CN114167063A - Latex enhanced competitive immunoturbidimetric assay kit and method and application of tigogenin - Google Patents
Latex enhanced competitive immunoturbidimetric assay kit and method and application of tigogenin Download PDFInfo
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- CN114167063A CN114167063A CN202011616064.6A CN202011616064A CN114167063A CN 114167063 A CN114167063 A CN 114167063A CN 202011616064 A CN202011616064 A CN 202011616064A CN 114167063 A CN114167063 A CN 114167063A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a latex-enhanced competitive immunoturbidimetric assay kit, a latex-enhanced competitive immunoturbidimetric assay method and application of tigogenin. The latex-enhanced competitive immunoturbidimetric assay kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises a target detector-carrier compound and tigogenin, and the reagent R2 comprises latex microspheres coupled with an anti-target detector antibody. The latex enhanced competitive immunoturbidimetric assay kit has excellent accuracy and stability, and can be highly consistent with a gold standard HPLC detection value.
Description
Technical Field
The invention relates to the field of biomedical detection, in particular to a latex-enhanced competitive immunoturbidimetric assay kit, a latex-enhanced competitive immunoturbidimetric assay method and application of tigogenin.
Background
Immunoturbidimetry (Turbidimetric inhibition assay) is an antigen-antibody binding kinetic assay. The basic principle is as follows: when the antigen and antibody react in a special dilution system and the ratio is appropriate (generally, it is specified that the antibody is in excess), the formed soluble immune complex precipitates from the liquid phase under the action of a polymerization promoter (polyethylene glycol or the like) in the dilution system to form microparticles, and turbidity appears in the reaction solution. When the antibody concentration is fixed, the amount of the immunocomplex formed increases with the increase in the amount of the antigen in the sample, and the turbidity of the reaction solution also increases. The content of the antigen in the sample can be calculated by measuring the turbidity of the reaction solution and comparing with a series of standard products.
Different from the common immunoturbidimetry, the latex-enhanced competitive immunoturbidimetry is mainly applied to the detection of small-molecule drugs and comprises two modes: target detection object coupling microspheres and antibody coupling microspheres. The basic principle of the antibody coupling microsphere is as follows: the method comprises the steps of coupling an anti-target detection object antibody on the surface of a latex microsphere to form the latex microsphere coupled with the antibody, wherein a target detection object in a sample and a target detection object-carrier compound in a kit compete to combine with the antibody coupled on the surface of the latex microsphere, the higher the concentration of the target detection object in the sample is, the higher the combination degree of the antibody on the surface of the latex microsphere is, the smaller the aggregation degree (turbidity) of latex which can be triggered by the target detection object-carrier compound is, the negative correlation between the turbidity of a reaction system and the concentration of the target detection object in the sample is, establishing a calibration curve by measuring the absorbance of different turbidity reaction systems, and calculating the concentration of the target detection object in the sample.
However, small molecule target detection substances in a sample to be tested often have a high binding rate with proteins. For example, the binding rate of the posaconazole small-molecule drug and the protein is greater than 98%. In addition, impurities such as lipids are also present in the sample. These all have interference to latex enhancement competition immunoturbidimetry detection, influence the accuracy and the stability of testing result.
In order to solve the problem, the existing reports try to add triton or tween-20 into the detection reagent, but the actual effect is not ideal, the accuracy and the stability are still not good, and the problems cannot be completely solved.
Disclosure of Invention
The invention aims to overcome the defects of low accuracy and stability of the conventional latex-enhanced competitive immunoturbidimetric assay, and provides a latex-enhanced competitive immunoturbidimetric assay kit, a latex-enhanced competitive immunoturbidimetric assay method and application of tigogenin. The latex enhanced competitive immunoturbidimetric assay kit has excellent accuracy and stability, and can be highly consistent with a gold standard HPLC detection value.
One of the technical schemes provided by the invention is as follows: a latex-enhanced competitive immunoturbidimetric assay kit, comprising a reagent R1 and a reagent R2, wherein the reagent R1 comprises a target detector-carrier complex and tigogenin, and the reagent R2 comprises latex microspheres coupled with an anti-target detector antibody.
In the present invention, the concentration of the tigogenin in the reagent R1 is the conventional concentration of the surfactant, and is preferably 0.1 to 1.0 wt%, more preferably 0.4 to 0.8 wt%, and even more preferably 0.6 wt%.
