CN113189343A

CN113189343A - Kit for simultaneously detecting retinol binding protein in serum and urine

Info

Publication number: CN113189343A
Application number: CN202110308217.9A
Authority: CN
Inventors: 王艳新; 吴鸣月; 张望; 常缘荣
Original assignee: Beijing Diagreat Biotechnology Co Ltd
Current assignee: Beijing Diagreat Biotechnology Co Ltd
Priority date: 2021-03-23
Filing date: 2021-03-23
Publication date: 2021-07-30

Abstract

The application discloses a kit for simultaneously detecting retinol binding protein in serum and urine, belonging to the technical field of medical in-vitro diagnosis. The kit comprises a reagent R1 and a reagent R2; the reagent R1 comprises the following components: buffer solution, coagulant, sodium chloride, surfactant, protective agent, preservative, retinol binding protein biotin labeled antibody; the reagent R2 comprises the following components: buffer solution, avidin, latex microspheres, surfactant and preservative. The application applies the principles of high-affinity firm combination between biotin and avidin and multi-stage amplification effect, and greatly improves the sensitivity of the detection kit. The detection linear range of the kit is 0.5-150mg/L, the highest detectable concentration is 200mg/L of blood samples, and the lowest detectable concentration is 0.02mg/L of urine samples.

Description

Kit for simultaneously detecting retinol binding protein in serum and urine

Technical Field

The application relates to the technical field of medical in-vitro diagnosis, in particular to a kit for simultaneously detecting retinol binding protein in serum and urine.

Background

Retinol Binding Protein (RBP) is a low molecular weight Protein (21000kD) secreted by the liver, is a transport Protein for vitamins in the blood, and is widely distributed in blood, cerebrospinal fluid, urine and other body fluids.

The retinol binding protein can be used for early detecting the functional damage of the renal tubules, sensitively reflecting the damage degree of the renal proximal convoluted tubules and being used as the index of early damage of the renal functions and monitoring treatment. Under normal conditions, the RBP has strong stability in urine, is not easy to decompose, is not interfered by pH and blood pressure, and has very little discharge (100 mug/d). About 90% of RBP in the plasma is combined with the thyroxine-binding pre-protein to form a high molecular protein complex, so that the RBP is not filtered by a glomerular filtration membrane, and when the retinol is transported to target cells, the RBP is dissociated into the plasma, is rapidly filtered by the glomerulus, and is almost completely reabsorbed by the renal proximal convoluted tubule to be decomposed. However, in the case of renal proximal tubular injury, the urine discharge of RBP is obviously increased, so that the increase of the urine discharge of RBP can be used as a marker of renal proximal tubular injury. Reduced plasma RBP is often seen in vitamin a deficiency, hypoproteinemia, malabsorption syndrome, liver disease (excluding hyperalimentation fatty liver), obstructive jaundice, hyperthyroidism, infection, trauma, and the like. Therefore, the simultaneous detection of blood and urine RBP can judge the type of part of kidney diseases and dynamically monitor the damage degree, and the combined examination with other items can provide accurate diagnosis basis for the damage position and degree of the kidney.

At present, the clinical detection method for RBP4 is mainly based on immunology, and utilizes the specific binding of antigen and antibody to quantitatively detect the content of RBP4 in blood, such as enzyme-linked immunosorbent assay, chemiluminescence assay and immunoturbidimetry. With the progress of detection technology and the increase of clinical detection requirements, the former two methods gradually fade out of clinical markets due to the disadvantages of high cost, tedious operation, long period and the like. Along with the popularization of full-automatic biochemical analyzers, the biochemical turbidimetry is a clinical mainstream detection method by virtue of the advantages of short reaction time, good precision, easiness in automation and the like. The conventional immunoturbidimetry has the defects of false negative, false positive and the like and is low in sensitivity, while the latex enhanced immunoturbidimetry is adopted, although the sensitivity is improved to a certain extent by virtue of a latex medium coated with specific rabbit polyclonal antibody, the cascade amplification effect is lacked, and when RBP in urine is detected, the RBP content in the urine is much lower than that in serum, the accuracy of low value detection cannot be ensured, so that a detection reagent with higher sensitivity is required.

