CN114295840B - Kit for high-sensitivity quantitative determination of adiponectin and preparation method thereof - Google Patents
Kit for high-sensitivity quantitative determination of adiponectin and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kit for high-sensitivity quantitative determination of adiponectin, which comprises a reagent R1 and a reagent R2, wherein the reagent R2 comprises a latex microsphere coated with an adiponectin monoclonal antibody, and the preparation process of the latex microsphere coated with the adiponectin monoclonal antibody comprises the following steps: BSA is added simultaneously in the process of labeling the latex microspheres and the adiponectin monoclonal antibody. According to the invention, the latex microspheres with large particle sizes are selected, so that the technical problem of low value and low precision of the adiponectin detection kit is solved, and meanwhile, the problem of poor detection linear range caused by the use of the microspheres with large particle sizes is solved by adding BSA (bovine serum albumin) in the process of labeling the latex microspheres and antibodies. The improvement measures are matched with the reaction system, so that the detection performance of the adiponectin latex kit is effectively improved, and the clinical requirements are fully met.
Description
Technical Field
The invention relates to the field of medical inspection, in particular to a kit for high-sensitivity quantitative determination of adiponectin and a preparation method thereof.
Background
Adiponectin is an endogenous bioactive polypeptide or protein secreted by adipocytes, and as a specific adipokine, adiponectin plays an important role in the development of insulin resistance, type ii diabetes, cardiovascular diseases and metabolic syndrome. Researches find that the serum adiponectin level of patients with type I diabetes and type II diabetes has significant difference, and the detection of the plasma adiponectin level can help to distinguish the type I diabetes from the type II diabetes. Low levels of adiponectin are common in obese patients, type ii diabetics, lipodystrophy patients, hypertensive patients, and the like. At the same time, the serum adiponectin level is in a positive correlation with the insulin sensitivity of the body. Therefore, the monitoring of adiponectin content is clinically significant.
The ADPN level content in human body is relatively high, and the plasma concentration of adiponectin is 5-30 mg/mL. In response to the particularity of ADPN-related diseases, such as diabetes, cardiovascular diseases and inflammation, the adiponectin concentration is negatively correlated, and the content of adiponectin decreases during the occurrence of the diseases. Therefore, it is necessary to improve the low-value sensitivity of the reagent for detecting ADPN as much as possible, and it is also necessary to achieve a satisfactory detection range. At the same time, since adiponectin exists in serum as a series of multimeric complexes, three major oligomeric forms, low molecular weight adiponectin (LMW-trimer), medium molecular weight adiponectin (MMW-hexamer), and high molecular weight adiponectin (HMW-multimer), which exert physiological roles through different signaling pathways, are formed by their collagen domains. The adiponectin recombinant monoclonal antibody has weak recognition ability on adiponectin monomers or dimers and low reactivity; the polymer recognition ability is strong, but in the buffer solution, the conventional recombinant polymer is easy to degrade, and the stability is extremely poor.
The current commercial kit for detecting ADPN in human body mainly adopts latex enhanced immunoturbidimetry, and the plasma concentration of adiponectin determines that the latex microspheres are usually small-particle-size microspheres (100-150 nm), but the microspheres in the particle size range cannot completely meet the low-value sensitivity required by detection. On the contrary, if the microspheres with large particle size are adopted, although the sensitivity of the reagent can be obviously improved, a better detection linear range cannot be achieved, and finally the problem that the detection of the existing kit is difficult to be compatible in low-value precision and linear range is caused. Meanwhile, due to the problems that the conventional adiponectin recombinant polymer is easy to degrade and has poor stability, manufacturers in the market mostly adopt a freeze-dried powder form to store the calibrator, so that the preparation cost and the process complexity of the reagent are increased.
Disclosure of Invention
According to the invention, the latex microspheres with large particle size are selected, so that the technical problem of low value and low precision of the adiponectin detection kit is solved, and meanwhile, the problem of poor detection linear range caused by the use of the microspheres with large particle size is solved by adding BSA (bovine serum albumin) in the process of labeling the latex microspheres and the antibody. The improvement measures are matched with the reaction system, so that the detection performance of the adiponectin latex kit is effectively improved, and the clinical requirements are fully met.
