CN112763724A

CN112763724A - Reagent combination and kit for simultaneously determining content of retinol binding protein in serum and urine

Info

Publication number: CN112763724A
Application number: CN202011570098.6A
Authority: CN
Inventors: 杨玉莹
Original assignee: Beijing Antu Bioengineering Co ltd
Current assignee: Beijing Antu Bioengineering Co ltd
Priority date: 2020-12-26
Filing date: 2020-12-26
Publication date: 2021-05-07

Abstract

The invention relates to the field of biological detection, in particular to a reagent combination and a kit for simultaneously determining the content of retinol binding protein in serum and urine. The kit provided by the invention can ensure that the reagent has excellent linearity and precision when the human serum and urine are measured simultaneously by preparing the reagent by a large and small microsphere combined method; the urine and the blood use the same parameter, so that the detection operation is simplified, and the efficiency is improved.

Description

Reagent combination and kit for simultaneously determining content of retinol binding protein in serum and urine

Technical Field

The invention relates to the field of biological detection, in particular to a reagent combination and a kit for simultaneously determining the content of retinol binding protein in serum and urine.

Background

Retinol Binding Protein (RBP) is a small molecular weight lipocalin synthesized by the liver, with a molecular weight of 21KD, which plays a role in transporting retinol from hepatocytes to body tissues and cells in vivo, and serum RBP transports retinol by binding with Prealbumin (PA) and retinol to form a macromolecular complex which cannot pass through the filter membrane of the glomerulus and thus exists in the blood of the human body; when the compound reaches the target cells, retinol is absorbed by the target cells, RBP is dissociated into a free state with small molecular weight and enters the serum, the free state can be filtered by glomeruli and enters the renal tubules to be absorbed by epithelial cells to be converted into amino acid for the organism to reuse, and in addition, a very small part of RBP is not absorbed and is directly discharged out of the body along with urine.

Clinically, the RBP level in serum and urine can be used as an effective detection index for liver and kidney injuries of organisms and other syndromes such as diabetes and the like. RBP is synthesized in the liver, and if the liver synthesis function is damaged, the concentration of serum RBP is reduced, the degree of reduction is highly related to the condition of the damaged liver, the more serious the damage of the liver is, the greater the degree of reduction is, and the lower the degree of cirrhosis is. Because the half-life period of the RBP is short, the response change is sensitive, and the RBP is more suitable to be used as an index of early liver function damage.

Retinol binding protein is metabolized in the kidney, so urine RBP levels are of great importance for early diagnosis of renal disease. Some diseases (such as diabetes, hypertension and the like) cause the function of glomeruli of the body to be damaged, the filtration of the glomeruli is blocked, and RBP is accumulated in blood to cause the content of serum RBP to be increased; in addition, certain kidney diseases (such as virus infection) can cause the reabsorption function of renal tubules of a body to be damaged, and RBP in body fluid can not be reabsorbed after entering the kidney and is completely discharged out of the body along with urine, so that the content of the RBP in the urine is increased.

Besides liver and kidney diseases, RBP is also closely linked to other diseases. Research shows that when the insulin resistance is increased, the RBP concentration in blood is increased, so that the body is obese; in addition, serum RBP can reflect the nutritional status of the organism and can be used as a sensitivity index for the early and sub-clinical nutritional deficiency detection of the organism.

At present, clinical diagnosis methods aiming at retinol binding protein mainly comprise an enzyme-linked immunosorbent assay, an immune transmission turbidimetry, a radioimmunoassay, an immunoelectrophoresis method, a latex enhanced immunoturbidimetry and the like.

The enzyme-linked immunoassay method is to detect the color development of enzyme-labeled secondary antibodies by utilizing the combination of antibodies and enzymes. The method has the advantages of high detection sensitivity, good specificity, simple and convenient operation and strong practicability as a detection method commonly used in immunology, but has the defects of weak anti-interference capability and easy influence of interference factors in a sample, so that the application of the method has certain limitation.

The immunoradiometric assay is a method in which a radionuclide-labeled antibody is combined with an antigen or hapten to form an antigen-antibody complex, the radioactivity of which is positively correlated with the amount of the added antigen, and the concentration of the antigen is determined by detecting the radioactivity. The method has the advantages of simple and convenient comparison and detection speed, high sensitivity and good specificity, but cross reaction, false positive reaction and the like are easy to occur, and the detection result is influenced.

