CN116413444A - Kit for detecting total triiodothyronine content and detection method thereof - Google Patents
Kit for detecting total triiodothyronine content and detection method thereof Download PDFInfo
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
The invention discloses a kit for detecting total triiodothyronine content and a detection method thereof, wherein the kit comprises a diluent for diluting a sample, a detection card and an ID card chip; the detection card comprises a PVC bottom plate, and a sample pad, a bonding pad, a nitrocellulose membrane and an absorption pad are arranged on the PVC bottom plate; the binding pad is provided with a fluorescent labeling antibody; the nitrocellulose membrane is provided with detection lines and quality control lines which are arranged at intervals, the detection lines are coated with T3-BSA conjugate, and the quality control lines are coated with chicken IgY antibody; the ID card chip is pre-stored with a detection standard curve. The kit and the detection aspect thereof provided by the invention have the advantages of high sensitivity, strong specificity, good stability of the used reagent, wider detection limit, capability of eliminating high background in conventional fluorescence detection, and the like.
Description
Technical Field
The invention relates to a technology for detecting the content of triiodothyronine in human blood, in particular to a kit for detecting the content of total triiodothyronine and a detection method thereof.
Background
Triiodothyronine (T3) is a hormone secreted by thyroid gland, has molecular weight of 650, and is a hapten small molecular substance. Most of T3 in blood binds to thyroid hormone binding protein (TBG), thyroid prealbumin and albumin, and only 0.3% of T3 exists in a free state, and is free T3 (FT 3) which exerts physiological activity. The total triiodothyronine (TT 3) refers to the total of free and bound triiodothyronine in serum or plasma, and is mainly used for clinically assisting diagnosis of hyperthyroidism (hyperthyroidism for short), hypothyroidism (hypothyroidism for short), evaluation of clinical curative effects thereof and the like. The sensitivity and specificity of the free T3 detection on disease diagnosis are better, but the total T3 detection result can more accurately reflect the in-vivo triiodothyronine state because the result is high or low in false caused by the interference of some diseases and medicines compared with the total T3. The main physiological functions of T3 are oxidation and thermogenesis, promotion of growth and development of organism, and regulation of anabolism of proteins, lipids and carbohydrates. The method is mainly used for clinically assisting in diagnosing hyperthyroidism (hyperthyroidism for short), hypothyroidism (hypothyroidism for short), evaluating clinical curative effects of the hyperthyroidism, etc.
Currently, the commonly used detection methods of T3 include an enzyme immunoassay method, a radioimmunoassay method, a chemiluminescent method and an electrochemiluminescent method. The existing detection methods have certain advantages and disadvantages, and are concretely as follows:
1) The enzyme immune method combines the amplification of enzyme catalytic reaction with the high specificity and specificity of antigen-antibody affinity reaction, and uses enzyme-marked antigen or antibody as main reagent to make immune test, so that it has high sensitivity. Together, the antigen in the sample (free antigen) and the antigen purified and immobilized on the solid support (immobilized antigen) compete for the same antibody, and when the more free antigen in the sample, the more antibody can be bound, while the immobilized antigen can only bind to fewer antibodies, and vice versa. After washing, the complex of free antigen and antibody is washed off, and only the complex of fixed antigen and antibody is left, which is compared with the result of the control group with fixed antigen, and the antigen content in the sample can be calculated according to the difference of color difference. Relatively impure samples are applicable and the data reproducibility is high. However, the disadvantage is that the sensitivity and specificity of the whole is poor; the operation process is somewhat complicated, and the reaction requires a long time.
2) The radioimmunoassay has high specificity of immune response and high sensitivity of radioactive measurement, so that various very trace substances with immunological activity can be accurately measured. However, only because of the use of biological reagents, the stability is affected by a plurality of factors, and a complete set of quality control measures is required to ensure the reliability of the results. Sensitivity is limited by the principle of operation of the method itself, and certain substances with particularly low contents in the body cannot be detected. Since radioimmunoassay is a competitive reaction, neither the test nor the standard can fully participate in the reaction, and the measured values are relative rather than absolute. In addition, there are problems of radiation irradiation, pollution, and the like.
