CN114805545A - Capture antigen for detecting anti-asialoglycoprotein receptor antibody and detection method - Google Patents
Capture antigen for detecting anti-asialoglycoprotein receptor antibody and detection method Download PDFInfo
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- CN114805545A CN114805545A CN202111615338.4A CN202111615338A CN114805545A CN 114805545 A CN114805545 A CN 114805545A CN 202111615338 A CN202111615338 A CN 202111615338A CN 114805545 A CN114805545 A CN 114805545A
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- asialoglycoprotein receptor
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- asgpr
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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Abstract
The invention discloses a capture antigen for detecting an anti-asialoglycoprotein receptor antibody and a detection method, and relates to the technical field of biological detection.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a capture antigen for detecting an anti-asialoglycoprotein receptor antibody and a detection method.
Background
Asialoglycoprotein receptor (ASGPR) is a liver-specific transmembrane glycoprotein with liver-specificity and species-specificity that closely matches the histopathological changes of autoimmune hepatitis (AIH) (massive lymphocyte infiltration, putrescence debris around the regions of the manifold) at specific sites of distribution in the liver, and thus ASGPR is presumed to be the major target antigen for attracting sensitized liver-infiltrating lymphocytes. Anti-asialoglycoprotein receptor antibody (anti-ASGPR antibody) is an autoantibody to AIH, is closely related to the activity of AIH disease, and can be used as an index for judging disease activity, monitoring therapeutic effect, and judging prognosis. The most commonly used method for anti-asialoglycoprotein receptor antibodies is currently enzyme-linked immunosorbent assay. According to the enzyme-linked immunosorbent assay (ELISA) principle, an antigen is coated on a solid-phase carrier ELISA plate, a sample is added into the ELISA plate, the antibody can be specifically combined with the antigen, an enzyme-labeled anti-human IgA or IgG antibody is added to form a compound, a chromogenic substrate is added after thorough washing, and an ELISA reader reads a chromogenic OD value to judge the concentration of the antibody, so that an unknown antibody can be identified. However, the antigen for detection at present is generally derived from protein expressed by ASGPR pronucleus, and does not contain protein glycosylation sites and spatial structures, and the methods have the technical problem of low detection rate.
Hitherto, few asialoglycoprotein receptor antigens are known and only a few parts are disclosed, so that a certain detection obstacle exists in the detection of asialoglycoprotein receptor antibodies, and how to optimize a detection method to quickly and effectively monitor the asialoglycoprotein receptor antibodies is one of the technical problems to be solved urgently.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a capture antigen for detecting an anti-asialoglycoprotein receptor antibody and a detection method.
The invention is realized by the following steps:
in a first aspect, embodiments of the present invention provide a capture antigen for use in the detection of an anti-asialoglycoprotein receptor antibody, comprising: ASGPR antigens and liver extracts.
In a second aspect, embodiments of the present invention provide a kit for the detection of an anti-asialoglycoprotein receptor antibody, comprising a capture antigen for the detection of an anti-asialoglycoprotein receptor antibody as described in the preceding embodiments.
In a third aspect, the present invention provides a method for detecting an anti-asialoglycoprotein receptor antibody, which includes mixing the capture antigen for detecting the anti-asialoglycoprotein receptor antibody as described in the previous embodiment with a sample to be detected, and detecting; the detection method is not directed towards the treatment or diagnosis of a disease.
The invention has the following beneficial effects:
the liver extract is added on the basis of ASGPR antigen to be used as detection antigen, the liver extract overcomes the defects that a single antigen lacks glycosylation epitope and space epitope is insufficient, the detection of the anti-asialoglycoprotein receptor antibody can be realized more quickly and effectively, the detection specificity is high, the stability is good, and a new way is provided for the detection of the anti-asialoglycoprotein receptor antibody.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The embodiment of the invention provides a capture antigen for detecting an anti-asialoglycoprotein receptor antibody, which comprises the following components: ASGPR antigens and liver extracts.
The kit is applied to the detection of the anti-asialoglycoprotein receptor antibody, during detection, the ASGPR antigen and the liver extract are mixed to be used as a capture antigen for capturing a target antibody in a sample, the liver extract overcomes the defects that a single ASGPR antigen lacks glycosylation epitopes and insufficient space epitopes, the detection of the anti-asialoglycoprotein receptor antibody can be realized more quickly and effectively, and the detection specificity is high and the stability is good.