In order to achieve the purpose, the invention adopts the following technical means: a kit for high-sensitivity quantitative determination of adiponectin is characterized by comprising a reagent R1 and a reagent R2;
the reagent R1 comprises the following components in percentage by weight: 50-150mmol/L buffer solution, 0.1-1mol/L inorganic salt ion and 2-10g/L surfactant;
the reagent R2 comprises the following components in percentage by weight: 70-120mmol/L buffer solution, 0.1-1mol/L inorganic salt ions and latex microspheres coated with adiponectin monoclonal antibodies;
the preparation process of the latex microsphere coated with the adiponectin monoclonal antibody comprises the following steps: BSA is added simultaneously in the process of labeling the latex microspheres and the adiponectin monoclonal antibody.
Preferably, the reagent R1 also comprises 5-20g/L coagulant and 0.5-1.5g/L preservative; the reagent R2 also comprises 20-80g/L of blocking protein and 10-30g/L of carbohydrate.
Preferably, the latex microspheres have a particle size of 150-250nm, preferably 200-230nm.
Preferably, the adiponectin monoclonal antibody is a recombinant monoclonal antibody.
Preferably, in the preparation process of the latex microspheres coated with the adiponectin monoclonal antibody, the mass ratio of the latex microspheres to the adiponectin monoclonal antibody to the BSA is 1 (0.01-0.1): (0.01-0.1), preferably 1 (0.05-0.1): (0.05-0.1).
Preferably, the buffer solution in the reagent R1 is selected from at least one of citric acid monohydrate, MOPS, glycine, tris or HEPES, the inorganic salt ions are selected from at least one of sodium chloride or potassium chloride, the coagulant is selected from at least one of PEG6000 or PEG8000, the surfactant is selected from at least one of Tween-20, brij58, TX-100 or A90, and the preservative is selected from at least one of PC-300 or sodium azide; the buffer solution in the reagent R2 is selected from at least one of MOPS, glycine, TRIS or HEPES, the inorganic salt ions are selected from at least one of sodium chloride or potassium chloride, the blocking protein is selected from at least one of BSA or casein, and the carbohydrate is selected from at least one of trehalose or sucrose.
Preferably, the pH of the reagent R1 is between 5.0 and 7.0, preferably between 6.0 and 7.0; the pH of the reagent R2 is from 5.0 to 7.0, preferably from 6.0 to 7.0.
A preparation method of a kit for high-sensitivity quantitative determination of adiponectin is characterized by comprising the following steps:
(1) Preparation of reagent R1
The reagent R1 is prepared according to the following formula:
buffer solution 50-150mmol/L, inorganic salt ion 0.1-1.0mmo/L, coagulant 5-20g/L, surfactant 2-10g/L and preservative 0.5-1.5g/L;
the buffer solution is selected from at least one of citric acid monohydrate, MOPS, glycine, tris or HEPES, the inorganic salt ions are selected from at least one of sodium chloride or potassium chloride, the coagulant is selected from at least one of PEG6000 or PEG8000, the surfactant is selected from at least one of Tween-20, brij58, TX-100 or A90, and the preservative is selected from at least one of PC-300 or sodium azide;
(2) Preparation of reagent R2
(1) Preparation of R2 labeling buffer
The R2 labeling buffer solution is prepared according to the following formula:
buffer solution 70-120mmoL, inorganic salt ion 0.1-1mol/L and saccharide substance 10-30g/L;
the marking buffer is at least one of boric acid buffer, glycine buffer or Tris buffer, the inorganic salt ions are at least one of sodium chloride or potassium chloride, and the saccharide substances are at least one of trehalose or sucrose;
(2) preparation of reagent R2
Adding the latex microsphere latex into a marking buffer solution, then adding an activating agent EDC, uniformly mixing, and incubating for 20-60min; simultaneously adding the adiponectin monoclonal antibody, the latex microspheres and BSA, uniformly mixing, and incubating for 5-20h; adding a blocking agent BSA, incubating for 0.5-2h, centrifuging to remove supernatant, and performing ultrasonic treatment to obtain a reagent R2;
preferably, in the step (2), the mass ratio of the latex microspheres to the adiponectin monoclonal antibody to the BSA is 1 (0.01-0.1): (0.01-0.1), preferably 1 (0.05-0.1): (0.05-0.1).
Preferably, the adiponectin monoclonal antibody is a recombinant monoclonal antibody.