The immunoelectrophoresis is a technology for separating different proteins by utilizing the difference of charges and the difference of molecular weights of the proteins in serum or urine, agarose is used as a carrier, and a method for generating specific precipitation lines, arcs or peaks by combining agarose zone electrophoresis and two-way diffusion is utilized by utilizing an immunological theory. The method is high in sensitivity, but is complex to operate and is not suitable for detection of large batches of clinical specimens.

The principle of the immunotransmission turbidimetry is that a soluble antigen is specifically combined with a corresponding antibody to form an insoluble immune complex, when light with a certain wavelength passes through the antigen-antibody complex, the turbidity of the antigen-antibody complex changes, and the concentration of the corresponding antigen can be determined by measuring the absorbance value of the antigen-antibody complex. The method has high accuracy and simple operation, and can be widely applied to clinical medical diagnosis.

Compared with the immunoelectrophoresis method which is complex in operation and not suitable for clinical automatic analysis, the enzyme-linked immunosorbent assay can only be used for qualitative or semi-quantitative analysis, the radioimmunoassay has the problems of radioactive pollution, safety and the like, and the immunotransmission turbidimetry is the most worthy clinical automatic detection method for popularization due to the advantages of simplicity and convenience in operation, high accuracy, good reagent stability, strong anti-interference capability, low detection cost and the like.

A detection method with higher sensitivity is established on the basis of an immunoturbidimetry method, namely a latex enhanced immunoturbidimetry method. The basic principle is that specific antigen or antibody is coated on the surface of latex microsphere, when the corresponding antibody or antigen is detected in the sample, the two antibodies or antigen are subjected to specific immunoreaction, and then the immune latex microsphere is agglomerated, so that the turbidity of the sample is increased. The same principle as the immunoturbidimetry detection principle is adopted, and the content of the protein to be detected in the sample can be calculated by detecting the turbidity of the solution according to the proportion of the content of the component to be detected in the sample solution to the turbidity of the sample. The method can directly detect the solution after immunoreaction by using a full-automatic biochemical analyzer, and has simple operation and quick detection.

Most of the commercially available RBP kits use microspheres of a single particle size to prepare reagent 2, but it is difficult to satisfy both high linearity and low-value high sensitivity. In some inventions, the big and small balls are connected with different antibodies respectively, because the antibodies are different, the specificity is different, and the production process is complicated by adopting a separate coupling process. Therefore, the kit capable of simultaneously determining the retinol binding protein content in human serum and urine has important practical significance.

Disclosure of Invention

In view of the above, the present invention provides a reagent combination and a kit for simultaneously determining the retinol binding protein content in serum and urine.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a reagent combination for simultaneously measuring the content of retinol binding protein in serum and urine, which comprises a reagent I and a reagent II;

wherein, the first reagent comprises dextran sodium sulfate;

the reagent two comprises latex particles marked with anti-human retinol binding protein polyclonal antibody; the latex particles are prepared by coupling an anti-human retinol binding protein polyclonal antibody and latex microspheres;

the latex microspheres comprise first latex microspheres and second latex microspheres; the particle size of the first latex microsphere is 80 nm; the particle size of the second latex microspheres is 200 nm.

In some embodiments of the invention, the weight ratio of the first latex microspheres to the second latex microspheres is 1: 3.

In some embodiments of the present invention, the method for preparing the latex particles labeled with polyclonal antibodies against human retinol binding protein comprises:

step 1, microsphere activation: taking first latex microspheres with the particle size of 80nm and second latex microspheres with the particle size of 200nm, mixing the first latex microspheres with the second latex microspheres in a ratio of 1:3, diluting the mixture to the concentration of 2% (w/w) by 10mM MES buffer solution with the pH value of 5, adding 0.5mg/ml EDC in a volume ratio of 120ml/L, and oscillating and activating the mixture for 30min at 20-30 ℃ to prepare a microsphere system;

step 2, antibody dilution: adding an anti-human retinol binding protein polyclonal antibody and casein to 50mmol MOPS buffer solution with pH7.0, wherein the anti-human retinol binding protein polyclonal antibody, the casein and the MOPS buffer solution are mixed according to the ratio of 2: 1: 45, and preparing a diluted antibody;