In alternative embodiments, the ASGPR antigen may be selected from among the ASGPR antigens already disclosed in the art. Preferably, the ASGPR antigen comprises: at least one of H1 protein and H2 protein.
Specifically, both H1 and H2 belong to glycoproteins, and the epitopes thereof include, in addition to linear epitopes composed of primary amino acid sequences, glycosylation sites, phosphorylation sites, which are epitopes present in the natural tertiary structure. The epitopes in the tertiary structure of proteins such as glycosyl are difficult to completely obtain by gene recombination, so when the recombinant protein is simply used as a self antigen, the epitopes aiming at the tertiary structure cannot be detected, and although the primary sequence of the ASGPR antigen is greatly different from that of a primate, the non-primate has the same or similar functions, and the glycosyl, glycosylation and phosphorylation modes are similar. This is one of them.
Secondly, because 1 or even a plurality of undegraded autoantigens exist, when H1 and H2 are simply used as the autoantigens, the detection rate is influenced by insufficient epitopes, and the defect is effectively compensated by adding the non-primate tissue extract.
Preferably, the amino acid sequence of the H1 protein comprises at least one of SEQ ID Nos. 1-4. Preferably, the amino acid sequence of the H1 protein comprises at least one of SEQ ID No. 1-3. SEQ ID No. 1-3 are antigen epitope sequences obtained after optimized screening, and compared with SEQ ID No.4, the method has more effective and stable detection effect.
Preferably, the amino acid sequence of the H2 protein is at least one of SEQ ID No. 5-9;
the amino acid sequence of the H2 protein is at least one of SEQ ID No. 5-8. SEQ ID No. 5-8 are antigen epitope sequences obtained after optimized screening, and compared with SEQ ID No.9, the method has more effective and stable detection effect.
TABLE 1 sequence information
The liver extract refers to a product obtained by cracking liver tissue or liver cells. Preferably, the liver extract is selected from at least one of a liver tissue extract and a liver tumor cell extract.
Preferably, the liver extract is a liver tissue extract, and the liver tissue extract is used as a liver extract for detecting the anti-asialoglycoprotein receptor antibody, compared with a liver tumor cell extract, and has a better technical effect.
Preferably, the liver tissue extract is an extract obtained after extraction with a glycoprotein extraction reagent.
Preferably, the step of extracting the liver tissue extract comprises: homogenizing the liver tissue until the tissue is cracked, centrifuging and taking supernatant; filtering the supernatant, and extracting with a glycoprotein extraction reagent to obtain the liver tissue extract;
preferably, the protein extraction reagent comprises a protease inhibitor.
Preferably, the liver tissue extract is selected from non-primate liver tissue extracts.
Preferably, the antigen and the liver tissue extract are in a packaged state or a mixed state.
The embodiments of the present invention also provide a kit for the detection of an anti-asialoglycoprotein receptor antibody, comprising a capture antigen for the detection of an anti-asialoglycoprotein receptor antibody as described in any of the preceding embodiments.
In alternative embodiments, the kit further comprises a detection reagent for performing the detection, the type of the reagent being different based on different detection methods, which may include any one of membrane strip method, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, luminescent immunoassay, immunoturbidimetry, and immunochromatography.
In addition, the embodiment of the invention also provides a detection method of the anti-asialoglycoprotein receptor antibody, which comprises the step of mixing the capture antigen for detecting the anti-asialoglycoprotein receptor antibody and a sample to be detected for detection.
The detection method is not directed to the treatment or diagnosis of a disease, for example, when the sample to be tested is an environmental sample containing a substance to be tested, the detection method is not directed to the treatment or diagnosis of a disease. For another example, when further acquisition or development of new anti-asialoglycoprotein receptor antibodies is desired, the detection method is not aimed at directly treating or diagnosing the disease.
Preferably, when the ASGPR antigen and liver extract in the kit are in a separate form, the step of detecting comprises: mixing ASGPR antigen and liver extract to be used as capture antigen, and capturing target antibody in a sample to be detected;
when the ASGPR antigen and liver extract in the kit are in a mixed state, the step of detecting comprises: the target antibody in the sample to be tested is captured using the mixed ASGPR antigen and liver extract as capture antigens.