In the invention, the target detection substance refers to various small molecule compounds in a biological or non-biological sample which can be detected by latex enhanced competitive immunoturbidimetry detection, such as clozapine, olanzapine, posaconazole (posaconazole), vancomycin, melamine, clenbuterol and the like, and posaconazole is preferred.
In the present invention, the carrier used in the target detector-carrier complex is a small molecule hapten carrier conventional in the art, such as a protein or polysaccharide, specifically Bovine Serum Albumin (BSA), Ovalbumin (OVA) or Keyhole Limpet Hemocyanin (KLH), etc. The target detection object-carrier compound is preferably posaconazole-bovine serum albumin.
In the present invention, the concentration of the target detector-carrier complex in the reagent R1 is generally 200-600ng/ml, preferably 300-500ng/ml, such as 400ng/ml, as is conventional in the art.
In the invention, the anti-target detection object antibody used in the latex microsphere coupled with the anti-target detection object antibody is protein which can generate specific immunological binding reaction with the target detection object and has the specific structural characteristics of the antibody.
In the present invention, the latex microspheres conjugated with the anti-target detection substance antibodies can be prepared by conventional methods in the art. The latex microspheres can be selected from latex microspheres which are commonly used in the field of latex-enhanced competitive immunoturbidimetric assay and can be coupled with antibody proteins, such as polystyrene latex microspheres with amino groups on the surface or polystyrene latex microspheres with carboxyl groups on the surface, and preferably polystyrene latex microspheres with carboxyl groups on the surface. The average particle diameter of the polystyrene latex microspheres containing carboxyl groups on the surface is preferably 70-470nm, more preferably 150-350nm, and further preferably 200nm or 240 nm. The surface carboxyl density of the polystyrene latex microspheres containing carboxyl on the surface is preferably 0.03-0.30mmol/g, more preferably 0.05-0.18mmol/g, and further preferably 0.1mmol/g or 0.134 mmol/g.
In the present invention, the concentration of the anti-target detection substance antibody-conjugated latex microspheres in the reagent R2 can be as conventional in the art, and is generally 0.05-0.16 wt%, preferably 0.09-0.13 wt%.
The reagent R1 and the reagent R2 in the present invention are in the form of buffer solutions as is conventional in the art. The buffer used in the buffer solution may be selected conventionally in the art, for example, a buffer pair consisting of any of the following reagents and its conjugate acid or its conjugate base: 2- (N-morphine) ethanesulfonic acid (MES), Tris (hydroxymethyl) aminomethane (Tris), 3- (N-morphino) propanesulfonic acid (MOPS), Triethanolamine (TRA), and Diethanolamine (DEA). The pH of the buffer solution is preferably 5.0 to 9.0, more preferably 5.5 to 7.5. The concentration of the buffer may be selected as is conventional, and is generally preferably in the range of 15 to 100 mM.
Preferably, the buffer solution comprises an alkali metal salt, which may be selected as is conventional in the art, for example comprising one or more of the following alkali metal salts: na (Na)2HPO3、NaH2PO3、NaCl、KCl、K2HPO3And KH2PO3(ii) a The concentration of the alkali metal salt is preferably 0.1 to 0.2mol/L, more preferably 0.14 to 0.18 mol/L.
The buffer solution may further comprise one or more of a stabilizer, a blocking agent, and a preservative.
Wherein the stabilizer is selected as conventional in the art, such as D-trehalose and/or sucrose, preferably D-trehalose.
Among these, the preservative may be selected as is conventional in the art, such as Proclin 300 and/or sodium azide, preferably sodium azide.
Wherein, the blocking agent can be selected according to the routine selection in the field, such as one or more selected from bovine serum albumin, CE210 and CE510, and bovine serum albumin and CE510 are preferred.
Preferably, the reagent R1 further contains methanol, which can further improve the accuracy and stability of the detection. The concentration of methanol in the reagent R1 is preferably 5 to 20 vol%, more preferably 10 vol%.
Preferably, the latex-enhanced competitive immunoturbidimetric assay kit of the present invention may further comprise a standard for the target assay for use in calibration.
Preferably, the latex-enhanced competitive immunoturbidimetric assay kit of the present invention comprises reagent R1 and reagent R2, wherein:
the reagent R1 comprises MES-NaOH buffer solution, bovine serum albumin, NaCl, D-trehalose, tigogenin, methanol, sodium azide and posaconazole-carrier compound,
the reagent R2 comprises HEPES-HCl buffer solution, a sealant, NaCl, D-trehalose, sodium azide, bovine serum albumin and latex microspheres coupled with anti-posaconazole antibodies.