Disclosure of Invention

Aiming at the problem that the sensitivity of a related retinol binding protein detection reagent is low, the application provides a kit for simultaneously detecting retinol binding protein in serum and urine.

The application provides a kit for simultaneously detecting retinol binding protein in serum and urine, which is realized by the following technical scheme.

A kit for simultaneously detecting retinol binding protein in serum and urine comprises a reagent R1 and a reagent R2; the reagent R1 comprises the following components:

0.1-0.2mg/ml of retinol binding protein biotin labeled antibody;

the reagent R2 comprises the following components:

preferably, the ratio of the concentration of the biotin labeled antibody in reagent 1 to the concentration of avidin in reagent 2 is 4-6: 1.

By adopting the technical scheme, the biotin-avidin system is further introduced on the basis of enhancing the sensitivity of the immunoturbidimetry by means of a latex medium, so that the cascade amplification of reaction signals is realized, and the sensitivity and the accuracy of the detection kit are improved.

Specifically, biotin in the reagent R1 of the present application is bound to an retinol binding protein antibody to form a biotin-labeled retinol binding protein antibody, and the bound antibody not only retains the original biological activity of a macromolecular substance, but also has high specificity and multivalence. Meanwhile, each avidin molecule in the latex microsphere coupled with avidin in the reagent R2 has four biotin binding sites, and can be simultaneously combined with a biotinylated flavonol binding protein antibody in a multivalent form, when retinol binding protein in a sample is combined with the flavonol binding protein antibody, the volume of the combination is amplified in multiple stages, and after light passes through the combination, the intensity change of transmitted light and scattered light is more obvious, so that the sensitivity of the detection method is greatly improved.

In addition, the binding between avidin and biotin has extremely high affinity, and the reaction is highly specific. Thus, the multi-level amplification of the biotin-avidin system increases sensitivity without increasing non-specific interference. Furthermore, the binding properties of the biotin-avidin system are not affected by the high dilution of the reagents, and the nonspecific effects of the reagents can be minimized. In the aspect of stability, the affinity constant of avidin combined with biotin can be millions of times of that of antigen-antibody reaction, and the dissociation constant of a complex formed by combining the avidin and the biotin is very small and shows irreversible reactivity; and the combination of acid, alkali, denaturant, proteolytic enzyme and organic solvent is not affected. Therefore, the product stability of the biotin-avidin system is high, so that the operation error can be reduced, and the accuracy of the determination can be improved.

To sum up, the invention provides a latex enhanced turbidimetric immunoassay kit which has the advantages of high sensitivity, strong specificity, good repeatability, high accuracy, simple and quick operation and low cost by applying the principles of high affinity firm combination and multistage amplification effect between biotin and avidin, the kit can simultaneously determine the content of retinol binding protein in blood and urine, the detection linear range is 0.5-150mg/L, the highest detectable concentration is a blood sample of 200mg/L, and the lowest detectable concentration is a urine sample of 0.02 mg/L.

Optionally, the kit further comprises a calibrator and a quality control product, wherein the calibrator and the quality control product are samples containing retinol binding protein antigens with different concentrations.

By adopting the technical scheme, the calibrator in the kit is used for drawing a calibration curve, when a detection sample is detected, a full-automatic biochemical analyzer is adopted to determine the absorbance change value of the sample and bring the absorbance change value into the calibration curve, so that the concentration of the sample to be detected can be obtained, and the detection method is simple; the quality control material in the kit is used for indicating the effectiveness of the reagents R1 and R2 in the kit, so that the detection result has better repeatability, and the accuracy and reliability of the detection result are improved.

Optionally, in the calibrator and the quality control product, the retinol binding protein antigen diluent comprises the following components:

by adopting the technical scheme, the retinol binding protein antigen diluent adopting the components can dilute the retinol binding protein antigen to a proper working concentration on one hand, and can provide a stable storage and working environment for the retinol binding protein antigen on the other hand, thereby reducing the incidence rate of retinol binding protein antigen denaturation, further drawing a more accurate calibration curve and more real effectiveness degree of a reaction reagent.

