 to 7 for homocysteine detection and/or vitamin B12 detection.
9. The use of claim 8, wherein said detecting comprises qualitative detection and quantitative detection.
10. The use of claim 9, wherein the assay is a quantitative assay.
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| CN115144598B (en) * | 2022-09-02 | 2023-03-10 | 山东子峰生物技术有限公司 | Quantum dot fluorescence detection method for fetal fibronectin and insulin growth factor binding protein-1 and application |
| CN117214445B (en) * | 2023-11-09 | 2024-01-26 | 山东子峰生物技术有限公司 | Quantum dot fluorescence detection reagent for joint inspection of folic acid, vitamin B12 and ferritin and application thereof |
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