It should be noted that the capture antigen is understood as an antigen for capturing an antibody.
Preferably, the mixing mass ratio of the ASGPR antigen to the liver extract can be (0.5-1.5): (0.5-1.5), including but not limited to 0.5: 1. 0.7: 1. 0.9: 1. 1: 1. 1: 0.9, 1: 0.7 and 1: 0.5 in any ratio.
Preferably, the sample to be tested is selected from any one of a biological sample and an environmental sample containing the biological sample, and the biological sample is selected from any one of a serum sample, a plasma sample and a whole blood sample. The environmental sample refers to a sample derived from the environment, but the sample itself contains a biological sample.
Preferably, the method of detection is selected from: the method of detection is selected from: membrane strip method, enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, luminescence immunoassay, immunoturbidimetry, and immunochromatography. In the case where the antigen for detection is defined, the specific steps of each detection method can be performed by referring to the existing steps, and will not be described again.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
A capture antigen of an anti-asialoglycoprotein receptor antibody comprising the following steps.
(1) Obtaining a non-primate liver tissue extract
1. Protein extraction step of animal liver tissue
Weighing the tissue, cutting into small pieces and placing into a tube; preparing a protein extraction reagent containing an inhibitor (50 mul of protease inhibitor mixed solution, 50 mul of PMSF and 50 mul of phosphatase mixed solution are added into 10ml of the extraction reagent); adding pre-cooled protein extraction reagent containing inhibitor (1 ml extraction reagent is added into 250mg tissue); homogenizing with homogenizer at low speed for 30 s each time, and ice bath for 1 min each time until the tissue is completely cracked; the lysate was centrifuged at 14,000 Xg for 15 minutes in a precooled centrifuge. Immediately transferring the supernatant into a new centrifuge tube for storage for later use;
2. purifying to obtain extract
Transferring the collected supernatant into an ultrafiltration tube with a 0.22 mu m filter membrane; centrifuging at 10000 Xg for 20 min to remove lipid and residual tissue debris; and collecting the centrifuged protein solution, and taking a small amount of the protein solution to measure the concentration of the protein solution.
3. Glycoprotein acquisition
Preparing a concanavalin A affinity column; using 10 column volumes of chromatography buffer (20 mmol/L, pH 8.0 Tris-HCl, 0.15 mol/L NaCl, 1 mmol/L MnCl) 2 ，1 mmol/L CaCl 2 30 mmol/L caprylic glucoside) in a well-balanced affinity column; protein solution of step 2 buffer 1:1, diluting; loading the adjusted protein solution to an affinity column; washing the column with chromatography buffer until A280 value returns to baseline; eluting with 5 column volumes of chromatography buffer containing 10 mmol/L methyl-alpha-D-mannose; continuing to elute with 5 column volumes of chromatography buffer containing 20, 50, 100, 250 and 500 mmol/L methyl-alpha-D-mannose, respectively; the protein component of interest was verified with ASGPR specific antibodies. After obtaining the target protein, removing carbohydrate by dialysis.
(2) Obtaining ASGPR antigens
Synthesizing sequences of SEQ ID No. 1-4 according to the H1 protein shown in Table 1, wherein SEQ ID No.4 is a full-length sequence of the H1 protein, and the polypeptides of SEQ ID No. 1-3 are mixed by equal mass to obtain the H1 mixed polypeptide.
Synthesizing sequences of SEQ ID No. 5-9 according to the H2 protein shown in Table 1, wherein SEQ ID No.9 is a full-length sequence of the H2 protein, and the polypeptides of SEQ ID No. 5-8 are mixed by equal mass to obtain the H2 mixed polypeptide.
3. Capturing antigens
The liver tissue extract obtained in step 1 was mixed with ASGPR antigen obtained in step 2 to obtain capture antigen, and the mixed volume was referred to table 2.
Examples 2 to 6
A capture antigen for an anti-asialoglycoprotein receptor antibody, substantially the same as in example 1, except that the capture antigen is different, the capture antigens are in turn as shown in Table 2.
TABLE 2 Capture antigens
Example 7
And (4) enzyme-linked immunosorbent assay comparison.
A sample to be detected: 50 serum samples from autoimmune liver disease and healthy controls.