Optionally, the buffer solution is one or more of tris buffer solution, triethanolamine, borax borate and HEPES buffer solution. Preferably, reagent R1 and reagent R2 are boric acid borax buffer solution with pH value of 7.2-7.4; the retinol binding protein antigen diluent is tris buffer solution with pH 7.2.

By adopting the technical scheme, the buffer solution can well maintain the stability of the solution, the service life of the kit is prolonged, and the accuracy of a detection result is improved.

Optionally, the coagulant is one or more selected from PEG6000, PEG8000 and PEG 10000. Preferably, the coagulant is PEG 6000.

By adopting the technical scheme, the coagulant can adopt the polyethylene glycol with the molecular weight, and the polyethylene glycol is added into the reagent R1 and can act together with the surfactant, the protective agent and the preservative, so that the stability and the uniformity of the reagent R1 are improved.

Optionally, the surfactant is one or more of tween 20, tween 80, triton 100 and BrijTML23 polyoxyethylene lauryl ether. Preferably, the surfactant is tween 20.

By adopting the technical scheme, one or more of the surfactants are added into the reagent R1 and the reagent R2, and the addition of the surfactants can fully disperse all components in the reagent, improve the uniformity of the reagent and reduce the formation amount of precipitates.

Optionally, the protective agent is one or more of sucrose, trehalose, PEG6000 and bovine serum albumin. Preferably, the protective agent in the reagent R1 is sucrose; the protective agent in the retinol binding protein antigen diluent is bovine serum albumin.

By adopting the technical scheme, the protective agent is added into the reagent R1 and the retinol binding protein antigen diluent, so that the stability of the retinol binding protein biotin labeled antibody and the retinol binding protein antigen can be effectively improved, the reduction and even inactivation of the activity of the antigen and the antibody can be prevented, and a foundation is laid for improving the sensitivity of the kit.

Optionally, the preservative is one or more of proclin300, sodium azide and thimerosal. Preferably, proclin300 is selected as the preservative.

By adopting the technical scheme, the preservative is added into the reagent R1, the reagent R2 and the retinol binding protein antigen diluent, the effectiveness of the reagent can be prolonged by adding the preservative, the service life of the kit is prolonged, the incidence rate of false positive results is reduced, and the detection result is accurate and reliable.

Optionally, the latex microspheres are carboxyl latex microspheres with the diameter of 70-150 nm. Preferably, the latex microspheres have a diameter of 120 nm.

By adopting the technical scheme, the diameter of the latex microsphere is further limited, the specific surface area of the microsphere with the diameter of 70-150nm is large, and the surface of the latex microsphere is coupled with avidin as much as possible, so that the cascade amplification effect of reaction signals is realized to the maximum extent, and the detection sensitivity is improved.

Optionally, in the reaction system, the volume ratio of the blood sample to be detected to the reagent R1 and the reagent R2 is 3:225: 75; the volume ratio of the urine sample to be detected to the reagent R1 and the reagent R2 is 10:225: 75; the detection wavelength was 600 nm.

By adopting the technical scheme, the reaction system and the detection condition are further limited, the sample is detected under the reaction system and the detection condition, the low-value sample and the high-value sample can be detected simultaneously, and the linear range of detection is wider.

In summary, the present application has the following beneficial effects:

1. the application applies the principles of high-affinity firm combination between biotin and avidin and multi-stage amplification effect, and greatly improves the sensitivity of the detection kit. The detection linear range of the kit is 0.5-150mg/L, the highest detectable concentration is 200mg/L of blood samples, and the lowest detectable concentration is 0.02mg/L of urine samples;

2. the kit has the advantages of strong specificity, good repeatability, good stability, high accuracy, simplicity, convenience and rapidness in operation, low cost and suitability for clinical large-scale popularization.

Drawings

FIG. 1 is a graph of the linear correlation of the kit of example 1 of the present application;

FIG. 2 is a graph of the linear dependence of the kit of example 2 of the present application.

Detailed Description

The present application will be described in further detail with reference to the following drawings and examples.

