CN111579799A - Preparation method of fluorescence immunochromatographic test paper for detecting Alzheimer disease related neurofilament protein in urine - Google Patents
Preparation method of fluorescence immunochromatographic test paper for detecting Alzheimer disease related neurofilament protein in urine Download PDFInfo
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Abstract
The invention discloses a preparation method of fluorescence immunochromatographic test paper for detecting Alzheimer disease related neurofilament protein in urine, which comprises the steps of purifying AD7c-NTP monoclonal antibody 1; preparing a monoclonal antibody binding pad: spraying fluorescent microspheres coupled with the AD7c-NTP monoclonal antibody 1 on a polyester film to prepare a monoclonal antibody binding pad; preparation of cellulose nitrate coating film: spraying AD7c-NTP monoclonal antibody 2 and labeled antibody IgG on a nitrocellulose membrane to respectively serve as a detection zone and a quality control zone antibody; a sample pad was prepared. The invention can realize quick and single quantitative detection, has high sensitivity, small error and low detection limit, and provides great convenience for clinical use.
Description
Technical Field
The invention belongs to the technical field of preparation methods of immunofluorescence chromatography test paper for Alzheimer's disease, and particularly relates to a preparation method of fluorescence immunochromatographic test paper for detecting Alzheimer's disease-related neurofilament protein in urine.
Background
Alzheimer's Disease (AD), also called senile dementia), is a chronic neurodegenerative disease with an insidious and progressive onset of disease and a serious intellectual disability. With the rapid development of aging of the population, AD has become the fourth leading cause of death in the elderly following heart disease, cancer, stroke, and is one of the three most costly diseases (second only to tumors and heart disease). The patient experiences several years or even decades from mild memory and cognitive impairment to the last vegetative state, which is a painful process for both the patient and the family. AD is not natural aging, is a disease, is early discovered, intervenes and treats, and is of great importance.
The Alzheimer's disease related neurofibrillary protein (AD-associated neurological protein, AD7C-NTP) is a safe and effective biomarker, has important significance in AD diagnosis and has wide application prospect. AD7C-NTP belongs to the neurofilament protein family, and is transmembrane phosphoprotein with the relative molecular mass of 41000. AD7C-NTP is expressed from neuron cell body, widely exists in axon generated by nerve cell, and largely appears in NFTs in AD patient brain, and is expressed in secretory up-regulation. Studies have shown that the deposition of AD7C-NTP in neurons occurs early in neuronal degeneration and that AD7C-NTP can be detected in brain tissue extracts, cerebrospinal fluid, urine of early AD patients before NFTs are formed. Meanwhile, the content level of AD7C-NTP is in positive correlation with the severity of dementia and is gradually increased along with the prolongation of the disease course, probably because the NTP immunocompetence and messenger RNA expression of brain tissues of AD patients are enhanced, so that the apoptosis of neuron cells is increased, and then a great amount of AD7C-NTP is released by dead cells. The pathological processes such as the loss of neuron soma and nerve synapse, the functional damage of neuron structural protein and the like can lead the expression of AD7C-NTP to be increased.
Although the blood detection of A beta and Tau protein is minimally invasive, rapid and convenient, the sensitivity and specificity of the protein are difficult to meet the diagnosis requirements so far.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
As one aspect of the invention, the invention provides a preparation method of a fluorescence immunochromatographic test strip for detecting Alzheimer disease-related neurofilament protein in urine.
In order to solve the technical problems, the invention provides the following technical scheme: a preparation method of a fluorescence immunochromatographic test paper for detecting Alzheimer disease related neurofilament protein in urine comprises the following steps,
purifying AD7c-NTP monoclonal antibody 1;
preparing a monoclonal antibody binding pad: spraying fluorescent microspheres coupled with the AD7c-NTP monoclonal antibody 1 on a polyester film to prepare a monoclonal antibody binding pad;
preparation of cellulose nitrate coating film: spraying AD7c-NTP monoclonal antibody 2 and labeled antibody IgG on a nitrocellulose membrane to respectively serve as a detection zone and a quality control zone antibody;
a sample pad was prepared.
