CN108776231B

CN108776231B - Human urinary albumin latex-enhanced secondary antibody competitive immunoturbidimetry detection kit and manufacturing and using methods thereof

Info

Publication number: CN108776231B
Application number: CN201811038368.1A
Authority: CN
Inventors: 文建新; 刘株; 漆乐新
Original assignee: Changsha Wenhan Biotechnology Co ltd
Current assignee: Changsha Wenhan Biotechnology Co ltd
Priority date: 2018-09-06
Filing date: 2018-09-06
Publication date: 2020-12-29
Anticipated expiration: 2038-09-06
Also published as: CN108776231A

Abstract

The invention discloses a human urinary albumin latex enhanced secondary antibody competitive immunoturbidimetric assay kit and a manufacturing and using method thereof. When no antigen exists in a measurement reaction system, the secondary antibody connected to the latex particles generates agglutination reaction with the primary antibody in the system, and the agglutination effect reaches the maximum; when the antigen is measured to be present in the reaction system, the antigen is combined with the primary antibody to form an antigen-antibody (primary antibody) immune complex, the combination blocks the combining sites of the primary antibody and the secondary antibody and competes with the primary antibody and the secondary antibody for immune agglutination reaction, if the amount of the primary antibody is limited, the antigen in the sample competitively inhibits the agglutination effect of the primary antibody-secondary antibody immune complex, and therefore, the agglutination effect of the primary antibody and the secondary antibody-latex reaction to form the immune complex is reduced as the amount of the free antigen in the reaction system is increased.

Description

Human urinary albumin latex-enhanced secondary antibody competitive immunoturbidimetry detection kit and manufacturing and using methods thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a human urinary albumin latex enhanced secondary antibody competitive immunoturbidimetric assay kit and a manufacturing and using method thereof.

Background

The immunoturbidimetric assay is widely developed clinically, dozens of kits are applied clinically, and the discovery of new biomarkers can continuously promote the generation of the new kits. At present, there are two types, one is that antibody is directly added into reaction system, under a certain condition, antigen and antibody react to form immune complex, agglutination or precipitation is generated to change turbidity of the system, the turbidity change has positive correlation with the antigen concentration in the system, and accordingly the antigen concentration in the sample can be determined, the method is generally called turbidimetry or common turbidimetry; the second method is developed on the basis of the first method, in which antibodies are bound to solid particles to enhance agglutination or precipitation, thereby improving assay sensitivity, and this method is generally called enhanced immunoturbidimetry.

In the homogeneous immunoassay process of enhancing immunoturbidimetry, it is very important to maintain the proper ratio of antigen and antibody, otherwise, when the antigen is excessive, the effect of the antigen-antibody immune complex generated precipitation or agglutination will be reduced on the contrary, that is, in the immunoturbidimetry analysis, the so-called hook effect, because the hook effect exists, when the excessive antigen is detected, the effect of the immune complex formed precipitation is reduced on the contrary, a lower concentration value can be detected, a 'false negative' result can be obtained, and the false negative result can often cause clinical misjudgment. Urinary microalbumin is a very sensitive indicator of glomerular disease and kidney injury. Especially, the method has special significance for understanding the damage condition of the kidney in the early stage of the occurrence of diseases such as diabetes, hypertension and the like, the content of albumin in human urine is lower than 20mg/L in the normal state, and when the kidney is damaged, the content of trace albumin can be as high as 1000mg/L or even higher. In the process of detecting albumin in urine, the false negative result generated by excessive antigen detection needs to be considered for clinical misjudgment, and the false negative result caused by excessive antigen is avoided. In addition, the effective method is to use a qualitative method to predict the albumin concentration in the sample, for example, to use urine protein test paper to predict, to screen out high concentration sample, and to dilute the high value sample for further determination.

The problems of the above immunoturbidimetric method are mainly as follows:

1. the existing latex-enhanced urine microalbumin immunoturbidimetry detection method is designed into a so-called forward immunoturbidimetry kit for clinical detection by connecting an antibody with latex, the detection range of the method is usually 0-100ug/L, and when the concentration of a sample exceeds 100ug/L, the detection result may be false negative due to the hook effect.

