CN116413445A - Detection card, kit and detection method for detecting total thyroxine content - Google Patents
Detection card, kit and detection method for detecting total thyroxine content 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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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- 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
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses a detection card, a kit and a detection method for detecting total thyroxine content, 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 reaction membrane and an absorption pad are arranged on the PVC bottom plate; the binding pad is provided with a fluorescent labeling antibody; the reaction film is provided with detection lines and quality control lines which are arranged at intervals, the detection lines are coated with TT4 antigen, 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 the technical field of biomedical detection, in particular to a detection card, a kit and a detection method for detecting total thyroxine content.
Background
3,5,3',5' tetraiodothyronine, thyroxine (T4), is the major hormone secreted by the thyroid gland, with a molecular weight of 777 and a half-life of 6-7 days. The T4 hormone, together with the cellular metabolism, regulates many biological processes, which are critical to the development of the nervous system. It is estimated that more than 99.9% of total T4 hormone in the blood binds to the transport proteins, i.e. thyroxine-binding globulin (TBG), thyro-pre-binding albumin (TBPA) and albumin (Alb), protein-binding T4. Thus, only 0.02-0.04% of T4 exists in free (unbound) form, free T4 being currently considered biologically active. The T4 combined with the protein and the free T4 hormone keep dynamic balance, so that the normal function of thyroid is ensured. The level of T4 may vary with changes in TBG concentration, for example, pregnancy or contraceptive use, chronic hepatitis and biliary sclerosis etc. may increase TBG levels, and renal disease and androgen therapy etc. may decrease. Free T4 enters body tissue where T4 is desired to be used. T4 is also converted to another thyroid hormone, triiodothyronine (T3); thus, T4 is an indicator of any hormonal changes in thyroid dysfunction. Although T3 is a thyroid hormone that is more active than T4, T4 is circulating in serum in far excess of T3. Both T4 and T3 play a role in regulating various biochemical processes in the body, which are essential for normal physiological metabolism and nervous system activity.
The concentration of serum total thyroxine (TT 4) has a close relation with thyroid function, and the detection of TT4 is an important clinical index for evaluating thyroid function and pathological state and is also an effective method for diagnosing congenital hypothyroidism of newborns. Therefore, a detection technique having high sensitivity and reliability for total thyroxine TT4 detection and capable of reducing detection cost has yet to be developed.
Total thyroxine is a hormone secreted by the thyroid gland, and is the total amount of protein-bound T4 and free T4 in serum, with elevated TT4 concentrations leading to hyperthyroidism and reduced TT4 concentrations leading to hypothyroidism. TT4 concentration is a sensitive index of in-vitro thyroid function test, can reflect thyroid function state to a certain extent, and provides basis for clinical diagnosis of hyperthyroidism and hypothyroidism. The main methods for total thyroxine determination in vivo are: radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescent immunoassay, etc., are described in detail below.
1) Protein Binding Iodination (PBI). The method is the earliest thyroxine detection method, and cannot be used as a conventional detection method of thyroid hormone due to a plurality of interference factors.
2) Radioimmunoassay (RIA). The method is realized by marking thyroxine hapten with I, the synthesis process is complex, the validity period is short, certain pollution is caused to the environment, and the factors influencing the detection result are more, so that the method basically exits from the market at present.
3) Immune colloidal gold method (ICA). Colloidal gold with high electron density is used as a marker to be marked on an antibody or an antigen, and when the colloidal gold is aggregated at a corresponding ligand, the colloidal gold generates macroscopic color, so that the colloidal gold can be used in qualitative or semi-quantitative detection research. The method is simple and quick to operate, but the problem of low sensitivity is not solved all the time, and the method is also determined by the technology.
4) Enzyme-linked immunosorbent assay (ELISA). The method is a common technology, and the common combination modes are a double antibody sandwich method and an indirect method through the combination of solid-phase antigen or antibody and enzyme-labeled antigen and antibody. The ELISA method adopts a double antibody sandwich method to detect, and the coated antibody is adsorbed on the surface of a solid phase carrier and is used for combining with a specific antigen, so that when the specific antigen is contained in an object to be detected, an antigen-antibody complex is formed with the object to be detected. Another enzyme-labeled antibody is added as a detection antibody, which can retain the enzyme activity and the immunological activity of the antibody and is used for improving the sensitivity of the ELISA method. The antibody after enzyme labeling can be combined with different epitopes of antigen in the to-be-detected object to form a double antibody sandwich compound of antibody-antigen-antibody, the formed compound is also combined on a solid phase carrier, after a substrate for enzyme reaction is added, the substrate is catalyzed into a colored product by the enzyme, and the concentration of the antigen in the to-be-detected object can be judged by the color depth of the colored product, so that qualitative or quantitative experiments can be carried out. The method has low detection sensitivity, narrow detection range and long operation time, is not suitable for emergency detection, and can only be applied to batch detection. In addition, enzymes themselves are unstable in nature and are susceptible to interference from biological samples.