As a preferred scheme of the preparation method of the fluorescence immunochromatographic test paper for detecting Alzheimer disease related neurofilament protein in urine, the invention comprises the following steps: the purified AD7c-NTP monoclonal antibody 1 is subjected to dialysis purification treatment on the AD7c-NTP monoclonal antibody 1 by using a phosphoric acid buffer solution with the concentration of 0.05mol/L and the pH value of 7.2-7.6 at the temperature of 4 ℃; the AD7c-NTP monoclonal antibody 1 is selected from a commercial antibody ADJ 700912.
As a preferred scheme of the preparation method of the fluorescence immunochromatographic test paper for detecting Alzheimer disease related neurofilament protein in urine, the invention comprises the following steps: the fluorescent microspheres are polystyrene spheres wrapped with fluorescent dye and modified with amino or carboxyl on the surface, and the average particle size of the fluorescent microspheres is 200 nm.
As a preferred scheme of the preparation method of the fluorescence immunochromatographic test paper for detecting Alzheimer disease related neurofilament protein in urine, the invention comprises the following steps: firstly, washing the fluorescent microspheres by using MES (methyl amino acid) flushing solution with the concentration of 0.05mol/L and the pH value of 7.4, adjusting the mass concentration of the fluorescent microspheres to 1%, and then labeling the AD7c-NTP monoclonal antibody 1 on the fluorescent microspheres in a carbodiimide and N-hydroxysuccinimide covalent coupling mode; then using a quantitative film spraying instrument to spray the film at a speed of 4 mu l/cm2The monoclonal antibody 1 labeled with AD7c-NTP is sprayed on a polyester film, and the mass ratio of the antibody to the microsphere is 1: and (3) drying for 1 hour at 35-38 ℃ under the condition of keeping out of the sun for 20-80 ℃ to prepare the monoclonal antibody binding pad, and adding a drying agent for sealing and storing for later use.
As a preferred scheme of the preparation method of the fluorescence immunochromatographic test paper for detecting Alzheimer disease related neurofilament protein in urine, the invention comprises the following steps: the preparation of the nitrocellulose coating film comprises the steps of respectively diluting AD7c-NTP monoclonal antibody 2 and rabbit anti-mouse IgG antibody to the concentration of 1mg/ml by using a phosphoric acid buffer solution containing 1 wt% of sucrose and having the concentration of 0.05mol/L and the pH value of 7.4, and then using a quantitative film spraying instrument to spray 1 mul/cm2The two are sprayed on a nitrocellulose membrane at intervals and dried at 37 DEG C1 hour, adding a drying agent and sealing for later use; the AD7c-NTP monoclonal antibody 2 is selected from a commercial antibody ADH 800134.
As a preferred scheme of the preparation method of the fluorescence immunochromatographic test paper for detecting Alzheimer disease related neurofilament protein in urine, the invention comprises the following steps: the sample pad is prepared by soaking a glass cellulose membrane for 1h with a phosphoric acid buffer solution containing 1 wt% BSA, 0.1 wt% triton x-100, the concentration of which is 0.02mol/L, and the pH value of which is 7.4, and drying the glass cellulose membrane for 3h at 37 ℃.