3) Chemiluminescence refers to the phenomenon that a luminescent agent of a substance participating in chemical change radiates by using absorbed chemical energy to generate light, and mainly comprises direct chemiluminescence and enzymatic chemiluminescence. The most common direct chemiluminescence is an acridinium ester/hydrogen peroxide system, and the sensitivity is relatively high, but the defects of short luminous time and the like are overcome. Enzymatic chemiluminescence primarily includes horseradish peroxidase (HRP) -luminol systems, alkaline phosphatase (ALP) -AMPPD systems, and the like. The advantage is that the light-emitting signal is strong and stable, and the light-emitting time is long, but the problem that the working curve drifts along with time may exist. The chemiluminescent method has low detection precision and high cost of instruments and equipment.
4) Electrochemiluminescence is the product of a combination of electrochemistry and chemiluminescence. It refers to a phenomenon in which an excited state is generated by an electron transfer reaction of the electrode surface, and light radiation is generated when it returns to a ground state. Electrochemiluminescence goes through two processes: electrochemical reactions and chemiluminescent reactions. The tripropylamine is used as electron donor, and the terpyridyl ruthenium is used for labeling antibody (antigen) to react chemically in electric field environment. Compared with photoluminescence analysis, the electrochemical luminescence is electrically started, an excitation light source is not needed, the luminescence signal is stable, the luminescence time is long, the interference caused by stray light and light source impurity is effectively avoided, and the analysis sensitivity is greatly increased. But has the defects of complex measurement mode, high instrument maintenance cost and the like.
5) The fluorescent quantitative immunochromatography technology is a novel quantitative detection technology which combines the immunofluorescence technology (Immunofluorescence technique) with the traditional immunochromatography technology to develop innovation. The technology not only maintains the advantages of simple operation, rapid detection and strong portability of the colloidal gold immunochromatography technology, but also realizes the accurate quantification of the detection result by the fluorescent tracing enhancement technology.
Disclosure of Invention
Based on the problems, the invention aims to provide a kit for detecting the total triiodothyronine content, which is simple and convenient to operate, rapid to detect and high in portability by adopting a fluorescent quantitative immunochromatography technology, and a detection method thereof.
The present invention is achieved from two technical aspects.
The first technical scheme of the invention is as follows:
a kit for detecting the content of total triiodothyronine comprises a diluent for diluting a sample, a detection card and an ID card chip; the detection card comprises a strip-shaped PVC bottom plate, a sample pad, a bonding pad, a nitrocellulose membrane and an absorption pad are sequentially arranged on one surface of the PVC bottom plate along the length direction of the PVC bottom plate, two ends of the nitrocellulose membrane are respectively overlapped with the absorption pad and the bonding pad, and the other end of the bonding pad is overlapped with one end of the sample pad; the combination pad is provided with a fluorescent marked T3 antibody and a fluorescent marked goat anti-chicken IgY antibody; the nitrocellulose membrane is provided with detection lines and quality control lines which are arranged at intervals, the detection lines are coated with T3-BSA conjugate, and the quality control lines are coated with chicken IgY antibody; the ID card chip is pre-stored with a detection standard curve.
In one embodiment, the kit comprises the diluent components including 0.01M PBS, 1% PVP, 1% sodium chloride, and 1% Tween.
In one embodiment, in the kit, the biological source of the fluorescent-labeled T3 antibody is murine monoclonal IgG.
In one embodiment, in the kit, the biological source of the goat anti-chicken IgY antibody is a goat-derived antibody.
In one embodiment, the antigen biological source of the coated T3-BSA conjugate is a recombinant antigen.
In one embodiment, in the kit, the labeled antibody biological source of the coated chicken IgY antibody is chicken-derived antibody.
In one embodiment, in the kit, the goat anti-chicken IgY antibodies are labeled with fluorescent microspheres.
The second technical scheme of the invention is as follows:
the method for detecting the total triiodothyronine content by using the kit comprises the following steps of:
preparing whole blood, plasma and serum samples;
sucking 100 mu L of whole blood, plasma and serum samples by a pipette, and respectively adding diluent for dilution for later use;
and respectively sucking diluted whole blood, plasma and serum sample liquid by using a pipette, respectively dripping the diluted whole blood, plasma and serum sample liquid onto detection cards in three kits, and detecting in an immunofluorescence detector to respectively obtain the total triiodothyronine content in the whole blood, the plasma and the serum.
