CN113358871A - Colloidal gold test strip for detecting Tg-anti-Tg antibody compound - Google Patents
Colloidal gold test strip for detecting Tg-anti-Tg antibody compound Download PDFInfo
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention relates to a colloidal gold test strip capable of detecting a binding complex of Tg and an anti-Tg antibody, which is provided with a detection line; the colloidal gold bonding pad contains colloidal gold particles which are bonded with antibody markers; the detection line is anchored with a capture antibody, the combined antibody is an anti-Tg polyclonal antibody, and the capture antibody is a mouse anti-human IgG antibody. According to the invention, by using Tg to immunize an animal which is far away from the genetic relationship with human, unique antiserum is obtained, and the antiserum and a mouse anti-human IgG antibody can be combined to prepare a colloidal gold test strip for detecting a Tg-anti-Tg antibody compound. By using the specific antiserum combination, the problem that the test strip prepared from single antiserum is poor in correlation in the quantitative detection process is solved. The test strip also has the function of detecting whole blood, and by using the colloidal gold test strip, the Tg-anti-Tg antibody compound and the content in the peripheral blood of a detected person can be quickly detected, so that a convenient tool is provided for monitoring the recurrence of a differentiated thyroid cancer rehabilitation person.
Description
Technical Field
The invention relates to the field of thyroid cancer diagnosis, in particular to a colloidal gold test strip for detecting a Tg-anti-Tg antibody compound.
Background
Thyroglobulin (Tg) is a glycoprotein secreted by thyroid follicular epithelial cells, has a molecular weight of about 660kD, and is a major component of thyroid follicular glia. The Tg content in patients with Differentiated Thyroid Cancer (DTC) is elevated, and therefore Tg is a key serological indicator of DTC and can assist in the initial and definitive diagnosis of DTC. After recovery of DTC patients, anti-Tg antibodies (TgAb) were induced by the immune system due to the presence of high concentrations of Tg in the serum. Therefore, when DTC convalescent relapses, Tg is rapidly bound by the antibody and forms a complex due to the presence of TgAb in vivo, although the secretion of Tg increases. In this case, no serum Tg content could be detected using a conventional Tg detection kit. Doctors often make a misjudgment according to the situation, and the Tg content in the body of the examinee does not rise, so that the relapse does not occur.
Therefore, there is a need for a kit for detecting Tg-anti Tg antibody complexes, thereby obtaining more accurate detection information.
Disclosure of Invention
In order to solve the problems, the invention provides a colloidal gold test strip capable of detecting a binding complex of Tg and an anti-Tg antibody, a bottom plate, and a sample pad, a colloidal gold binding pad, a nitrocellulose membrane and a water absorption pad which are sequentially connected with each other and arranged on the bottom plate, wherein a detection line is arranged on the nitrocellulose membrane; the colloidal gold bonding pad contains colloidal gold particles which are bonded with antibody markers; the detection line is anchored with a capture antibody, characterized in that the binding antibody is an anti-Tg polyclonal antibody.
In a specific embodiment, the anti-Tg polyclonal antibody comprises serum obtained from fish immunized with human Tg.
In a specific embodiment, the anti-Tg polyclonal antibody comprises human Tg immune pelteobagrus fulvidraco serum.
In a specific embodiment, the anti-Tg polyclonal antibody further comprises human Tg immune goat serum and human Tg immune duck serum.
In a specific embodiment, the anti-Tg polyclonal antibody human Tg immune pelteobagrus fulvidraco serum, human Tg immune duck serum and human Tg immune sheep serum are in a volume ratio of 10:2: 1.
In a specific embodiment, the capture antibody is a murine anti-human IgG antibody.
In a specific embodiment, a quality control line is further arranged on the nitrocellulose membrane, and the quality control line is farther away from the colloidal gold bonding pad than the detection line.
In a specific embodiment, the quality control line is anchored with a murine anti-pelteobagrus fulvidraco IgM antibody, a murine anti-duck IgG antibody and a murine anti-sheep IgG antibody.
