CN115711933A - Detection reagent, detection kit and application thereof - Google Patents
Detection reagent, detection kit and application thereof Download PDFInfo
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- CN115711933A CN115711933A CN202211403259.1A CN202211403259A CN115711933A CN 115711933 A CN115711933 A CN 115711933A CN 202211403259 A CN202211403259 A CN 202211403259A CN 115711933 A CN115711933 A CN 115711933A
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Abstract
The invention relates to the field of biological detection, in particular to a detection reagent, a detection kit and application thereof. The present invention provides a detection reagent comprising: SAA internal standard, acetonitrile, sinapic acid, and trifluoroacetic acid. The invention is based on the principle of mass spectrometry, and can effectively identify different subtypes of SAA and qualitative variants thereof. Meanwhile, the pretreatment step is simple, and the method does not depend on antibodies, so that the instability of detection caused by the difference between batches of the antibodies can be avoided.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a detection reagent, a detection kit and application thereof.
Background
Rheumatoid Arthritis (RA) is an autoimmune inflammatory disease mainly manifested by invasion of joints, and the cause of the disease is not completely clear at present. The joint swelling and pain appear in the early stage of RA patients, and the synovial inflammation continues or repeatedly attacks, which results in bone destruction, dysfunction and higher disability rate.
RA is a chronic disease and cannot be completely cured, the current core strategy of RA treatment is standard treatment (T2T), and if RA patients with long disease course, low disease activity can be selected as a substitute target. Therefore, it is an indispensable part of the diagnosis and treatment of RA to determine the disease activity of RA patients.
The 28 joint disease activity scoring method (DAS 28) is a clinically common diagnosis method of RA disease activity, and is often combined with blood sedimentation (ESR) or CRP concentration (DAS 28-ESR or DAS28-CRP, respectively) to evaluate the remission degree of RA. The DAS28-CRP can break through some limitations of DAS28-ESR, such as large influence of age and sex, slow response to disease activity, and the like. However, some biologic therapies, such as Tumor Necrosis Factor (TNF) antagonists, can lower CRP levels, resulting in decreased CRP concentrations in patients without decreased disease activity, leading to false negative disease diagnosis.
SAA (serum amyloid a) also shows great potential as a systemic acute phase reactant in the diagnosis of RA. Compared to CRP, patient SAA levels were not affected by TNF antagonist treatment. In addition, there is increasing evidence that SAA concentrations are more sensitive to changes in disease activity, rising at higher levels than CRP in the presence of disease and falling more rapidly in remission.
However, the current assay for SAA is based on the gross detection of antigen-antibody affinity principles. Actually, SAA has a plurality of subtypes such as SAA1 and SAA2, each subtype also has a plurality of truncations and glycosylation modification variants, and the like, so that the change conditions of the plurality of subtypes (including variants) such as SAA1 and SAA2 cannot be reflected, and simultaneously, based on the principle of immunoaffinity, the SAA is easily influenced by antibody performance, and batch consistency and chamber consistency are difficult to guarantee.
Disclosure of Invention
In view of the above, the invention provides a detection reagent, a detection kit and application thereof. The invention is based on the principle of mass spectrometry, and can effectively identify different subtypes of SAA and qualitative variants thereof. Meanwhile, the pretreatment step is simple, and the method does not depend on antibodies, so that the instability of detection caused by the difference between batches of the antibodies can be avoided.
In order to achieve the above object, the present invention provides the following technical solutions:
the present invention provides a detection reagent comprising: SAA internal standard, acetonitrile, sinapic acid, and trifluoroacetic acid.
In some embodiments of the invention, the detection reagent comprises reagents I to III;
the reagent I comprises: the trifluoroacetic acid;
the reagent II comprises: the SAA internal standard;
the reagent III comprises: the acetonitrile, the sinapic acid, and the trifluoroacetic acid.
In some embodiments of the present invention, the concentration of the trifluoroacetic acid in the detection reagent is 0.05 to 0.2% (v/v).
In some embodiments of the invention, the concentration of the trifluoroacetic acid in the detection reagent is 0.05% (v/v).
In some embodiments of the invention, the concentration of the trifluoroacetic acid in the detection reagent is 0.1% (v/v).
In some embodiments of the invention, the concentration of the trifluoroacetic acid in the detection reagent is 0.2% (v/v).
