CN112485433A - Serum amyloid protein A immunochromatographic test strip, kit and preparation method thereof - Google Patents

Serum amyloid protein A immunochromatographic test strip, kit and preparation method thereof Download PDF

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CN112485433A
CN112485433A CN202011179062.5A CN202011179062A CN112485433A CN 112485433 A CN112485433 A CN 112485433A CN 202011179062 A CN202011179062 A CN 202011179062A CN 112485433 A CN112485433 A CN 112485433A
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serum amyloid
pad
sample
quality control
antibody
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邓艳珍
王伟
岑赞询
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Healvet Guangzhou Medical Technology Co ltd
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Healvet Guangzhou Medical Technology Co ltd
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein

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Abstract

The invention discloses a serum amyloid A immunochromatographic test strip, which comprises a sample pad, a combination pad, a reaction membrane and a water absorption pad, wherein the reaction membrane is provided with a detection line and a quality control line which are mutually spaced, the combination pad is coated with a first serum amyloid A monoclonal antibody and a quality control antibody which are respectively marked by fluorescent microspheres, the detection line is coated with a second serum amyloid A monoclonal antibody, the quality control line is coated with an antigen which can be specifically combined with the quality control antibody, the surface area of the sample pad is calculated, and the sample pad is coated with an antigen with the concentration of 1μg/cm2～16μg/cm2Antibody of non-detection sample species, 0.2. mu.g/cm2～16μg/cm2Lectin and 1. mu.g/cm2～16μg/cm2A blocking agent for heterophilic antibodies in a test sample. The invention also discloses a preparation method of the serum amyloid A immunochromatographic test strip. The invention also discloses a kit.

Description

Serum amyloid protein A immunochromatographic test strip, kit and preparation method thereof
Technical Field
The invention relates to a test paper, in particular to a serum amyloid A immunochromatographic test paper and a preparation method thereof.
Background
Serum amyloid a (saa) is an acute phase protein secreted by the liver and binds to High Density Lipoprotein (HDL) in plasma. When the body is in an inflammatory state, the concentration of SAA can rise rapidly within hours; at the same time, the half-life of SAA is very short, and the concentration level of SAA is rapidly reduced along with the elimination of an inflammation source. In veterinary medicine, SAA is a sensitive marker of inflammation and tissue damage. The existing SAA detection method mainly comprises an enzyme-linked immunosorbent assay, a chemiluminescence method, a colloidal gold immunochromatography method and the like. At present, the TSHD detection method mainly comprises the traditional radioimmunoassay, enzyme-linked immunosorbent assay and chemiluminescence assay. Among them, the radio-immunity method and the enzyme-linked immunosorbent method have low sensitivity, narrow detection range, serious lack of anti-interference capability and long detection time, so that the clinical value is greatly limited and the method is basically out of the market; the chemiluminescence method has high sensitivity and high detection speed, but has high cost, large serum sample amount and complex operation, and is only suitable for detection in large hospitals.
Chinese patent 201820668071.2 is used for detecting cat serum amyloid A's immunofluorescence chromatography detection card the kit includes the bottom plate, links up sample pad, combination pad, nitrocellulose membrane and the pad that absorbs water in proper order on the bottom plate, be equipped with fluorescent microsphere mark's SAA monoclonal antibody and fluorescent microsphere mark's goat anti-chicken IgY on the combination pad, the nitrocellulose membrane be equipped with a detection T line of peridium SAA monoclonal antibody to and a quality control C line of peridium chicken IgY. However, the kit needs 10 minutes to obtain a result, and is not suitable for the requirement of emergency detection.
Disclosure of Invention
Therefore, the serum amyloid A immunochromatographic test strip and the preparation method thereof are needed to solve the problem that the traditional SAA detection card cannot meet the requirement of emergency treatment, the result can be obtained quickly, the sampling amount is small, and the detection requirement of animals, especially small animals with damaged body functions can be met.
A serum amyloid A immunochromatographic test strip comprises a sample pad, a binding pad, a reaction membrane and a water absorption pad, wherein the reaction membrane is provided with a detection line and a quality control line which are mutually spaced, the detection line is closer to the binding pad than the quality control line, the binding pad is coated with a first serum amyloid A monoclonal antibody and a quality control antibody which are respectively marked by fluorescent microspheres, the detection line is coated with a second serum amyloid A monoclonal antibody which is matched with the first serum amyloid A monoclonal antibody, the quality control line is coated with an antigen which can be specifically bound with the quality control antibody, and the sample pad is coated with an antigen with the concentration of 1 mu g/cm calculated according to the surface area of the sample pad2～16μg/cm2Antibody of non-detection sample species, 0.2. mu.g/cm2～16μg/cm2Lectin and 1. mu.g/cm2～16μg/cm2A blocking agent for heterophilic antibodies in a test sample.
In one embodiment, the antibody of the non-test sample species, the lectin and the blocking agent are distributed at least at the end of the sample pad remote from the conjugate pad.
In one embodiment, the antibody of the non-test sample species, the lectin, and the blocking agent are uniformly distributed in the sample pad.
In one embodiment, the test sample source is feline and the antibodies of the non-test sample species are selected from non-feline IgG.
In one embodiment, the quality control antibody is selected from IgG of the same generic origin as the first serum amyloid a monoclonal antibody.
In one embodiment, the blocking agent is selected from any one or more of normal serum and Scantibodies HBR that are not directed against the target analyte.
In one embodiment, the first serum amyloid A monoclonal antibody is coated with fluorescent microsphere on the conjugate pad as calculated from the surface area of the conjugate padThe concentration is 0.5 mug/cm2～2μg/cm2The concentration of the quality control antibody marked by the fluorescent microspheres is 0.05 mu g/cm2～0.5μg/cm2。
In one embodiment, the fluorescent microspheres have a diameter of 350nm to 500 nm.
In one embodiment, the sample pad, the binding pad, the detection line and the quality control line are coated with at least one of sugar, BSA and casein, respectively and independently.
In one embodiment, the length ratio of the sample pad, the binding pad, the reaction membrane and the water absorption pad in the serum amyloid A immunochromatographic test strip is 1.6 cm-2.8 cm, calculated as a structure after lapping; the serum amyloid immunochromatographic test strip is 7 cm-8 cm in length, 3 mm-4 mm in width and 0.1 mm-1 mm in thickness.