step 3, antibody-microsphere coupling: adding the diluted antibody prepared in the step 2 into the microsphere system prepared in the step 1 at a constant speed of 6ml/min, and reacting for 18 hours;

step 4, ultrasound: ultrasonic power: 50%, time interval: 5s, probe diameter: 25 mm; the ultrasonic time length is as follows: 30 min/l reagent;

and step 5, centrifugation: centrifuging at 15000rpm for 15min, and collecting precipitate;

step 6, ultrasonic dispersion: ultrasonic power: 50%, time interval: 5s, probe diameter: 25 mm; the ultrasonic time length is as follows: 30 min/l reagent; dispersing the precipitate in a storage solution to prepare a latex particle solution marked with an anti-human retinol binding protein polyclonal antibody;

the components of the storage solution include:

in some embodiments of the present invention, the concentration of the latex particles labeled with the polyclonal antibody against human retinol binding protein in the latex particle solution labeled with the polyclonal antibody against human retinol binding protein is 0.4%.

In some embodiments of the present invention, the dextran sulfate sodium in the reagent one has a molecular weight of 1500-50000 and a concentration of 0.1-2% (w/v).

In some embodiments of the present invention, the first reagent further comprises 50-100 mM MOPS buffer, 0.1-1% (w/w) BSA, 1-5% NaCl, 0.1-1% Tween, 0.5-5% EDTA, and the pH of the first reagent is 5.0-8.0.

In some embodiments of the invention, the volume ratio of the first reagent to the second reagent is 3: 1.

Based on the research, the invention also provides application of the reagent combination in preparing a kit for simultaneously determining the content of the retinol binding protein in serum and urine.

The invention also provides a kit for simultaneously determining the content of the retinol binding protein in serum and urine, which comprises the reagent combination and acceptable auxiliary agents or carriers.

The invention also provides a detection method for non-disease diagnosis based on the reagent combination or the kit, and the detection method adopts an end-point determination method, and the reaction direction is increased; the main wavelength and the auxiliary wavelength are 604/NONE, the reading point is 18-19/31-33, and the volume ratio of the sample amount to the reagent combination is (6-20): 200.

Most of the commercially available RBP kits use microspheres of a single particle size to prepare reagent 2, but it is difficult to satisfy both high linearity and low-value high sensitivity. Therefore, the invention adopts microspheres with two particle sizes to prepare the latex reagent, thereby achieving high linearity and excellent low-value sensitivity.

In some inventions, the big and small balls are connected with different antibodies respectively, because the antibodies are different, the specificity is different, and the production process is complicated by adopting a separate coupling process. Aiming at the problem, the large and small ball coupled antibodies are the same, and are coupled at the same time, so that the process is simple.

In the preparation process of the reagent II, the antibody and the casein are added into the microsphere together, so that the antibody can be more uniformly connected with the activating groups on the microsphere, and meanwhile, the casein can also seal redundant activating groups on the microsphere, so that the stability of the reagent is improved.

Generally, the reagent has a larger influence on the linearity of the reagent, and the invention improves the linearity and the anti-interference performance of the reagent by adding a dextran sodium salt into the reagent I.

Because the RBP is easy to generate matrix effect when measuring a urine sample so as to influence the accuracy of a measured value, the invention eliminates the matrix effect by adding EDTA (ethylene diamine tetraacetic acid) into the reagent I to chelate metal ions in the urine sample.

Although some reagents in the market can simultaneously measure hematuria, different parameters are used, and the hematuria in the experiment can be detected in the same channel by using the same parameter.

The kit provided by the invention can ensure that the reagent has excellent linearity and precision when the human serum and urine are measured simultaneously by preparing the reagent by a large and small microsphere combined method; the urine and the blood use the same parameter, so that the detection operation is simplified, and the efficiency is improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a calibration curve of the kit provided in example 1 of the present invention;

FIG. 2 shows the effect of the kit without dextran sodium sulfate on linearity;

FIG. 3 shows the effect of the dextran sulfate sodium addition kit on linearity;

FIG. 4 is a linear curve of a serum sample detected by the kit provided in example 1 of the present invention;

FIG. 5 shows a linear curve of a control reagent assay serum sample;

FIG. 6 shows the correlation analysis result of the serum sample detected by the kit provided in example 1 of the present invention and the control reagent;

FIG. 7 shows the correlation analysis result of the urine sample detected by the kit provided in example 1 of the present invention and the control reagent;

FIG. 8 shows the matrix effect of a sample diluted in deionized water;

figure 9 shows the matrix effect of a urine diluted sample.