5) Chemiluminescent immunoassay (CLIA). The method combines a chemiluminescent assay technology with high sensitivity and a high-specificity immune reaction, and is used for detection and analysis technologies of various antigens, hapten, antibody, hormone, enzyme, fatty acid, vitamin, medicine and the like. In recent years, the development of chemiluminescent immunoassay technology is rapid, the sensitivity, specificity and automation degree reach or exceed the RIA level, and in particular, the stability of the marker and no environmental pollution are incomparable with the RIA method. Full-automatic chemiluminescence immunoassay systems are imported in large and medium hospitals in recent years, but instruments and reagents are expensive. At present, a plurality of patent applications for detecting thyroxine by using a magnetic particle chemiluminescence method are applied, namely, a luminol luminescent marker is used for marking thyroxine antibody, the detection sensitivity is low, and the stability is poor.
Disclosure of Invention
Based on the problems, one of the problems to be solved by the invention is to provide a detection card which adopts a fluorescent quantitative immunochromatography technology and can detect the total thyroxine content with simple operation, rapid detection and strong portability.
The second problem to be solved by the invention is to provide a kit for detecting the total thyroxine content.
The third problem to be solved by the invention is to provide a detection method for detecting the total thyroxine content by using the kit
The first technical scheme of the invention is as follows:
the detection card comprises a strip-shaped PVC bottom plate, a sample pad, a bonding pad, a reaction 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 reaction membrane are respectively overlapped with one end of the absorption pad and one end of 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 TT4 antibody and a fluorescent marked goat anti-chicken IgY antibody; the reaction membrane is provided with detection lines and quality control lines which are arranged at intervals, the detection lines are coated with TT4 antigen, and the quality control lines are coated with chicken IgY antibody.
In one embodiment, in the detection card, the biological source of the fluorescent-labeled TT4 antibody is murine monoclonal IgG.
In one embodiment, in the detection card, the biological source of the goat anti-chicken IgY antibody is a goat-derived antibody.
In one embodiment, in the test card, the biological source of the coated TT4 antigen is a recombinant antigen.
In one embodiment, in the test card, the biological source of the coated chicken IgY antibody is chicken-derived antibody.
In one embodiment, in the detection card, the goat anti-chicken IgY antibody and the TT4 antibody are both labeled with fluorescent microspheres.
In one embodiment, in the test card, the final concentration of the labeled antibody of the TT4 antigen on the test card is 0.5mg/mL; the final concentration of the TT4 coating antigen on the detection card is 0.5mg/mL.
In one embodiment, in the detection card, the final concentration of the chicken IgY antibody for the quality control line on the detection card is 0.5mg/mL; the final concentration of the coated chicken IgY antibody used for the quality control line on the detection card is 1mg/mL.
The invention also provides a kit for detecting the total thyroxine content, which comprises any one of the detection cards, a diluent for diluting a sample and an ID card chip; the ID card chip is pre-stored with a detection standard curve; the diluent component is phosphate buffer solution.
The invention also provides a detection method for detecting the total thyroxine content in human serum, plasma and whole blood by using the kit; the method 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, 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 thyroxine content in the whole blood, the plasma and the serum.
Compared with the prior art, the detection card, the kit or the detection method provided by the invention has the following advantages:
1. the accuracy and stability of the measurement result are good;
2. has higher sensitivity and shorter detection time than ELISA kit method;
3. the whole blood sample in the anticoagulation tube can be directly detected, pretreatment of the blood sample to be detected is not needed, the operation steps of total thyroxine detection are simplified, and the detection time is saved;
4. the detection kit only needs to mix a sample with a diluent, add the mixed solution into a detection card and read data by using a fluorescent instrument, and various matched reagents or expensive instruments are not needed.
5. The detection method 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 invention provides a kit for quantitatively determining total thyroxine (TT 4) in human serum, plasma and whole blood samples by using lanthanide immune fluorescent latex microspheres coated as markers and using a fluorescent detector to detect fluorescent signal intensity based on the principles of immunology and time-resolved fluorescence immunochromatography, which has the advantages of high sensitivity, strong specificity, simplicity and rapidness in operation and the like.
