CN115639371A - Kit for reflecting mass concentration of adiponectin and using method thereof - Google Patents
Kit for reflecting mass concentration of adiponectin and using method thereof Download PDFInfo
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- CN115639371A CN115639371A CN202211284748.XA CN202211284748A CN115639371A CN 115639371 A CN115639371 A CN 115639371A CN 202211284748 A CN202211284748 A CN 202211284748A CN 115639371 A CN115639371 A CN 115639371A
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Abstract
The invention discloses a kit for reflecting the mass concentration of adiponectin and a use method thereof, belonging to the crossing field of in vitro diagnostic reagents and bioengineering technology, wherein the kit disclosed by the invention consists of a reagent R1 and a reagent R2, can dissociate adiponectin with HMW and MMW molecular weights so as to accurately determine the total adiponectin content in a sample, and compared with a commercially available adiponectin turbidimetric reagent, the kit disclosed by the invention has small detection deviation and can truly reflect the actual adiponectin concentration in the sample.
Description
Technical Field
The invention relates to the crossing field of in-vitro diagnostic reagents and bioengineering technology, in particular to a kit capable of truly reflecting the mass concentration of adiponectin and a use method thereof.
Background
Adiponectin is an endogenous plasma hormone protein secreted specifically by adipocytes. Human adiponectin is glycosylated after transcription and translation in adipocytes, and adiponectin genes are regulated by insulin and glucose in human adipose tissue. Adiponectin is a protective adipocyte factor with anti-inflammatory effect, plays an important role in the process of regulating insulin sensitivity and glucose metabolism, and can be used as a novel index for early risk prediction of diabetes. Prospective studies of adiponectin show that high adiponectin levels are associated with low risk of type 2 diabetes, low adiponectin levels are associated with high risk of type 2 diabetes, low adiponectin levels can predict the risk of progression to diabetes, and increasing adiponectin levels can effectively reduce the risk of occurrence of type 2 diabetes.
The adiponectin structure consists of a single-chain trimer, a variable n-terminal domain, a collagen domain, and a c-terminal globular domain homologous to the immune complement Clq. Adiponectin has the form of a trimer (about 90kda, lmw form), a hexamer (about 180kda, mmw form), or a multimer (molecular weight greater than 400kda, hmw form).
Studies have shown that a decrease in LMW adiponectin levels, in addition to total adiponectin levels and HMW adiponectin levels, may also be associated with diabetes. Epidemiological studies have mainly measured total adiponectin concentration, but there are also analytical methods for detecting adiponectin molecular weight subtypes. Chinese patent publication nos. CN103777023A, CN 11239421A, CN111337692A, and CN104237522A all provide methods for detecting adiponectin, but these methods are all based on the principle of specific binding of adiponectin polyclonal antibody or monoclonal antibody to adiponectin, and because adiponectin is in multimeric form, all antibodies used are antibodies recognizing certain fragments on monomers, and because adiponectin exists in plasma in the form of HMW, MMW, and LMW molecules in trimer units, adiponectin in different molecular forms has the same binding ability with antibodies, and all of the above detection methods express the detected adiponectin levels of different molecular weights as mass concentrations (e.g., mg/L, ng/mL). However, the antibodies in the commercial total adiponectin detection kit cannot distinguish between HMW, MMW and LMW, that is, if the sample contains LMW, MMW and HMW, the commercial total adiponectin detection kit has great deviation in detecting the mass concentration of total adiponectin, and obviously, the method for detecting the number of moles of total adiponectin and representing adiponectin polymers with different molecular weights in a mass concentration mode is not suitable, so that the real concentration of adiponectin in the blood sample of a patient cannot be truly reflected, and the clinical diagnosis is disturbed.
Disclosure of Invention
In order to solve the problems, the invention provides a kit reflecting the mass concentration of adiponectin and a using method thereof.