In one embodiment, in the detection method, the detection card includes a labeled antibody of a T3 antigen, a coated antigen of a T3 antigen, a labeled antibody of a quality control line, and a coated antibody of a quality control line.
In one embodiment, in the detection method, the final concentration of the labeled antibody of the T3 antigen on the detection card is 0.8-1mg/mL; the final concentration of the T3 antigen coated antigen on the detection card is 0.8-1mg/mL; the final concentration of the labeled antibody for the quality control line on the detection card is 0.8-1mg/mL; the final concentration of the coated antibody for the quality control line on the detection card is 0.8-1mg/mL.
The kit for detecting the total triiodothyronine content and the detection method thereof adopt fluorescent quantitative immunochromatography technology, directly detect excitation fluorescent signals based on a matched quantitative instrument, and calculate the content of an object to be detected in a sample through a standard curve built in the instrument. Besides the advantages of retaining colloidal gold, there are the following advantages: (1) compared with a colloidal gold method, the fluorescent quantitative immunochromatography has higher detection sensitivity; (2) the result is accurate, the anti-matrix interference is strong, and the reagent stability is good; (3) the instrument realizes quantitative calculation and eliminates the influence of artificial factors on results; (4) compared with the traditional quantitative detection technology, the technology has the advantages of rapidness, low price and the like, and is more suitable for timely and quantitative rapid detection beside a bed. The invention quantitatively detects the TT3 content in serum by using a competition method in a fluorescence immunochromatography technology. An effective TT3 fluorescence immunochromatography assay kit is established by optimizing the technological conditions, evaluating the performance, clinically applying and the like.
The detection method provided by the invention adopts a fluorescent quantitative immunochromatography technology, is a quantitative detection method based on an immunological principle, and has the characteristics of rapidness, simplicity and convenience in operation, low cost and wide application prospect.
Drawings
FIG. 1 is a schematic diagram of a detection card structure in a kit provided by the invention;
fig. 2 is a schematic diagram of the structure of a cartridge (containing a detection card) in the kit provided by the invention.
Detailed Description
The preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
The fluorescent quantitative immunochromatography technology is a novel quantitative detection technology which combines the immunofluorescence technology (Immunofluorescence technique) with the traditional immunochromatography technology to develop innovation. The technology not only maintains the advantages of simple operation, rapid detection and strong portability of the colloidal gold immunochromatography technology, but also realizes the accurate quantification of the detection result by the fluorescent tracing enhancement technology.
The invention provides a fluorescent immunochromatography kit for quantitatively determining total triiodothyronine content in serum, plasma and whole blood and a detection method. The method has the advantages of high sensitivity, strong specificity, good stability of the used reagent, wider detection limit, capability of eliminating high background in conventional fluorescence measurement, and the like.
The technical scheme of the invention is as follows:
the kit for detecting the total triiodothyronine content in serum, plasma and whole blood samples is used for detecting the concentration or the content of the total triiodothyronine in serum, plasma and whole blood. The kit comprises a diluent for diluting a sample, a detection card and an ID card chip; the ID card chip pre-stores a detection standard curve, namely, the detection standard curve comprises the standard curve information of the batch of reagents; the diluent (also known as TT3 diluent) components included 0.01M PBS, 1% PVP, 1% sodium chloride, and 1% Tween.
As shown in fig. 1, the detection card comprises an elongated PVC base plate 7, a sample pad 1, a bonding pad 2, a nitrocellulose membrane 4 and an absorbent pad 6 are sequentially arranged on one surface of the PVC base plate 7 along the length direction thereof, two ends of the nitrocellulose membrane 4 are respectively overlapped with one end of the absorbent pad 6 and one end of the bonding pad 2, and the other end of the bonding pad 2 is overlapped with one end of the sample pad 1; the combination pad is provided with a fluorescent marked T3 antibody and a fluorescent marked goat anti-chicken IgY antibody; the nitrocellulose membrane is provided with a detection line 3 and a quality control line 5 which are arranged in parallel at intervals, the detection line 3 is coated with a T3-BSA conjugate, and the quality control line 5 is coated with a chicken IgY antibody.