2. The existing immunoturbidimetric immunoassay method usually uses an antibody to connect latex, is designed into a so-called positive immunoturbidimetric immunoassay kit for clinical detection, and aims at different analytes, different types of antibody latex connectors need to be prepared, the quality of the latex-antibody connectors is a key factor of the quality of kit products, and the quality of the latex-antibody connectors is influenced by the self-property of the latex, the antibody property, the connection method and the like. The latex properties include latex material and synthesis method, latex particle size, latex surface properties, etc.; antibody properties including antibody type, antigenic determinant, antibody purity, etc.; the coupling method includes a physical method and a chemical method, the physical adsorption coupling method is influenced by the surface properties, the size, the antibody purity and the adsorption process of the particles, and the chemical method is influenced by the surface active groups of the particles, a cross-linking agent, the coupling process and the like. The above factors are influenced, and due to the diversity of antibody proteins and latex particles, even if the same latex and antibody are prepared by a physical method or a chemical method, different manufacturers cannot obtain the same kit product due to the fact that the preparation methods of different latex-antibody conjugates are not completely consistent. The clinical results are affected by the product factors of the kit and are difficult to standardize, so that different hospitals cannot commonly use the same detection result, the phenomenon of multiple detections is caused, and the detection cost of patients is increased.

3. In the existing immunoturbidimetric method, for each analyte, a corresponding latex-antibody conjugate needs to be prepared, and the research and development process of the kit needs to research the types of antibodies, the types of latex, the sizes of latex and the conjugation method to obtain the optimal detection scheme so as to prepare the kit; biotechnology is changing day by day, new markers which are clinically valuable are continuously discovered, and new kits which need to be developed are continuously increased, which increases huge workload for the development of the kits.

Disclosure of Invention

In order to solve the problems, the invention discloses a human urinary albumin latex enhanced secondary antibody competitive immunoturbidimetric assay kit and a manufacturing method thereof. As shown in FIG. 4, the method for enhancing immunosuppressive turbidimetry of the present invention overcomes the defects of the existing method for enhancing immunosuppressive turbidimetry in principle of immune reaction,it utilizes the excess antigen curve portion of the agglutination of the immunoreaction, and the secondary antibody (Ab) attached to the latex particle when no antigen is measured in the reaction system₂) Will neutralize the first antibody (Ab) in the system₁Anti-albumin antibody) to form latex agglutinate, under the condition of a certain antibody concentration, the agglutination effect reaches the maximum, when the antigen exists in the measurement reaction system, the antigen reacts with the first antibody to form soluble immune complex, due to the steric hindrance effect of macromolecules, the antigen blocks the binding site of the secondary antibody on the first antibody and forms a competitive inhibition effect, the antigen in the sample competitively inhibits the agglutination effect of the primary-secondary antibody immune complex, therefore, as the amount of the free antigen in the reaction system increases, the agglutination effect of the immune complex formed by the reaction of the primary antibody and the secondary antibody-latex is reduced, when the amount of the antigen in the measurement system reaches a certain amount, the free antigen tends to completely inhibit the agglutination effect of the primary-secondary antibody reaction, if the nonspecific binding of the latex and other substances in the sample can be overcome, the turbidity change will tend to be zero during the reaction.

Although the method cannot expand the high concentration range infinitely, false negative detection results cannot occur, the high concentration albumin sample passing through the line can be effectively screened out, and when necessary, the high concentration albumin sample can be diluted and measured again to obtain accurate results.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a human urinary albumin latex enhanced secondary antibody competitive immunoturbidimetric assay kit comprises a reagent 1 and a reagent 2; the reagent 1 comprises a support on which an antibody to be used with a primary antibody, i.e., a secondary antibody, is supported; the secondary antibody is rabbit anti-mouse IgG or goat anti-rabbit IgG; the reagent 2 comprises a primary antibody to be used, wherein the primary antibody to be used is a mouse anti-human albumin monoclonal antibody or a rabbit anti-human albumin polyclonal antibody.

In a further refinement, the support is latex particles.

In a further improvement, the latex particles are carboxyl nano latex particles.

In a further improvement, the carboxyl nano latex particles have a particle size range of 40-500 nm.

In a further improvement, the reagent 1 and the reagent 2 are respectively prepared, and the method specifically comprises the following steps:

reagent 1: taking a nano latex particle solution, diluting the nano latex particle solution by using an acidic buffer solution, adding an activating agent for activation, adding a secondary antibody to be used for a linking reaction, adding a sealing agent, a preservative and a surfactant after the linking reaction is finished, and diluting the secondary antibody to a working volume by using a neutral buffer solution to prepare a reagent 1 containing the secondary antibody-latex;

reagent 2: adding inorganic salt, preservative and surfactant into neutral buffer solution, filtering to obtain diluent, and diluting with the diluent until the primary antibody reaches working concentration to obtain the reagent.