The invention has the beneficial effects that: according to the invention, the large-particle-size microspheres are adopted, and the recombinant monoclonal antibody and the small-molecule protein BSA are simultaneously marked, so that the precision of the reagent is greatly improved while the linear range of the reagent is ensured, and especially the detection precision of a low-value sample is improved. Meanwhile, compared with the traditional ascites antibody, the recombinant monoclonal antibody has the advantages of higher purity, less foreign protein, higher yield and the like, so that the difference between raw material batches is small, the difference between reagent batches can be strictly controlled, and the kit has better batch difference and detection stability.
Drawings
FIG. 1 is a graph showing the analysis of the linear correlation between the test values of the kits of group A and group B and the theoretical values of the samples according to example 3 of the present invention;
FIG. 2 is a graph showing the analysis of the linear correlation between the test values of the kits of group A, group B, group C and group D and the theoretical value of the sample according to example 5 of the present invention;
FIG. 3 is a graph showing the analysis of linear correlation between the test values of the kits of group A, group B, group C and group D and the theoretical value of the sample according to example 6 of the present invention.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following examples are included to more clearly and clearly illustrate the technical solutions of the present invention by way of illustration. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. The specific embodiments of the present invention are merely illustrative of the invention and are not intended to limit the invention in any way.
EXAMPLE 1 preparation of adiponectin detection kit
The Adiponectin (ADPN) detection kit comprises a reagent R1 and a reagent R2 which are independent of each other and are in double liquid components.
1. Preparation of reagent R1
The preparation method comprises the following steps of fully stirring, uniformly mixing, and storing at 2-8 ℃.
2. Preparation of reagent R2
(1) Preparation of labeling buffer
(2) Preparation of R2 reagent label:
(1) measuring 1.25mL of a labeling buffer solution (boric acid buffer solution and glycine), and adding 100 mu L of latex microspheres (solid content is 10%);
(2) adding 8 mu L of activating agent EDC, quickly mixing uniformly, incubating for 30min, and activating the latex microspheres;
(3) adding an antibody with a final labeling concentration of 0.4mg/mL and BSA of 0.4mg/mL, quickly and uniformly mixing, and then placing on a shaking table for a crosslinking reaction (more than 6 h);
(4) adding 800 mu L of 40g/L BSA, and blocking for 1h;
(5) centrifuging at 12000rpm for 20min to remove supernatant, and ultrasonically dispersing;
(6) and (5) after the preparation of the reagent is finished, putting the reagent into an oven for aging for 2d to obtain a reagent R2.
Example 2 method of Using the kit
In this example, a fully automatic biochemical analyzer (Hitachi 7180) was used in combination with the kit of the present invention to perform sample detection.
(1) Instrument parameter setting
(2) Assay protocol
(3) Computing method
And (3) using a two-point linear calibration mode, taking a linear function as a calculation mode, and making a dose/response curve according to the value of the calibrator and the absorbance change value, wherein the content of the specific growth factor in the sample can be calculated on the dose/response curve according to the absorbance change value.
The detection principle of the invention is as follows: ADPN in a serum sample can be combined with an anti-ADPN antibody in a reagent to form an antigen-antibody-microsphere complex, a certain turbidity is generated, the turbidity is in direct proportion to the content of the antigen when a certain antibody exists, the turbidity is measured at a certain wavelength, and the ADPN can be quantitatively measured through a multi-point calibration curve.
ADPN (mg/L) = CS × Δ AT/Δ AS (mg/L) in sample
( In the formula: delta AT sample tube absorbance value with blank tube absorbance AS reference, delta AS calibration tube absorbance value with blank tube absorbance AS reference, and ADPN concentration in CS calibration solution )
EXAMPLE 3 Performance testing of the kits
In order to verify all performances of the kit, 4 groups of kits are arranged for performance verification:
group A: the kit prepared in the invention example 1;
group B: a commercially available kit;
group a kits were tested according to the method of use described in example 2, and group B was tested according to the instructions.
(1) Accuracy verification
And (3) respectively carrying out accuracy test on the clinical assignment samples by using two groups of kits, setting 2 times of the tests, reading signals by using a full-automatic biochemical analyzer (Hitachi 7180), and calculating the relative deviation between the measured mean value and the target value to carry out accuracy verification. The results are shown in the following table:
table 1 accuracy verification
From the above experimental results, the relative deviation between the test value 1 and the target value 1 of the two sets of kits is 0.80% and-3.80%, respectively, and the relative deviation between the test value 2 and the target value 2 is 0.88% and 3.50%, respectively. The detection accuracy of the kit (group A) prepared in the embodiment 1 of the invention is better than that of the kit (group B) sold in the market.