More preferably, the latex-enhanced competitive immunoturbidimetric assay kit comprises reagent R1 and reagent R2, wherein:
the reagent R1 comprises 20-30mM MES-NaOH buffer solution, 0.08-0.12 wt% bovine serum albumin, 0.14-0.18mol/L NaCl, 0.08-0.12 wt% D-trehalose, 0.1-1.0 wt% tigogenin, 5-20 vol% methanol, 0.08-0.12 wt% sodium azide and 0.2-0.6 mu g/ml posaconazole-carrier compound, the pH value of the reagent R1 is 5.5-6.0,
the reagent R2 comprises 48-52mM HEPES-HCl buffer solution, 0.08-0.12 vol% CE510, 0.14-0.18mol/L NaCl, 0.08-0.12 wt% D-trehalose, 0.08-0.12 wt% sodium azide, 0.08-0.12 wt% bovine serum albumin and 0.09-0.13 wt% anti-posaconazole antibody coupled polystyrene latex microspheres, and the pH of the reagent R2 is 7.4-7.8.
In a specific embodiment of the invention, the latex-enhanced competitive immunoturbidimetric assay kit comprises a reagent R1 and a reagent R2, wherein,
the reagent R1 comprises 25mM MES-NaOH buffer solution, 0.1 wt% bovine serum albumin, 0.17mol/L NaCl, 0.1 wt% D-trehalose, 10 vol% methanol, 0.1 wt% sodium azide, 0.4 mu g/ml posaconazole-bovine serum albumin complex and 0.6 wt% tigogenin, the pH value of the reagent R1 is 5.65,
the reagent R2 comprises 50mM HEPES-HCl buffer solution, 0.1 vol% CE510, 0.17mol/L NaCl, 0.1 wt% D-trehalose, 0.1 wt% sodium azide, 0.1 wt% bovine serum albumin and 0.11 wt% anti-posaconazole antibody coupled polystyrene latex microspheres, and the pH of the reagent R2 is 7.6.
In the invention, the reagent R1 and the reagent R2 in the latex-enhanced competitive immunoturbidimetric assay kit can be prepared by simply mixing according to a conventional method in the field.
The second technical scheme provided by the invention is as follows: the application of the tigogenin as an auxiliary agent in the preparation of the latex-enhanced competitive immunoturbidimetric assay kit according to one of the technical schemes.
The third technical scheme provided by the invention is as follows: a latex enhanced competitive immunoturbidimetric assay method uses the latex enhanced competitive immunoturbidimetric assay kit according to one of the technical schemes for detection.
As is conventional in the art, the latex-enhanced competitive immunoturbidimetric assay comprises the steps of:
(1) mixing the sample with the reagent R1 uniformly, incubating,
(2) and (2) uniformly mixing the mixed liquor obtained in the step (1) with the reagent R2, detecting the initial absorbance A1, detecting the absorbance A2 after incubation, calculating the absorbance difference delta A-A2-A1, and obtaining the content of the substance to be detected in the sample according to a standard curve.
Wherein the standard curve is obtained by detecting a standard substance according to the routine in the field.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: the latex enhanced competitive immunoturbidimetric assay kit can reduce the interference of impurities in samples when different samples are assayed, has excellent accuracy and stability compared with the existing latex enhanced competitive immunoturbidimetric assay kit, and the assay result can be highly consistent with the gold standard HPLC assay value. The addition of methanol can further improve the detection accuracy and stability of the latex enhanced competitive immunoturbidimetric assay kit.
Drawings
FIG. 1 is a plot of the latex-enhanced competitive immunoturbidimetry measurements of reagent R1 without surfactant as compared to the HPLC measurements of the reference example in the comparative example.
FIG. 2 is a plot of the latex-enhanced competitive immunoturbidimetry measurements for reagent R1 using triton versus the HPLC measurements of the reference example in the comparative example.
FIG. 3 is a plot of the latex-enhanced competitive immunoturbidimetry measurements for reagent R1 using Tween-20 in comparison to the HPLC measurements of the reference example.
FIG. 4 is a plot of the latex enhanced competitive immunoturbidimetry measurements of example 1 as fitted to the HPLC measurements of the reference example.