Detailed Description

The invention discloses a reagent combination and a kit for simultaneously measuring the content of retinol binding protein in serum and urine, and a person skilled in the art can realize the determination by appropriately improving process parameters by referring to the contents. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Preparation of RBP detection kit:

reagent 1: contains 50-100 mM MOPS buffer solution, 0.1-1% BSA, 1-5% NaCl, 0.1-1% Tween, 0.5-5% EDTA, 0.1-2% dextran sodium sulfate (molecular weight 1500-50000), and pH 5.0-8.0.

Reagent 2:

preparation of latex particles labeled with polyclonal antibodies against human retinol binding protein: 1. activation of microspheres: mixing small latex microspheres with the particle size of 80nm and large latex microspheres with the particle size of 200nm in a proportion of 1: (2-4), diluting with 10mM MES buffer (pH 5) to 2%, adding EDC (0.5mg/ml), and activating with shaking at room temperature for 30 min; 2. antibody dilution: antibodies and casein were mixed at a ratio of 2: 1 is added into MOPS buffer solution with 50mmPH (6.0-8.0) and mixed evenly; 3. antibody-microsphere coupling: adding the diluted antibody into a microsphere system at a constant speed for reaction; 4. ultrasonic: carrying out coupling reaction (at the temperature of 15-30 ℃ and at the rotating speed of 200-400) for 18 hours, and then carrying out ultrasonic treatment on the reaction system to enable the antibody physically adsorbed on the microspheres to fall off; 5. centrifuging: the centrifugation condition is 15000rpm, 15min, supernatant removal, 6 ultrasonic dispersion: the pellet after centrifugation was suspended in the stock solution and ultrasonically dispersed to a final concentration of 0.4%.

Detection parameters of the RBP:

the measuring instrument: toshiba FX8

TABLE 1

(remarks: standard sample amount for urine sample detection, sample amount for

serum sample detection

1 or 2; for example, 20. mu.l of sample is mixed with 120. mu.l of diluent, 12. mu.l of the mixture is taken, and then the mixture is added into a reaction system for reaction.)

Description of data in the table, for example, review of group 1: 20ul of sample is taken, 120ul of diluent is added, 12ul of sample is taken after the mixture is uniformly mixed, and then the mixture is added into a reaction system for reaction.

The beneficial effects of the invention include but are not limited to:

in the invention, the coupling is carried out after the large and small balls are mixed, the process is simple, and the physically coupled antibody can be effectively removed by carrying out ultrasonic treatment before the reagent II is centrifuged, so that the stability of the reagent is improved.

In the invention, the mixing ratio of the large latex particles and the small latex particles in the reagent II is high.

In the invention, the content and molecular weight of dextran sodium sulfate in the reagent II are shown.

In the present invention, the same parameters are used for serum and urine.

The reagent combination for simultaneously measuring the content of the retinol binding protein in the serum and the urine and the raw materials and the reagents used in the kit can be purchased from the market.

The contrast reagent is an RBP kit of domestic excellent manufacturers, and comprises the following components: r1: the main components are phosphate buffer solution, R2: mainly phosphate buffer solution and latex coated anti-human retinol binding protein antibody.

The invention is further illustrated by the following examples:

example 1 preparation of RBP detection kit

Reagent 1: contains 50mM MOPS buffer, 1% BSA, 5% NaCl, 1% Tween, 5% EDTA, 2% dextran sulfate sodium (molecular weight 1500), pH 5.0.