In a further improvement, the blocking agent is glycine, the preservative is Proclin300, and the surfactant is Tween 20; the acidic buffer solution and the neutral buffer solution are both phosphate buffer solutions; the inorganic salt is NaCl; the latex particles are carboxyl nano latex particles, the activating agent is dicyclohexylcarbodiimide, and the secondary antibody is rabbit anti-mouse IgG or goat anti-rabbit IgG.

In a further improvement, the mass-to-volume ratio of each component in the final reagent formula of the reagent 1 and the reagent 2 is as follows:

the mass-to-volume ratios of the ingredients in the final reagent formulations of reagent 1 and reagent 2 were as follows:

reagent 1: pH7.0,0.02M/L phosphate buffer

Secondary antibody-latex 0.01-0.05% g/L

Glycine 0.002M/L

Tween20 0.01％g/L

Proclin 300 0.1％g/L

Reagent 2: pH7.5,0.02M/L phosphate buffer

First resisting dilution according to working concentration

Glycine 0.002M/L

NaCl 0.15M/L

Tween20 0.001％g/L

Proclin 300 0.1％。g/L。

The further improvement comprises the following steps:

preparation of reagent 1: taking 0.2ml of 10% carboxyl nano latex particles with the particle size of 100nm, adding 0.8ml of pH 4.6 and 0.02M phosphate buffer, adding 20 mul of dicyclohexylcarbodiimide with the concentration of 20mg/M l, after 20 minutes of shaking table heat preservation reaction at 37 ℃ and activation, adding 0.5ml of phosphate buffer with the pH7.5 and 0.02M and 0.02ml of purified rabbit anti-mouse IgG antibody or 0.05ml of goat anti-rabbit IgG, carrying out 60 minutes of shaking table heat preservation reaction at 37 ℃, after the connection reaction is finished, adding 0.7.5 and 0.02M phosphate buffer to ensure that the volume of the total solution is 10ml, then adding 0.2M glycine 0.1ml and 1% tween 200.1 ml, carrying out 30 minutes of shaking table heat preservation reaction at 37 ℃ and sealing and stopping the reaction, and adding proclin300 according to 0.1%, thus preparing the reagent 1 secondary antibody containing the rabbit anti-mouse IgG or goat anti-rabbit IgG-latex connector;

preparation of reagent 2: diluting the primary antibody by water fold ratio, taking phosphate buffer solution with 0.02M, pH7.5 and containing NaCl 0.15M as R1 ', taking rabbit anti-mouse IgG or goat anti-rabbit IgG secondary antibody-latex conjugate as R2', and respectively determining the diluted primary antibody by each fold ratio on a biochemical analyzer according to the following parameter setting measuring programs; measuring wavelength: 546 nm; r₁’：R₂': the sample to be tested is 100: 100: 20, i.e. addition of R₁After' 100. mu.l, 20. mu.l of the sample to be tested was added, incubated at 37 ℃ for 5 minutes, and R was added₂' 100. mu.l, the light absorption values at 17 th and 34 th spots are read, and the light absorption difference between 34 th and 17 th spots is calculated: Δ a ═ a34-a 17; the dilution with the largest difference in light absorption was taken as the primary antibody dilution, and the primary antibody was diluted with 0.02M, pH7.5 and 0.15M NaCl-containing phosphate buffer according to the primary antibody dilution, and proclin300 and 0.01% tween20 were added at 0.1% to prepare reagent 2.

A use method of a human urinary albumin latex enhanced secondary antibody competitive immunoturbidimetric assay kit comprises the following steps:

the latex-enhanced secondary antibody competitive immunoturbidimetric assay kit comprises a load, wherein a secondary antibody of an antibody to be used is loaded on the load;

establishing a calibration curve: setting a measuring program according to the following parameters on a biochemical analyzer by using a reagent 1 and a reagent 2, namely R2, and establishing a calibration curve by using the concentration ranges of the human albumin calibrator as 0, 2.5, 5, 20, 50, 100 and 200mg/L respectively; measuring wavelength: 546 nm; reagent 1: reagent 2: calibration product 100: 100: 5, namely 1100. mu.l of the reagent is added, 5. mu.l of the calibrator is added, the mixture is incubated at 37 ℃ for 5 minutes, 2100. mu.l of the reagent is added, the light absorption values at 17 th and 34 th spots are read, and the difference between the light absorptions at 34 th and 17 th spots is calculated: A34-A17, and establishing a calibration curve according to the concentration of each calibrator and the corresponding Delta A by multipoint spline fitting; the reagent 1 contains a load on which an antibody to be used as a primary antibody, i.e., a secondary antibody, is loaded; the reagent 2 contains a primary antibody to be used;