(2) Precision verification
Selecting a low-value sample, a medium-value sample and a high-value sample of clinical adiponectin, testing the samples by using two sets of kits, respectively repeating the measurement for 10 times, reading signals by a full-automatic biochemical analyzer (Hitachi 7180), respectively calculating a measurement mean value and a standard deviation, and calculating a variation coefficient to perform precision investigation. The results are shown in the following table:
TABLE 2 precision verification
From the above experiment results, the coefficient of variation of the two sets of kits in the detection of the low value sample is 0.60% and 6.38%, the coefficient of variation of the median sample is 1.29% and 1.52%, and the coefficient of variation of the high value sample is 0.91% and 0.98%, respectively.
(3) Linear range verification
Selecting a clinical ultrahigh-value sample and a clinical low-value sample, then utilizing the high-value sample and the low-value sample to prepare each concentration gradient sample in proportion, using two groups of kits to test the samples respectively, repeatedly measuring each sample for 2 times, reading signals through a full-automatic biochemical analyzer (Hitachi 7180), and calculating a measured mean value respectively to perform linear range investigation. The results are shown in the following table:
TABLE 3 Linear Range verification
Note: the relative deviation is the relative deviation of the mean from the theoretical value.
From the above experimental results, it can be seen that the kits prepared in example 1 of the present invention (group A) and the commercial kits (group B) have small relative deviations between the detection values and the theoretical values in the linear range of the sample concentration of 1.5-52.4 mg/L. Meanwhile, the detection results of the two groups of kits and the theoretical value of the sample concentration are subjected to correlation analysis (as shown in the attached figure 1): wherein the correlation R of the group A detection values with the theoretical values 2 Is 0.9991 in group B R 2 Is 0.9985. The experimental results show that the kits prepared in example 1 of the present invention (group a) all have a better linear range than the commercially available kits (group B).
(4) Difference between batches
With the two batches of kits a and C of the present invention prepared in example 1, clinical assigned samples were selected, each sample was measured twice, signals were read by a full-automatic biochemical analyzer (hitachi 7180) for inter-batch difference investigation, and the test results are shown in the following table:
TABLE 4 verification of inter-batch differences
Note: ABS is the sample detection reactivity.
According to the experimental results, the batch-to-batch difference of the two batches of the kit A and the kit C prepared in the embodiment 1 can be controlled within 4%, and the experimental results show that the kit provided by the invention has small batch-to-batch difference and good manufacturing stability.
Example 4 influence of microsphere particle size on the detection Performance of the kit
In order to verify the influence of the particle size of the microspheres on the detection performance of the kit, 6 groups of kits are arranged:
group A: the kit prepared in the invention example 1;
group B: the kit differs from the kit of example 1 only in that the latex microspheres have a particle size of 100nm;
group C: the kit differs from the kit of example 1 only in that the latex microspheres have a particle size of 150nm;
group D: the kit differs from the kit of example 1 only in that the latex microspheres have a particle size of 230nm;
group E: the kit differs from the kit of example 1 only in that the latex microspheres have a particle size of 250nm;
and group F: the kit differed from the kit of example 1 only in that the latex microspheres had a particle size of 300nm.