The retinol binding protein antibody is a polyclonal antibody, and is purchased from Beijing Deolping Biotechnology GmbH;

biotin of the present application is NH₂Reactive Biotin, available from Wuhan Irelet Biotech, Inc.;

avidin in the present application is streptavidin, purchased from sigma corporation;

the latex microspheres of the present application were purchased from the JSR group of japan;

retinol binding protein antigens of the present application were purchased from beijing de oxepin biotechnology limited;

the full-automatic biochemical analyzer adopts a Hitachi 7180 full-automatic biochemical analyzer;

the commercially available hematuria simultaneous assay kit is purchased from Beijing nine strength Biotechnology Ltd;

preparation example 1

The preparation process of 2mg/mL retinol binding protein biotin labeled antibody comprises the following steps:

s1, dissolving biotin: add 30uL DMF to NH₂-standing in a reactionbiotin bottle for 10min until the biotin is fully dissolved to obtain biotin with the concentration of 10 mM;

s2, taking 1mg of retinol binding protein polyclonal antibody to be marked in a filter tube, adding a Labeling Buffer with a corresponding volume to ensure that the final concentration of the antibody is 2mg/mL, and centrifuging for 10min at 12,000g (note: the maximum volume of the filter tube is 0.5 mL);

s3, carrying out 13.3uL NH obtained in the step S1₂-Reactive Biotin and appropriate amount of laboratory Buffer were added to the centrifuged filter tube of step S2 to a final volume of 0.5mL and gently blown up and mixed; placing in 37 deg.C incubator, incubating in dark for 30min, and centrifuging at 12,000g for 10 min;

s4, adding a proper amount of Labeling Buffer into the filter tube centrifuged in the step S3 to enable the final volume to be 0.5mL, gently blowing and uniformly mixing, and centrifuging for 10min at 12,000 g; repeating the operation S4 once;

s5, adding 0.2mL of Labeling Buffer into the filter tube, and lightly blowing and beating; the filter element was placed in another centrifuge tube upside down and centrifuged at 6,000g for 10 min;

s6, collecting the solution in the centrifugal tube, namely the biotin-labeled retinol binding protein polyclonal antibody.

Preparation example 2

The method for coupling the latex microspheres with avidin comprises the following steps:

taking 1mL of latex microspheres with the mass fraction of 10% and the particle size of 120nm, adding the latex microspheres into a 100mL glass bottle containing 10mL of borax borate buffer solution with the pH value of 6.8, uniformly mixing, adding 15mg/mL EDC and 60mg/mL NHS, stirring for 15min in a constant-temperature incubator at 37 ℃, and activating the latex microspheres; then 5mg of avidin is added and mixed evenly for 2 hours; centrifuging, removing supernatant, resuspending with 10mL pH6.8 borax borate buffer solution, performing 20% power ultrasound for 10min to obtain avidin coupled microspheres with concentration of 1%, and refrigerating at 2-8 deg.C for use.

Preparation example 3

taking 1mL of latex microspheres with the mass fraction of 10% and the particle size of 150nm, adding the latex microspheres into a 100mL glass bottle containing 10mL of borax borate buffer solution with the pH value of 6.8, uniformly mixing, adding 15mg/mL of EDC and 60mg/mL of NHS, stirring for 15min in a constant-temperature incubator at 37 ℃, and activating the latex microspheres; then adding 2mg of avidin, and uniformly mixing for 2 hours; centrifuging, removing supernatant, resuspending with 10mL pH6.8 borax borate buffer solution, performing 20% power ultrasound for 10min to obtain avidin coupled microspheres with concentration of 1%, and refrigerating at 2-8 deg.C for use.

Example 1

Reagent R1

pH7.4 boric acid borax buffer solution 100 mmol/L;

PEG600010g/L；

9g/L of sodium chloride;

tween 201 g/L;

20g/L of sucrose;

proclin3000.5g/L；

0.2mg/ml of retinol binding protein biotin labeled antibody;

the retinol binding protein biotin-labeled antibody in this example is obtained by diluting the 2mg/ml retinol binding protein biotin-labeled antibody prepared in preparation example 1;

reagent R2

50mmol/L of borax borate buffer solution with the pH value of 7.2;

1g/L of latex microspheres;

50ug/ml of avidin;

tween 200.5 g/L;

proclin300 0.5g/L；

in the embodiment, the avidin and the latex microspheres are obtained by pretreating by adopting the method of the preparation example 2 and diluting;

in the examples of the present application, the ratio of the concentration of the biotin labeled antibody in reagent 1 to the concentration of avidin in reagent 2 was 4: 1.