A preparation method of a serum amyloid A immunochromatographic test strip comprises the following steps:
the concentration is 1 mu g/cm2～16μg/cm2Antibody of non-detection sample species, 0.2. mu.g/cm2～16μg/cm2Lectin and 1. mu.g/cm2～16μg/cm2A blocking agent for heterophilic antibodies of the test sample is coated on the sample pad;
coating a first serum amyloid A monoclonal antibody and a quality control antibody which are respectively marked by fluorescent microspheres on a combination pad;
coating a second serum amyloid A monoclonal antibody on a reaction membrane to form a detection line;
coating an antigen specifically bound with the quality control antibody on the reaction membrane to form a quality control line separated from the detection line;
and overlapping a sample pad, the binding pad coated with a first serum amyloid A monoclonal antibody and a quality control antibody which are respectively marked by fluorescent microspheres, the reaction film with the detection line and the quality control line and a water absorption pad.
A serum amyloid A immunochromatographic kit comprises the serum amyloid A immunochromatographic test strip and a sample diluent.
According to the serum amyloid A immunochromatographic test strip, the first serum amyloid A monoclonal antibody and the quality control antibody which are respectively marked by the fluorescent microspheres are coated on the combination pad, so that the signal quantity of a reaction system can be amplified, and the sensitivity and the accuracy are greatly improved. Endogenous heterophilic antibodies in plasma can be combined with immunoglobulins of other species, including heterologous antibodies as immunoassay reagents, which can interfere with an immunoassay system to cause false high detection values unrelated to actual analyte concentrations, possibly causing misdiagnosis, and the inventors found that antibodies of non-detection sample species, lectins and blocking agents against heterophilic antibodies of detection samples are coated on a sample pad at appropriate concentrations, the antibodies of non-detection sample species play a passive blocking role to ensure that the binding of heterophilic antibodies is prior to the analyte, and a part of heterophilic interference can be excluded, while the blocking agents against heterophilic antibodies of detection samples are active blocking roles against heterophilic antibodies, effectively neutralizing the interference thereof, and the combination of the two blocking roles is more effective in preventing bridging of non-analyte mediated antibodies. Experiments show that the detection efficiency can be obviously improved by coating the sample pad with the antibody and the lectin of the non-detection sample species with proper concentration and the blocking agent of the heterophilic antibody aiming at the detection sample, and the detection effects of safe and simple operation, small sampling amount (as low as 10 mu l), rapidness (as low as 3min) and high sensitivity are realized. Is particularly suitable for emergency detection, can quickly judge the state of an illness, shortens the curing time and really realizes instant detection.
Drawings
Fig. 1 is a schematic structural diagram of a serum amyloid a immunochromatographic test strip according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of the reaction time of the serum amyloid A immunochromatographic test strip according to one embodiment of the present invention;
FIG. 3 is a sample size selection diagram of a serum amyloid A immunochromatographic test strip according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of a linear fitting curve of the test strip for immunochromatography of serum amyloid A according to an embodiment of the present invention;
fig. 5 is a schematic diagram of the detection correlation of the serum amyloid a immunochromatographic test strip according to an embodiment of the present invention.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
"antibodies of non-test sample species" refer to antibodies of a different origin than the species of the sample to be tested. The antibodies of the non-test sample species are capable of binding to endogenous heterophilic antibodies in the sample to be tested.
"Heterophilic Antibodies (HA)" are a class of immunoglobulins produced by known or unknown antigenic substances that have sufficient titers of multiple specificities to bind immunoglobulins of various species with relatively weak degrees of binding. Such antibodies react with antigens unrelated to the original antigen, and such substances having a chemical structure different from that of the test substance but having similar activities are called heterophilic antibodies. Heterophilic antibodies can bind to fragments of many animal immunoglobulins, interfering with the assay, and making the assay result inconsistent with clinical performance, leading to false results.
Referring to fig. 1, an embodiment of the present invention provides a serum amyloid a immunochromatographic test strip, which includes a sample pad 1, a conjugate pad 2, a reaction membrane 4, and a water absorption pad 6, the reaction membrane 4 is provided with a detection line 3 and a quality control line 5 which are mutually spaced, the detection line 3 is closer to the combination pad 2 than the quality control line 5, the combination pad 2 is coated with a first serum amyloid A monoclonal antibody and a quality control antibody which are respectively marked by fluorescent microspheres, the detection line 3 is coated with a second serum amyloid A monoclonal antibody, the first serum amyloid A monoclonal antibody and the second serum amyloid A monoclonal antibody are respectively combined with different epitopes of serum amyloid A, the control line 5 is coated with an antigen that specifically binds to the control antibody and is capable of specifically binding to the control antibody.
The sample pad was coated with a concentration of 1. mu.g/cm, calculated as the surface area of the sample pad2～16μg/cm2Antibody of non-detection sample species, 0.2. mu.g/cm2～16μg/cm2Lectin and 1. mu.g/cm2～16μg/cm2A blocking agent for heterophilic antibodies in a test sample. Surface area
The serum amyloid A immunochromatographic test strip is coated on the binding pad 2 by the serum amyloid A monoclonal antibody and the quality control antibody marked by the fluorescent microspheres respectively, and can amplify the signal quantity of a reaction system, so that the sensitivity and the accuracy are greatly improved. Endogenous heterophilic antibodies in plasma can be combined with immunoglobulins of other species, including heterologous antibodies as immunoassay reagents, which can interfere with an immunoassay system to cause a false high detection value unrelated to the actual analyte concentration, possibly causing misdiagnosis, and the inventors found that antibodies of non-detection sample species, lectins, and blocking agents against heterophilic antibodies of detection samples are coated on the sample pad 1 at appropriate concentrations, and the antibodies of non-detection sample species play a passive blocking role to ensure that the binding of heterophilic antibodies is prior to the analyte, and can exclude a part of heterophilic interference, while the blocking agents against heterophilic antibodies of detection samples are active blocking roles against heterophilic antibodies, effectively neutralizing the interference thereof, and the combination of the two blocking roles more effectively prevents bridging of non-analyte-mediated antibodies. Experiments show that the sample pad 1 is coated with the antibody and the agglutinin of non-detection sample species with proper concentration and the blocking agent of heterophilic antibody aiming at the detection sample, so that the detection efficiency can be obviously improved, and the detection effects of safe and simple operation, small sampling amount (as low as 10 mu l), rapidness (as low as 3min) and high sensitivity are realized. Is particularly suitable for emergency detection, can quickly judge the state of an illness, shortens the curing time and really realizes instant detection.