Time resolved fluoroimmunoassay, nonradioactive ligand immunoassay with lanthanide chelates as tracers. The lanthanide ion chelate fluorescence has a longer decay time than the conventional fluorophore, 10 of that of conventional fluorescence 3 ～10 6 Multiple times. By time resolution, background fluorescence is greatly reduced, and a high signal-to-noise ratio is realized. The lanthanide series integration has a wider excitation light spectrum, the maximum excitation wavelength is 300-500 nm, and the sensitivity can be improved by increasing the excitation light energy. The emission spectrum band is very narrow, even less than 10nm, and a filter which only allows the emitted fluorescence to pass through can be adopted, so that background fluorescence is further reduced, and the narrow emission band is possible for multiple analysis. Therefore, the method has the characteristics of high sensitivity, stable marker, no interference of natural fluorescence of a sample, no radioactive pollution and the like, and is a microanalysis technology widely applied at present. The time-resolved fluorescence technology and the immunochromatography technology are combined to obtain the detection kit capable of rapidly, conveniently, accurately and sensitively carrying out total thyroxine.
The technical scheme of the invention is as follows:
the kit for detecting the total thyroxine content in serum, plasma and whole blood samples is used for detecting the concentration or the total thyroxine content 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 component is phosphate buffer solution.
As shown in fig. 1, the detection card comprises a strip-shaped PVC bottom plate 7, a sample pad 1, a bonding pad 2, a reaction membrane 4 and an absorption pad 6 are sequentially arranged on one surface of the PVC bottom plate 7 along the length direction, two ends of the reaction membrane 4 are respectively overlapped with one end of the absorption 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 2 is provided with a fluorescent marked TT4 antibody and a fluorescent marked goat anti-chicken IgY antibody; the reaction film 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 TT4 antigen, and the quality control line 5 is coated with chicken IgY antibody.
In the above-mentioned test card, the sample pad 1 and the bonding pad 2 are both made of glass cellulose film; the reaction membrane 4 is prepared by adopting a nitrocellulose membrane; the absorbent pad 6 is made of H-1 absorbent paper.
Further, as shown in fig. 2, the kit further includes a cartridge 8 made of a long strip-shaped structure and a hard material (such as plastic, hard paper, etc.), and the size of the cartridge is adapted to the size of the detection card, so as to accommodate the detection card, and a sample loading hole 9 and a detection result observation area 10 are provided on the cartridge. 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 reaction film, 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 inconvenient 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 a fluorescence-labeled total thyroxine TT4 antibody and a fluorescence-labeled goat anti-chicken IgY antibody.
A detection line 3 (T line) and a quality control line 5 (C line) are arranged on the reaction film, and the detection line 3 is coated with a total thyroxine TT4 antigen; and the quality control line 5 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, TT4 antigen in the samples is combined with a fluorescent-labeled TT4 antibody on a binding pad 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, and the fluorescent-labeled TT4 monoclonal antibody which is not combined with the to-be-tested object is captured by TT4 antigen coated on a nitrocellulose membrane detection line; the TT4 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 TT4 concentration in the sample according to a set standard curve.
In the above-mentioned kit for measuring total thyroxine in human serum, plasma and whole blood samples, the component elements and components contained in the test card are shown in Table 1.
TABLE 1 Total thyroxine detection card Assembly and Components
The biological source of TT4 antibody of the binding pad is mouse monoclonal IgG, which is marked with fluorescent microsphere, the biological source of TT4 coated antigen on the reaction film is recombinant antigen, the biological source of goat anti-chicken IgY (derived from the binding pad) antibody used for being combined with the quality control line (C line) antibody is goat antibody, the biological source of chicken IgY antibody used for being coated with the quality control line (C line) is chicken antibody.
The preparation and implementation steps of the kit for quantitatively determining total thyroxine in human serum, plasma and whole blood samples are as follows.
Step one, preparation of bond pad
1. Immunofluorescence microsphere activation:
taking a centrifuge tube, taking 100ul of immunofluorescent microspheres from each tube, suspending the immunofluorescent microspheres in 900ul of reaction buffer, and performing ultrasonic dispersion for 3min.
The activator EDC and the activator NHS were prepared at 5mg/ml each with the reaction buffer, and 100ul each was mixed.
Adding 30ul of the mixed activator into each microsphere (adding into the upper cover, immediately mixing, covering), rotating, mixing, activating for 30min, centrifuging at 12000r/min and 4deg.C for 15min, removing supernatant, and standing for precipitation.