In a specific embodiment, a blood filtering membrane is arranged at the junction of the sample pad and the colloidal gold combined pad.
According to the invention, by using Tg to immunize an animal which is far away from the genetic relationship with human, unique antiserum is obtained, and the antiserum and a mouse anti-human IgG antibody can be combined to prepare a colloidal gold test strip for detecting a Tg-anti-Tg antibody compound. And by using a specific antiserum combination, the problem that the test strip prepared from a single antiserum is poor in correlation in the quantitative detection process is solved. The test strip also has the function of detecting whole blood, and by using the colloidal gold test strip, the Tg-anti-Tg antibody compound and the content in the peripheral blood of a detected person can be quickly detected, so that a convenient tool is provided for monitoring the recurrence of a differentiated thyroid cancer rehabilitation person.
Drawings
FIG. 1 is a statistical graph of the binding of anti-Tg antisera from different species to a Tg-anti-Tg antibody complex;
FIG. 2 is a schematic structural diagram of the test strip of the present invention;
FIG. 3 is a top view of a nitrocellulose membrane of the quantitative determination test strip;
FIG. 4 is a schematic view of a sample pad, a blood filter membrane, and a gold gel pad;
FIG. 5 is a linear relationship curve formed by a quantitative test strip prepared by using the Pelteobagrus fulvidraco antiserum to test 100-250ng/ml Tg-anti-Tg antibody complexes;
FIG. 6 is a linear plot of the formation of 100-ml Tg-anti-Tg antibody complexes using a quantitative dipstick test prepared with grass carp antiserum;
FIG. 7 is a linear plot of the formation of 100-ml Tg-anti-Tg antibody complexes tested using a quantitative dipstick prepared with a combination of antisera.
The device comprises a base plate, a sample pad, a colloidal gold combined pad, a nitrocellulose membrane, a water absorption pad, a detection line, a quality control line and a blood filtering membrane, wherein the base plate is 1, the base plate is 2, the sample pad is 3, the colloidal gold combined pad is 4, the nitrocellulose membrane is 41, the detection line is 42, the quality control line is 5, and the water absorption pad is 6.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
1. Antibody screening and preparation
To investigate the detection antibodies of the Tg-anti-Tg antibody complex, we tried various approaches.
First, an affinity experiment using various anti-Tg monoclonal antibodies, respectively, with Tg-anti-Tg antibody complexes was attempted, and the results showed that all of the used commercially available anti-Tg monoclonal antibodies and anti-Tg monoclonal antibodies developed by us did not exhibit effective antibody titers.
We studied intensively for the reason that antiserum raised against Tg itself contains polyclonal antibodies against Tg, which form complexes with Tg and mask most of the epitopes on Tg, resulting in that the monoclonal antibodies against Tg are substantially unlikely to bind to the complex. Therefore, we need to find new specific antibodies.
To find new specific antibodies against Tg, we immunized animals of various species with human Tg full length protein and evaluated antiserum titers by using Tg-anti-Tg antibody complexes.
The result is shown in figure 1, and after dilution by 2560 times, the antiserum of the pelteobagrus fulvidraco and the grass carp has a higher binding value. Poultry antisera have a relatively low binding value compared to fish, and chicken antisera have a high binding value in poultry. The binding values of mammalian antisera are very low, with sheep antisera being relatively high. In general, fish antisera and avian antisera have a potency that can be used to detect Tg-anti-Tg antibody complexes. In mammals, sheep antiserum shows a certain positive reaction and can also be used for detecting the Tg-anti-Tg antibody compound.
2. Preparation of test paper strip
Based on the above experiments, we prepared colloidal gold test strips using a double antibody sandwich method. Antiserum of fish, poultry and the like is used as a binding antibody with colloidal gold. Since the anti-Tg antibody in the Tg-anti-Tg antibody complex is a human antibody, a murine anti-human IgG antibody was selected as the capture antibody.