(1) Precision verification
Selecting low-value clinical adiponectin samples, testing the samples by using six groups of kits, respectively repeating the measurement for 10 times for each sample, reading signals by a full-automatic biochemical analyzer (Hitachi 7180), respectively calculating a measurement mean value and a standard deviation, and calculating a variation coefficient to perform precision investigation. The results are shown in the following table:
TABLE 5 verification of precision
| Sample(s) | Group A | Group B | Group C | Group D | Group E | Group F |
| 1 | 2.12 | 2.73 | 2.03 | 2.10 | 2.22 | 1.98 |
| 2 | 2.13 | 2.34 | 2.22 | 2.09 | 2.14 | 2.32 |
| 3 | 2.13 | 2.89 | 2.15 | 2.13 | 2.09 | 2.43 |
| 4 | 2.09 | 2.12 | 2.00 | 2.08 | 2.06 | 2.22 |
| 5 | 2.12 | 2.23 | 2.03 | 2.14 | 2.24 | 2.09 |
| 6 | 2.11 | 2.34 | 2.11 | 2.03 | 2.12 | 2.04 |
| 7 | 2.12 | 2.53 | 2.00 | 2.10 | 2.53 | 2.42 |
| 8 | 2.12 | 2.32 | 2.27 | 2.15 | 2.34 | 2.31 |
| 9 | 2.10 | 2.22 | 2.13 | 2.05 | 2.11 | 2.07 |
| 10 | 2.13 | 2.13 | 2.07 | 2.11 | 2.20 | 2.20 |
| Mean value | 2.12 | 2.39 | 2.10 | 2.10 | 2.21 | 2.21 |
| SD | 0.01 | 0.26 | 0.09 | 0.04 | 0.14 | 0.16 |
| CV | 0.63％ | 10.73％ | 4.42％ | 1.81％ | 6.41％ | 7.25％ |
The experimental results show that the adiponectin detection kit prepared by using the latex microspheres with the particle size range of 150-230nm has good low-value precision, and particularly, the detection CV value of the low-value sample can be controlled within 2% when the particle size of the latex microspheres is within the range of 200-230nm.
Example 5 Effect of marker proteins on kit Performance
In order to verify the influence of the marker protein on the detection performance of the kit, 4 groups of kits are arranged:
group A: the kit prepared in example 1 of the present invention;
group B: the kit differs from the kit of example 1 only in that BSA is not added during labeling;
group C: the kit is different from the kit in the embodiment 1 only in that BSA is not added in the labeling process, and casein with the final concentration of 0.4mg/mL is added;
group D: the kit is different from the kit in the embodiment 1 only in that BSA (bovine serum albumin) is not added in the labeling process, and 0.4mg/mL of alanine is added;
(1) Linear range verification
Selecting a clinical ultrahigh-value sample and a clinical low-value sample, then preparing each concentration gradient sample by using the high-value sample and the low-value sample in proportion, respectively testing the samples by using the four groups of kits, respectively repeating the measurement for 2 times for each sample, reading signals by a full-automatic biochemical analyzer (Hitachi 7180), and respectively calculating a measurement mean value to perform linear range investigation. The results are shown in the following table:
TABLE 6 kit Linear Range verification
Note: the relative deviation is the relative deviation of the mean from the theoretical value.
And (3) carrying out correlation analysis on the detection results of the four groups of kits and the theoretical value of the sample concentration (as shown in the attached figure 2): correlation R of A group test detection values and theoretical values 2 Is 0.9997, group B R 2 Is 0.9879, group C R 2 Is 0.9927, group D R 2 Is 0.9994. The experimental result shows that BSA is added in the labeling process of the latex microspheres and the antibody, so that the technical problem of poor linear range caused by the use of the microspheres with large particle size can be effectively solved.
Example 6 influence of labeling ratio of latex microsphere, antibody and BSA on detection Performance of kit
In order to verify the influence of the labeling proportion of the latex microspheres, the antibodies and the BSA on the detection performance of the kit, the following 4 groups of kits are arranged:
group A: the kit prepared in the invention example 1;
group B: the kit is different from the kit in example 1 only in that in the latex microsphere antibody labeling process, the mass ratio of the latex microsphere to the antibody to the BSA is 1;
group C: the kit is different from the kit in example 1 only in that in the latex microsphere antibody labeling process, the mass ratio of the latex microspheres to the antibodies to the BSA is 1;
group D: the kit is different from the kit in example 1 only in that in the latex microsphere antibody labeling process, the mass ratio of the latex microspheres to the antibodies to the BSA is 1;
(1) Precision verification
Selecting a low-value sample, a medium-value sample and a high-value sample of clinical adiponectin, testing the samples by using four groups of kits, respectively repeating the measurement for 10 times, reading signals by a full-automatic biochemical analyzer (Hitachi 7180), respectively calculating a measurement mean value and a standard deviation, and calculating a variation coefficient to perform precision investigation. The results are shown in the following table:
TABLE 7 precision verification
From the above experimental results, the coefficient of variation of the four sets of kits in the low value sample detection is 0.75%, 10.75%, 1.25% and 7.81%, respectively, and the experimental results show that, in the process of labeling the latex microspheres and the antibodies, the mass ratio of the latex microspheres, the antibodies and the BSA is 1: (0.05-0.1): and when the concentration is within the range of (0.05-0.1), the low-value precision of the detection kit is highest.