In some embodiments, the test strip further includes a bottom plate 7, the sample pad 1, the binding pad 2, the reaction membrane 4 and the absorbent pad 6 are disposed on the bottom plate 7, two ends of the binding pad 2 are respectively overlapped with the sample pad 1 and the reaction membrane 4, and one end of the reaction membrane 4 away from the binding pad 2 is overlapped with the absorbent pad 6.
When the serum amyloid A immunochromatographic test strip works, a sample to be detected is contacted with the sample pad 1, and sequentially chromatographed to the binding pad 2 and the reaction membrane 4 through a chromatography effect.
If the sample to be detected contains serum amyloid A, the serum amyloid A in the sample is firstly combined with the first serum amyloid A monoclonal antibody marked by the fluorescent microspheres on the binding pad 2 to form a reaction complex, and then under the action of chromatography, the reaction complex moves forwards along the reaction membrane 4 and is captured by the second serum amyloid A monoclonal antibody coated on the detection line 3 in the reaction membrane 4. The more serum amyloid protein A in the sample to be detected, the more bound complex accumulated on the detection line 3, and the more obvious color development is. For example, when the labeled microspheres are selected from fluorescent microspheres, the intensity of the fluorescent signal reflects the amount of serum amyloid A captured. The reaction complex and the quality control antibody labeled by the microspheres are continuously chromatographed to the quality control line 5, and the quality control antibody on the combination pad 2 is combined with the antigen in the quality control line 5, which is specifically combined with the quality control antibody, for color development.
If the sample to be detected does not contain serum amyloid A, neither the first serum amyloid A monoclonal antibody labeled with fluorescent microspheres on the binding pad 2 nor the second serum amyloid A monoclonal antibody coated by the detection line 3 is bound with the serum amyloid A, so that the detection line 3 does not develop color, the quality control antibody labeled with microspheres is continuously chromatographed to the quality control line 5, and the quality control antibody on the binding pad 2 is bound with the antigen specifically bound with the quality control antibody in the quality control line 5 to develop color.
If the quality control line 5 does not develop color, the test strip of the serum amyloid A immunochromatography test strip is invalid.
In some embodiments, the antibody of the non-test sample species, the lectin, and the blocking agent for heterophilic antibodies to the test sample are distributed at least at the end of the sample pad 1 distal from the conjugate pad 2. In operation, the sample to be tested is preferably applied to the starting end of the sample pad 1 of the strip. Therefore, when the sample to be detected is applied to the sample pad 1, the lectin intercepts the erythrocyte membrane, and the non-specific binding is reduced by the antibody of the non-detection sample species and the blocking agent of the heterophilic antibody against the detection sample, so that the interfering substance in the detection sample can be intercepted, and the false positive interference of the interfering substance can be avoided.
In some embodiments, the antibody of the non-test sample species, the lectin, and the blocking agent for the heterophilic antibody of the test sample are distributed only at one end of the sample pad 1 remote from the conjugate pad 2, for example, at the first half of the sample pad 1 (the end remote from the conjugate pad 2). In other embodiments, the antibody of the non-test sample species, the lectin, and the blocking agent for heterophilic antibodies to the test sample are uniformly distributed in the sample pad 1.
The concentration of the antibody of the non-test sample species coated on the sample pad 1 may be 1. mu.g/cm2、2μg/cm2、5μg/cm2、8μg/cm2、10μg/cm2、12μg/cm2、14μg/cm2Or 16. mu.g/cm2。
The concentration of lectin coated on the sample pad 1 may be 0.2. mu.g/cm2、0.5μg/cm2、0.8μg/cm2、1μg/cm2、2μg/cm2、5μg/cm2、8μg/cm2、10μg/cm2、12μg/cm2、14μg/cm2Or 16. mu.g/cm2。
The concentration of the blocking agent for heterophilic antibodies to the test sample coated on the sample pad 1 may be 1. mu.g/cm2、2μg/cm2、5μg/cm2、8μg/cm2、10μg/cm2、12μg/cm2、14μg/cm2Or 16. mu.g/cm2。
The concentrations of the antibody of the non-test sample species, the lectin, and the blocking agent against the heterophilic antibody of the test sample coated on the sample pad 1 may be the same or different.
In a particular embodiment, the test sample species is of feline origin and the antibodies of the non-test sample species are selected from non-feline IgG, e.g., murine IgG. The antibodies of the non-test sample species are non-specific antibodies, and only the species source that defines the antibodies are the non-test sample species.
In a specific embodiment, the quality control antibody is selected from IgG of the same generic origin as the first serum amyloid a monoclonal antibody.
In a specific embodiment, the antibody of the non-test sample species is selected from murine IgG, the quality control antibody is selected from goat anti-rabbit IgG, and the antigen specifically binding to the quality control antibody is selected from rabbit IgG. Specifically, the rabbit IgG may be selected from rabbit anti-mouse IgG.
In some embodiments, the blocking agent for heterophilic antibodies in the test sample may be selected from any one or more of normal serum, normal IgG, monoclonal antibodies, and Scantibodies HBR that are not directed against the analyte of interest.
In some embodiments, the concentration of the fluorescent microsphere labeled serum amyloid A monoclonal antibody coated on the conjugate pad 2 is 0.5 μ g/cm, calculated as the surface area of the conjugate pad 22～2μg/cm2The concentration of the quality control antibody marked by the fluorescent microspheres is 0.05 mu g/cm2～0.5μg/cm2。
In some embodiments, the fluorescent microspheres have a diameter of 350nm to 500 nm. The specific diameter may be 350nm, 380nm, 400nm, 450nm, or 500 nm.
In some embodiments, the sample pad 1, the binding pad 2, the detection line 3, and the quality control line 5 are each independently coated with at least one of sugar, BSA, and casein.
The test strip contains sugar, macromolecular protein BSA and casein, which have the function of protecting protein, and for antigen and antibody, the reagents are small molecular substances, and the small molecular substances wrap the antigen and antibody and are not easy to oxidize.
In one embodiment, the bottom plate 7 may be selected from PVC plates.