FIG. 5 is a plot of the latex enhanced competitive immunoturbidimetry measurements of example 2 as fitted to the HPLC measurements of the reference example.
FIG. 6 is a plot of the latex enhanced competitive immunoturbidimetry measurements of example 3 as fitted to the HPLC measurements of the reference example.
FIG. 7 is a plot of the latex enhanced competitive immunoturbidimetry measurements of example 4 as fitted to the HPLC measurements of the reference example.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Reagent:
all standards in the examples were purchased from TRC (Toronto Research Chemicals) standards. Polystyrene latex microspheres (average particle size 240nm, density of surface carboxyl groups 0.134mmol/g) were purchased from JSR Life Sciences.
The preparation process of anti-posaconazole antibody and anti-posaconazole antibody-conjugated polystyrene latex microspheres is described in journal of cell and molecular immunology 2015, 31: at page 256, polyclonal antibodies to GP73 were prepared and a latex enhanced immunoturbidimetry of GP73 was established.
The posaconazole-bovine serum albumin complex is prepared by the following steps:
1. accurately weighing the following reagents into a clean ampoule: 20mg BSA and 3ml 50mM PBS buffer (pH 7.75. + -. 0.05); after dissolving, standing for 30-60min to obtain a product 1.
2. Weighing 10mg of carboxylated posaconazole, adding into a small clean penicillin bottle, adding 300 μ l of methanol, and adding 10mg of EDCI and 20mg of NHS under magnetic stirring; standing for 90min at room temperature (20-25 ℃) to obtain a product 2.
3. Dropwise adding the obtained product 2 into the product 1; after the dropwise addition is finished, the solution is clearer; reacting at room temperature for 10h to obtain a product 3.
4. Putting the product 3 into a dialysis bag, placing the bag in 4L 0.025M PB buffer solution (pH 7.75 +/-0.05, no NaCl), dialyzing for 3 x 28 hours, and changing the solution for 3 times to obtain a product 4; adding NaN into the product 43And obtaining the posaconazole-bovine serum albumin when the final concentration is 0.05 wt%.
Latex enhanced competitive immunoturbidimetry detection steps and conditions:
before detection, the parameters of the latex enhanced competitive immunoturbidimetric assay device (Hitachi 7180 automatic biochemical analyzer) are set according to the parameters in Table 1:
TABLE 1 latex enhanced competitive immunoturbidimetric assay parameters
Standard curve:
mixing 25 μ l posaconazole standard with reagent R1, incubating at 37 ℃ for 5 minutes, mixing with 40 μ l reagent R2, incubating at 37 ℃ for about 20 seconds, reading absorbance value a1, incubating for 5 minutes, reading absorbance value a2, calculating Δ a ═ a2-a1, and plotting a standard curve.
Sample detection:
and (3) uniformly mixing 25 mu l of the sample with the reagent R1, incubating at 37 ℃ for 5 minutes, uniformly mixing with 40 mu l of the reagent R2, incubating at 37 ℃ for about 20 seconds, reading the absorbance value A1, incubating for 5 minutes, reading the absorbance value A2, calculating delta A (A2-A1), and calculating the content of posaconazole in the sample according to a standard curve.
Example 1 latex enhanced competitive immunoturbidimetry for Posaconazole (0.6 wt% tigogenin +10 vol% methanol)
Reagent R1(100 μ l): 25mM MES-NaOH, 0.1 wt% bovine serum albumin, 0.17mol/L NaCl, 0.1 wt% D-trehalose, 10 vol% methanol, 0.1 wt% sodium azide, 0.4. mu.g/ml posaconazole-bovine serum albumin complex and 0.6 wt% tigogenin, pH of reagent R1 is 5.65;
reagent R2(40 μ l): 50mM HEPES-HCl, 0.1 vol% CE510, 0.17mol/L NaCl, 0.1 wt% D-trehalose, 0.1 wt% sodium azide, 0.1 wt% bovine serum albumin, 0.11 wt% anti-posaconazole antibody-coupled polystyrene latex microspheres (concentration of anti-posaconazole antibody in R2 is 0.2mg/ml), pH of reagent R2 is 7.6.
The 22 plasma samples were subjected to latex-enhanced competitive immunoturbidimetric assay using the above-described reagents R1 and R2.
The measurements of posaconazole concentration in 22 plasma samples are shown in table 2 and the fitted curve to the HPLC measurements is shown in fig. 4.