Reagent 2:

preparation of latex particles labeled with polyclonal antibodies against human retinol binding protein: 1. activation of microspheres: mixing small latex microspheres with the particle size of 80nm and large latex microspheres with the particle size of 200nm in a proportion of 1: 2, diluted to a concentration of 2% (by weight) with 10mM MES buffer PH5, followed by addition of 200 mg/l of R2 EDC (0.5mg/ml) and activation with shaking at room temperature (25 ± 5 ℃) for 30 min; 2. antibody dilution: antibodies and casein were mixed at a ratio of 2: 1 into 50mmPH6.0 MOPS buffer solution (the weight ratio of antibody to casein to buffer solution is 2: 1: 45), and mixing uniformly; 3. antibody-microsphere coupling: adding the diluted antibody into a microsphere system at a constant speed (6ml/min) for reaction; 4. ultrasonic: after the coupling reaction (stirring at 25 +/-5 ℃) for 18 hours, carrying out ultrasonic treatment on the reaction system (ultrasonic power: 50%, time interval: 5s, probe diameter: 25 mm; ultrasonic treatment time: 30 min/L reagent) to ensure that the antibody physically adsorbed on the microspheres falls off; 5. centrifuging: the centrifugation condition is 15000rpm, 15min, supernatant removal, 6 ultrasonic dispersion: the centrifuged pellet was suspended in the storage solution and dispersed by ultrasonic waves (ultrasonic power: 50%, time interval: 5s, probe diameter: 25 mm; ultrasonic time period: 30 min/liter of reagent) to give a final concentration of 0.4%.

Example 2 preparation of RBP detection kit

Reagent 1: contains 100mM MOPS buffer, 0.1% BSA, 1% NaCl, 0.1% Tween, 0.5% EDTA, 0.1% dextran sulfate sodium (molecular weight 50000), and pH 8.0.

Reagent 2:

preparation of latex particles labeled with polyclonal antibodies against human retinol binding protein: 1. activation of microspheres: mixing small latex microspheres with the particle size of 80nm and large latex microspheres with the particle size of 200nm in a proportion of 1:3, diluted to a concentration of 2% (by weight) with 10mM MES buffer PH5, followed by addition of 200 mg/l R2 of EDC (0.5mg/ml) and activation with shaking at room temperature (25 ± 5 ℃) for 30 min; 2. antibody dilution: antibodies and casein were mixed at a ratio of 2: 1 into 50mmPH7.0 MOPS buffer solution (the weight ratio of antibody to casein to buffer solution is 2: 1: 45), and mixing uniformly; 3. antibody-microsphere coupling: adding the diluted antibody into a microsphere system at a constant speed (6ml/min) for reaction; 4. ultrasonic: after the coupling reaction (stirring at 25 +/-5 ℃) for 18 hours, carrying out ultrasonic treatment on the reaction system (ultrasonic power: 50%, time interval: 5s, probe diameter: 25 mm; ultrasonic treatment time: 30 min/L reagent) to ensure that the antibody physically adsorbed on the microspheres falls off; 5. centrifuging: the centrifugation condition is 15000rpm, 15min, supernatant removal, 6 ultrasonic dispersion: the centrifuged pellet was suspended in the storage solution and dispersed by ultrasonic waves (ultrasonic power: 50%, time interval: 5s, probe diameter: 25 mm; ultrasonic time period: 30 min/liter of reagent) to give a final concentration of 0.4%.

Example 3 preparation of RBP detection kit

Reagent 1: contains 75mM MOPS buffer, 0.5% BSA, 3% NaCl, 0.5% Tween, 2.5% EDTA, 1% dextran sulfate sodium (molecular weight 25000), pH 7.0.

Reagent 2:

preparation of latex particles labeled with polyclonal antibodies against human retinol binding protein: 1. activation of microspheres: mixing small latex microspheres with the particle size of 80nm and large latex microspheres with the particle size of 200nm in a proportion of 1: 4, diluted to a concentration of 2% (by weight) with 10mM MES buffer PH5, followed by addition of 200 mg/l of R2 EDC (0.5mg/ml) and activation with shaking at room temperature (25 ± 5 ℃) for 30 min; 2. antibody dilution: antibodies and casein were mixed at a ratio of 2: 1 into 50mmPH8.0 MOPS buffer solution (the weight ratio of antibody to casein to buffer solution is 2: 1: 45), and mixing uniformly; 3. antibody-microsphere coupling: adding the diluted antibody into a microsphere system at a constant speed (6ml/min) for reaction; 4. ultrasonic: after the coupling reaction (stirring at 25 +/-5 ℃) for 18 hours, carrying out ultrasonic treatment on the reaction system (ultrasonic power: 50%, time interval: 5s, probe diameter: 25 mm; ultrasonic treatment time: 30 min/L reagent) to ensure that the antibody physically adsorbed on the microspheres falls off; 5. centrifuging: the centrifugation condition is 15000rpm, 15min, supernatant removal, 6 ultrasonic dispersion: the centrifuged pellet was suspended in the storage solution and dispersed by ultrasonic waves (ultrasonic power: 50%, time interval: 5s, probe diameter: 25 mm; ultrasonic time period: 30 min/liter of reagent) to give a final concentration of 0.4%.