(2) Linear range verification
Selecting a clinical ultrahigh-value sample and a clinical low-value sample, then preparing each concentration gradient sample by using the high-value sample and the low-value sample in proportion, respectively testing the samples by using the four groups of kits, respectively repeating the measurement for 2 times for each sample, reading signals by a full-automatic biochemical analyzer (Hitachi 7180), and respectively calculating a measurement mean value to perform linear range investigation. The results are shown in the following table:
TABLE 8 kit Linear Range verification
And (3) carrying out correlation analysis on the detection results of the 4 groups of kits and the theoretical value of the sample concentration (as shown in the attached figure 3): correlation R of A group of test values and theoretical values 2 Is 0.9997, group B R 2 Is 0.9895, group C R 2 Is 0.9958, group D R 2 Is 0.9782. The experimental results show that in the process of labeling the latex microspheres and the antibodies, the ratio of the latex microspheres to the antibodies to BSA is 1: (0.05-0.1): when the range is (0.05-0.1), the linear range of the detection kit is good.
Example 7 Effect of recombinant mAbs on kit run-to-run
In order to verify the influence of the recombinant monoclonal antibody on the detection performance of the kit, 3 groups of kits are arranged:
group A: the kit prepared in the invention example 1;
group B: the preparation method of the kit is different from that of the kit in the embodiment 1 only in that the kit does not contain a recombinant monoclonal antibody component and adopts an antibody prepared by conventional ascites injection;
group C: the preparation method of the kit is different from that of the kit in example 1 only in that the kit does not contain a recombinant monoclonal antibody component, and the antibody is prepared by adopting a conventional fermentation method.
(1) Difference between batches
The three groups of kits are used for measuring the same low-value clinical assigned sample and high-value clinical assigned sample, each level is repeatedly measured for three times, signals are read through a full-automatic biochemical analyzer (Hitachi 7180), batch-to-batch difference investigation is carried out, and experimental results show that the batch-to-batch difference of the kit A prepared in the embodiment 1 can be controlled within 4%, the batch-to-batch difference of the kit B can be controlled within 10%, the batch-to-batch difference of the kit C can be controlled within 8%, and the experimental results show that the adiponectin detection kit prepared by the recombinant monoclonal antibody can effectively improve the batch-to-batch difference of the kit and ensure the stability of the preparation of the kit.
In conclusion, the recombinant monoclonal antibody and the small molecular protein BSA are simultaneously marked by adopting the large-particle-size microspheres, so that the linear range of the reagent is ensured, the precision of the reagent is greatly improved, and particularly the detection precision of a low-value sample is improved. Meanwhile, compared with the traditional ascites antibody, the recombinant monoclonal antibody has the advantages of higher purity, less impure protein, higher yield and the like, so that the raw material difference is small, the reagent difference can be strictly controlled, and the kit has better difference and detection stability.
Although embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments without departing from the principles and spirit of the present invention.
Claims (8)
1. A kit for high-sensitivity quantitative determination of adiponectin is characterized by comprising a reagent R1 and a reagent R2;
the reagent R1 comprises the following components in percentage by weight: 50-150mmol/L buffer solution, 0.1-1mol/L inorganic salt ion 2-10g/L surfactant;
the reagent R2 comprises the following components in percentage by weight: 70-120mmol/L buffer solution, 0.1-1mol/L inorganic salt ions and latex microspheres coated with adiponectin monoclonal antibodies;
the preparation process of the latex microsphere coated with the adiponectin monoclonal antibody comprises the following steps: adding adiponectin monoclonal antibody, latex microspheres and BSA to coat the latex microspheres and the antibody, mixing uniformly, incubating for 5-20h, and adding blocking agent BSA;
the mass ratio of the latex microspheres to the adiponectin monoclonal antibody to the BSA is 1 (0.05-0.1): (0.05-0.1);
the adiponectin monoclonal antibody is a recombinant monoclonal antibody;
the particle size of the latex microsphere is 150-250nm.
2. The kit for high-sensitivity quantitative determination of adiponectin as claimed in claim 1, wherein said reagent R1 further comprises 5-20g/L coagulant, 0.5-1.5g/L preservative; the reagent R2 also comprises 20-80g/L of blocking protein and 10-30g/L of carbohydrate.