The kit for reflecting the mass concentration of the adiponectin, provided by the invention, comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises a steroid zwitterionic surfactant, and the steroid zwitterionic surfactant can dissociate adiponectin with HMW and MMW molecular weights; the reagent R2 comprises an anti-human adiponectin antibody marked by nano microspheres, and the anti-human adiponectin antibody can be specifically combined with adiponectin serving as a substance to be detected in a sample.
Preferably, the steroid zwitterionic surfactant is selected from one or more of 3- [3- (cholamidopropyl) dimethylammonium ] -1-propanesulfonic acid inner salt, 3- [ (3-cholesterylaminopropyl) dimethylamino ] -2-hydroxy-1-propanesulfonic acid and the like.
Preferably, the concentration of the steroid zwitterionic surfactant is 0.5-5g/L.
The steroid zwitterionic surfactant acts on a sample containing the adiponectin, so that HMW adiponectin and MMW adiponectin are dissociated into LMW adiponectin, and then the adiponectin antibody reacts with the LMW adiponectin, so that the immunoassay result can truly reflect the mass concentration of the total adiponectin.
Further, the reagent R1 comprises a buffer solution, a macromolecular polymer, salt, a preservative and an interference elimination substance, wherein the interference elimination substance is an anti-rheumatoid factor antibody.
Furthermore, the anti-human adiponectin antibody is an antibody prepared by immunizing animals with LMW adiponectin as an immunogen and a fragment thereof.
The monoclonal antibody is obtained by taking B cells from the spleen of an immunized animal to be fused with immortalized tumor cells in vitro, and subcloning the successfully fused hybridoma cells, so that corresponding cell strains are screened according to the application purpose, and then the antibody is further purified through cell culture or ascites collection; the polyclonal antibody is obtained by collecting animal antiserum and directly performing affinity purification.
Preferably, the nano-microsphere in the reagent R2 is selected from any one of a polystyrene microsphere, a colloidal gold microsphere, and a magnetic bead.
Further, the reagent R2 comprises buffer, protein, sugar or glycerol and preservative.
Furthermore, the kit for reflecting the mass concentration of the adiponectin, provided by the invention, further comprises a luminescent substance or enzyme-labeled adiponectin antibody, and the adiponectin antibody is a polyclonal antibody or a monoclonal antibody.
The luminescent substance or the enzyme-labeled adiponectin polyclonal antibody or monoclonal antibody is applied to the detection by a chemiluminescence method, the chemiluminescence or bioluminescence system is combined with an immunoreaction by the chemiluminescence method, and the corresponding antigen or antibody is detected according to the linear relation between the concentration of a substance to be detected in the detection system and the chemiluminescence intensity of the system under a certain condition.
Further, the use method of the kit for reflecting the mass concentration of the adiponectin provided by the invention comprises the following steps: and adding 160 ul of reagent R1 into 8 ul of sample, then incubating, reading and measuring the 1 st sample data A1 by the full-automatic biochemical analyzer, adding 40 ul of reagent R2, then reading and measuring the 2 nd sample data A2 by the full-automatic biochemical analyzer, and the mass concentration A = A2-A1 of the sample adiponectin.
The invention has the following beneficial effects: most surfactants have the capability of eliminating hydrophobic interaction, but most surfactants have weak capability of dissociating adiponectin multimers and cannot achieve the effect of complete dissociation, or have too strong capability of dissociating adiponectin to dissociate adiponectin trimers, or denature adiponectin trimers or denature antibodies to influence immunoassay.
Drawings
FIG. 1 shows the results of comparison of HMW and a commercially available adiponectin turbidimetric reagent in example 1 according to the embodiment of the present invention;
FIG. 2 shows the results of comparison of HMW and turbidimetric reagents of adiponectin sold on the market in example 2 according to the present embodiment;
FIG. 3 shows the results of comparison between HMW and a commercially available adiponectin turbidimetric reagent in example 3 according to the embodiment of the present invention;
FIG. 4 shows the results of comparison between HMW and a commercially available adiponectin turbidimetric reagent in example 4 according to the embodiment of the present invention;
FIG. 5 shows the results of comparison of HMW and a commercially available adiponectin turbidimetric reagent in example 5 according to the embodiment of the present invention;
FIG. 6 shows the alignment results of examples 1-5 according to the present invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, embodiments of the present invention are described in detail below. It should be noted that the following examples are only used to illustrate the implementation method and typical parameters of the present invention, and are not used to limit the scope of the parameters of the present invention, and therefore reasonable variations are still claimed in the scope of the claims of the present invention.