Example 2 latex-enhanced competitive immunoturbidimetry for the detection of posaconazole (0.1 wt% tigogenin +5 vol% methanol)
The components and the detection method in the reagents R1 and R2 were the same as in example 1 except that 0.1 wt% tigogenin and 5 vol% methanol were used.
The 22 plasma samples were subjected to latex-enhanced competitive immunoturbidimetric assay using the above-described reagents R1 and R2.
The measurements of posaconazole concentration in 22 plasma samples are shown in table 2 and the fitted curve to the HPLC measurements is shown in fig. 5.
Example 3 detection of posaconazole by latex-enhanced competitive immunoturbidimetry (1.0 wt% tigogenin +20 vol% methanol)
The components and the detection method in the reagents R1 and R2 were the same as in example 1 except that 1.0 wt% tigogenin and 20 vol% methanol were used.
The 22 plasma samples were subjected to latex-enhanced competitive immunoturbidimetric assay using the above-described reagents R1 and R2.
The measurements of posaconazole concentration in 22 plasma samples are shown in table 2 and the fitted curve to the HPLC measurements is shown in fig. 6.
Example 4 latex-enhanced competitive immunoturbidimetry for Posaconazole (0.6 wt% tigogenin +0 vol% methanol)
The components and detection methods in reagents R1 and R2 were the same as in example 1, except that 0.6 wt% tigogenin was used and no methanol was added.
The 22 plasma samples were subjected to latex-enhanced competitive immunoturbidimetric assay using the above-described reagents R1 and R2.
The measurements of posaconazole concentration in 22 plasma samples are shown in table 2 and the fitted curve to the HPLC measurements is shown in fig. 7.
Reference example HPLC detection of posaconazole
HPLC internal standard method
The operation process comprises the following steps: taking 150 mu L of a plasma sample, putting the plasma sample into a 1.5ml EP tube, adding 450 mu L of a pretreatment liquid (a diazepam acetonitrile solution, 0.5mg/L) containing an internal standard, covering the tube, uniformly mixing the tube on a vortex mixer for 1min, centrifuging the tube at 13000r/min for 10min, taking supernatant, filtering the supernatant into a sample feeding bottle by using a filter of 0.22 mu m (a nylon filter), and carrying out sample feeding analysis, wherein the sample feeding amount is 20 mu L. Chromatographic conditions are as follows:
a detection instrument: ultimate 3000 high performance liquid chromatograph.
And (3) analyzing the column: AgilentHC (2) -C18 (250X 4.6mm,5 μm)
Internal standard: diazepam;
column temperature: 35 ℃;
sample introduction amount: 20 mu l of the mixture;
mobile phase: phase A (water), phase B (acetonitrile), acetonitrile is chromatographic grade; the water is pure water, and the ultrasonic treatment is carried out for 10min after the filtration.
Gradient elution, elution procedure: 0-5min, VB: 37 percent;
5-6min，VB：37％-57％；
6-7min，57-60％；
7-8min，VB：60％；
8-9min，VB：60％-75％；
9-11min，VB：75％；
12min，VB：75％-65％；
12-15min，VB：65％；
15-16min，VB：65％-37％；
16-30min，VB：37％。
where VB is expressed as the volume fraction of phase B in the mobile phase.
Flow rate: 1 ml/min.
The concentration of posaconazole in each of the 22 plasma samples was determined according to the above procedure, and the results are shown in table 2.
Comparative example existing Immunoturbidimetric assay kit (containing triton, Tween-20 and no surfactant) for detecting posaconazole
Latex enhanced competitive immunoturbidimetric assay (no surfactant added to the reagents): the other components and the detection method are the same as those in example 1 except that the tigogenin is not added; the measurements of posaconazole concentration in 22 plasma samples are shown in table 2 and the fitted curve to the HPLC measurements is shown in fig. 1.
Latex enhanced competitive immunoturbidimetric assay (triton added to reagent): the components and the detection method are the same as those in example 2 except that the sisal saponin is changed to 0.1 wt% of triton; the measurements of posaconazole concentration in 22 plasma samples are shown in table 2, and the fitted curve to the HPLC measurements is shown in fig. 2.
Latex enhanced competitive immunoturbidimetric assay (reagent with tween-20): the components and the detection method are the same as those in example 3 except that the weight percent of the tigogenin is changed to 0.05 weight percent of the tween-20, and the weight percent of the tigogenin is 0.1 to 1.2 weight percent; the measurements of posaconazole concentration in 22 plasma samples are shown in table 2 and the fitted curve to the HPLC measurements is shown in fig. 3.