In some embodiments, the length ratio of the sample pad 1, the binding pad 2, the reaction membrane 4, and the absorbent pad 6 in the test strip for serum amyloid a immunochromatography is 1.6cm to 2.8cm, calculated as a structure after lapping.
In some embodiments, the serum amyloid immunochromatographic test strip has a length of 7cm to 8cm, a width of 3mm to 4mm, and a thickness of 0.1mm to 1 mm.
The serum amyloid immunochromatographic test strip can reduce non-specific interference, and the type of a detected sample can support whole blood, plasma or serum, thereby providing multiple choices for users. The method can quantitatively detect the content of the amyloid A in the sample, only needs a small instrument (a dry type immunofluorescence detector), has high sensitivity, wide linear range and high accuracy, has correlation R2 with the clinical sample of the Korean agile reagent of 0.98, and is suitable for being popularized in small and medium-sized pet hospitals.
The embodiment of the invention also provides a serum amyloid A immunochromatographic kit, which comprises the serum amyloid A immunochromatographic test strip and a sample diluent.
In one embodiment, the sample diluent includes surfactants S9, NaCl, triton, PC300, and PB. The mass concentration of the surfactant S9 can be 0.5-2%, the molar concentration of NaCl can be 0.2-0.5M, the mass concentration of triton can be 0.5-2%, the mass concentration of PC300 can be 0.2-0.5%, and the molar concentration of PB can be 0.015-0.025M.
The serum amyloid A immunochromatographic kit can comprise a card shell for holding the serum amyloid A immunochromatographic test strip.
The embodiment of the invention also provides a preparation method of the serum amyloid immunochromatographic test strip, which comprises the following steps:
the concentration is 1 mu g/cm2～16μg/cm2Antibody of non-detection sample species, 0.2. mu.g/cm2～16μg/cm2Lectin and 1. mu.g/cm2～16μg/cm2A blocking agent against heterophilic antibodies of the test sample is coated on the sample pad 1;
coating a first serum amyloid A monoclonal antibody and a quality control antibody which are respectively marked by fluorescent microspheres on a binding pad 2;
coating a second serum amyloid A monoclonal antibody on a reaction membrane 4 to form a detection line 3;
coating an antigen specifically bound with the quality control antibody on the reaction membrane 4 to form a quality control line 5 separated from the detection line 3, wherein the antigen specifically bound with the quality control antibody can be specifically bound with the quality control antibody;
arranging a sample pad 1, a water absorption pad 6, the combination pad 2 coated with a first serum amyloid A monoclonal antibody and a quality control antibody which are respectively marked by microspheres, and the reaction membrane 4 provided with the detection line 3 and the quality control line 5 on a bottom plate 7 to obtain the serum amyloid A detection immune test paper, wherein two ends of the combination pad 2 are respectively lapped with the sample pad 1 and the reaction membrane 4, one end of the reaction membrane 4, far away from the combination pad 2, is lapped with the water absorption pad 6, and the detection line 3 is closer to the combination pad 2 than the quality control line 5.
In some of these embodiments, the sample pad 1 can be prepared as follows: preparing a sample pad 1 treatment solution: contains 0.5-2 mg/mL of antibody of non-detection sample species, 0.1-2 mg/mL of agglutinin and 0.5-2 mg/mL of blocking agent of heterophilic antibody aiming at the detection sample. The sample pad 1 treatment solution was sprayed on the glass fiber. Further, the sample pad 1 includes evans blue in a volume fraction of 0.5% to 1% in the treatment liquid. Further, the sample pad 1 treatment solution contains 0.5-3% of tween-20, 0.5-3% of sodium caseinate, 2-5% of BSA, 0.2-0.5% of PC300 and 0.015-0.025M PB by volume fraction.
Wherein, the combination pad 2 coated with the first serum amyloid A monoclonal antibody and the quality control antibody which are respectively marked by the fluorescent microspheres is formed by adopting the following method: the first serum amyloid A monoclonal antibody and the quality control antibody which are respectively marked by the fluorescent microspheres are coated on the binding pad 2.
In some embodiments, the specific steps include:
and (3) respectively adding the first serum amyloid A monoclonal antibody or the quality control antibody into the fluorescent microsphere solution for mixing, separating and taking the precipitate, and carrying out sealing treatment.
Further, the blocking solution for blocking treatment was 0.5% to 2% BSA in 0.01M Tris buffer.
In some examples, the fluorescent microspheres comprise the steps of washing, activating, and terminating activation of the fluorescent microspheres prior to mixing with the first serum amyloid a monoclonal antibody or quality control antibody.
Specifically, the washing solution used in the washing step was TRIS buffer, and the concentration thereof was 0.01M. Further, the cleaning step employs ultrasonic cleaning.
Specifically, the reagents used in the activation step comprise 5-10mg/mL of an activating agent A and 5-10mg/mL of an activating agent B, wherein the activating agent A is N-hydroxysuccinimide, and the activating agent B is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
Specifically, the stop solution used in the activation stop step is 0.1-0.5% methanol in 0.1M MES buffer.
The preparation of the fluorescent microsphere solution comprises the following steps: mixing the fluorescent microspheres with a microsphere diluent. Specifically, the microsphere diluent contains 0.5-3% of tween-20, 2-5% of BSA, 5-20% of sucrose, 0.5-2% of sodium caseinate, 0.2-0.5% of PC300 and 0.02M PB buffer solution by volume fraction.
Compared with colloidal gold nanoparticles, the fluorescent latex particles are used as markers, quantitative detection of cat serum amyloid A can be realized by means of a quantitative analyzer, the blank that no fluorescence immunochromatography quantitative detection of cat serum amyloid A exists in the current market is filled, meanwhile, the detection sensitivity and accuracy are improved, the quantitative detection time is greatly shortened, and the method has the advantages of being rapid, simple, on-site, small in interference and the like.
The reaction membrane 4 with the detection line 3 and the quality control line 5 can be prepared by the following method: coating a second serum amyloid A monoclonal antibody on a reaction membrane 4 to form a detection line 3; an antigen specifically binding to the quality control antibody is coated on the reaction membrane 4 to form a quality control line 5 spaced from the detection.
In some embodiments, the absorbent pad 6 is prepared by: the absorbent paper was cut to a gauge of 30cm by 2.4cm per strip.