It is noted that the endpoints of the ranges and any values disclosed herein are not limited to the precise range or value and that such ranges or values are understood to encompass values close to such ranges or values. For numerical ranges, each range between its endpoints and individual point values, and each individual point value can be combined with each other to give one or more new numerical ranges, and such numerical ranges should be construed as specifically disclosed herein.
In view of the fact that the existing adiponectin detection kits cannot accurately detect the actual mass concentration of adiponectin in a sample due to the existence of LMW, MMW and HMW adiponectin in the plasma in the form of trimers and the same binding force between different polymer forms and antibodies as described in the background art, embodiments of the present invention provide a century kit and a detection method that can reflect the mass concentration of adiponectin, and dissociate adiponectin multimers into LMW adiponectin by steroid zwitterions to further determine the total adiponectin concentration. The method has the advantages of good dissociation effect and small detection deviation.
Example 1
An adiponectin immunoassay reagent comprises the following raw material components in parts by weight:
a reagent R1:100mmol/L HEPES (pH7.4), 2.0g/L polyethylene glycol 6000, 2.0g/L LBSA, 50.0 g/L sodium chloride, 1g/L sheep anti-human IgM antibody, 1g/L sodium azide, 2.0g/L Tween 20;
a reagent R2:20mmol/L HEPES (pH7.4), 2.5g/L polystyrene microsphere labeled anti-human adiponectin monoclonal antibody, 2.0g/L BSA, and 1g/L sodium azide.
Example 2
An adiponectin immunoassay reagent comprises the following raw material components in parts by weight:
a reagent R1:100mmol/L HEPES (pH7.4), 2.0g/L polyethylene glycol 6000, 2.0g/LBSA, 50.0 g/L sodium chloride, 1g/L sheep anti-human IgM antibody, 1g/L sodium azide, 2.0g/L triton X-100;
and (3) reagent R2:20mmol/L HEPES (pH 7.4), 2.5g/L polystyrene microsphere labeled anti-human adiponectin monoclonal antibody, 2.0g/L BSA, 1g/L sodium azide.
Example 3
An adiponectin immunoassay reagent comprises the following raw material components in parts by weight:
reagent R1:100mmol/L HEPES (pH7.4), 2.0g/L polyethylene glycol 6000, 2.0g/L LBSA, 50.0 g/L sodium chloride, 1g/L sheep anti-human IgM antibody, 1g/L sodium azide, 2.0g/L sodium dodecyl sulfate;
a reagent R2:20mmol/L HEPES (pH7.4), 2.5g/L polystyrene microsphere labeled anti-human adiponectin monoclonal antibody, 2.0g/L BSA, and 1g/L sodium azide.
Example 4
An adiponectin immunoassay reagent comprises the following raw material components in parts by weight:
reagent R1:100mmol/L HEPES (pH 7.4), 2.0g/L polyethylene glycol 6000, 2.0g/L LBSA, 50.0 g/L sodium chloride, 1g/L sheep anti-human IgM antibody, 1g/L sodium azide, 2.0 g/L3- [ (3-cholesterylaminopropyl) dimethylamino ] -2-hydroxy-1-propanesulfonic acid;
a reagent R2:20mmol/L HEPES (pH 7.4), 2.5g/L polystyrene microsphere labeled anti-human adiponectin monoclonal antibody, 2.0g/L BSA, 1g/L sodium azide.