Effects of the embodiment
The results of the tests of examples 1 to 4, reference examples and comparative examples are shown in Table 2, and the results of the tests of examples 1 to 4 and comparative examples are fitted to the values measured in the reference examples, respectively, and are shown in FIGS. 1 to 7.
According to Table 2 and FIGS. 1 to 7, the fitting curve R of the results of the detection in examples 1 to 4 of the present invention and the results of the HPLC detection in the reference example2Reaches 0.977 and above, and the fitting curve R of the detection result of the comparative example and the HPLC detection result of the reference example2Not higher than 0.9509. Therefore, compared with the existing latex enhanced competitive immunoturbidimetric assay kit without surfactant or containing triton or tween-20, the latex enhanced competitive immunoturbidimetric assay kit containing the tigogenin has the advantages of obviously improved accuracy and stability, and even can be highly consistent with a gold standard HPLC detection value.
As can be seen from the comparison between FIG. 4 and FIG. 7, the addition of methanol can further improve the accuracy and stability of the detection of the latex-enhanced competitive immunoturbidimetric assay kit of the present invention.
TABLE 2 detection values of latex enhanced competitive immunoturbidimetry for examples 1-4, reference examples, and comparative examples
Note: all measurements in the table are in units of μ g/ml.
Claims (10)
1. The kit is characterized by comprising a reagent R1 and a reagent R2, wherein the reagent R1 comprises a target detector-carrier compound and tigogenin, and the reagent R2 comprises latex microspheres coupled with an anti-target detector antibody.
2. The latex-enhanced competitive immunoturbidimetric assay kit according to claim 1, wherein the concentration of tigogenin in said reagent R1 is 0.1-1.0 wt%, preferably 0.4-0.8 wt%, more preferably 0.6 wt%.
3. The latex-enhanced competitive immunoturbidimetric assay kit of claim 1, wherein the target analyte is posaconazole;
and/or the target detector-carrier complex is posaconazole-bovine serum albumin;
and/or the concentration of the target detection object-carrier complex in the reagent R1 is 200-600ng/ml, preferably 300-500 ng/ml.
4. The latex-enhanced competitive immunoturbidimetric assay kit according to claim 1, wherein the latex microspheres are polystyrene latex microspheres with amino groups on the surface or polystyrene latex microspheres with carboxyl groups on the surface;
the average particle size of the polystyrene latex microspheres with carboxyl groups on the surface is preferably 70-470nm, more preferably 150-350nm, and further preferably 200nm or 240 nm;
the surface carboxyl density of the polystyrene latex microspheres with carboxyl on the surface is preferably 0.03-0.30mmol/g, more preferably 0.05-0.18mmol/g, and further preferably 0.1mmol/g or 0.134 mmol/g;
and/or the concentration of the anti-target detection object antibody-conjugated latex microspheres in the reagent R2 is 0.05-0.16 wt%, preferably 0.09-0.13 wt%.
5. The latex-enhanced competitive immunoturbidimetric assay kit according to claim 1, wherein said reagent R1 and said reagent R2 are buffer solutions, and the buffer in said buffer solutions is selected from a buffer pair consisting of any one of the following reagents and its conjugate acid or its conjugate base: 2- (N-morpholino) ethanesulfonic acid, tris, 3- (N-morpholino) propanesulfonic acid, triethanolamine, and diethanolamine; the pH of the buffer solution is preferably 5.0 to 9.0, more preferably 5.5 to 7.5; the concentration of the buffer is preferably 15-100 mM;
and/or, the buffer solution comprises one or more of the following alkali metal salts: na (Na)2HPO3、NaH2PO3、NaCl、KCl、K2HPO3And KH2PO3(ii) a The concentration of the alkali metal salt is preferably 0.1 to 0.2mol/L, more preferably 0.14 to 0.18 mol/L.
6. The latex-enhanced competitive immunoturbidimetric assay kit of claim 5, wherein said buffer solution comprises one or more of a stabilizer, a blocking agent, and a preservative;
the stabilizer is preferably D-trehalose and/or sucrose;
the preservative is preferably Proclin 300 and/or sodium azide;
the blocking agent is preferably selected from one or more of bovine serum albumin, CE210 and CE 510.