The capture antigens provided in examples 1-6 are used for detecting the asialoglycoprotein receptor antibody of the sample to be detected by an enzyme-linked immunosorbent assay. Respectively diluting the capture antigens coated ELISA plates obtained in the embodiments 1-6 to 10 mu g/mL by adopting CBS buffer solution, coating the plates by adopting 100 mu L/hole, standing the plates overnight at 0-4 ℃, washing the plates by using washing liquid PBST for three times, and drying the plates by beating each time; blocking with 200. mu.L/well blocking solution (1% BSA, PBS), standing at room temperature for 3 hours, washing the plate three times, and drying each time; add 100. mu.L/well 1: 100 diluted serum samples were left at room temperature for 1 hour, washed three times and patted dry each time; adding an enzyme-labeled anti-human IgA or IgG antibody with the working concentration of 100 mu L/hole, standing for 1 hour at room temperature, washing the plate for three times, and drying by beating each time; adding 100 μ L/hole substrate color developing solution, adding acid to terminate reaction, measuring luminescence value, and determining that the luminescence value is more than 2 times of negative hole as positive. The results are shown in Table 3.
TABLE 3 test results
Example 8
And (4) comparing immunochromatography methods.
The capture antigens provided in examples 1 to 6 were used for detecting asialoglycoprotein receptor antibodies in a sample to be tested (same as example 7) by an immunochromatography method, and the tissue immunofluorescence method was the same as example 7.
The immunochromatography method comprises the following steps:
1) preparing gold-labeled antigen protein: adjusting the pH value of the prepared colloidal gold to 7.5-9.0 by using 0.1M potassium carbonate, slowly adding 4-25 mu g of capture antigen into each ml of colloidal gold solution, stirring for 10-30 min, then adding BSA (bovine serum albumin) to the final concentration of 0.5-1%, stirring for 10-30 min, centrifuging, discarding supernatant, washing the precipitate for 3 times by using washing liquid, re-suspending the precipitate by using one tenth of the initial volume of preservation liquid at the last time, and standing at 4 ℃ for later use;
2) detecting a T line: the obtained capture antigen protein was mixed with 10mM PB buffer solution having a pH of 7.2 to prepare a mixed solution having a concentration of 0.5mg/ml, which was sprayed on a nitrocellulose membrane in an amount of 10. mu.L/cm 2 . Quality control band C line: mixing goat anti-mouse IgG with 10mM PB buffer solution with pH of 7.2 to obtain a mixed solution with a concentration of 0.1mg/mL, spraying on nitrocellulose membrane with a coating amount of 10 μ L/cm 2 。
3) Treatment of the bonding pad: the bonding pad is made of glass fiber paper or polyester film, and is soaked in prepared bonding pad treating solution in an amount of 100 μ L/cm 2 Then drying at 37 deg.C for 1h, and mixing the antibody or protein at 80 μ L/cm 2 The dosage of the composition is sprayed on a pretreated polyester film, and the polyester film is dried for 1 hour at 37 ℃ to obtain a bonding pad, and the bonding pad is placed in an environment of 2-8 ℃ for later use; the composition of the combined pad treatment solution is as follows: pH 7.2 in 10mM PB buffer containing 1% BSA, 10% sucrose, 0.1% Tween-20.
4) Preparation of sample pad
The sample pad is made of glass fiber paper or polyester film, and is soaked in prepared sample pad treating solution in an amount of 100 μ L/cm 2 And then dried at 37 ℃ for 1h, and the composition of the sample pad buffer solution is as follows: a10 mM PB buffer solution at pH 7.2 containing 1% BSA, 10% sucrose, 0.1% Tween-20.
The results are shown in Table 4.
TABLE 4 test results
Example 9
Magnetic particle chemiluminescence method comparison.
The capture antigens provided in examples 1 to 6 were subjected to anti-asialoglycoprotein receptor antibody detection on a sample to be detected (same as example 7) by a magnetic particle chemiluminescence method, and the tissue immunofluorescence method was the same as example 7.