The preparation method of the retinol binding protein antigen diluent comprises the following steps: weighing 6.06g of tris base, 9g of sodium chloride, 5g of bovine serum albumin and 3000.5 g of proclin, adding water to 950mL, adjusting the pH value of the solution to 7.2, and finally fixing the volume to 1L.

A calibrator dilution method: retinol binding protein antigen was diluted to 5 levels of 150mg/L, 100mg/L, 50mg/L, 25mg/L, 0mg/L using retinol binding protein antigen diluent, each level was measured twice and the average value was taken.

The quality control product dilution method comprises the following steps: retinol binding protein antigen was diluted to 40mg/L, 80mg/L, 2 levels in total, using retinol binding protein antigen diluent.

Adding 1225 mu L of reagent R, 275 mu L of reagent R and 3 mu L of sample to be detected into the reaction system, measuring the absorbance of the sample to be detected by adopting a full-automatic biochemical analyzer, wherein the detection wavelength is 600nm, and reading the readings of 19-34 points by a two-point end-point method and calculating.

1. Drawing of calibration curve

And (3) carrying out absorbance detection on the calibrator in a full-automatic biochemical analyzer, wherein specific detection data are shown in table 1.

TABLE 1 calibration Curve results

Level of	S1	S2	S3	S4	S5
						Concentration of calibrator	0	25	50	100	150
1	16	1706	2929	4799	5393
						2	10	1716	2916	4790	5364
Mean value	13	1711	2922.5	4794.5	5378.5

2. Linearity detection

Linear high-value samples are selected and diluted into five levels of 150mg/L, 120mg/L, 60mg/L, 30mg/L and 10mg/L by low-value samples; and (3) carrying out absorbance detection on each sample for three times by using a full-automatic biochemical analyzer, introducing a calibration curve to convert into a detection concentration value, and calculating relative deviation and a correlation coefficient, wherein specific experimental results are shown in table 2. With the theoretical concentration as the abscissa and the average value of the detected concentration as the ordinate, a linear correlation chart is drawn, see fig. 1.

TABLE 2 linearity test results

As can be seen from Table 2 and FIG. 1, when the kit of the present embodiment is used for detecting a sample simulating the retinol binding protein antigen content in blood, the obtained detection concentration is very close to the theoretical concentration, the linearity is very good, the linearity range is between 10mg/L and 150mg/L, the correlation coefficient R is not less than 0.999, and the detection result is accurate and reliable.

3. Repeatability detection

And (3) carrying out absorbance detection on the quality control substances of two concentration levels by using a full-automatic biochemical analyzer, detecting each level for 10 times, introducing a calibration curve to convert into a detection concentration value, and calculating a standard deviation and a variation coefficient, wherein specific experimental results are shown in a table 3.

TABLE 3 results of repeated measurements

As can be seen from Table 3, the kit of the present embodiment is adopted to perform repeated detection on samples of 40mg/L and 80mg/L, and the coefficient of variation CV of the obtained result is less than 5%, so that the kit meets the requirements, has excellent repeatability, and has high accuracy of the detection result.

4. Highest detected concentration

The method comprises the steps of diluting high-value retinol binding protein antigen by using antigen diluent to form different concentration gradients, carrying out absorbance detection on samples with different concentration levels by using a full-automatic biochemical analyzer, carrying out detection for 3 times for each concentration, converting the concentration into a calibration curve, comparing the obtained average detection concentration value with a theoretical concentration, and finally obtaining the highest detection concentration of the kit in the embodiment, wherein specific experimental results are shown in table 4.

Table 4: highest detected concentration

As can be seen from Table 4, when the sample was examined at a theoretical concentration of 250mg/L, the concentration to be examined was lower than the theoretical concentration, but when the sample was examined at a theoretical concentration of 200mg/L, the theoretical concentration coincided with the concentration to be examined. The experimental result shows that the highest detection concentration of the kit is 200mg/L, the detection result is credible, and the hook effect cannot occur.