and (3) sample determination: setting a measuring program on a biochemical analyzer by using a reagent 1 and a reagent 2 according to the following parameters, and taking human urine as a measuring sample; measuring wavelength: 546 nm; r₁：R₂: sample 100: 100: 5, i.e. addition of R₁After 100. mu.l, 5. mu.l of the sample was added, incubated at 37 ℃ for 5 minutes, and R was added₂100 μ l, reading the light absorption values at 17 th point and 34 th point, and calculating the light absorption difference between 34 th point and 17 th point: the concentration of albumin in the urine sample was calculated from Δ a as a34-a17 according to the calibration curve established above.

The invention has the following advantages: 1. the latex immunoturbidimetric kit on the market at present is mainly a so-called forward immunoturbidimetric kit, namely latex is connected with a first antibody to form a latex-antibody reagent, an antigen in a sample and the latex-antibody (primary antibody) form an immune complex to generate turbidity, so that the antigen concentration in the sample is measured.

2. Comparison of the results of the assay on 100 urine samples with the method of the present invention used in clinical practice (currently available kit method) indicates the use of monoclonal antibody (A) or polyclonal antibody (B)The method for establishing the secondary antibody competition turbidimetric method for the primary antibody has better correlation with the urine sample determination result of the current commercial kit, and the regression equation of the method (secondary antibody competition A) established by adopting the mouse anti-human albumin monoclonal antibody as the primary antibody and the rabbit anti-mouse IgG as the secondary antibody and the determination result of the commercial kit is as follows: y is_{Contrast kit}＝1.043X_{This method A}-0.2072，(R²0.9796) as shown in fig. 2-2. The regression equation of the method (secondary antibody competition B) established by taking rabbit anti-human albumin polyclonal antibody as a primary antibody and goat anti-rabbit IgG as a secondary antibody and the measurement result of the commercial kit is as follows: y is_{Contrast kit}＝1.001X_{This method B}-0.2253，(R²0.9897), as in fig (2-1); the two methods established by the human albumin monoclonal antibody and the polyclonal antibody have better correlation, and the regression equation of the measurement results of the two methods is as follows: y is_{This method A}＝0.9503X_{This method B}+0.2063，(R²0.9895), the mean and standard deviation of the results of the three methods are as follows: x_{This method A}＝19.75±19.28，X_{This method B}＝21.00±20.18，Y_{Contrast kit}20.67 ± 20.19, variance analysis three groups of mean differences were not significant (F0.114, P0.893 > 0.05).

TABLE (I) correlation analysis of results of three methods for determining 100 human urine samples

Measurement method	Intercept of a beam	Slope of	R2
				The method is a competitive A and contrast kit for the second antibody	0.2072	1.043	0.9796
This method is two to compete B and contrast kit	0.2253	1.001	0.9897
				The second antibody of the method competes with the first antibody of the method to compete with the second antibody of the method to compete with the first antibody of the method to compete	0.2063	0.9503	0.9895

3. The positive latex immunoturbidimetric assay, which usually has the so-called "hook" effect, can produce false negative results when the antigen is excessive, and the secondary antibody competition assay method can effectively avoid the false negative, the secondary antibody competition assay method of the present invention, when the albumin concentration in the sample exceeds 200mg/L, the measured value of the sample has a larger deviation from the actual value, but the measurement result below 200mg/L can not be produced, the sample concentration is increased to 5000mg/L, and the false negative results can not be produced, as shown in the table (III) and the figure (4). Comparing 1000-5000mg/L, determining results, 5 groups of high concentration determining results, and the overall mean value: AV 326.1, standard deviation: SD-20.64, no significance of mean differences (F-3.84, P-0.52 > 0.05). By adopting the method, the high-concentration sample can be effectively screened, but the measured value of the high-concentration sample cannot reflect the real result, and if clinically needed, the sample can be diluted to obtain an accurate measurement result.