The Tg test strip has a structure shown in figures 3 and 4, and comprises a rectangular base plate 1, and a sample pad 2, a colloidal gold bonding pad 3, a nitrocellulose membrane 4 and a water absorption pad 5 which are sequentially overlapped on the base plate 1. In a preferred embodiment, the interface between the sample pad 2 and the gold colloidal conjugate pad 3 is increased to improve the sample diffusion efficiency. Preferably, the boundary between the sample pad 2 and the gold colloid bonding pad 3 is made into a shape with a protruding upper part, the boundary between the gold colloid bonding pad 3 and the sample pad 2 is made into a shape with a protruding lower part, and the two parts are mutually matched to increase the contact surface, and because of the boundary, the sample pad 2 is on the upper part, the gold colloid bonding pad 3 is on the lower part, and the sample is favorably diffused from the sample pad 2 to the gold colloid bonding pad 3 under the action of gravity.
The sample pad 2 is a glass fiber membrane treated with phosphate buffer solution containing 1-5% BSA and a surfactant.
The gold colloidal conjugate pad 3 is a glass fiber membrane coated with gold colloidal particles to which antibodies are conjugated. The colloidal gold binding pad 3 contains colloidal gold particles, and anti-Tg polyclonal antibodies (yellow catfish antiserum, grass carp antiserum, chicken antiserum, duck antiserum or goose antiserum) are bound on the colloidal gold particles. If the Tg-anti-Tg antibody complex is present in the sample dropped on the sample pad 2, the complex can pass through the colloidal gold particles after being diffused into the colloidal gold conjugate pad 3. The composite with the colloidal gold particles continues to diffuse toward the nitrocellulose membrane 4 and is displayed by aggregation on the nitrocellulose membrane 4.
The method for labeling the conjugated antibody with the colloidal gold particles is as follows:
1) preparing colloidal gold: diluting 1% chloroauric acid solution to 0.01% with double distilled water, boiling, adding trisodium citrate solution, boiling until the liquid is bright red, stopping heating, and adding water lost due to boiling to obtain colloidal gold;
2) adding antiserum into the colloidal gold, uniformly mixing, standing, centrifuging to obtain precipitate, and washing twice to obtain the colloidal gold particles marked with AP 8. The colloidal gold particles labeled with antiserum were sprayed on a glass fiber membrane to prepare a colloidal gold conjugate pad 3.
In a preferred embodiment, as shown in fig. 4, a blood filtration membrane 6 is disposed at the interface between the sample pad 2 and the gold gel conjugate pad 3. This arrangement allows the test strip of the present invention to be used to test a whole blood sample, rather than being limited to serum or tissue eluate.
In a preferred embodiment, anti-erythrocyte antibodies (RBC) are marked on the blood filtering membrane, so that erythrocytes can be trapped, and other substances in the blood smoothly pass through the blood filtering membrane and move towards the colloidal gold pad under the driving of electrostatic attraction. The whole blood sample is used for rapid diagnosis and detection, and one difficulty is that the target substance can pass through the sample pad without being influenced, and blood cells can be completely isolated, so that the final detection result is not interfered by the color of the blood cells, and the peripheral blood is directly used for detection without extracting serum from the peripheral blood and then used for detection.
The nitrocellulose membrane 4 is provided with a detection line 411. The detection line 411 is anchored with a coated mouse anti-human IgG antibody, when the complex with the colloidal gold particles diffuses on the nitrocellulose membrane 4 and meets the detection line 411, the complex is combined with the capture antibody anchored in the detection line 411 and gathers on the detection line 411, and the more the complex with the colloidal gold particles gathers on the detection line, the darker the color development.