Example 2

Reagent R1

0.1mg/ml of retinol binding protein biotin labeled antibody;

reagent R2

The avidin and the latex microspheres in the embodiment are obtained by pretreating and diluting the avidin and the latex microspheres by the method of the preparation example 3;

in the examples of the present application, the ratio of the concentration of the biotin labeled antibody in reagent 1 to the concentration of avidin in reagent 2 was 5: 1.

A calibrator dilution method: retinol binding protein antigen was diluted to 5 levels of 20mg/L, 10mg/L, 5mg/L, 2.5mg/L, 0mg/L using retinol binding protein antigen diluent, each level was measured twice and the average value was taken.

The quality control product dilution method comprises the following steps: retinol binding protein antigen was diluted to 0.8mg/L, 5mg/L, 2 levels in total, using retinol binding protein antigen diluent.

Adding 1225 mu L of reagent R, 275 mu L of reagent R and 10 mu L of sample to be detected into the reaction system, measuring the absorbance of the sample to be detected by adopting a full-automatic biochemical analyzer, wherein the detection wavelength is 600nm, and reading the readings of 19-34 points by a two-point end-point method and calculating.

1. Drawing of calibration curve

And (3) carrying out absorbance detection on the calibrator in a full-automatic biochemical analyzer, wherein specific detection data are shown in table 5.

TABLE 5 calibration Curve results

Level of	S1	S2	S3	S4	S5
						Concentration of calibrator	0	2.5	5	10	20
1	10	305	410	660	1366
						2	11	302	412	665	1365
Mean value	10.5	303.5	411	662.5	1365.5

2. Linearity detection

Linear high-value samples are selected and diluted into five levels of 20mg/L, 10mg/L, 5mg/L, 2.5mg/L and 0.5mg/L by low-value samples; and (3) carrying out three times of absorbance detection on each sample by using a full-automatic biochemical analyzer, introducing a calibration curve to convert into a detection concentration value, and calculating relative deviation and a correlation coefficient, wherein specific experimental results are shown in table 6. With the theoretical concentration as the abscissa and the average value of the detected concentration as the ordinate, a linear correlation chart is drawn, see fig. 2.

TABLE 6 results of linearity measurements

As can be seen from Table 6 and FIG. 2, when the kit of the present embodiment is used for detecting a sample simulating the retinol binding protein antigen content in urine, the obtained detection concentration is very close to the theoretical concentration, the linearity is very good, the linearity range is between 0.5mg/L and 20mg/L, the correlation coefficient R is not less than 0.999, and the detection result is accurate and reliable.

3. Repeatability detection

And (3) carrying out absorbance detection on the quality control substances of two concentration levels by using a full-automatic biochemical analyzer, detecting each level for 10 times, introducing a calibration curve to convert into a detection concentration value, and calculating a standard deviation and a variation coefficient, wherein specific experimental results are shown in a table 7.

TABLE 7 results of repeated measurements

As can be seen from Table 7, the kit of the present embodiment is used for repeated detection of 0.8mg/L and 5mg/L samples, and the coefficient of variation CV of the obtained result is less than 5%, so that the kit meets the requirements, has excellent repeatability, and has high accuracy of the detection result.

4. Minimum detection limit

Gradually diluting the sample with the concentration of 20mg/L by 2 times, detecting the absorbance of the sample with different concentration levels by using a full-automatic biochemical analyzer, bringing the sample into a calibration curve to convert the sample into a detection concentration value, and comparing the obtained detection concentration value with a theoretical concentration to finally obtain the lowest detection limit of the kit in the embodiment, wherein the specific experimental result is shown in Table 8.

TABLE 8 determination of detection limits

Dilution factor	Theoretical value	Detection value
			2	10	10.5
4	5	5.1
			8	2.5	2.5
16	1.25	1.24
			32	0.625	0.61
64	0.313	0.31
			128	0.156	0.14
256	0.078	0.07
			512	0.039	0.03
1024	0.020	0.02
			2048	0.010	0.02

As can be seen from Table 8, when the 20mg/L sample is diluted by 1024 times, the theoretical concentration is identical to the detection concentration, and the experimental result is accurate; however, when the 20mg/L sample is diluted 2048 times, the theoretical concentration is greatly different from the detection concentration. Therefore, the minimum detection limit of the kit is 0.02mg/L, the sensitivity is high, even if the sample (urine) to be detected contains a low content of retinol binding protein antigen, the kit can still detect the retinol binding protein antigen, the probability of the occurrence of false negative detection results can be effectively reduced, and the risk of delaying the disease condition of a patient is reduced.