In some embodiments of the invention, the sinapic acid concentration in the detection reagent is 10-20 mg/mL
In some embodiments of the invention, the sinapic acid concentration in the above detection reagent is 20mg/mL.
In some embodiments of the invention, the sinapic acid concentration in the above detection reagent is 15mg/mL.
In some embodiments of the invention, the sinapic acid concentration in the above detection reagent is 10mg/mL.
In some embodiments of the present invention, the SAA internal standard substance in the above detection reagent comprises SAA isotopic label or SAA with protein purification tag, and the concentration of the SAA internal standard substance is 1-10 μ g/mL.
In some embodiments of the present invention, the SAA internal standard substance in the above detection reagent is SAA with histidine tag modification at the N-terminal.
In some embodiments of the invention, the concentration of the SAA internal standard in the above detection reagent is 1. Mu.g/mL.
In some embodiments of the invention, the concentration of the SAA internal standard in the above detection reagents is 5. Mu.g/mL.
In some embodiments of the invention, the concentration of the SAA internal standard in the above detection reagents is 10. Mu.g/mL.
In some embodiments of the present invention, the concentration of acetonitrile in the above-mentioned detection reagent is 30% to 40 (v/v).
In some embodiments of the present invention, the concentration of acetonitrile in the above-mentioned detection reagent is 30% (v/v).
In some embodiments of the present invention, the concentration of acetonitrile in the above-mentioned detection reagent is 33% (v/v).
In some embodiments of the present invention, the concentration of acetonitrile in the above-mentioned detection reagent is 40% (v/v).
The invention provides a detection kit, which comprises the detection reagent and other auxiliary agents or carriers.
The invention provides application of the detection reagent or the detection kit in preparation of products for detecting SAA qualitative variants.
The invention provides application of the detection reagent or the detection kit in preparation of a product for detecting the abundance of the SAA qualitative variant.
The invention provides an application of the detection reagent or the detection kit in preparation of a product for detecting rheumatoid arthritis.
The invention also provides a detection method of the SAA qualitative variant, and a sample is mixed with the detection reagent or the detection kit for detection.
In some embodiments of the invention, in the above detection method, the detection comprises mass spectrometry.
The present invention provides a detection reagent comprising: SAA internal standard, acetonitrile, sinapic acid, and trifluoroacetic acid.
The beneficial effects of the invention include:
(1) The method is based on accurate identification of molecular weight of a mass spectrum platform, so that the detection of SAA can overcome the dependence on a special antibody;
(2) The method can realize the relative content measurement of the total SAA, more importantly, can measure the relative content of different variants of the SAA, and can avoid the defect of total amount detection by an immunological affinity method.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 illustrates a technical flow diagram;
FIG. 2 shows the SAA qualitative variant profiles of examples 1-4; wherein: ABCD shows the profiles of four patient serum samples, respectively; wherein in the A picture, SAA1.1, SAA1.3, SAA2.2, des-RSAA1.1, des-R SAA2.2 show the position of SAA qualitative variant peak, and SAA-His shows the SAA internal standard peak;
FIG. 3 is a graph showing a comparison result of the methodology;
FIG. 4 shows the content distribution of SAA and its qualitative variations in different groups of example 5; wherein: a shows the measurement condition of the total SAA content in each group, and the total SAA content is increased along with the exacerbation of the disease; b shows that the total SAA content was not significantly different between the relieved group and the healthy group, all at very low levels; c shows the measurement condition of the total content of SAA1 in each group, the total content of SAA1 and the total content of SAA have better consistency, but the total content of SAA1 and the total content of SAA are not completely consistent in the high activity group; d shows the determination conditions of the total content of SAA1 in the H group, RA0 group and RA1 group; e shows the total content of SAA2 in each group of measurement, in the active group and high active group in RA, the frequency of the detection of SAA2 is obviously increased, 7 samples in the active group detect SAA2, and 16 samples in the high active group in RA detect SAA2; f shows the determination conditions of the total content of SAA2 in the H group, the RA0 group and the RA1 group, the frequency of the SAA2 in the healthy group, the RA low activity group and the remission group is very low, and the SAA2 is detected only once in the RA remission group; wherein: h: a healthy control group and RA0-RA3 sequentially represent a rheumatoid arthritis remission group (RA 0), a low activity group (RA 1), a medium activity group (RA 2) and a high activity group (RA 3); wherein: * Data in the two groups showed significant statistical differences (. P <0.05,. P < 0.01), and ns showed no statistical significance for the difference in the two groups.