As a preferred scheme of the preparation method of the fluorescence immunochromatographic test paper for detecting Alzheimer disease related neurofilament protein in urine, the invention comprises the following steps: the covalent coupling is carried out by the following steps,
preparing 50mM MES reaction buffer solution with the pH value of 6.0;
dissolving the monoclonal antibody 1 in a reaction buffer solution, wherein the protein concentration is 10 mg/ml;
diluting the microspheres with a reaction buffer to a concentration of 1% (w/v);
mixing the monoclonal antibody 1 with the microspheres, and incubating at room temperature for 20 minutes;
preparing EDAC solution with concentration of 10mg/ml by using deionized water;
measuring an EDAC solution and adding the EDAC solution into a mixed solution of the monoclonal antibody 1 and the microspheres;
adjusting the pH value of the reaction solution to 6.5 by using 0.1M sodium hydroxide solution, mixing, and then culturing for 2 hours on a table concentrator at room temperature; unbound antibody was removed and placed in a refrigerator at 4 ℃ for future use.
The invention has the beneficial effects that: the fluorescence immunochromatographic reagent strip for detecting AD7c-NTP provided by the invention takes the fluorescent microspheres as a signal source, can realize rapid and single quantitative detection, has high sensitivity and small error, and provides great convenience for clinical use. The fluorescence immunochromatographic test strip for detecting AD7c-NTP provided by the invention is simple in preparation method and can realize batch production.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a schematic diagram of the structure of a reagent strip for detecting AD7c-NTP in the invention shown in FIG. 1;
FIG. 2 is a front view of a test strip for detecting AD7c-NTP according to the present invention disposed on a base;
FIG. 3 is a side view of the test strip of the present invention.
The reference numbers in the figures denote: 1-base, 2-card lid, 3-test paper strip, 4-bottom plate, 5-sample pad, 6-monoclonal antibody binding pad, 7-nitrocellulose coated membrane, 8-absorbent paper, 9-detection band, 10-observation port, 11-quality control band, and 12-sample port.
FIG. 4 is a standard graph of the reagent for detecting AD7c-NTP according to the present invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
In the invention, AD7c-NTP monoclonal antibody 1, AD7c-NTP monoclonal antibody 2, rabbit anti-mouse lgG antibody, phosphate buffer solution, Bovine Serum Albumin (BSA), polyethylene glycol octylphenyl ether (TritonX-100), 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution, polystyrene spheres wrapped with fluorescent dye and modified with amino or carboxyl on the surface, carbodiimide (EDC), N-hydroxysuccinimide (NHS), sucrose, polyester film, nitrocellulose film, glass cellulose film and high-strength absorbent paper are commercially available.
As shown in fig. 1 to 3, the invention provides a fluorescence immunochromatographic test strip 3 for detecting AD7c-NTP, wherein the test strip 3 comprises a bottom plate 4, and a water-absorbent paper 8, a cellulose nitrate coated film 7, a monoclonal antibody binding pad 6 and a sample pad 5 which are sequentially adhered to the bottom plate 4 along the length direction of the test strip 3;
a cellulose nitrate coating film 7 is adhered to the middle part of the bottom plate 4, and is provided with a detection belt 9 and a quality control belt 11 which are spaced from each other, wherein the detection belt 9 is close to the monoclonal antibody binding pad 6, and the quality control belt 11 is close to the absorbent paper 8;
the monoclonal antibody binding pad 6 is sprayed with a polyester film of fluorescent microspheres with the surfaces modified with AD7c-NTP monoclonal antibody 1;
the absorbent paper 8, the nitrocellulose coating film 7, the monoclonal antibody binding pad 6, and the sample pad 5 are in contact with and partially overlap with adjacent sites in this order.
The test strip for detecting AD7c-NTP comprises a test strip 3 and a shell for coating the test strip 3; the shell further comprises a base 1 and a card cover 2, wherein the card cover 2 is provided with an observation port 10 and a sample adding port 12 so as to expose a local area of the test strip 3; the sample addition port 12 is opened at the upper part of the sample pad 5 to expose part or all of the area of the sample pad 5; the observation port 10 is opened on the upper side of the nitrocellulose coating film 7 to expose all of the detection zone 9 and the quality control zone 11.
The sample pad 5 is a glass cellulose membrane. The detection zone 9 is an AD7c-NTP monoclonal antibody 2 zone coated on the nitrocellulose, and the quality control zone 11 is a rabbit anti-mouse lgG antibody zone coated on the nitrocellulose-coated membrane 7.