Comparative experiment

The kit of the embodiment 1 and the commercially available hematuria simultaneous assay kit are adopted to simultaneously detect the concentration of retinol binding protein in 40 clinical serum samples and 40 clinical urine samples, and the detection results are shown in table 9.

TABLE 9 comparative experimental results

As can be seen from Table 9, the kit of the present application and the commercially available hematuria simultaneous assay kit are used for simultaneously detecting 40 serum samples, and the correlation coefficient of the detection results of the two kits is greater than 0.98; and simultaneously detecting 40 urine samples, wherein the correlation coefficient of the detection results of the two kits is more than 0.99. The test results show that the kit has extremely high correlation with the test results of the current clinical large-scale used kit, meets the clinical test requirements, and can be applied to large-scale clinical application.

The embodiments of the present invention are preferred embodiments of the present application, and the scope of protection of the present application is not limited by the embodiments, so: all equivalent changes made according to the structure, shape and principle of the present application shall be covered by the protection scope of the present application.

Claims

1. A kit for simultaneously detecting retinol binding protein in serum and urine is characterized in that: comprises a reagent R1 and a reagent R2;

the reagent R1 comprises the following components:

50-100mmol/L buffer solution;

10-30g/L of coagulant;

9g/L of sodium chloride;

0.5-1g/L of surfactant;

20-50g/L of protective agent;

0.5-1g/L of preservative;

0.1-0.2mg/ml of retinol binding protein biotin labeled antibody;

the reagent R2 comprises the following components:

50-100mmol/L buffer solution;

20-60ug/ml of avidin;

1g/L of latex microspheres;

0.5-1g/L of surfactant;

0.5-1g/L of preservative.

2. The kit for simultaneously detecting retinol binding protein in serum and urine as claimed in claim 1, wherein: the kit also comprises a calibrator and a quality control product, wherein the calibrator and the quality control product are samples containing retinol binding protein antigens with different concentrations.

3. The kit for simultaneously detecting retinol binding protein in serum and urine as claimed in claim 2, wherein: in the calibrator and the quality control product, the retinol binding protein antigen diluent comprises the following components:

50-100mmol/L buffer solution;

9g/L of sodium chloride;

5-10g/L of protective agent;

0.5-1g/L of preservative.

4. A kit for the simultaneous detection of retinol binding protein in serum and urine according to any of claims 1-3, wherein: the buffer solution is one or more of tris buffer solution, triethanolamine, borax borate and HEPES buffer solution.

5. A kit for the simultaneous detection of retinol binding protein in serum and urine according to any of claims 1-3, wherein: the coagulant is one or more selected from PEG6000, PEG8000 and PEG 10000.

6. A kit for the simultaneous detection of retinol binding protein in serum and urine according to any of claims 1-3, wherein: the surfactant is one or more of Tween 20, Tween 80, Triton 100, and Brij-L23 polyoxyethylene lauryl ether.

7. A kit for the simultaneous detection of retinol binding protein in serum and urine according to any of claims 1-3, wherein: the protective agent is one or more of sucrose, trehalose, PEG6000 and bovine serum albumin.

8. A kit for the simultaneous detection of retinol binding protein in serum and urine according to any of claims 1-3, wherein: the preservative is one or more of proclin300, sodium azide and thimerosal.

9. A kit for the simultaneous detection of retinol binding protein in serum and urine according to any of claims 1-3, wherein: the latex microspheres are carboxyl latex microspheres with the diameter of 70-150 nm.

10. A kit for the simultaneous detection of retinol binding protein in serum and urine according to any of claims 1-3, wherein: in the reaction system, the volume ratio of the blood sample to be detected to the reagent R1 and the reagent R2 is 3:225: 75; the volume ratio of the urine sample to be detected to the reagent R1 and the reagent R2 is 10:225: 75; the detection wavelength was 600 nm.