In some embodiments, the reaction membrane 4, the absorbent pad 6, the sample pad 1 and the binding pad 2 are adhered to the bottom plate 7 to form the fluorescence immunochromatographic test strip for serum amyloid a.
The following are specific examples (% each means a mass fraction).
The embodiment provides a fluorescence immunochromatographic assay kit for quantitatively detecting cat serum amyloid A, including card shell, reagent strip, sample diluent, be equipped with the immunochromatographic test strip that is used for detecting cat serum amyloid A in the card shell, refer to fig. 1, the test strip is equipped with by the lower to last time: the kit comprises a PVC plate, a sample pad 1, a marker combination pad 2, a reaction membrane 4 and a water absorption pad 6, wherein the marker combination pad 2 is adsorbed with a cat serum amyloid A monoclonal antibody (a first serum amyloid A monoclonal antibody) and a goat anti-rabbit IgG which are marked by fluorescent microspheres, and the diameter of the fluorescent microspheres is 400 nm; the reaction membrane 4 is provided with a detection line 3(T line) for coating cat serum amyloid A (second serum amyloid A monoclonal antibody) and a quality control line 5(C line) for coating rabbit IgG. The sample diluent was a buffer containing 0.5% S9, 0.5M NaCl, 1% triton, 0.5% PC300, 0.02M PB.
Coating treatment of the reaction film 4:
diluting cat serum amyloid A monoclonal antibody to 0.5mg/mL with 0.01M PB (pH 7.4) containing 5% trehalose as a working solution for detection line 3(T line);
rabbit IgG was diluted to 0.5mg/mL with 5% trehalose in 0.01M PB (pH 7.4) as working solution of quality control line 5 (C-line);
and sticking the reaction film 4 on a PVC plate, scribing a detection line 3 and a quality control line 5 on the film, wherein the liquid outlet amount of the scribed film is 0.5-2uL/cm, and placing the film at 50 ℃ for drying for 24 h.
Preparation of sample pad 1:
A. preparing a sample pad 1 diluent: mouse IgG 1mg/mL, Evans 1%, lectin 1mg/mL, 1mg/mL blocker against heterophilic antibodies of the test sample, 1% Tween-20, 3% BSA, 0.4% PC300, 0.02M PB buffer;
B. and D, uniformly spraying the diluent of the sample pad 1 in the step A on glass fiber, and drying overnight.
Preparation of the conjugate pad 2:
preparing fluorescent microsphere diluent containing 15% of fluorescent microsphere labeled cat serum amyloid A monoclonal antibody and goat anti-rabbit IgG, spraying the fluorescent microsphere diluent on glass fibers at the speed of 8ul/cm, wherein the fluorescent microsphere diluent contains 2% of tween-20, 5% of BSA, 10% of sucrose, 1% of casein sodium salt, 0.4% of PC300 and 0.02M PB buffer solution, and drying overnight.
The preparation method of the cat serum amyloid A monoclonal antibody and the goat anti-rabbit IgG marked by the fluorescent microspheres comprises the following steps:
1. cleaning the fluorescent microspheres: sucking 1mL of fluorescent microspheres by using a pipette gun, centrifuging at 12000rpm for 15min, discarding the supernatant, adding 1mL of cleaning buffer, and ultrasonically mixing uniformly.
2. Activating the fluorescent microspheres: add 1mL of activation buffer to the centrifuge tube in step 1 and mix on a rotary mixer for 20 min.
3. And (3) stopping activation: and (3) centrifuging the fluorescent microspheres in the step (2) at 15000rpm for 15min, discarding the supernatant, adding 1mL of stop solution, and ultrasonically mixing uniformly.
4. Labeling the antibody: and (3) adding 0.5mg of anti-cat serum amyloid A monoclonal antibody and goat anti-rabbit IgG into the fluorescent microspheres in the step (3), and uniformly mixing for 80min on a rotary mixing machine.
5. And (3) sealing: and (4) adding the fluorescent microspheres in the step (4) into 1mL of confining liquid, ultrasonically mixing uniformly, and mixing uniformly on a rotary mixer for 30 min.
6. Final washing: and (4) centrifuging the fluorescent microspheres in the step (4) at 15000rpm for 15min, discarding the supernatant, adding 1mL of fluorescent microsphere diluent, and ultrasonically mixing uniformly.
The diameter of the fluorescent microsphere is 400nm, and the fluorescent microsphere wraps fluorescein.
The washing buffer solution is 0.01M Tris buffer solution; the activation buffer is 0.01M Tris buffer containing 5-10mg/mL of an activating agent A and 5-10mg/mL of an activating agent B, wherein the activating agent A is N-hydroxysuccinimide, and the activating agent B is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; the stop solution is 0.1M MES buffer solution containing 0.4% methanol; the blocking solution is 0.01M Tris buffer containing 1% BSA; the fluorescent microsphere diluent contains 2% of tween-20, 5% of BSA, 10% of sucrose, 1% of sodium caseinate, 0.4% of PC300 and 0.02M PB buffer.
1. Selection of reaction time of the detection kit:
the kit prepared by the method is used for measuring four concentrations of SAA reference substances, namely 0mg/L,5mg/L, 160mg/L and 320mg/L, testing the same concentration at 2 minutes, 3 minutes, 5 minutes, 10 minutes and 15 minutes respectively, comparing the sensitivity and linearity of T/C and the concentration, and selecting the optimal testing time, wherein the detection values are represented by the ratio T/C as shown in the following table 1 and figure 2.
TABLE 1
Concentration mg/L 0 5 160 320
Time of measurement T/C T/C T/C T/C
2min 0.0221 0.0345 1.7231 3.2311
3min 0.0438 0.1264 2.4410 4.8655
5min 0.0441 0.1121 2.3413 4.5079
10min 0.0431 0.1289 2.4611 4.7139
15min 0.0401 0.1257 2.9643 4.0490
The results show that: according to the sensitivity and the linear range of 5-300mg/L, when the reagent is tested at 2min, 3min, 5min, 10min and 15min, compared with the sensitivity, the sensitivity requirement can be met within 3min, and the time does not need to be prolonged to 10 min.