Example 4 detection method

Detection parameters of the RBP:

the measuring instrument: toshiba FX8

TABLE 2

(remarks: standard sample size for urine sample detection; sample size for serum sample detection in either review 1 or review 2.)

Effect example 1

The calibration curve of the kit provided by the invention is shown in figure 1.

Effect example 2 effect of dextran sulfate on anti-interference properties of reagents:

for example, to prepare a serum sample containing 2g/L hemoglobin, a 20g/L hemoglobin aqueous solution is prepared, then 20ul of the hemoglobin aqueous solution is added to 180ul of the serum to prepare a 2g/L serum sample, and so on, the blank serum is 180ul of the serum to which 20ul of water is added.

The sample size can be checked by referring to the parameter settings in Table 1, and the sample size is checked by using the sample size of check 1

TABLE 3

As can be seen from the data in Table 3, the dextran sodium sulfate added into the kit provided by the invention has an obvious effect on resisting the interference of chyle.

Effect example 3 effect of dextran sulfate on reagent linearity:

the experiment is mainly to verify that the linearity of the reagent is improved by adding dextran sulfate into R1, namely R1 without adding dextran and R1 with adding dextran are matched with the same R2 respectively, and the linearity is detected, wherein the linear sample is diluted by water multiple ratio, 1/2 refers to half of common serum half of water, 1/4 refers to one part of serum, three parts of water and the like.

As shown in fig. 2 to 3 and tables 4 to 5:

TABLE 4

Theoretical value	Gradient of gradient	Sodium dextran sulfate is not added	Fitting value	Deviation of fitting value	Deviation of theoretical value
						0.00	0	-0.1	8.837	-8.90	-0.07
5.16	1/32	6.0	12.092	-6.05	0.88
						10.31	1/16	12.2	15.347	-3.19	1.84
20.63	1/8	23.9	21.857	9.3％	15.8％
						41.25	1/4	46.1	34.876	32.1％	11.7％
82.50	1/2	76.1	60.915	24.9％	-7.8％
						165.00	1	102.7	112.994	-9.1％	-37.7％

TABLE 5

Theoretical value	Gradient of gradient	Adding dextran sodium sulfate	Fitting value	Deviation of fitting value	Deviation of theoretical value
						0.0	0	-0.1	0.7	-0.73	-0.06
5.2	1/32	5.3	5.7	-0.41	0.10
						10.3	1/16	10.4	10.7	-0.24	0.10
20.6	1/8	20.6	20.6	-0.4％	-0.3％
						41.3	1/4	41.1	40.6	1.3％	-0.3％
82.5	1/2	82.6	80.5	2.5％	0.1％
						165.0	1	159.2	160.4	-0.7％	-3.5％

The above results show that: dextran sodium sulfate has obvious effect on improving linearity.

Effect example 4 Performance test of serum sample by kit

(1) Linearity

The RBP high-value serum sample is diluted by deionized water according to the proportion of 1, 3/4, 1/2, 1/4, 1/8, 1/16, 1/32 and 0 in a multiple ratio, each concentration is repeatedly measured for 3 times according to the measuring method and conditions of example 4, the average value is calculated, the measured value of the sample and the dilution ratio are subjected to relevant comparison, a regression equation is solved, the theoretical value of the sample is calculated through the regression equation, and the result is shown in table 6 and figures 4 to 5.

The linear deviation is calculated according to the theoretical value and the detection value, and the result shows that the upper limit of the detection of the serum sample RBP by the kit can reach 170 mg/L.