7. The latex-enhanced competitive immunoturbidimetric assay kit according to any of claims 1-6, wherein said reagent R1 comprises methanol; the concentration of the methanol in the reagent R1 is preferably 5-20 vol%, more preferably 10 vol%;
and/or, the latex-enhanced competitive immunoturbidimetric assay kit further comprises a standard substance of the target detection substance.
8. The latex-enhanced competitive immunoturbidimetric assay kit of claim 1, wherein the latex-enhanced competitive immunoturbidimetric assay kit comprises reagents R1 and R2:
the reagent R1 comprises MES-NaOH buffer solution, bovine serum albumin, NaCl, D-trehalose, tigogenin, methanol, sodium azide and posaconazole-carrier compound,
the reagent R2 comprises a HEPES-HCl buffer solution, a sealant, NaCl, D-trehalose, sodium azide, bovine serum albumin and emulsion microspheres coupled with anti-posaconazole antibodies;
preferably, the latex-enhanced competitive immunoturbidimetric assay kit comprises a reagent R1 and a reagent R2:
the reagent R1 comprises 20-30mM MES-NaOH buffer solution, 0.08-0.12 wt% bovine serum albumin, 0.14-0.18mol/L NaCl, 0.08-0.12 wt% D-trehalose, 0.1-1.0 wt% tigogenin, 5-20 vol% methanol, 0.08-0.12 wt% sodium azide and 0.2-0.6 mu g/ml posaconazole-carrier compound, the pH of the reagent R1 is 5.5-6.0,
the reagent R2 comprises 48-52mM HEPES-HCl buffer solution, 0.08-0.12 vol% CE510, 0.14-0.18mol/L NaCl, 0.08-0.12 wt% D-trehalose, 0.08-0.12 wt% sodium azide, 0.08-0.12 wt% bovine serum albumin and 0.09-0.13 wt% anti-posaconazole antibody coupled polystyrene latex microspheres, and the pH of the reagent R2 is 7.4-7.8;
more preferably, the latex-enhanced competitive immunoturbidimetric assay kit comprises reagent R1 and reagent R2:
the reagent R1 comprises 25mM MES-NaOH buffer solution, 0.1 wt% bovine serum albumin, 0.17mol/L NaCl, 0.1 wt% D-trehalose, 10 vol% methanol, 0.1 wt% sodium azide, 0.4 mu g/ml posaconazole-bovine serum albumin complex and 0.6 wt% tigogenin, the pH value of the reagent R1 is 5.65,
the reagent R2 comprises 50mM HEPES-HCl buffer solution, 0.1 vol% CE510, 0.17mol/L NaCl, 0.1 wt% D-trehalose, 0.1 wt% sodium azide, 0.1 wt% bovine serum albumin and 0.11 wt% anti-posaconazole antibody coupled polystyrene latex microspheres, and the pH of the reagent R2 is 7.6.
9. Use of tigogenin as an adjuvant in the preparation of a latex-enhanced competitive immunoturbidimetric assay kit according to any of claims 1-8.
10. A latex-enhanced competitive immunoturbidimetric assay method using the latex-enhanced competitive immunoturbidimetric assay kit of any one of claims 1 to 8.
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| US20140227717A1 (en) * | 2013-02-13 | 2014-08-14 | Ark Diagnostics, Inc. | Posaconazole Immunoassays |
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| CN108614108A (en) * | 2016-12-12 | 2018-10-02 | 上海复星长征医学科学有限公司 | The kit and preparation method thereof of amikacin content in a kind of detection blood |
| CN110441514A (en) * | 2019-06-18 | 2019-11-12 | 北京利德曼生化股份有限公司 | A kind of preparation method, product and its application of latex microsphere and antibody complex |
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| US20140227717A1 (en) * | 2013-02-13 | 2014-08-14 | Ark Diagnostics, Inc. | Posaconazole Immunoassays |
| CN104198725A (en) * | 2014-08-14 | 2014-12-10 | 上海睿康生物科技有限公司 | Anti-cyclic citrullinated peptide (CCP) antibody detection kit |
| CN108614108A (en) * | 2016-12-12 | 2018-10-02 | 上海复星长征医学科学有限公司 | The kit and preparation method thereof of amikacin content in a kind of detection blood |
| CN110441514A (en) * | 2019-06-18 | 2019-11-12 | 北京利德曼生化股份有限公司 | A kind of preparation method, product and its application of latex microsphere and antibody complex |
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