Further, as shown in fig. 2, the kit further includes a card case 8 made of a hard material and having a long-shaped structure, and the size of the card case is adapted to the size of the test card, so as to accommodate the test card, and a sample loading hole 9 and a test result observation area 10 are provided on the card case. After the detection card is arranged in the card shell 8, the sample adding hole 9 is matched with the sample pad 1, the detection result observation area 10 is matched with the nitrocellulose membrane, and the color change of the T line and the C line can be observed. During detection, the clamping shell 8 is used for avoiding unchanged operation caused by flexible bending of the detection card.
Still further, to prevent contamination and moisture from the test card, the test card is also hermetically wrapped in an aluminum foil bag with a desiccant inside the bag.
The binding pad is coated with immunofluorescence cross-linked triiodothyronine T3 antibody and sheep anti-chicken IgY fluorescent antibody.
A detection line 3 (T line) and a quality control line 5 (C line) are arranged on the nitrocellulose membrane, and the detection line T is coated with a triiodothyronine T3-BSA conjugate; and the quality control line C is coated with chicken IgY antibody.
The kit provided by the invention is realized by utilizing the technical principle of fluorescence immunochromatography, and the specific realization principle is as follows:
during testing, serum, plasma and whole blood samples are respectively and uniformly mixed with diluent, and are dripped into a sample adding hole on a detection card in a kit, chromatography is carried out under the capillary effect, TT3 antigen in the samples is combined with a fluorescent-labeled TT3 antibody to form a reaction complex, at the moment, a to-be-tested object-fluorescent-labeled antibody complex in the liquid coexists with an unbound fluorescent-labeled antibody, the fluorescent-labeled TT3 monoclonal antibody which is not combined with the to-be-tested object is diffused to a testing area, and the TT3 monoclonal antibody is captured by TT3 coupling BSA antigen coated on a nitrocellulose membrane detection line; the TT3 concentration in the sample is inversely proportional to the fluorescence intensity of the compound on the detection line, and the immunofluorescence detector converts the fluorescence signal value into the TT3 concentration in the sample according to a set standard curve. The concentration of serum, plasma and total triiodothyronine in the sample can be detected by using a fluorescence immunoassay analyzer, and the default measurement result of the analyzer is taken nmol/L as a unit.
In the kit for measuring total triiodothyronine in human serum, plasma and whole blood samples, the components and components contained in the test card are shown in Table 1.
TABLE 1 Total triiodothyronine detection card Assembly and Components
| Numbering device | Component (A) | Main component of the composition |
| a | Sample pad | Glass cellulose film |
| b | Bonding pad | Fluorescent-labeled T3 antibody and fluorescent-labeled goat anti-chicken antibody IgY antibody |
| c | Nitrocellulose membrane | T3-BSA conjugate coated, quality control line coated chicken IgY antibody |
| d | Absorbent pad | H-1 absorbent paper |
| e | PVC bottom plate | |
| f | Upper (lower) cover of clamping shell |
The fluorescent-labeled antibody biogenesis is murine monoclonal IgG, fluorescent microspheres are labeled, the coated antigen biogenesis of the T3-BSA conjugate is recombinant antigen, the labeled goat anti-chicken IgY (derived from a binding pad) antibody biogenesis of a quality control line (C line) is goat antibody, the fluorescent microspheres are labeled, and the coated chicken IgY antibody biogenesis of the quality control line (C line) is chicken antibody; wherein, the fluorescent microsphere is convenient to combine with buffer solution to obtain the time-resolved fluorescence-antibody complex.
The diluent comprises 0.01M PBS, 1% PVP, 1% sodium chloride and 1% Tween according to the content of each component (1 g PVP, 1g sodium chloride and 1g Tween are contained in each 100mL diluent).
The detection card comprises a specific antibody and a coupling antigen for measuring total triiodothyronine (TT 3) in human serum, plasma and whole blood samples; for example, T3 antibody biological source of the binding pad is shown as Ab1, T3-BSA conjugate coating antigen biological source on the nitrocellulose membrane is shown as Ab2, labeled goat anti-chicken IgY antibody biological source for C line quality control is shown as Ab3, and chicken IgY coating antibody biological source for C line quality control is shown as Ab 4.
The biological origin of the antibodies to total triiodothyronine (TT 3) was quantitatively determined as shown in Table 2 below:
TABLE 2 detection of the biological origins of antibodies and antigens to Total triiodothyronine (TT 3)
The preparation and implementation steps of the kit for quantitatively determining the total triiodothyronine in human serum, plasma and whole blood samples are as follows.