The soleplate 4 is a plastic soleplate. The particle size of the fluorescent microspheres is 10-1000 nm, and preferably 200 nm. The fluorescent microspheres are polystyrene spheres wrapped with fluorescent dye and modified with murine monoclonal antibodies; the absorbent paper 8 is preferably high-strength absorbent paper; the base 4 is a plastic base, preferably a polyvinyl chloride (PVC) plate.
Example 1:
the preparation method of the fluorescence immunochromatographic test strip for detecting AD7c-NTP comprises the following steps:
S2 purified AD7c-NTP monoclonal antibody 1. Commercial AD7c-NTP monoclonal antibody 1(ADJ700912, No-Sn AoRuidong Biotechnology Co., Ltd.) was selected, and the AD7c-NTP monoclonal antibody 1 was dialyzed and purified at 4 ℃ with a phosphate buffer solution having a concentration of 0.05mol/L and a pH of 7.4.
S3 monoclonal antibody conjugate pad 6 was prepared. The fluorescent microspheres are polystyrene spheres (microspheres are purchased from Thermo Scientific, a cargo number of mTRFIA-200) wrapped with fluorescent dye and modified with amino or carboxyl on the surface, and the particle size is 200 nm; firstly, washing fluorescent microspheres by MES flushing solution with the concentration of 0.05mol/L and the pH value of 7.4, adjusting the mass concentration of the fluorescent microspheres to 1%, and then labeling the AD7c-NTP monoclonal antibody 1 obtained by processing in the step S1 on the fluorescent microspheres in a covalent coupling mode of carbodiimide (EDC) and N-hydroxysuccinimide (NHS); the steps of covalent coupling are:
a. preparing 50mM MES reaction buffer solution with the pH value of 6.0;
b. dissolving the monoclonal antibody 1 in a reaction buffer solution, wherein the protein concentration is 10 mg/ml;
c. diluting the microspheres with a reaction buffer solution to a concentration of 1% (W/V);
d. mixing the monoclonal antibody 1 with the microspheres, and incubating at room temperature for 20 minutes;
e. preparing EDAC solution with concentration of 10mg/ml by using deionized water;
f. measuring an EDAC solution and adding the EDAC solution into a mixed solution of the monoclonal antibody 1 and the microspheres;
g. adjusting the pH value of the reaction solution to 6.5 by using 0.1M sodium hydroxide solution, mixing, and then culturing for 2 hours on a table concentrator at room temperature;
h. unbound antibody was removed and placed in a refrigerator at 4 ℃ for future use.
Then using a quantitative film spraying instrument to spray the film at a speed of 4 mu l/cm2The monoclonal antibody 1 marked with AD7c-NTP is sprayed on a polyester film (the mass content of the microsphere is 1 percent, and the mass ratio of the antibody to the microsphere is 1: 60), dried for 1 hour at 37 ℃ in a dark condition to prepare a monoclonal antibody binding pad 6, and added with a drying agent for sealing and storage for later use.
S4 preparation of nitrocellulose coating film 7. AD7c-NTP monoclonal antibody 2 (detection-labeled antibody, ADH800134) and rabbit anti-mouse IgG antibody (quality control-labeled antibody) were each diluted to a concentration of 1mg/ml using a phosphate buffer solution containing 1 wt% sucrose at a concentration of 0.05mol/L and a pH of 7.4, and then 1. mu.l/cm using a quantitative membrane-spraying apparatus2Spraying the two on a nitrocellulose membrane at intervals, drying at 37 ℃ for 1 hour to prepare a nitrocellulose coating membrane 7 comprising a detection band 9 and a quality control band 11, and adding a drying agent for sealing and storing for later use.