The magnetic particle chemiluminescence method comprises the following steps:
1) biotinylation of mixed antigens
1mg of the captured antigen was put into a 5 mL centrifuge tube, 50. mu.L of 10mg/mL activated biotin (Sulfo-NHS-LC-Bintin) was added thereto, 0.02M PBS was added thereto to make the volume of 1mL, the mixture was reacted at room temperature for 1 hour, and the reaction mixture was taken out and dialyzed overnight against 0.02M PBS. The biotinylated antigen was removed and preserved by adding 0.03% Proclin300 at 4 ℃.
2) Acridinium ester labeled anti-human IgA or IgG
2mg of anti-human IgA or IgG was taken as antibody: and (3) adding the activated acridinium ester according to the molar ratio of 1: 10-50, and reacting for 30min at room temperature. Dialyzing the reaction solution with 0.05M CB solution, taking out, adding glycerol with the same volume, and storing at-20 ℃ for later use.
3) Detection step
The method realizes antibody detection by utilizing acridinium ester chemiluminescence immunoassay technology-combined biotin-avidin magnetic particle separation technology. The detection principle is as follows: and mixing the sample (diluted by 20 times) with streptavidin magnetic particles and biotinylated antigen to obtain a streptavidin magnetic bead-biotinylated antigen-sample compound, washing, adding acridinium ester labeled anti-human IgA or IgG for reaction to form the streptavidin magnetic bead-biotinylated antigen-sample-acridinium ester labeled anti-human IgA or IgG compound. Adding luminous excitation liquid, measuring luminous intensity, comparing with the value of standard or reference, and judging the content of the anti-antibody in the sample to be detected.
The results are shown in Table 5.
TABLE 5 test results
Example 10
Latex enhanced immunoturbidimetric method comparison.
The capture antigens provided in examples 1-6 were used for anti-asialoglycoprotein receptor antibody detection of a sample to be tested (same as example 7) by using a latex-enhanced immunoturbidimetric method, which is the same as example 7.
The latex enhanced immunoturbidimetric method comprises the following steps:
1) marked microspheres
Diluting the polystyrene microspheres to 2% by MES buffer solution, dissolving EDC & HCl to 10mg/ml concentration by pure water, quickly adding into the 2% latex solution (the volume ratio of 2% polystyrene microspheres to 10mg/ml EDC & HCl solution is 1000: 12), stirring and mixing uniformly, and stirring and reacting for 15 minutes at 30 ℃ after 5 minutes of ultrasonic treatment by an ultrasonic pulverizer.
Diluting the capture antigen to the concentration of 1mg/ml by using a working solution MES buffer solution, adding the latex solution prepared in the same volume, uniformly mixing at 30 ℃, and stirring for reacting for 3 hours.
2) Sealing of
The marked polystyrene microspheres are put into a centrifuge tube, the volume of the polystyrene microspheres does not exceed 3/5 of the volume of the centrifuge tube, the polystyrene microspheres are centrifuged for 45 minutes at the rotating speed of 19500rpm according to the standard operation procedure of a centrifuge, supernatant is completely absorbed, the polystyrene microspheres are added into an equal volume of 1% BSA phosphate buffer solution ultrasonic pulverizer to be completely monodisperse, and then the polystyrene microspheres are mixed uniformly at room temperature (30 ℃) and stirred for reaction for 45 minutes.
3) Cleaning of
And (3) putting the sealed polystyrene microspheres into a centrifuge tube, wherein the volume of the polystyrene microspheres does not exceed 3/5 of the volume of the centrifuge tube, centrifuging for 45 minutes at the rotating speed of 19500rpm according to the standard operation procedure of the centrifuge, completely absorbing the supernatant, and adding an isovolumetric 0.4% glycine solution into an ultrasonic pulverizer to perform ultrasonic treatment until complete monodispersion is achieved. The washing step was repeated 2 times.
4) Blending
The labeled antibody microspheres were adjusted to 0.7% concentration with MOPS buffer.
) Detection of
MOPS buffer solution is used as R1 reagent, prepared microspheres are used as R2 reagent, and the reagent is loaded on a biochemical detector for sample detection, wherein the sample is detected by using a Meyer BS-240 full-automatic biochemical detector. The results are shown in Table 6.