Detailed Description
The invention discloses a detection reagent, a detection kit and application thereof.
It is understood that one or more of the expressions "… …" individually includes each stated object after the expression and various different combinations of two or more of the stated objects, unless otherwise understood from context and usage. The expression "and/or" in connection with three or more of the stated objects shall be understood to have the same meaning unless otherwise understood from the context.
The use of the terms "comprising," "having," or "containing," including grammatical equivalents thereof, are generally to be construed as open-ended and non-limiting, e.g., without excluding other unstated elements or steps, unless specifically stated otherwise or otherwise understood from context.
It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Further, two or more steps or actions may be performed simultaneously.
The use of any and all examples, or exemplary language such as "for example" or "including" herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Moreover, the numerical ranges and parameters setting forth the invention are approximations that may have numerical values that are within the numerical ranges specified in the specific examples. Any numerical value, however, inherently contains certain standard deviations found in their respective testing measurements. Accordingly, unless expressly stated otherwise, it is understood that all ranges, amounts, values and percentages used in this disclosure are by weight modified by "about". As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.
The invention provides a detection reagent, which comprises:
reagent I: is water solution (0.05-0.2%, v/v) containing trifluoroacetic acid, which can be trifluoroacetic acid, formic acid, etc.;
reagent II: the SAA internal standard substance is an aqueous solution containing an SAA internal standard substance, the SAA internal standard substance can be an SAA isotope label or a derivative with a His label and other modifications, the content of the internal standard substance is 1-10 mug/mL, and the preferred concentration range is 5-10 mug/mL;
reagent III: is an aqueous solution containing 10-20 mg/mL sinapic acid (preferably 10-15 mg/mL), 30-40% (v/v) acetonitrile (preferably 30-34%), and 0.05-0.2% (v/v) trifluoroacetic acid (preferably 0.1-0.2%).
The invention also provides a detection method, and a flow chart of the detection method is shown in figure 1.
In examples 1 to 7 and effect examples 1 to 3 of the present invention, the raw materials and reagents used were commercially available.
The invention is further illustrated by the following examples:
EXAMPLE 1 reagent composition
Reagent I: an aqueous trifluoroacetic acid solution containing 0.05% (v/v);
and (2) reagent II: the SAA internal standard substance is SAA with histidine tag modification at the N end, and the concentration is 5 mu g/mL;
reagent III: an aqueous solution containing 10mg/mL sinapic acid, 30% (v/v) acetonitrile and 0.05% (v/v) trifluoroacetic acid.
EXAMPLE 2 reagent composition
Reagent I: an aqueous trifluoroacetic acid solution containing 0.1% (v/v);
and (2) reagent II: the SAA internal standard substance is SAA with histidine tag modification at the N end, and the SAA internal standard substance is 1 mu g/mL;
and (3) reagent III: an aqueous solution containing 15mg/mL sinapic acid, 33% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid.
EXAMPLE 3 reagent composition
Reagent I: an aqueous solution containing 0.2% (v/v) of trifluoroacetic acid;
reagent II: the SAA internal standard substance is SAA with histidine tag modification at the N end, and the concentration is 10 mu g/mL;
and (3) reagent III: an aqueous solution containing 20mg/mL sinapic acid, 40% (v/v) acetonitrile and 0.2% (v/v) trifluoroacetic acid.
EXAMPLE 4 reagent composition
Reagent I: an aqueous solution containing 0.2% (v/v) of trifluoroacetic acid;
and (2) reagent II: the SAA internal standard substance is SAA with histidine tag modification at the N end, and the content is 10 mu g/mL;
and (3) reagent III: an aqueous solution containing 10mg/mL sinapic acid, 34% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid.
EXAMPLE 5 reagent composition
Reagent I: an aqueous solution containing 0.2% (v/v) of trifluoroacetic acid;
and (2) reagent II: the SAA internal standard substance is SAA with histidine tag modification at the N end, and the content is 10 mu g/mL;
and (3) reagent III: an aqueous solution containing 10mg/mL sinapic acid, 33% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid.