Step one, preparing a sample pad:
preparing a treatment fluid: 0.1M BB,1% sucrose, 1% trehalose, 1% PVPK3O (polyvinylpyrrolidone), 0.1mg/mLRBC (human anti-erythrocyte antibody).
Sample pad treatment: the treatment solution is uniformly smeared on a sample pad according to the volume of 50 mL/piece (width: 25 and length: 30 cm), and is dried overnight (16-24) under the conditions of the temperature (18-26) DEG C and the relative humidity less than or equal to 30 percent. Cutting the upper and lower long sides of the dried sample pad by 5mm respectively at the temperature of 18-28 ℃ and the relative humidity of less than or equal to 30%, and then cutting the sample pad into the size of (23+/-1) mm x (300+/-10) mm for later use.
Step two, preparing a bonding pad:
activation of immunofluorescent microspheres: taking 100uL of microsphere suspension with solid content of 1%, adding into a centrifuge tube, adding 1mL of 2- (N-morpholino) ethanesulfonic acid (MES) buffer solution, uniformly mixing, weighing 30uL of EDC (1-ethyl-carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) (1:1) activation solution, mixing with latex in the centrifuge tube, performing rotary reaction on the mixture for 30min on a rotary mixer, performing ultrasonic treatment on the reacted suspension to enable microspheres on the tube wall to be resuspended in aqueous solution, centrifuging the microsphere suspension, centrifuging at 12000r/min for 15min, discarding supernatant, adding 1mL of 2- (N-morpholino) ethanesulfonic acid (MES) buffer solution, and performing ultrasonic dispersion uniformly;
immunofluorescent microsphere cross-linking T3 antibodies: adding 0.5mg of T3 antibody into the activated microsphere suspension, rotating for 2 hours, adding 10% Bovine Serum Albumin (BSA) for blocking for 30 minutes after the reaction is completed, and respectively centrifuging the blocked microspheres at the temperature of 4 ℃ for 15 minutes at the speed of 12000 r/min. After centrifugation, the supernatant was discarded, the pellet was left, 1mL of Tris buffer (Tris) was added, sonicated for 3min, followed by 12000r/min, centrifugation at 4℃for 15min, and this step was repeated once. Adding 1mL microsphere complex solution after removing supernatant, performing ultrasonic treatment for 3min, centrifuging for 3min at 2000r/min, removing precipitate, and preserving at 4deg.C in dark place.
Immunofluorescence microsphere cross-linked goat anti-chicken IgY antibody: adding 0.5mg of goat anti-chicken IgY antibody into the activated microsphere suspension, carrying out rotary reaction for 2 hours, adding 10% Bovine Serum Albumin (BSA) for blocking for 30 minutes after the reaction is finished, and respectively centrifuging the blocked microspheres at 4 ℃ at the speed of 12000r/min for 15 minutes. After centrifugation, the supernatant was discarded, the pellet was left, 1mL of Tris buffer (Tris) was added, sonicated for 3min, followed by 12000r/min, centrifugation at 4℃for 15min, and this step was repeated once. Adding 1mL microsphere complex solution after removing supernatant, performing ultrasonic treatment for 3min, centrifuging for 3min at 2000r/min, removing precipitate, and preserving at 4deg.C in dark place.
Mixing the immunofluorescence microsphere crosslinked T3 antibody and the immunofluorescence microsphere crosslinked goat anti-chicken IgY antibody: the marked immunofluorescence microsphere crosslinked T3 antibody and the immunofluorescence microsphere crosslinked goat anti-chicken IgY antibody are mixed according to the following weight ratio of 1:1 (V: V) are uniformly mixed, 90W ultrasonic is performed, the work is performed for 2 seconds, the interval is 5 seconds, and the process is repeated for 3 times.
Spraying pad: glass fiber 8964 was cut to (10.+ -. 0.5) mm× (300.+ -. 10) mm. Loading the fluorescent microsphere marked antibody solution into a metal spraying scribing machine, and spraying the fluorescent microsphere marked antibody solution onto the cut bonding pad according to the spraying amount of 4.0 mu L/cm; and (3) placing the sprayed bonding pad at the temperature of (37+/-2) ℃ and drying overnight (16-24) in an environment with the humidity of less than or equal to 30 percent.