TABLE 6 test results
The results of the above examples 7-10 prove that the detection rate of the combination of the non-primate liver tissue extract protein with H1 and H2 is significantly higher than that of the single combination of the tissue extract protein and the human protein integrated with the expression for the autoimmune liver disease.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Suzhou & Rui Biotechnology Ltd
<120> a capture antigen for detecting anti-asialoglycoprotein receptor antibody and detection method
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 31
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Tyr Gln Asp Leu Gln His Leu Asp Asn Glu Glu Ser Asp His His Gln
1 5 10 15
Leu Arg Lys Gly Pro Pro Pro Pro Gln Pro Leu Leu Gln Arg Leu
20 25 30
<210> 2
<211> 60
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gln Leu Gln Glu Glu Leu Arg Gly Leu Arg Glu Thr Phe Ser Asn Phe
1 5 10 15
Thr Ala Ser Thr Glu Ala Gln Val Lys Gly Leu Ser Thr Gln Gly Gly
20 25 30
Asn Val Gly Arg Lys Met Lys Ser Leu Glu Ser Gln Leu Glu Lys Gln
35 40 45
Gln Lys Asp Leu Ser Glu Asp His Ser Ser Leu Leu
50 55 60
<210> 3
<211> 34
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Pro Trp Lys Trp Val Asp Gly Thr Asp Tyr Glu Thr Gly Phe Lys Asn
1 5 10 15
Trp Arg Pro Glu Gln Pro Asp Asp Trp Tyr Gly His Gly Leu Gly Gly
20 25 30
Gly Glu
<210> 4
<211> 291
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Thr Lys Glu Tyr Gln Asp Leu Gln His Leu Asp Asn Glu Glu Ser
1 5 10 15
Asp His His Gln Leu Arg Lys Gly Pro Pro Pro Pro Gln Pro Leu Leu
20 25 30
Gln Arg Leu Cys Ser Gly Pro Arg Leu Leu Leu Leu Ser Leu Gly Leu
35 40 45
Ser Leu Leu Leu Leu Val Val Val Cys Val Ile Gly Ser Gln Asn Ser
50 55 60
Gln Leu Gln Glu Glu Leu Arg Gly Leu Arg Glu Thr Phe Ser Asn Phe
65 70 75 80
Thr Ala Ser Thr Glu Ala Gln Val Lys Gly Leu Ser Thr Gln Gly Gly
85 90 95
Asn Val Gly Arg Lys Met Lys Ser Leu Glu Ser Gln Leu Glu Lys Gln
100 105 110
Gln Lys Asp Leu Ser Glu Asp His Ser Ser Leu Leu Leu His Val Lys
115 120 125
Gln Phe Val Ser Asp Leu Arg Ser Leu Ser Cys Gln Met Ala Ala Leu
130 135 140
Gln Gly Asn Gly Ser Glu Arg Thr Cys Cys Pro Val Asn Trp Val Glu
145 150 155 160
His Glu Arg Ser Cys Tyr Trp Phe Ser Arg Ser Gly Lys Ala Trp Ala
165 170 175
Asp Ala Asp Asn Tyr Cys Arg Leu Glu Asp Ala His Leu Val Val Val
180 185 190
Thr Ser Trp Glu Glu Gln Lys Phe Val Gln His His Ile Gly Pro Val
195 200 205
Asn Thr Trp Met Gly Leu His Asp Gln Asn Gly Pro Trp Lys Trp Val
210 215 220
Asp Gly Thr Asp Tyr Glu Thr Gly Phe Lys Asn Trp Arg Pro Glu Gln
225 230 235 240
Pro Asp Asp Trp Tyr Gly His Gly Leu Gly Gly Gly Glu Asp Cys Ala
245 250 255
His Phe Thr Asp Asp Gly Arg Trp Asn Asp Asp Val Cys Gln Arg Pro
260 265 270
Tyr Arg Trp Val Cys Glu Thr Glu Leu Asp Lys Ala Ser Gln Glu Pro
275 280 285
Pro Leu Leu
290
<210> 5
<211> 50
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Phe Gln Asp Ile Gln Gln Leu Ser Ser Glu Glu Asn Asp His Pro Phe