EXAMPLE 6 procedure
1) Taking 2 mu L of patient serum, adding 16 mu L of the reagent I obtained in the embodiments 1 to 5, and quickly and uniformly mixing to obtain a solution A;
2) Taking 5 mu L of the solution A obtained in the step 1), adding 5 mu L of the reagent II obtained in the examples 1 to 5 and 10 mu L of the reagent III obtained in the examples 1 to 5, and quickly and uniformly mixing to obtain a solution B;
3) Dripping 2.5 mu L of the solution B obtained in the step 2) on a target plate, and naturally volatilizing the solvent or heating at 39 ℃ to promote the volatilization of the solvent;
4) SAA1 and SAA2 and their heterogeneous variants were detected using MALDI-TOF-MS.
Example 7 data analysis
1) Identifying peaks through software configured by a MALDI-TOF-MS instrument manufacturer, and setting the signal-to-noise ratio to be more than or equal to 3;
2) Recording the mass-to-charge ratio (m/z) of the peak in the range of 11000-12000 Da, and comparing the mass-to-charge ratio with the theoretical molecular weight of the SAA mass variant or the molecular weight data reported in the literature to judge the type of the SAA mass variant (shown in figure 2);
3) The peak areas of the total SAA, total SAA1 and total SAA2 mass variants obtained in example 6 were calculated separately and labeled A T 、A 1 And A 2 ；
4) Internal standard peak with SAA (A) IR ) For reference, the relative contents (C) of total SAA, total SAA1, total SAA2, and each of the modifications were calculated according to the following formulas, respectively;
C i ＝A i /A IR
effect example 1
The experimental results of examples 1-4, respectively, are shown in fig. 2 (fig. 2A corresponds to the data of example 1, fig. 2B corresponds to the data of example 2, fig. 2C corresponds to the data of example 3, and fig. 2D corresponds to the data of example 4) by the procedure of example 6 and the data analysis of example 7, which shows that under the adjusted experimental conditions, SAA can be detected in serum samples of different patients by the MALDI-TOF-MS platform, and simultaneously, a plurality of SAA qualitative variants, such as SAA1.1, SAA1.3, SAA2.2, des-R SAA1.1, des-R SAA2.2, can be detected.
Effect example 2 test results
A comparison of the ELISA with MALDI-TOF-MS of the present invention is shown in Table 1, table 2, and FIG. 3. It can be seen that ELISA can only quantitatively detect SAA1 or SAA2 alone, while MALDI-TOF-MS mass spectrometry can detect protein variants, and can simultaneously detect total amount of SAA, SAA1, SAA2 and each protein variant (such as SAA1.1, SAA1.2, SAA2.1, SAA 2.2), etc., and obtain the ratio of each type.
TABLE 1 comparison of methodological rationales
TABLE 2MALDI-TOF-MS Mass Spectrometry
As shown in FIG. 3, table 1 and Table 2, the molecules of native SAA1 (SAA 1.1) and SAA2 (SAA 2.1) are 11682.7 and 11647.7Da, respectively. The SAA molecular weight after mutation is changed due to the loss of terminal amino acid residues or the mutation of middle part amino acid sites, and shows multiple peaks between 11300 and 11800 Da. SAA is structurally highly polymorphic due to the coding gene, allele, and post-translational modification. The representative profile of the SAA qualitative variants detected by this method, from which it can be seen that SAA has a very high complexity in structure.
Effect example 3
The experimental results of test example 5 are shown in fig. 4, using the procedure of example 6 and the data analysis of example 7.
1) The method realizes the determination of the relative content of different qualitative variants of SAA by adding an SAA internal standard substance. Overall, total SAA levels increased with increasing disease (as shown in fig. 4A, fig. 4B and table 3). The total content of SAA in the RA high activity group was significantly higher than that in the medium activity group, which was significantly higher than that of the low activity and remission groups. Total SAA levels were not significantly different between the remission group and the healthy group, all at very low levels (< 0.110), and no significant SAA was detected in the vast majority of samples (17 out of 20 samples in the healthy group, 12 out of 19 samples in the remission group).
2) The total SAA1 content has better agreement with the total SAA content in the active, low active and relief groups in RA, but not completely in the high active group (as shown in fig. 4C, 4D and table 3). While SAA2 appears very infrequently in the healthy, RA low activity and remission groups, and is detected only once in the RA remission group. While SAA2 was detected with significantly higher frequency in the active group and the high active group in RA, SAA2 was detected in 7 samples in the active group and SAA2 was detected in 16 samples in the high active group in RA (see fig. 4E, fig. 4F and table 3).