2. Selecting the sample size of the detection kit:
the kit prepared by the method is used for measuring four concentrations of 0mg/L,5mg/L, 160mg/L and 320mg/L of SAA reference substances, respectively measuring the concentrations at 5ul, 10ul, 20ul and 40ul, testing the same concentration, comparing the sensitivity and linearity of T/C and the concentration, and selecting the optimal testing time for 3min, wherein the detection values are represented by the ratio T/C as shown in the following table 2 and figure 3.
TABLE 2
Concentration mg/L 0 5 160 320
Amount of test sample T/C T/C T/C T/C
5ul 0.0421 0.1345 2.8231 3.2311
10ul 0.0438 0.1264 2.4410 4.8655
20ul 0.0351 0.0313 2.3452 4.5551
40ul 0.0333 0.3289 2.4611 4.7139
The results show that: according to the sensitivity and the linear range of 5-300mg/L, the sample size is selected to be 10ul, and the high sensitivity requirement can be met.
3. Fitting a linear equation:
the kit prepared by the method is characterized in that the cat serum amyloid A reference substance is prepared into different concentrations by using negative diluent, the concentrations are respectively diluted into 0mg/L,5mg/L,15mg/L,40mg/L,70mg/L,120mg/L,160mg/L and 320mg/L, 10 microliters of each calibrator is respectively taken and added into 400 microliters of diluent to be uniformly mixed, 75 microliters of the calibrator is taken and dripped into a sample adding hole by using a pipetting gun to be measured, the concentration of the cat serum amyloid A reference substance, the corresponding detection limit signal value and the corresponding quality control line 5 signal value are shown in table 1, wherein the detection limit signal value and the quality control line 5 signal value are respectively represented by T and C, the average value of the T/C of each concentration is calculated, the average value of the T/C is used as a vertical coordinate, the cat serum amyloid A reference substance is used as a horizontal coordinate, equations were set up and linear regression curves were fitted as shown in table 3 below and fig. 4.
TABLE 3 detection limit signal value and quality control line 5 signal value corresponding to different concentrations of cat serum amyloid A calibrator
Figure BDA0002749574900000141
The results of the linear fit equation show: the reagent is in the detection range of 5-300mg/L, and the correlation coefficient R of SAA linear fitting is more than 0.99.
4. Precision:
randomly drawing the kit of the same batch number, taking two levels of 15mg/L and 70mg/L reference substances, and calculating the mean value of the measurement results of the samples and the precision variation Coefficient (CV) in the batch by 10 reagent cards in each concentration test, as shown in Table 4.
TABLE 4
Figure BDA0002749574900000151
The results show that: the kit has good batch precision, and the batch precision variation Coefficient (CV) of the reference substances for detecting the two concentrations is less than 15%.
5. Accuracy:
SAA reference samples were measured at concentrations of 8mg/L, 70mg/L, and 160mg/L, and the mean and relative deviation of the sample measurements were calculated for 3 measurements per concentration, as shown in Table 5.
TABLE 5
Figure BDA0002749574900000152
The results show that: the kit has good accuracy, and the relative deviation Bias% of the reference products with 3 detected concentrations is within +/-15%.
6. Data of detection performed by the kit stored at normal temperature at different times:
the kit is stored at normal temperature for 3 months, 6 months, 12 months, 18 months and 24 months, the calibrators are respectively detected to be 8mg/L, 40mg/L and 160mg/L, and the stability detection results are shown in Table 6.
TABLE 6
Figure BDA0002749574900000161
And (3) knotting: after the kit is stored for 24 months at normal temperature, the relative deviation Bias% is within +/-15%, and the kit can be stored for 2 years at normal temperature.
7. Reliability:
under the same test conditions, samples were tested with the kit of the present invention and a kit (dry fluoroimmunoassay kit) and compared with the Korea agile Vcheck reagent (the kit of fSAA of Vcheck is a well-known test kit in the pet industry, and the precision and the results are confirmed to be reliable), as shown in Table 7 and FIG. 5.
TABLE 7
Sample numbering Vcheck(ug/ml) The test value of the reagent is mg/L
1 5 6.5
2 13.48 23.7
3 5 5.4
4 50.47 58.59
5 5 7.5
6 11.5 25.8
7 12.48 22.5
8 110.48 120.45
9 15.65 25.47
10 50.48 55.01
11 108.78 118.59
12 50.26 55.89
13 5 11.5
14 68.91 65.48
15 117.9 120
16 200 224.18
17 92.15 91.07
18 134 142
19 5 6.14
20 5 7.8
21 55.69 69.15
22 5 5.18
23 5 6.5
24 5 5.5
25 99.47 95.48
26 178.48 201.2
27 108.15 111.45
28 6.5 15.2
29 5.9 11.48
30 5.1 5
31 85.14 89.59
32 65.48 55.18
33 44.5 43.65
34 5 5.5
35 110.5 123.47
36 200 230.14
37 200 198.17
38 200 195
39 14.58 26.14
40 5 5.2
And (3) knotting: the correlation coefficient of the test result of the kit of the invention and the test result of the Korea agile Vcheck instrument and the matched kit is R20.9839, the correlation is better, and the method can be used for clinical diagnosis.
Comparative example 1
Comparative example 1 is substantially the same as the example except that the sample pad 1 is sprayed with the diluent uniformly on the conjugate pad 2 without spraying the sample pad 1 with the diluent. Namely, the method of preparing the conjugate pad 2: preparing fluorescent microsphere diluent containing a cat serum amyloid A monoclonal antibody marked by 15% fluorescent microspheres and goat anti-rabbit IgG, and spraying the fluorescent microsphere diluent on glass fibers at the speed of 8ul/cm for drying to form a binding pad 2; the sample pad 1 dilution is then sprayed evenly onto the conjugate pad 2 and subsequently dried.
The results of selecting the reaction time and the sample amount of the detection kit are shown in tables 8 and 9, respectively.
TABLE 8
Concentration mg/L 0 5 160 320
Time of measurement T/C T/C T/C T/C
2min 0.0211 0.0215 0.9241 1.5251
3min 0.0238 0.0204 0.9540 1.1580
5min 0.0210 0.0202 1.3413 1.9079
10min 0.0331 0.1238 2.3 611 4.5139
15min 0.0401 0.1257 2.5643 4.5490
TABLE 9
Concentration mg/L 0 5 160 320
Amount of test sample T/C T/C T/C T/C
5ul 0.0128 0.0104 0.8241 1.1251
10ul 0.0299 0.0200 1.9754 2.0580
20ul 0.0300 0.1219 2.5230 4.5079
40ul 0.0401 0.1289 2.4561 4.8139
Comparative example 1 test results: table 8 according to the sensitivity and the linear range of 5-300mg/L, the reagent can meet the sensitivity requirement only within at least 10min when tested for 2min, 3min, 5min, 10min and 15 min; table 9 the sample size was selected to be at least 20ul to achieve high sensitivity depending on the sensitivity and the linear range 5-300 mg/L.