1 5 10 15
His Gln Gly Glu Gly Pro Gly Thr Arg Arg Leu Asn Pro Arg Arg Gly
20 25 30
Asn Pro Phe Leu Lys Gly Pro Pro Pro Ala Gln Pro Leu Ala Gln Arg
35 40 45
Leu Cys
50
<210> 6
<211> 62
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
His Arg Gly Ala Gln Leu Gln Ala Glu Leu Arg Ser Leu Lys Glu Ala
1 5 10 15
Phe Ser Asn Phe Ser Ser Ser Thr Leu Thr Glu Val Gln Ala Ile Ser
20 25 30
Thr His Gly Gly Ser Val Gly Asp Lys Ile Thr Ser Leu Gly Ala Lys
35 40 45
Leu Glu Lys Gln Gln Gln Asp Leu Lys Ala Asp His Asp Ala
50 55 60
<210> 7
<211> 26
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Pro Val Asp Leu Arg Phe Val Ala Cys Gln Met Glu Leu Leu His Ser
1 5 10 15
Asn Gly Ser Gln Arg Thr Cys Cys Pro Val
20 25
<210> 8
<211> 35
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Gly Ser Trp Lys Trp Val Asp Gly Thr Asp Tyr Arg His Asn Tyr Lys
1 5 10 15
Asn Trp Ala Val Thr Gln Pro Asp Asn Trp His Gly His Glu Leu Gly
20 25 30
Gly Ser Glu
35
<210> 9
<211> 311
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Met Ala Lys Asp Phe Gln Asp Ile Gln Gln Leu Ser Ser Glu Glu Asn
1 5 10 15
Asp His Pro Phe His Gln Gly Glu Gly Pro Gly Thr Arg Arg Leu Asn
20 25 30
Pro Arg Arg Gly Asn Pro Phe Leu Lys Gly Pro Pro Pro Ala Gln Pro
35 40 45
Leu Ala Gln Arg Leu Cys Ser Met Val Cys Phe Ser Leu Leu Ala Leu
50 55 60
Ser Phe Asn Ile Leu Leu Leu Val Val Ile Cys Val Thr Gly Ser Gln
65 70 75 80
Ser Glu Gly His Arg Gly Ala Gln Leu Gln Ala Glu Leu Arg Ser Leu
85 90 95
Lys Glu Ala Phe Ser Asn Phe Ser Ser Ser Thr Leu Thr Glu Val Gln
100 105 110
Ala Ile Ser Thr His Gly Gly Ser Val Gly Asp Lys Ile Thr Ser Leu
115 120 125
Gly Ala Lys Leu Glu Lys Gln Gln Gln Asp Leu Lys Ala Asp His Asp
130 135 140
Ala Leu Leu Phe His Leu Lys His Phe Pro Val Asp Leu Arg Phe Val
145 150 155 160
Ala Cys Gln Met Glu Leu Leu His Ser Asn Gly Ser Gln Arg Thr Cys
165 170 175
Cys Pro Val Asn Trp Val Glu His Gln Gly Ser Cys Tyr Trp Phe Ser
180 185 190
His Ser Gly Lys Ala Trp Ala Glu Ala Glu Lys Tyr Cys Gln Leu Glu
195 200 205
Asn Ala His Leu Val Val Ile Asn Ser Trp Glu Glu Gln Lys Phe Ile
210 215 220
Val Gln His Thr Asn Pro Phe Asn Thr Trp Ile Gly Leu Thr Asp Ser
225 230 235 240
Asp Gly Ser Trp Lys Trp Val Asp Gly Thr Asp Tyr Arg His Asn Tyr
245 250 255
Lys Asn Trp Ala Val Thr Gln Pro Asp Asn Trp His Gly His Glu Leu
260 265 270
Gly Gly Ser Glu Asp Cys Val Glu Val Gln Pro Asp Gly Arg Trp Asn
275 280 285
Asp Asp Phe Cys Leu Gln Val Tyr Arg Trp Val Cys Glu Lys Arg Arg
290 295 300
Asn Ala Thr Gly Glu Val Ala
305 310
Claims (10)
1. A capture antigen for use in the detection of an antibody against the asialoglycoprotein receptor, comprising: ASGPR antigens and liver extracts.
2. The capture antigen for anti-asialoglycoprotein receptor antibody detection according to claim 1, wherein said ASGPR antigen comprises: at least one of ASGPR H1 subunit and H2 subunit proteins.