S7 assembles a fluorescence immunochromatographic test strip for detecting AD7 c-NTP. The test strip 3 prepared in the step S6 is arranged in the center of the base 1, the bottom plate 4 is connected with the base 1 and is buckled with the card cover 2, the observation port 10 is over against the nitrocellulose coating film 7, and the sample addition port 12 is over against the sample pad 5; or a drying agent can be added into a hollow cavity formed after the base 1 and the card cover 2 are buckled.
When the kit is used, a sample liquid is dripped onto the sample pad 5 through the sample adding port 12, the sample liquid flows towards one side of the absorbent paper 8 under the capillary action, when the sample liquid contains AD7c-NTP, the AD7c-NTP and the AD7c-NTP monoclonal antibody 1 on the fluorescent microsphere form an antigen-antibody complex, the antigen-antibody complex continuously flows towards one side of the absorbent paper 8 along with the chromatography action and reaches the AD7c-NTP monoclonal antibody 1 for identifying different antigen epitopes, namely a detection band 9 to form an antibody-antigen-antibody complex, and the fluorescent microsphere with the antibody-antigen-antibody complex is gathered at the detection band 9. And the fluorescent microspheres which are not combined with the monoclonal antibody 1 continue to swim to one side of the absorbent paper 8, when the fluorescent microspheres reach the quality control zone 11, the rabbit anti-mouse lgG antibody is combined with the mouse-derived monoclonal antibody on the fluorescent microspheres, and the aggregation of the fluorescent microspheres is produced at the quality control zone 11.
After the reaction is completed, the fluorescence immunochromatographic test strip for detecting AD7c-NTP is placed in a fluorescence detector, fluorescence signals at two positions of the detection zone 9 and the quality control zone 11 are read, and the read value is compared with a preset standard value to calculate a quantitative result.
143 healthy negative samples and 98 patient samples were taken for repeated detection, and the intra-and inter-batch differences, CV, sensitivity, specificity, Cut off values, were calculated. Calculated intra-batch difference CV was 7.68%, inter-batch difference CV was 11.23%, sensitivity was 93.2%, specificity was 86.7%, and Cut off value was 1.5 ng/mL. The detection limit is 0.10ng/mL, the linear range is 0.1-10 ng/mL, and the correlation coefficient r is greater than 0.98.
The fluorescence immunochromatographic test strip for detecting AD7c-NTP can realize rapid and single quantitative detection, has high sensitivity and small error in batch and between batches, and provides great convenience for clinical use; moreover, the volume is small, the carrying is convenient, the storage is easy, and the application and the popularization of a primary hospital are facilitated; in addition, the preparation method is simple and can realize batch production.
Comparative example:
the difference between the example and the example 1 is that the colloidal gold particles are adopted, and the colloidal gold particles coupled with the antibody are fixed on the binding pad, and the research of the invention finds that the detection effect of the method is obviously inferior to that of the example 1, mainly shows that the sensitivity is insufficient, the detection limit is 0.4ng/ml, and meanwhile, the detection sensitivity is obviously reduced.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (7)
1. A preparation method of a fluorescence immunochromatographic test paper for detecting Alzheimer disease related neurofilament protein in urine is characterized in that: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
purifying AD7c-NTP monoclonal antibody 1;
preparing a monoclonal antibody binding pad: spraying fluorescent microspheres coupled with the AD7c-NTP monoclonal antibody 1 on a polyester film to prepare a monoclonal antibody binding pad;
preparation of cellulose nitrate coating film: spraying AD7c-NTP monoclonal antibody 2 and labeled antibody IgG on a nitrocellulose membrane to respectively serve as a detection zone and a quality control zone antibody;
a sample pad was prepared.
2. The method for preparing the fluorescence immunochromatographic test strip for detecting Alzheimer's disease-associated neurofilament protein in urine according to claim 1, which is characterized in that: the purified AD7c-NTP monoclonal antibody 1 is subjected to dialysis purification treatment on the AD7c-NTP monoclonal antibody 1 by using a phosphoric acid buffer solution with the concentration of 0.05mol/L and the pH value of 7.2-7.6 at the temperature of 4 ℃; the AD7c-NTP monoclonal antibody 1 is selected from a commercial antibody ADJ 700912.