The nitrocellulose membrane 4 is also provided with a quality control line 42, and the quality control line 42 is far away from the colloidal gold combined pad 3 than the detection line 41. Secondary antibodies (e.g., murine anti-pelteobagrus fulvidraco IgM, murine anti-grass carp IgM, murine anti-chicken IgG, murine anti-duck IgG, or murine anti-goose IgG) that bind to the antibodies are anchored on the quality control line 412. When the gold particles diffuse to the position of the control line 42, they gather on the control line 42. Therefore, in the diffusion process, the gold colloidal particles first encounter the detection line 41, the gold colloidal particles bound with the Tg-anti-Tg antibody complex are gathered on the detection line 41, the gold colloidal particles not bound with the complex continue to diffuse forward, and when they encounter the quality control line 42, they are gathered on the quality control line 42. Since the colloidal gold particle abundance is much greater than the complex, the control line 42 will develop color regardless of the presence of the Tg-anti-Tg antibody complex in the sample.
The preparation method of the nitrocellulose membrane 4 is as follows:
1) sealing the nitrocellulose membrane in a sealing solution containing 1% BSA and 0.1% Tween-20 in 0.01M phosphate buffer (pH 7.0) for 60 min;
2) and respectively adding the capture antibody and the quality control antibody into the spotting membrane diluent to obtain a capture antibody spotting fluid and a quality control antibody spotting fluid, and respectively spraying the capture antibody spotting fluid and the quality control antibody spotting fluid on a detection line and a quality control line which are separated by 5mm according to the amount of 2 mu L/cm. Wherein the dot-membrane dilution solution is 0.01M phosphate buffer (pH 7.4) containing 0.15M sodium chloride, 10mM ethylenediaminetetraacetic acid, 1g/L sodium azide and 25g/L methanol. The concentration of the capture antibody spotting fluid is 3.5 mug/mL, and the concentration of the quality control antibody spotting fluid is 1.5 mug/L.
In qualitative detection, 50 μ L of peripheral blood, serum or tissue eluate sample is added into 50 μ L of sample pretreatment solution, stirred and mixed uniformly, dropped on the sample loading region of the sample pad of the detection test strip, and chromatographed at 20-30 deg.C for 5 min. Whether the sample contains the complex can be determined by observing the detection line and the quality control line. That is, when both the detection line and the quality control line develop color, the sample contains the complex; when the detection line is not colored and the quality control line is colored, the Tg is not contained in the sample.
In one embodiment, the sample pretreatment solution is 0.1M phosphate buffer containing 0.3-0.5% ammonium chloride, 0.1% potassium bicarbonate. The sample pretreatment solution can be used for cracking red blood cells in a whole blood sample, and is beneficial to the detection of the whole blood sample.
In quantitative detection, a color value reading system is required to be prepared. And (3) placing the test strip after the chromatographic reaction under a scanning device of a color value-concentration system for scanning, processing the scanned image by a color value reading system to obtain a detection value, and obtaining the concentration of the compound according to the corresponding relation between the detection value and the standard curve.
In the actual use process, because the existence period of the Tg-anti-Tg antibody compound in the peripheral blood of the detected object is short, in order to improve the precision, the peripheral blood can be taken and detected at intervals of 20 minutes in the sampling process, so that the window period of the existence of the compound in the peripheral blood is prevented from being missed.
3. Qualitative test paper result
We performed qualitative assays using the test strips described above after treating various concentrations of Tg (1000, 500, 250, 100, 50, 20ng/ml) with human antiserum (excess) containing Tg induction.
The assay was performed using the quantitative test strips described above. The results are shown in table 1, and the detection limit of the test paper prepared from the pelteobagrus fulvidraco antiserum is lower than 50ng/ml and higher than 30 ng/ml. The detection limit of test paper prepared from grass carp antiserum is lower than 100ng/ml and higher than 50 ng/ml. The detection limit of test paper prepared by chicken antiserum is lower than 500ng/ml and higher than 250 ng/ml. The detection limit of test paper prepared from duck and goose antiserum is lower than 250ng/ml and higher than 100 ng/ml. As the normal value of the Tg of the serum in a human body is 3-40ng/ml, the test strip prepared from the Pelteobagrus fulvidraco antiserum and the test strip prepared from the grass carp antiserum can be used for qualitative detection.