3. The capture antigen for detecting the anti-asialoglycoprotein receptor antibody according to claim 2, wherein the amino acid sequence of the ASGPR H1 subunit protein comprises at least one of seq id nos. 1 to 4;
preferably, the amino acid sequence of the ASGPR H1 subunit protein comprises at least one of SEQ ID No. 1-3.
4. The capture antigen for detecting the anti-asialoglycoprotein receptor antibody according to claim 2, wherein the amino acid sequence of the ASGPR H2 subunit protein is at least one of seq id nos. 5 to 9;
the amino acid sequence of the ASGPR H2 protein is at least one of SEQ ID No. 5-8.
5. The capture antigen for anti-asialoglycoprotein receptor antibody detection according to any one of claims 1 to 4, wherein said liver extract is selected from the group consisting of: at least one of a liver tissue extract and a liver tumor cell extract;
preferably, the liver extract is a liver tissue extract;
preferably, the liver tissue extract is an extract obtained after extraction with a glycoprotein extraction reagent;
preferably, the step of extracting the liver tissue extract comprises: homogenizing the liver tissue until the tissue is cracked, centrifuging and taking supernatant; filtering the supernatant, and extracting with a glycoprotein extraction reagent to obtain the liver tissue extract;
preferably, the liver tissue extract is selected from non-primate liver tissue extracts.
6. A kit for the detection of an anti-asialoglycoprotein receptor antibody, comprising the capture antigen for the detection of an anti-asialoglycoprotein receptor antibody according to any one of claims 1 to 5.
7. A method for detecting an anti-asialoglycoprotein receptor antibody, which is characterized by comprising the steps of mixing the capture antigen for detecting the anti-asialoglycoprotein receptor antibody according to any one of claims 1 to 5 with a sample to be detected, and then detecting;
the detection method is not directed towards the treatment or diagnosis of a disease.
8. The method for detecting an antibody against asialoglycoprotein receptor according to claim 7, wherein the capture antigen comprises a mixture of ASGPR antigen and liver extract in a mass ratio of (0.5 to 1.5): (0.5 to 1.5).
9. The method for detecting an antibody against asialoglycoprotein receptor according to claim 7, wherein said sample to be tested is selected from any one of a biological sample selected from any one of a serum sample, a plasma sample and a whole blood sample, and an environmental sample containing said biological sample.
10. The method for detecting an anti-asialoglycoprotein receptor antibody according to any one of claims 7 to 9, wherein said method for detecting is selected from the group consisting of: membrane strip method, enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, luminescence immunoassay, immunoturbidimetry, and immunochromatography.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111615338.4A CN114805545A (en) | 2021-12-28 | 2021-12-28 | Capture antigen for detecting anti-asialoglycoprotein receptor antibody and detection method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111615338.4A CN114805545A (en) | 2021-12-28 | 2021-12-28 | Capture antigen for detecting anti-asialoglycoprotein receptor antibody and detection method |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130059335A1 (en) * | 2010-05-06 | 2013-03-07 | Charite-Universitatsmedizin Berlin | Use of a genetically modified cell line expressing functional asialoglycoprotein receptor in the production of sialylated glycoproteins |
| CN104418950A (en) * | 2013-08-28 | 2015-03-18 | 中国人民解放军第二军医大学 | Human asialoglycoprotein receptor (ASGPR) monoclonal antibody as well as preparation method and application thereof |
-
2021
- 2021-12-28 CN CN202111615338.4A patent/CN114805545A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130059335A1 (en) * | 2010-05-06 | 2013-03-07 | Charite-Universitatsmedizin Berlin | Use of a genetically modified cell line expressing functional asialoglycoprotein receptor in the production of sialylated glycoproteins |
| CN104418950A (en) * | 2013-08-28 | 2015-03-18 | 中国人民解放军第二军医大学 | Human asialoglycoprotein receptor (ASGPR) monoclonal antibody as well as preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| TANAKA H, ET AL.: "Genbank accession number: NP_001172.1", GENBANK, pages 1 - 2 * |
| ULRICH TREICHEL, ET AL.: "High-Yield Purification and Characterization of Human Asialoglycoprotein Receptor", PROTEIN EXPRESSION AND PURIFICATION, vol. 6, pages 251 - 255 * |
| XIE B, ET AL.: "Genbank accession number: NP_001662.1", GENBANK, pages 1 - 2 * |
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