Step three: preparation of immunochromatography test strips:
coating diluent preparation: 0.05M Tris (Tris), 1% sucrose.
And (3) film drawing: and (3) sticking the NC film on a PVC plate, diluting the coating concentration of the T3-BSA on an NC film detection line to 1mg/mL by using the coating diluent, and controlling the coating concentration of the goat anti-rabbit IgG on the quality control line to 1mg/mL. A metal spraying scribing instrument is used to scribe at a predetermined position on the NC film of the specification. Preferably, the amount of the film is 1. Mu.L/cm, the length of the film is 300mm, the distance between the C line and the T line is (5.+ -. 0.5) mm, the distance between the C line and the lower edge of the nitrocellulose film is (10.+ -. 0.5) mm, and the distance between the T line and the upper edge of the nitrocellulose film is (10.+ -. 0.5) mm. After the film is divided, the NC film is placed in a drying oven with the temperature of 37+/-2 ℃ and the humidity of less than or equal to 30 percent and dried for 16-24 hours.
Preparation of absorbent pad: cutting H-1 absorbent paper into pieces of (23+ -1) mm× (300+ -10) mm size
Assembling a test card sample: the reaction plate is paved on the working table surface, the protective film at the pasting position of the absorption pad on the reaction plate is uncovered, the absorption pad is adhered to the reaction plate, the upper part of the absorption pad is aligned with the top end of the bottom plate, and the other side of the absorption pad is partially covered on the NC film by 2-3 mm. The reaction plate is paved on the workbench surface, the protective film at the adhesion part of the bonding pad and the sample pad on the reaction plate is uncovered, the bonding pad is adhered to the upper end of the reaction plate, preferably the bonding pad faces upwards, and one end of the bonding pad is covered on the NC film by 1-2 mm. And adhering a sample pad to the PVC base plate, aligning the lower end of the sample pad with the adhering part of the sample pad of the PVC base plate, and covering the upper end of the sample pad on the bonding pad.
Standard test card assembly: cutting the prepared large plate into test strips with the width of (3.85+/-0.05) mm; and (3) loading each test strip into the lower cover of the fluorescent card shell, covering the upper cover after loading, placing the test strip on a card pressing machine, and adjusting the height of the card pressing machine to be 3.5mm to tightly combine the upper cover and the lower cover of the fluorescent card shell. Each reagent card is filled into aluminum foil bags, each bag is added with 1 bag of drying agent, and the bags are sealed to be used as standard detection cards.
Fourth, quality judgment of detection effect:
sample diluent preparation: 0.01M PB,1% PVP (polyvinylpyrrolidone), 1% sodium chloride, 1% Tween.
Sample inspection: taking 100uL of serum, plasma or whole blood sample to be detected, adding the 100uL of serum, plasma or whole blood sample into 300uL of the sample diluent, and dripping the mixture onto an immunochromatography reagent card after the mixture is uniformly mixed for carrying out an immunochromatography reaction; fluorescence detection is then performed under a fluorescence detector using the wavelength of the emitted light corresponding to the fluorescent latex particles. In the case of fluorescence detection under a fluorescence detector, the detection result can be confirmed to be effective only by detecting a signal on a quality control line.
The invention also provides a detection method for detecting the total triiodothyronine content of serum, plasma and whole blood samples by using the kit, which comprises the following steps:
collecting venous blood of a patient, shaking the venous blood in an anticoagulant tube, taking part of whole blood to separate plasma and serum, and respectively preparing whole blood, plasma and serum samples for later use;
sucking 100 mu L of whole blood, plasma and serum samples by a pipette, and respectively adding diluent for dilution for later use;
and respectively sucking diluted whole blood, plasma and serum sample liquid by using a pipette, dripping the diluted whole blood, plasma and serum sample liquid onto detection cards in the three kits, and detecting in an immunofluorescence detector to respectively obtain the total triiodothyronine content in the whole blood, the plasma and the serum.
Preferably, the diluent comprises a marked antibody of the T3 antigen, a coated antigen of the T3 antigen, a marked antibody of the quality control line and a coated antibody of the quality control line; wherein the final concentration of the labeled antibody of the T3 antigen on the detection card is 0.8-1mg/mL, the final concentration of the coated antigen of the T3 antigen on the detection card is 0.8-1mg/mL, the final concentration of the labeled antibody for the C line quality control on the detection card is 0.8-1mg/mL, and the final concentration of the coated antibody for the C line quality control on the detection card is 0.8-1mg/mL.