2. Antibody coupling: adding 1mL MES into the centrifuge tube, carrying out vortex, carrying out ultrasonic treatment for 3min, respectively adding 0.05mg TT4 antibody and 0.05mg chicken IgY antibody, and carrying out rotary reaction for 2h to obtain the fluorescent microsphere-antibody complex.
3. Latex sealing and preservation: 100uL of 10% BSA (bovine serum albumin) solution was added to the centrifuge tube and blocked for 30min,12000r/min, and centrifuged at 4℃for 15min. The supernatant was removed, 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 of microsphere complex solution into the supernatant, performing ultrasonic treatment for 3min, centrifuging for 3min at 2000r/min, removing precipitation, and keeping the precipitate at 4 ℃ in a dark place to obtain fluorescent latex particle solution.
4. The bonding pad is prepared by: and uniformly spraying the prepared fluorescent latex particle solution on 1.5-1.8cm glass fiber at the speed of 6uL/cm, and drying at 55 ℃ for 3-4 hours.
Step two, preparation and treatment of sample pad
1. Preparing a treatment fluid: 10g of bovine serum albumin, 5mL of Tween-20 and 1000mL of 0.2M borax borate buffer (pH 8.0).
2. Sample pad treatment: uniformly coating the treating fluid on a sample pad of 0.8-1.0cm according to 3-4 mL/strip (width: 17-18, length: 30 cm), and drying at 55deg.C for 6-8 h.
Step three, preparation of reaction film
1. Coating liquid preparation: 1g of sucrose, 100mL 0.01M pH7.4PBS buffer.
2. Spraying pad: and sticking a nitrocellulose membrane (NC membrane) on a PVC plate, coating TT4 antigen by using the coating liquid, so that the coating concentration of the TT4 antigen on an NC membrane detection line is 0.5mg/mL, the coating concentration of chicken IgY antibody on a quality control line is 1mg/mL, and the drying time of the coating is selected to be 48 hours.
Step four, assembling test paper strips
And (3) sequentially attaching the treated sample pad, the bonding pad and the absorbent paper on a PVC plate, and cutting the assembled PVC plate into the immunochromatographic test strip with the width of 3.85-4.00mm by a slitter.
Step five, sample inspection and result judgment
Sample inspection: taking 100uL of serum, plasma or whole blood sample to be detected, adding the 100uL into 300uL of the sample diluent, and dripping 100uL onto an immunochromatographic reagent card after the mixture is uniformly mixed for carrying out immunochromatographic 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, a signal must be detected on a quality control line to prove that the detection result is effective.
The following is a further detailed description of several examples
Example 1 composition, package and quantity (25 parts/box) of kit for rapid detection of total thyroxine (TT 4) in human serum, plasma and whole blood samples are shown in Table 2.
Table 2 composition, package and amount of kit
Example 2 blank Performance evaluation experiment of kit
And adding 100uL of negative bovine serum into 300uL of sample diluent, uniformly mixing, adding 100uL into a sample adding hole, detecting by adopting a fluorescence detector after 10min, and repeating for 20 times. The relative T/C average value (M) and Standard Deviation (SD) are compared to obtain a numerical value corresponding to M+2SD, the numerical value is put into a standard curve equation, a corresponding concentration value is obtained, namely a blank limit, and the test result is shown in Table 3. The results show that the blank limit of the kit is not more than 10ng/mL.
TABLE 3 blank test results
Example 3 test for evaluation of Linear Performance of kit
Diluting a high-value sample close to the upper limit of a linear interval into a standard substance solution of 240ng/mL, 120ng/mL, 60ng/mL, 30ng/mL and 10ng/mL according to a certain proportion, wherein the sample with low-value concentration is close to the lower limit of the linear interval, taking 100uL of standard substance, adding 300uL of sample diluent, uniformly mixing, taking 100uL of sample diluent, adding the sample diluent into a sample adding hole, detecting by adopting a fluorescence detector after 10min, repeatedly detecting the sample with each concentration for 3 times, calculating the average value of the sample, performing linear fitting on the average value of the measured concentration and the theoretical concentration or the dilution proportion by using a least square method, and calculating a linear correlation coefficient r according to a formula (1). The result shows that the linear correlation coefficient r is more than 0.9900, the detection limit is 10ng/mL, the detection variation coefficient of each concentration is less than 10%, and the test result is as follows
Table 4 shows the results.