Example 5
An adiponectin immunoassay reagent comprises the following raw material components in parts by weight:
a reagent R1:100mmol/L HEPES (pH 7.4), 2.0g/L polyethylene glycol 6000, 2.0g/L LBSA, 50.0 g/L sodium chloride, 1g/L sheep anti-human IgM antibody, 1g/L sodium azide, 2.0 g/L3- [3- (cholamidopropyl) dimethylammonium ] -1-propanesulfonic acid inner salt;
and (3) reagent R2:20mmol/L HEPES (pH7.4), 2.5g/L polystyrene microsphere labeled anti-human adiponectin monoclonal antibody, 2.0g/L BSA, and 1g/L sodium azide.
Example 6
An adiponectin immunoassay reagent comprises the following raw material components in parts by weight:
a reagent R1:100mmol/L HEPES (pH 7.4), 2.0g/L polyethylene glycol 6000, 2.0g/L LBSA, 50.0 g/L sodium chloride, 1g/L sheep anti-human IgM antibody, 1g/L sodium azide, 0.5 g/L3- [3- (cholamidopropyl) dimethylammonium ] -1-propanesulfonic acid inner salt;
and (3) reagent R2:20mmol/L HEPES (pH7.4), 2.5g/L polystyrene microsphere labeled anti-human adiponectin monoclonal antibody, 2.0g/L BSA, and 1g/L sodium azide.
Example 7
An adiponectin immunoassay reagent comprises the following raw material components in parts by weight:
a reagent R1:100mmol/L HEPES (pH 7.4), 2.0g/L polyethylene glycol 6000, 2.0g/L LBSA, 50.0 g/L sodium chloride, 1g/L sheep anti-human IgM antibody, 1g/L sodium azide, 1.0 g/L3- [3- (cholamidopropyl) dimethylammonium ] -1-propanesulfonic acid inner salt;
a reagent R2:20mmol/L HEPES (pH7.4), 2.5g/L polystyrene microsphere labeled anti-human adiponectin monoclonal antibody, 2.0g/L BSA, and 1g/L sodium azide.
Example 8
An adiponectin immunoassay reagent comprises the following raw material components in parts by weight:
reagent R1:100mmol/L HEPES (pH 7.4), 2.0g/L polyethylene glycol 6000, 2.0g/L LBSA, 50.0 g/L sodium chloride, 1g/L sheep anti-human IgM antibody, 1g/L sodium azide, 5.0 g/L3- [3- (cholamidopropyl) dimethylammonium ] -1-propanesulfonic acid inner salt;
and (3) reagent R2:20mmol/L HEPES (pH 7.4), 2.5g/L polystyrene microsphere labeled anti-human adiponectin monoclonal antibody, 2.0g/L BSA, 1g/L sodium azide.
Example 9
An adiponectin immunoassay reagent comprises the following raw material components in parts by weight:
reagent R1:100mmol/L HEPES (pH 7.4), 2.0g/L polyethylene glycol 6000, 2.0g/L LBSA, 50.0 g/L sodium chloride, 1g/L sheep anti-human IgM antibody, 1g/L sodium azide, 10.0 g/L3- [3- (cholamidopropyl) dimethylammonium ] -1-propanesulfonic acid inner salt;
and (3) reagent R2:20mmol/L HEPES (pH7.4), 2.5g/L polystyrene microsphere labeled anti-human adiponectin monoclonal antibody, 2.0g/L BSA, and 1g/L sodium azide.
Example 10
Examples 1-9 human serum total adiponectin, HMW molecular weight adiponectin assay, and a commercial adiponectin latex-enhanced immunoassay kit comparison.
The method for detecting the human serum sample by the kit comprises the following steps: adding a sample or a calibration substance into a first reagent, incubating at 37 ℃ for 5min, reading the 1 st sample data A1 by a full-automatic biochemical analyzer, adding a second reagent, incubating at 37 ℃ for 3-5min, reading the 2 nd sample data A2 by the full-automatic biochemical analyzer, and obtaining the sample/calibration substance mass concentration data A = A2-A1; the analysis parameters were: dominant wavelength 570nm, secondary wavelength 800nm, sample or calibrator 8 μ l, R1:160 μ l, R2:40 μ l.