3. The method for preparing the fluorescence immunochromatographic test strip for detecting Alzheimer's disease-associated neurofilament protein in urine according to claim 1 or 2, which is characterized in that: the fluorescent microspheres are polystyrene spheres wrapped with fluorescent dye and modified with amino or carboxyl on the surface, and the average particle size of the fluorescent microspheres is 200 nm.
4. The method of preparing the fluorescent immunochromatographic strip for detecting Alzheimer's disease-associated neurofilament protein in urine according to claim 3, which is characterized in that: firstly, washing the fluorescent microspheres by using MES (methyl amino acid) flushing solution with the concentration of 0.05mol/L and the pH value of 7.4, adjusting the mass concentration of the fluorescent microspheres to 1%, and then labeling the AD7c-NTP monoclonal antibody 1 on the fluorescent microspheres in a carbodiimide and N-hydroxysuccinimide covalent coupling mode; then using a quantitative film spraying instrument to spray the film at a speed of 4 mu l/cm2The monoclonal antibody 1 labeled with AD7c-NTP is sprayed on a polyester film, and the mass ratio of the antibody to the microsphere is 1: and (3) drying for 1 hour at 35-38 ℃ under the condition of keeping out of the sun for 20-80 ℃ to prepare the monoclonal antibody binding pad, and adding a drying agent for sealing and storing for later use.
5. The method for preparing the fluorescence immunochromatographic test strip for detecting Alzheimer's disease-associated neurofilament protein in urine according to claim 1, which is characterized in that: the preparation of the nitrocellulose coating film comprises the steps of respectively diluting AD7c-NTP monoclonal antibody 2 and rabbit anti-mouse IgG antibody to the concentration of 1mg/ml by using a phosphoric acid buffer solution containing 1 wt% of sucrose and having the concentration of 0.05mol/L and the pH value of 7.4, and then using a quantitative film spraying instrument to spray 1 mul/cm2Spraying the two on a nitrocellulose membrane at intervals, drying for 1 hour at 37 ℃, adding a drying agent, and sealing for later use; the AD7c-NTP monoclonal antibody 2 is selected from a commercial antibody ADH 800134.
6. The method for preparing the fluorescence immunochromatographic test strip for detecting Alzheimer's disease-associated neurofilament protein in urine according to claim 1, which is characterized in that: the sample pad is prepared by soaking a glass cellulose membrane for 1h with a phosphoric acid buffer solution containing 1 wt% BSA, 0.1 wt% triton x-100, the concentration of which is 0.02mol/L, and the pH value of which is 7.4, and drying the glass cellulose membrane for 3h at 37 ℃.
7. The method of preparing the fluorescent immunochromatographic strip for detecting Alzheimer's disease-associated neurofilament protein in urine according to claim 4, which is characterized in that: the covalent coupling is carried out by the following steps,
preparing 50mM MES reaction buffer solution with the pH value of 6.0;
dissolving the monoclonal antibody 1 in a reaction buffer solution, wherein the protein concentration is 10 mg/ml;
diluting the microspheres with a reaction buffer to a concentration of 1% (w/v);
mixing the monoclonal antibody 1 with the microspheres, and incubating at room temperature for 20 minutes;
preparing EDAC solution with concentration of 10mg/ml by using deionized water;
measuring an EDAC solution and adding the EDAC solution into a mixed solution of the monoclonal antibody 1 and the microspheres;
adjusting the pH value of the reaction solution to 6.5 by using 0.1M sodium hydroxide solution, mixing, and then culturing for 2 hours on a table concentrator at room temperature; unbound antibody was removed and placed in a refrigerator at 4 ℃ for future use.
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