TABLE 6

Dilution gradient	Contrast agent	The invention
			0	-0.14	-0.05
1/32	6.03	5.33
			1/16	12.43	12.56
1/8	25.1	26.77
			1/4	49.63	52.36
1/2	97.55	103.49
			3/4	143.43	150.26
1	159.04	175.38

(2) And (3) correlation analysis:

according to the determination method and conditions of example 4, the kit provided by the invention and the control kit detect 20 serum samples at the same time, and the results are shown in table 7 and fig. 6:

TABLE 7

Effect example 5 test of Performance of urine sample by Using reagent kit

(1) Sensitivity test

A high value urine sample was diluted in multiple proportions to prepare samples of different concentrations (0.125, 0.25, 0.5, 1mg/L), and the measurements were repeated 10 times for each sample according to the measurement method and conditions of example 4, and the mean and standard deviation were calculated, the results are shown in Table 8.

The detection is carried out with reference to the parameter settings in Table 1, and the sample amount is determined using the standard sample amount

As can be seen from Table 8, the detection sensitivity of the kit of the present invention for urine RBP was 0.125 mg/L.

TABLE 8

(2) Correlation analysis

According to the determination method and conditions of example 4, the kit provided by the invention and the control kit can simultaneously detect 20 urine samples, and the results are shown in fig. 7 and table 9:

TABLE 9

(3) Matrix effect

A high value urine sample was diluted with deionized water and a low value urine sample at the same time in multiple ratios and simultaneously detected with a control reagent and the kit of the present invention, respectively (according to the assay method and conditions of example 4). In which fig. 8 shows a deionized water diluted sample, and fig. 9 shows a urine diluted sample. The samples diluted by the two methods tested by the invention have good linearity, which shows that the urine sample has no matrix effect.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The reagent combination for simultaneously measuring the content of the retinol binding protein in serum and urine is characterized by comprising a reagent I and a reagent II;

wherein, the first reagent comprises dextran sodium sulfate;

2. The reagent combination of claim 1, wherein the weight ratio of the first latex microspheres to the second latex microspheres is 1: 3.

3. The reagent combination according to claim 1 or 2, wherein the latex particles labeled with polyclonal antibodies against human retinol binding protein are prepared by the method comprising:

step 4, ultrasound: ultrasonic power: 50%, time interval: 5s, probe diameter: 25mm, ultrasonic duration: 30 min/l reagent;

step 6, ultrasonic dispersion: ultrasonic power: 50%, time interval: 5s, probe diameter: 25mm, ultrasonic duration: 30 min/l reagent; dispersing the precipitate in a storage solution to prepare a latex particle solution marked with an anti-human retinol binding protein polyclonal antibody;

the storage solution includes:

4. the reagent combination according to any one of claims 1 to 3, wherein the concentration of the latex particles labeled with the polyclonal antibody against human retinol binding protein in the solution of the latex particles labeled with the polyclonal antibody against human retinol binding protein is 0.4%.

5. The reagent combination of any one of claims 1 to 4, wherein the dextran sulfate sodium in reagent one has a molecular weight of 1500-50000 and a concentration of 0.1-2% (w/v).

6. The reagent combination of any one of claims 1 to 5, wherein the reagent one further comprises 50 to 100mM MOPS buffer, 0.1 to 1% (w/w) BSA, 1 to 5% NaCl, 0.1 to 1% Tween,0.5 to 5% EDTA, and the pH of the reagent one is 5.0 to 8.0.

7. The reagent combination of any one of claims 1 to 6, wherein the volume ratio of the first reagent to the second reagent is 3: 1.

8. Use of a combination of reagents according to any one of claims 1 to 7 for the manufacture of a kit for the simultaneous determination of the retinol binding protein content in serum and urine.

9. Kit for the simultaneous determination of the retinol binding protein content in serum and urine, comprising a combination of reagents according to any one of claims 1 to 7 together with acceptable auxiliaries or carriers.

10. Detection method for non-disease diagnostic purposes based on a combination of reagents according to any one of claims 1 to 7 or a kit according to claim 9, characterized in that the direction of reaction is ascending using an end-point assay; the main wavelength and the auxiliary wavelength are 604/NONE, the reading point is 18-19/31-33, and the volume ratio of the sample amount to the reagent combination is (6-20): 200.