In the method for detecting the total triiodothyronine content, the sample is prepared as follows:
the sample collection method comprises collecting venous blood of a subject with an anticoagulant tube containing heparin lithium, EDTA-K2 or sodium citrate anticoagulant, and shaking the collected blood sample for use; collecting blood plasma by using an anticoagulant tube containing heparin lithium, EDTA-K2 or sodium citrate anticoagulant to collect venous blood of a tested person, and separating the blood plasma as soon as possible after blood collection so as to avoid hemolysis; serum collection serum tubes or rapid serum tubes containing coagulants collect venous blood from subjects and serum should be separated as soon as possible after collection to avoid hemolysis.
Sample types include: whole blood, plasma, serum.
Preferably, the different samples should be used as immediately as possible after collection. If the detection can not be carried out in time, the serum/plasma/whole blood can be stored for 3 hours at room temperature; the serum/plasma can be preserved for 7 days at 2-8deg.C, and the whole blood can be preserved for 3 days at 2-8deg.C; serum/plasma can be stored for 90 days at-20+ -5deg.C, and whole blood sample cannot be frozen. The refrigerated and frozen samples are fully dissolved and evenly mixed before detection, and can be tested after being restored to room temperature without repeated freezing and thawing.
The specific operation of the method for detecting the total triiodothyronine content is as follows:
1. opening the instrument, and inserting a chip identical to the reagent batch number;
2. sucking 100 mu L of sample by a pipette, adding the sample into the diluent, and mixing the sample upside down;
3. opening an aluminum foil bag, taking out the detection card, and horizontally placing the detection card on a tabletop;
4. sucking 100 mu L of the diluted and uniformly mixed sample by using a pipette, and adding the 100 mu L of the diluted and uniformly mixed sample into a sample adding hole of a detection card;
5. selecting a sample type of plasma, serum or whole blood on a companion instrument;
6. and (3) testing in real time: after reacting for 15min at room temperature, placing the detection card into an instrument card slot, selecting an 'instant test' mode, and clicking a 'test'; standard test: placing the detection card into an instrument card slot, selecting a standard test mode, clicking a test mode, automatically timing the instrument, automatically testing and displaying a result after timing is finished;
7. clicking "print" may print the test result report.
The present invention will be clearly described with reference to specific examples.
Example 1
The composition, package and amount (1/10/25/50 parts/box) of the total triiodothyronine kit are shown in Table 3.
TABLE 3 composition, package and quantity of kit
Example 2
Linear range performance assessment experiment of the kit
And diluting a high-value sample near the upper limit of the linear interval to 0.61nmol/L, 1.5nmol/L, 3nmol/L, 6nmol/L and 9.22nmol/L according to a certain proportion, respectively taking 100 mu L of each sample, respectively adding the 100 mu L of each sample into the diluent, uniformly mixing, respectively taking 100 mu L of each mixed liquid, adding the 100 mu L of each mixed liquid into a TT3 detection card sample adding hole, and repeating each reference sample for 3 times. After the reaction was allowed to stand for 15 minutes, the concentration value was read on a fluorescence immunoassay analyzer. And calculating the average value of the detection results of each reference product for 3 times, and then adopting a least square method to perform linear fitting to obtain a linear correlation coefficient r. From the experimental data shown in Table 4, it can be seen that the correlation coefficient r > 0.9900.
Table 4 results of the Linear Range detection of the kit
Example 2
Blank limit detection experiment of kit
Blank limit: repeated 20 times with 0nmol/L TT3 reference, relative T/C average
And Standard Deviation (SD), give +.>
And the corresponding numerical value is brought into a linear equation to obtain a corresponding concentration value, namely a blank limit. Shown by table 5. The result shows that the blank limit of the kit is not higher than 0.4nmol/L.