TABLE 3MALDI-TOF-MS method for determining the relative content of SAA and its plastid variants in a sample
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (11)
1. A detection reagent, comprising: SAA internal standard, acetonitrile, sinapic acid, and trifluoroacetic acid.
2. The detection reagent according to claim 1, comprising reagents I to III;
the reagent I comprises: the trifluoroacetic acid;
the reagent II comprises: the SAA internal standard;
the reagent III comprises: the acetonitrile, the sinapic acid, and the trifluoroacetic acid.
3. The detection reagent according to claim 1 or 2, wherein the concentration of trifluoroacetic acid is 0.05 to 0.2% (v/v).
4. The detection reagent according to any one of claims 1 to 3, wherein the sinapic acid concentration is between 10 and 20mg/mL.
5. The detection reagent of any one of claims 1 to 4, wherein the SAA internal standard comprises SAA isotopic label or SAA with a protein purification tag, and the concentration of the SAA internal standard is 1-10 μ g/mL.
6. The detection reagent according to any one of claims 1 to 5, wherein the acetonitrile is at a concentration of 30 to 40% (v/v).
7. A test kit comprising a test reagent according to any one of claims 1 to 6 and other auxiliary agents or carriers.
8. Use of a detection reagent according to any one of claims 1 to 6 or a detection kit according to claim 7 in the manufacture of a product for detecting a modification of SAA.
9. Use of a detection reagent according to any one of claims 1 to 6 or a detection kit according to claim 7 in the manufacture of a product for detecting the abundance of a variant of SAA.
10. Use of a detection reagent according to any one of claims 1 to 6 or a detection kit according to claim 7 in the manufacture of a product for detecting rheumatoid arthritis.
A method of detecting a mutant SAA, wherein a sample is mixed with the detection reagent according to any one of claims 1 to 6 or the detection kit according to claim 7 and detected.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002046448A2 (en) * | 2000-11-20 | 2002-06-13 | Eastern Virginia Medical School | Methods and devices for the quantitative detection of prostate specific membrane antigen and other prostatic markers |
| US20030027216A1 (en) * | 2001-07-02 | 2003-02-06 | Kiernan Urban A. | Analysis of proteins from biological fluids using mass spectrometric immunoassay |
| US6734023B1 (en) * | 2000-04-28 | 2004-05-11 | Duke University | Quantitative, high-throughput screening method for protein stability |
| US20060178306A1 (en) * | 2004-12-02 | 2006-08-10 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | Modulation of the neuroendoctrine system as a therapy for motor neuron disease |
| US20090104602A1 (en) * | 2005-05-23 | 2009-04-23 | Delmiro Fernandez-Reyes | Diagnosis of Tuberculosis |
| US20130217630A1 (en) * | 2010-10-26 | 2013-08-22 | ARIZONA BOARD OF REGENTS, a body corporate of the State of Arizona, acting for an on behalf of Arizo | Parathyroid hormone variants and assays related to disease |
| CN111948404A (en) * | 2020-08-03 | 2020-11-17 | 融智生物科技（青岛）有限公司 | Characteristic protein marker composition for screening thalassemia, mass spectrum model and application thereof |
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2022
- 2022-11-10 CN CN202211403259.1A patent/CN115711933A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6734023B1 (en) * | 2000-04-28 | 2004-05-11 | Duke University | Quantitative, high-throughput screening method for protein stability |
| WO2002046448A2 (en) * | 2000-11-20 | 2002-06-13 | Eastern Virginia Medical School | Methods and devices for the quantitative detection of prostate specific membrane antigen and other prostatic markers |
| US20030027216A1 (en) * | 2001-07-02 | 2003-02-06 | Kiernan Urban A. | Analysis of proteins from biological fluids using mass spectrometric immunoassay |
| US20060178306A1 (en) * | 2004-12-02 | 2006-08-10 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | Modulation of the neuroendoctrine system as a therapy for motor neuron disease |
| US20090104602A1 (en) * | 2005-05-23 | 2009-04-23 | Delmiro Fernandez-Reyes | Diagnosis of Tuberculosis |
| US20130217630A1 (en) * | 2010-10-26 | 2013-08-22 | ARIZONA BOARD OF REGENTS, a body corporate of the State of Arizona, acting for an on behalf of Arizo | Parathyroid hormone variants and assays related to disease |
| CN111948404A (en) * | 2020-08-03 | 2020-11-17 | 融智生物科技（青岛）有限公司 | Characteristic protein marker composition for screening thalassemia, mass spectrum model and application thereof |
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