Comparative example 2
Comparative example 2 is substantially the same as example except that the sample pad 1 diluent is not sprayed on the sample pad 1, a reinforcing pad is disposed between the sample pad 1 and the conjugate pad 2, and the sample pad 1 diluent is uniformly sprayed on the glass fiber at a speed of 8ul/cm and dried to form the reinforcing pad.
The results of selecting the reaction time and the sample amount of the detection kit are shown in tables 10 and 11, respectively.
Watch 10
Concentration mg/L 0 5 160 320
Time of measurement T/C T/C T/C T/C
2min 0.0101 0.0234 0.8451 0.8251
3min 0.0292 0.0205 1.5650 1.8580
5min 0.0251 0.0200 1.4530 1.5029
10min 0.0242 0.1238 1.4611 1.7122
15min 0.0361 0.1201 2.5643 4.5249
TABLE 11
Concentration mg/L 0 5 160 320
Amount of test sample T/C T/C T/C T/C
5ul 0.02108 0.0210 1.8241 1.9251
10ul 0.02302 0.0200 2.1754 2.5358
20ul 0.0318 0.1312 2.4445 4.5179
40ul 0.0458 0.1398 2.5561 4.7139
Comparative example 2 test results: according to the sensitivity and the linear range of 5-300mg/L, the reagent can meet the sensitivity requirement only within at least 15min when tested for 2min, 3min, 5min, 10min and 15 min; table 11 shows that the high sensitivity requirement can be achieved only by selecting at least 20ul of sample size according to the sensitivity and the linear range of 5-300 mg/L.
Comparative example 3
Comparative example 3 is substantially the same as example except that the sample pad 1 was not sprayed with the diluent, a reinforcing pad was disposed between the conjugate pad 2 and the reaction membrane 4, and the sample pad 1 diluent was uniformly sprayed on the glass fiber at a rate of 8ul/cm and dried to form the reinforcing pad.
The results of selecting the reaction time and the sample size of the detection kit are shown in tables 12 and 13, respectively.
TABLE 12
Concentration mg/L 0 5 160 320
Time of measurement T/C T/C T/C T/C
2min 0.0101 0.0234 1.1451 1.1781
3min 0.0292 0.0205 1.5650 1.4598
5min 0.0251 0.0500 1.453 1.4784
10min 0.0242 0.08089 1.8611 1.9122
15min 0.0461 0.12645 2.5643 4.5490
Watch 13
Concentration mg/L 0 5 160 320
Amount of test sample T/C T/C T/C T/C
5ul 0.0228 0.02135 1.1281 1.1251
10ul 0.0222 0.02021 1.8454 1.5758
20ul 0.0110 0.01030 2.1445 3.5179
40ul 0.0356 0.1285 2.5461 4.5139
Comparative example 3 test results: table 12 according to the sensitivity and the linear range of 5-300mg/L, the reagent can meet the sensitivity requirement only after at least 15min when tested for 2min, 3min, 5min, 10min and 15 min; table 13 shows that the high sensitivity requirement can be achieved only by selecting at least 40ul of sample size according to the sensitivity and the linear range of 5-300 mg/L.
Comparative example 4
Comparative example 4 is substantially the same as in example except that the sample pad 1 dilution does not contain a blocking agent for heterophilic antibodies to the test sample.
The results of selecting the reaction time and the sample amount of the detection kit are shown in tables 14 and 15, respectively.
TABLE 14
Concentration mg/L 0 5 160 320
Time of measurement T/C T/C T/C T/C
2min 0.5321 0.5155 3.7231 3.8711
3min 0.5338 0.5354 3.5111 4.8655
5min 0.5061 0.5147 3.3413 4.9807
10min 0.5453 0.5362 3.3641 4.7993
15min 0.5201 0.5025 3.1643 4.519
Watch 15
Concentration mg/L 0 5 160 320
Amount of test sample T/C T/C T/C T/C
5ul 0.5016 0.5214 3.1231 3.2311
10ul 0.5342 0.5580 3.3410 3.8655
20ul 0.5005 0.5121 3.2752 3.5551
40ul 0.5148 0.5285 3.3211 3.7139
Comparative example 4 test results: under the condition that the reagent sample pad solution does not contain a blocking agent aiming at heterophilic antibodies of a detection sample, endogenous heterophilic antibodies in blood plasma can be combined with other species of immunoglobulin, including heterologous antibodies used as immunoassay reagents, the antibodies can interfere an immunoassay system to cause a false high detection value unrelated to the actual analyte concentration, and the linear range of the table 14 within 2-15 min of the test time cannot reach the linear range of the reagent by 5-300 mg/L; in Table 15, the sample size of 5 ul-40 ul cannot reach the linear range of 5-300mg/L of the reagent.
Comparative example 5
Comparative example 5 is substantially the same as example except that the sample pad 1 dilution does not contain mouse IgG.
The results of selecting the reaction time and the sample amount of the detection kit are shown in tables 16 and 17, respectively.
TABLE 16
Concentration mg/L 0 5 160 320
Time of measurement T/C T/C T/C T/C
2min 0.6421 0.6215 3.6209 3.7990
3min 0.6338 0.6254 4.5148 4.8651
5min 0.6261 0.6121 4.365 4.8070
10min 0.6113 0.6289 4.35411 4.8993
15min 0.6101 0.6125 4.3643 4.799
TABLE 17
Concentration mg/L 0 5 160 320
Amount of test sample T/C T/C T/C T/C
5ul 0.6216 0.6145 3.6231 3.9390
10ul 0.6379 0.6348 4.4541 4.7655
20ul 0.6515 0.6221 4.5752 4.5551
40ul 0.6458 0.65995 4.5211 4.7139
Comparative example 4 test results: in the case that the reagent sample pad solution does not contain mouse IgG, endogenous heterophilic antibodies in plasma can be combined with other species of immunoglobulin, including heterologous antibodies used as immunoassay reagents, and the antibodies can interfere with an immunoassay system to cause a false high detection value unrelated to the actual analyte concentration, and the linear range of the test time of 2 min-15 min of the table 16 is 5-300mg/L which cannot reach the reagent; in Table 17, the sample size of 5 ul-40 ul cannot reach the linear range of 5-300mg/L of the reagent.