TABLE 1 results of detection of complexes containing Tg-anti-Tg antibodies at different concentrations using test strips with different binding antibodies
4. Optimization of quantitative test strip
In the quantitative test strip experiment process, a new problem is found, and the test strip prepared by using the antiserum of the pelteobagrus fulvidraco or the grass carp cannot obtain a good linear relation in quantitative detection. Running quantitative test strips with sample concentrations of 100, 125, 150, 175, 200, 225, 250 ng/ml. The test strip was then used in a color-concentration system for standard curve establishment. The results are shown in fig. 5 and 6, and the correlation coefficient R of the test strip prepared by using the pseudobagrus fulvidraco antiserum or the grass carp antiserum is shown in the figure2Are all less than 0.95, it failed to be measured as a concentration.
Without being bound by the present theory, we speculate that the reason may be that human serum contains antibodies covering most epitopes of Tg proteins, but each Tg protein has a certain randomness in binding to its antibodies after incubation with serum, resulting in the exposed epitope of each Tg-anti-Tg antibody complex being not identical. Some complexes may expose epitopes that the pelteobagrus fulvidraco antiserum or the grass carp antiserum can bind to, and some complexes may not expose, so that the linear correlation is limited.
To this end, we tested various antisera combinations for the preparation of test strips. It was found that the use of antiserum combinations of the species with closer relativity (e.g. pelteobagrus fulvidraco antiserum + grass carp antiserum) could not compensate for the drawbacks of the correlation. After testing various combinations, we find that the test strip prepared by combining the pelteobagrus fulvidraco antiserum, the duck antiserum and the sheep antiserum in a volume ratio of 10:2:1 is used as a binding antibody. As shown in FIG. 7, the combination can obtain a linear correlation coefficient higher than 0.99, greatly improves the correlation between the detection value and the actual value, and can be used as a quantitative detection test strip.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A colloidal gold test strip capable of detecting a binding complex of a Tg-anti-Tg antibody comprises a bottom plate (1), and a sample pad (2), a colloidal gold binding pad (3), a nitrocellulose membrane (4) and a water absorption pad (5) which are sequentially connected with each other and arranged on the bottom plate (1), wherein a detection line (41) is arranged on the nitrocellulose membrane (4); the colloidal gold bonding pad (3) contains colloidal gold particles which are bonded with antibody labels; the detection line (41) has anchored therein a capture antibody, characterized in that the binding antibody is an anti-Tg polyclonal antibody.
2. The colloidal gold test strip of claim 1, wherein the anti-Tg polyclonal antibody comprises sera obtained by immunizing multiple species with human Tg, respectively.
3. The colloidal gold test strip of claim 2, wherein the anti-Tg polyclonal antibody contains human Tg immune Pelteobagrus fulvidraco serum.
4. The colloidal gold test strip of claim 3, wherein the anti-Tg polyclonal antibody further comprises human Tg immune goat serum and human Tg immune duck serum.
5. The colloidal gold test strip of claim 4, wherein the anti-Tg polyclonal antibody human Tg immune Pelteobagrus fulvidraco serum, human Tg immune duck serum and human Tg immune sheep serum are in a volume ratio of 10:2: 1.
6. The colloidal gold test strip of claim 1, wherein the capture antibody is a murine anti-human IgG antibody.
7. The colloidal gold test strip of any one of claims 1-6, wherein the nitrocellulose membrane (4) is further provided with a quality control line (42), and the quality control line (42) is further away from the colloidal gold conjugate pad (3) than the detection line (41).
8. The test strip of claim 7, wherein the quality control line (42) is anchored with a murine anti-Pelteobagrus fulvidraco IgM antibody, a murine anti-duck IgG antibody and a murine anti-sheep IgG antibody.
9. The test strip according to any one of claims 1 to 6, wherein a blood filter (6) is arranged at the interface of the sample pad (2) and the colloidal gold conjugate pad (3).
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