Meanwhile, human serum samples were detected using a human-derived polymer adiponectin ELISA kit (manufactured by R & D systems, cat. PDHWAD 0) and a commercially available adiponectin latex-enhanced immunoassay kit of one company, respectively. The results are shown in FIGS. 4 and 5.
As can be seen from the results, the correlation between the HMW adiponectin concentration level and the detection result of the commercially available adiponectin latex-enhanced immunoassay kit is poor, the correlation between the HMW adiponectin concentration level and the detection result of the commercially available adiponectin latex-enhanced immunoassay kit is good, the correlation between the HMW adiponectin concentration level and the detection result of the adiponectin latex-enhanced immunoassay kit is good, and the results of the tests of the examples 4 and 5 are higher, which indicates that the test result of the examples is closer to the total adiponectin concentration level.
As can be seen from FIGS. 1 to 5, the steroid zwitterionic surfactant as a dissociating agent is used for testing that the concentration levels of the total adiponectin and the HMW adiponectin have a positive correlation, but other surfactants have no correlation with the HMW adiponectin, which shows that the steroid zwitterionic surfactant as a dissociating agent can better dissociate multimeric adiponectin.
As can be seen from FIG. 6, the dissociation effect is the best when the concentration of the steroid zwitterionic surfactant is 5g/L, and the measured value of adiponectin is the highest.
Although the present disclosure has been described above, the scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and such changes and modifications will fall within the scope of the present invention.
Claims (9)
1. A kit for reflecting the mass concentration of adiponectin, comprising a reagent R1 and a reagent R2, wherein the reagent R1 comprises a steroidal zwitterionic surfactant capable of dissociating adiponectin at HMW and MMW molecular weights; the reagent R2 comprises an anti-human adiponectin antibody marked by nano microspheres, and the anti-human adiponectin antibody can be specifically combined with adiponectin serving as a substance to be detected in a sample.
2. The kit for reflecting the mass concentration of adiponectin according to claim 1, wherein said steroidal zwitterionic surfactant is selected from one or more of 3- [3- (cholamidopropyl) dimethylammonium ] -1-propanesulfonic acid inner salt, 3- [ (3-cholesterylaminopropyl) dimethylamino ] -2-hydroxy-1-propanesulfonic acid, and the like.
3. The kit for reflecting the mass concentration of adiponectin according to claim 1 or claim 2, wherein the concentration of the steroid zwitterionic surfactant is 0.5 to 5g/L.
4. The kit for reflecting the mass concentration of adiponectin according to claim 1, wherein the reagent R1 comprises a buffer, a macromolecular polymer, a salt, a preservative, and an interfering substance-eliminating substance that is an anti-rheumatoid factor antibody.
5. The kit for reflecting the mass concentration of adiponectin as claimed in claim 1, wherein said anti-human adiponectin antibody is an antibody or a fragment thereof prepared by immunizing an animal with LMW molecular weight adiponectin as an immunogen.
6. The kit for reflecting the mass concentration of adiponectin according to claim 1, wherein the nanospheres are selected from any one of polystyrene microspheres, colloidal gold microspheres and magnetic beads.
7. The kit for reflecting the mass concentration of adiponectin according to claim 1, wherein the reagent R2 includes a buffer, a protein, a sugar or glycerol, and a preservative.
8. The kit for reflecting the mass concentration of adiponectin as claimed in claim 1, further comprising a luminescent substance or an enzyme-labeled adiponectin antibody, wherein said adiponectin antibody is a polyclonal antibody or a monoclonal antibody.
9. A method for using the kit for reflecting the mass concentration of adiponectin according to any one of claims 1 to 8, comprising the steps of: and adding 160 ul of reagent R1 into 8 ul of sample, incubating, reading the 1 st sample data A1 by a full-automatic biochemical analyzer, adding 40 ul of reagent R2, reading the 2 nd sample data A2 by the full-automatic biochemical analyzer, and obtaining the sample adiponectin mass concentration A = A2-A1.
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