Comparative example 6
Comparative example 6 is substantially the same as example except that the sample pad 1 does not contain lectin in the dilution liquid.
The results of selecting the reaction time and the sample amount of the detection kit are shown in tables 18 and 19, respectively.
Watch 18
Concentration mg/L 0 5 160 320
Time of measurement T/C T/C T/C T/C
2min 0.7321 0.7305 2.6991 2.9711
3min 0.7338 0.7354 3.4825 4.9655
5min 0.7561 0.7021 3.34013 4.8880
10min 0.7453 0.7389 3.36001 4.8749
15min 0.7601 0.7025 3..3643 4.6541
Watch 19
Concentration mg/L 0 5 160 320
Amount of test sample T/C T/C T/C T/C
5ul 0.7016 0.7105 2.2231 2.2291
10ul 0.7342 0.7308 3.4410 4.5555
20ul 0.7148 0.7121 3.3550 4.6551
40ul 0.7158 0.7285 3.3451 4.7852
Comparative example 4 test results: in the condition that the reagent sample pad solution does not contain agglutinin, endogenous heterophilic antibodies in blood plasma can be combined with immunoglobulins of other species, including heterologous antibodies used as immunoassay reagents, the antibodies can interfere an immunoassay system to cause a false high detection value irrelevant to the actual analyte concentration, and the linear range of the table 18 within 2-15 min of the test time cannot reach the linear range of the reagent by 5-300 mg/L; in Table 19, the sample size of 5 ul-40 ul cannot reach the linear range of 5-300mg/L of the reagent.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (12)

1. The serum amyloid A immunochromatographic test strip is characterized by comprising a sample pad, a binding pad, a reaction membrane and a water absorption pad, wherein the reaction membrane is provided with a detection line and a quality control line which are mutually spaced, the detection line is closer to the binding pad than the quality control line, the binding pad is coated with a first serum amyloid A monoclonal antibody and a quality control antibody which are respectively marked by fluorescent microspheres, the detection line is coated with a second serum amyloid A monoclonal antibody, the quality control line is coated with an antigen capable of being specifically bound with the quality control antibody, and the sample pad is coated with an antigen with the concentration of 1 mu g/cm calculated by the surface area of the sample pad2～16μg/cm2Antibody of non-detection sample species, 0.2. mu.g/cm2～16μg/cm2Lectin and 1. mu.g/cm2～16μg/cm2A blocking agent for heterophilic antibodies in a test sample.
2. The serum amyloid a immunochromatographic test strip according to claim 1, wherein the antibody of the non-test sample species, the lectin and the blocking agent are distributed at least at one end of the sample pad away from the binding pad.
3. The serum amyloid a immunochromatographic test strip according to claim 2, wherein the antibody of the non-test sample species, the lectin and the blocking agent are uniformly distributed in the sample pad.
4. The serum amyloid a immunochromatographic test strip according to claim 1, wherein the detection sample species is of feline origin and the antibody of the non-detection sample species is selected from non-feline IgG.
5. The serum amyloid A immunochromatographic test strip according to claim 4, wherein the quality control antibody is selected from IgG of the same species origin as the first serum amyloid A monoclonal antibody.
6. The serum amyloid a immunochromatographic test strip according to claim 1, wherein the blocking agent is selected from any one or more of normal serum and Scantibodies HBR, which are not directed against the target analyte.
7. The serum amyloid A immunochromatographic test strip according to any one of claims 1 to 6, wherein the concentration of the first serum amyloid A monoclonal antibody labeled with fluorescent microspheres coated on the conjugate pad is 0.5 μ g/cm, calculated on the surface area of the conjugate pad2～2μg/cm2The concentration of the quality control antibody marked by the fluorescent microspheres is 0.05 mu g/cm2～0.5μg/cm2。
8. The serum amyloid A immunochromatographic test strip according to claim 7, wherein the diameter of the fluorescent microsphere is 350nm to 500 nm.
9. The test strip for immunochromatography of serum amyloid A according to any one of claims 1 to 6, wherein the sample pad, the binding pad, the detection line and the quality control line are each independently coated with at least one of sugar, BSA and casein.
10. The serum amyloid A immunochromatographic test strip according to any one of claims 1 to 6, wherein the length ratio of the sample pad, the binding pad, the reaction membrane, and the water absorption pad in the serum amyloid A immunochromatographic test strip is 1.6cm to 2.8cm, calculated as a structure after lapping; the serum amyloid immunochromatographic test strip is 7 cm-8 cm in length, 3 mm-4 mm in width and 0.1 mm-1 mm in thickness.
11. A preparation method of a serum amyloid A immunochromatographic test strip is characterized by comprising the following steps:
the concentration is 1 mu g/cm2～16μg/cm2Antibody of non-detection sample species, 0.2. mu.g/cm2～16μg/cm2Lectin and 1. mu.g/cm2～16μg/cm2A blocking agent for heterophilic antibodies of the test sample is coated on the sample pad;
coating a first serum amyloid A monoclonal antibody and a quality control antibody which are respectively marked by fluorescent microspheres on a combination pad;
coating a second serum amyloid A monoclonal antibody on a reaction membrane to form a detection line;
coating an antigen specifically bound with the quality control antibody on the reaction membrane to form a quality control line separated from the detection line;
and overlapping a sample pad, the binding pad coated with a first serum amyloid A monoclonal antibody and a quality control antibody which are respectively marked by fluorescent microspheres, the reaction film with the detection line and the quality control line and a water absorption pad.
12. A serum amyloid A immunochromatographic kit, comprising the serum amyloid A immunochromatographic test strip according to any one of claims 1 to 10, and further comprising a sample diluent.
CN202011179062.5A 2020-10-29 2020-10-29 Serum amyloid protein A immunochromatographic test strip, kit and preparation method thereof Pending CN112485433A (en)

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Application publication date: 20210312