Table 5 results of blank test for kit
Example 3
Accuracy performance assessment test of the kit
Taking 100uL of a reference sample of an enterprise with the concentration of about 1.5nmol/L and 6nmol/LTT3, fully mixing with sample diluent in the kit, then taking 100uL of mixed solution, adding the mixed solution into a sample adding hole of a reagent card, standing for 15min, inserting the mixed solution into a fluorescence immunoassay analyzer, and reading a fluorescence signal T/C value to obtain the corresponding sample concentration. From the test data shown in Table 6, it can be seen that the relative deviation of all samples is no more than.+ -. 15%.
TABLE 6 accuracy test results
According to the results in the table, the accuracy detection result coincidence rate of each quality control product is 100%, which shows that the accuracy detection of the kit meets the requirements.
Example 4
Repeatability performance evaluation test of the kit of the present application
The TT3 enterprise references of 1.5nmol/L and 3nmol/L are detected and repeated 10 times, the average value M and the standard deviation SD of 10 times of measurement results are calculated respectively, and the Coefficient of Variation (CV) is obtained according to a formula. Taking 100uL of reference sample and sufficiently mixing with sample diluent in the kit, taking 100uL of mixed solution, adding the mixed solution into a sample adding hole of the reagent card, standing for 15min, then inserting the mixed solution into a fluorescence immunoassay analyzer, and reading a fluorescence signal T/C value to obtain corresponding sample concentration. Shown by table 7. As can be seen from the test data, the variation coefficient of the sample is not more than +/-15%.
TABLE 7 results of the kit reproducibility test
It is to be understood that the foregoing description of the preferred embodiments of the invention is not to be considered as limiting the scope of the invention, which is defined by the appended claims.
Claims (10)
1. The kit for detecting the total triiodothyronine content is characterized by comprising a diluent for diluting a sample, a detection card and an ID card chip; the detection card comprises a strip-shaped PVC bottom plate, a sample pad, a bonding pad, a nitrocellulose membrane and an absorption pad are sequentially arranged on one surface of the PVC bottom plate along the length direction of the PVC bottom plate, two ends of the nitrocellulose membrane are respectively overlapped with the absorption pad and the bonding pad, and the other end of the bonding pad is overlapped with one end of the sample pad; the binding pad is provided with a fluorescent-labeled T3 antibody and a fluorescent-labeled goat anti-chicken IgY antibody; the nitrocellulose membrane is provided with detection lines and quality control lines which are arranged at intervals, the detection lines are coated with T3-BSA conjugate, and the quality control lines are coated with chicken IgY antibody; the ID card chip is pre-stored with a detection standard curve.
2. The kit of claim 1, wherein the diluent components comprise 0.01M PBS, 1% pvp, 1% sodium chloride, and 1% tween.
3. The kit of claim 1, wherein the biological source of the fluorescently labeled T3 antibody is murine monoclonal IgG.
4. The kit of claim 1, wherein the biological source of the goat anti-chicken IgY antibody is a goat-derived antibody.
5. The kit of claim 1, wherein the antigen biological source of the coated T3-BSA conjugate is a recombinant antigen.
6. The kit of claim 1, wherein the labeled antibody biological source of the coated chicken IgY antibody is a chicken-derived antibody.
7. The kit of claim 1, wherein the goat anti-chicken IgY antibodies are labeled with fluorescent microspheres.
8. A method for detecting the total triiodothyronine content using the kit according to any one of claims 1 to 7, characterized in that it comprises the following steps:
preparing whole blood, plasma and serum samples;
sucking 100 mu L of whole blood, plasma and serum samples by a pipette, and respectively adding diluent for dilution for later use;
and respectively sucking diluted whole blood, plasma and serum sample liquid by using a pipette, respectively dripping the diluted whole blood, plasma and serum sample liquid onto detection cards in the three kits, and detecting in an immunofluorescence detector to respectively obtain the total triiodothyronine content in the whole blood, the plasma and the serum.
9. The method according to claim 8, wherein the detection card comprises a labeled antibody of T3 antigen, a coated antigen of T3 antigen, a labeled antibody of a quality control line, and a coated antibody of a quality control line.
10. The method of claim 9, wherein the final concentration of the labeled antibody to the T3 antigen on the test card is 0.8-1mg/mL; the final concentration of the T3 antigen coated antigen on the detection card is 0.8-1mg/mL; the final concentration of the labeled antibody for the quality control line on the detection card is 0.8-1mg/mL; the final concentration of the coated antibody for the quality control line on the detection card is 0.8-1mg/mL.
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