3. The kit for high-sensitivity quantitative determination of adiponectin as claimed in claim 2, wherein the latex microspheres have a particle size of 200-230nm.
4. The kit for high-sensitivity quantitative determination of adiponectin as claimed in claim 3, wherein the buffer in the reagent R1 is at least one selected from citric acid monohydrate, MOPS, glycine, tris and HEPES, the inorganic salt ion is at least one selected from sodium chloride and potassium chloride, the coagulant is at least one selected from PEG6000 or PEG8000, the surfactant is at least one selected from Tween-20, brij58, TX-100 or A90, and the preservative is at least one selected from PC-300 and sodium azide; the buffer solution in the reagent R2 is selected from at least one of MOPS, glycine, tris or HEPES, the inorganic salt ions are selected from at least one of sodium chloride or potassium chloride, the blocking protein is selected from at least one of BSA or casein, and the carbohydrate is selected from at least one of trehalose or sucrose.
5. The kit for the high-sensitivity quantitative determination of adiponectin as claimed in claim 4, wherein said reagent R1 has a pH of 5.0 to 7.0 and said reagent R2 has a pH of 5.0 to 7.0.
6. The kit for the high-sensitivity quantitative determination of adiponectin as claimed in claim 5, wherein said reagent R1 has a pH of 6.0 to 7.0, and said reagent R2 has a pH of 6.0 to 7.0.
7. A method for preparing a kit for the high sensitivity quantitative determination of adiponectin according to any one of claims 1 to 6, comprising the steps of:
(1) Preparation of reagent R1
The reagent R1 is prepared according to the following formula:
50-150mmol/L buffer solution, 0.1-1.0 mmol/L inorganic salt ion, 5-20g/L coagulant, 2-10g/L surfactant and 0.5-1.5g/L preservative;
the buffer solution is selected from at least one of citric acid monohydrate, MOPS, glycine, tris or HEPES, the inorganic salt ions are selected from at least one of sodium chloride or potassium chloride, the coagulant is selected from at least one of PEG6000 or PEG8000, the surfactant is selected from at least one of Tween-20, brij58, TX-100 or A90, and the preservative is selected from at least one of PC-300 or sodium azide;
(2) Preparation of reagent R2
(1) Preparation of R2 labeling buffer
The R2 labeling buffer solution is prepared according to the following formula:
buffer solution 70-120mmoL, inorganic salt ion 0.1-1mol/L and saccharide substance 10-30g/L;
the marking buffer is at least one of boric acid buffer, glycine buffer or Tris buffer, the inorganic salt ions are at least one of sodium chloride or potassium chloride, and the saccharide substances are at least one of trehalose or sucrose;
(2) preparation of reagent R2
Adding the latex microsphere latex into a marking buffer solution, then adding an activating agent EDC, uniformly mixing, and incubating for 20-60min; simultaneously adding the adiponectin monoclonal antibody, the latex microsphere and BSA, uniformly mixing, and incubating for 5-20h; adding a blocking agent BSA, incubating for 0.5-2h, centrifuging to remove supernatant, and performing ultrasonic treatment to obtain a reagent R2;
the mass ratio of the latex microspheres to the adiponectin monoclonal antibody to the BSA is 1 (0.05-0.1): (0.05-0.1).
8. The method for preparing the kit for the high-sensitivity quantitative determination of adiponectin according to claim 7, wherein the adiponectin monoclonal antibody is a recombinant monoclonal antibody.
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| CN202066863U (en) * | 2010-10-29 | 2011-12-07 | 厦门大学附属中山医院 | Particle-enhanced turbidimetric immunoassay kit for adiponectin detection |
| CN101968492B (en) * | 2010-10-29 | 2013-01-09 | 厦门大学附属中山医院 | Particle-enhanced turbidimetric immune assay kit for detecting adiponectin and preparation method thereof |
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| CN111239421A (en) * | 2020-02-19 | 2020-06-05 | 安徽大千生物工程有限公司 | Latex-enhanced immunoturbidimetry kit for quantitatively detecting adiponectin ADPN and preparation and application methods thereof |
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| CN113447662A (en) * | 2021-07-05 | 2021-09-28 | 北京万泰德瑞诊断技术有限公司 | Latex immunoturbidimetry kit for improving detection capability of high molecular weight adiponectin |
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