Wherein:
n-determining the number of samples;
xi-determining the concentration of the sample;
yi-3 repeated measurements of the mean of the measured values corresponding to the measured sample concentrations;
r-linear correlation coefficient.
TABLE 4 results of test for different concentration standards
Example 4 accuracy Performance assessment experiment of kit
A certain amount of total thyroxine standard is added into negative calf serum, so that the total thyroxine final concentration in the serum is respectively 60ng/mL and 120ng/mL, and 6 total thyroxine standard samples are repeated. And (3) fully mixing 100uL of sample to be detected with sample diluent in a 300uL kit for 1min, then adding the 100uL of mixed solution into a sample adding hole of a reagent card, horizontally placing the reagent card for 10min, inserting the reagent card into a fluorescence quantitative detector, reading a fluorescence signal T/C value, and calculating corresponding sample concentration according to a standard curve. As shown in Table 5, the ratio of the mean value of the measured values to the theoretical value is between 0.850 and 1.150 as can be seen from the test data in Table 5.
TABLE 5 accuracy test experiment results
Example 5 assay for specific Performance assessment of kit
A sample without any analyte was added with a specific reference of triiodothyronine (TT 3) at a concentration of 500ng/mL and anti-triiodothyronine (rT 3) at a concentration of 50ng/mL, each of which was repeated 6 times. And (3) fully mixing 100uL of sample to be tested with sample diluent in a 300uL kit for 1min, then adding the 100uL of mixed solution into a sample adding hole of a reagent card, horizontally placing the reagent card for 10min, inserting the reagent card into a fluorescence quantitative detector, reading a fluorescent signal T/C value, taking an average value, and taking a test result as shown in a table 6, wherein the test data of the table 6 show that the cross reaction value of a specific reference sample and the reagent is less than 10.0ng/mL.
TABLE 6 specificity test results
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 detection card for detecting the total thyroxine content is characterized by comprising a strip-shaped PVC bottom plate, wherein a sample pad, a bonding pad, a reaction 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 reaction membrane are respectively overlapped with one end of the absorption pad and one end of 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 TT4 antibody and a fluorescent marked goat anti-chicken IgY antibody; the reaction membrane is provided with detection lines and quality control lines which are arranged at intervals, the detection lines are coated with TT4 antigen, and the quality control lines are coated with chicken IgY antibody.
2. The test card of claim 1, wherein the biological source of the fluorescent-labeled TT4 antibody is murine monoclonal IgG.
3. The test card of claim 1, wherein the biological source of the goat anti-chicken IgY antibody is a goat-derived antibody.
4. The test card of claim 1, wherein the biological source of the coated TT4 antigen is a recombinant antigen.
5. The test card of claim 1, wherein the biological source of the coated chicken IgY antibodies is chicken-derived antibodies.
6. The test card of claim 1, wherein the goat anti-chicken IgY antibody and the TT4 antibody are each labeled with fluorescent microspheres.
7. The test card of claim 1, wherein the labeled antibody to the TT4 antigen has a final concentration on the test card of 0.5mg/mL; the final concentration of the TT4 coating antigen on the detection card is 0.5mg/mL.
8. The test card of claim 1, wherein the final concentration of the chicken IgY antibodies for the quality control line on the test card is 0.5mg/mL; the final concentration of the coated chicken IgY antibody used for the quality control line on the detection card is 1mg/mL.
9. A kit for detecting total thyroxine content, comprising a detection card according to any one of claims 1 to 8, a diluent for diluting a sample and an ID card chip; the ID card chip is pre-stored with a detection standard curve; the diluent component is phosphate buffer solution.
10. A method of detecting total thyroxine content in serum, plasma and whole blood using the kit of claim 9, comprising the 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, dripping the diluted whole blood, plasma and serum sample liquid onto detection cards in the three kits, and placing the three detection cards in an immunofluorescence detector for detection to respectively obtain the total thyroxine content in the whole blood, the plasma and the serum.
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| CN117169519A (en) * | 2023-10-26 | 2023-12-05 | 艾康生物技术(杭州)有限公司 | Dissociation agent and kit for detecting TT3 and/or TT4 in sample |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117169519A (en) * | 2023-10-26 | 2023-12-05 | 艾康生物技术(杭州)有限公司 | Dissociation agent and kit for detecting TT3 and/or TT4 in sample |
| CN117169519B (en) * | 2023-10-26 | 2024-01-30 | 艾康生物技术(杭州)有限公司 | Dissociation agent and kit for detecting TT3 and/or TT4 in sample |
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