4. The latex enhanced secondary antibody competition immunoturbidimetry method overcomes the defects of the prior enhanced immunoturbidimetry method in the principle of immunoreaction, utilizes the competition principle of immunoreaction agglutination, when no antigen exists in a measurement reaction system, a rabbit anti-mouse IgG or a goat anti-rabbit IgG secondary antibody connected on latex particles is combined with an anti-human albumin primary antibody (monoclonal antibody or polyclonal antibody) to generate agglutination or precipitation reaction, and the agglutination or precipitation effect is maximum, when the antigen exists in the measurement reaction system, because the primary antibody and the secondary antibody have limited amounts, the primary antibody and the antigen are combined to form an immune complex, the combining sites of the primary antibody and the secondary antibody are closed competitively, and a competitive reaction is formed relative to the combination of the primary antibody and the secondary antibody, thereby reducing the agglutination or precipitation effect, when the free antigen is excessive, the combination of the primary antibody and the secondary antibody is completely inhibited in competition, therefore, as the amount of antigen in the reaction system increases, the agglutination or precipitation effect of the primary-secondary antibody immunocomplex decreases, and when the amount of antigen in the measurement system reaches a certain amount, the free antigen will completely inhibit the binding of the primary antibody to the secondary antibody on the latex, and if the nonspecific binding of the latex and the excessive antigen and other substances in the sample can be overcome, the turbidity change in the reaction process will be zero. Although the method cannot expand the high concentration range infinitely, false negative detection results cannot occur, the high concentration sample of the line can be effectively screened out, and when the need is certain, the high concentration sample can be diluted and measured again to obtain accurate results.

5. By adopting the secondary antibody-latex establishing method, the influence of different latexes, different antibodies, different prepared latex-antibody conjugates and other factors on the product quality can be avoided, the standardization of clinical detection results is facilitated, the universality of the detection results is achieved, and the detection cost of patients is reduced.

6. If the universal second antibody-latex conjugate is adopted, the development method can avoid the influence of various antigens, and can achieve the purpose of measuring different antigens by using the same second antibody-latex conjugate, namely, the same second antibody-latex forms kits of different antigen types, so that the development time of the kits can be greatly saved, and the development workload can be reduced.

Drawings

FIG. 1 shows an anti-titration assay (1, 2, 3, 4, 5, 6, 7) with titers (1: 8),

1：16，1：32，1：64，1：128，1：256，0)；

FIG. 2 shows the secondary antibody competition calibration curves of HSA monoclonal antibody (A) and polyclonal antibody (B);

FIG. 3-1 shows the result of the second antibody competition method A compared with the result of the comparative kit for determining urine;

3-2 comparing the results of the second antibody competition method B with the results of the comparative kit;

3-3 two antibody competition method A and B urine results comparison;

fig. 4 is a schematic diagram of the present invention.

Detailed Description

Example 1

Preparation of Rabbit anti-mouse IgG Secondary antibody (A) or goat anti-Rabbit IgG (B) -latex conjugate

A commercial carboxyl nano latex particle with the particle size of 100nm and the concentration of 10 percent is 0.2ml, 0.6 pH and 0.02M phosphate buffer solution is added, 20 mul of dicyclohexylcarbodiimide with the concentration of 20mg/ml is added, after 20 minutes of shaking table heat preservation reaction at 37 ℃ and activation, 0.5ml, pH7.5,0.02M phosphate buffer solution and 0.02ml of purified rabbit anti-mouse IgG antibody or 0.05ml of goat anti-rabbit IgG (secondary antibody) are added, the shaking table heat preservation reaction at 37 ℃ is carried out for 60 minutes, after the connection reaction is finished, 0.0 pH7.0 and 0.02M phosphate buffer solution are added to ensure that the volume of the total solution is 10ml, 0.1ml of 0.2M glycine, 200.1 ml of 1 percent Tween, 3000.1 ml, 30 minutes of shaking table heat preservation reaction at 37 ℃ is carried out, the reaction is closed and stopped, and proclin300 is added according to 0.1 percent, thus respectively obtaining the rabbit anti-mouse IgG (A) or goat anti-rabbit IgG (B) -rabbit anti-rabbit latex conjugate.

Example 2

And (3) determining the titer of the mouse anti-human albumin monoclonal antibody or the rabbit anti-human albumin polyclonal antibody:

diluting a mouse anti-human albumin monoclonal antibody or a rabbit anti-human albumin polyclonal antibody by a water-fold ratio, taking a phosphate buffer solution with 0.02M and pH7.0 (containing NaCl 0.15M) as R1, respectively preparing a rabbit anti-mouse IgG secondary antibody-latex continuous conjugate by the method as R2, setting a measuring program on a Hitachi 7170S full-automatic biochemical analyzer according to the following parameters, and measuring primary antibody solutions with different dilutions; measuring wavelength: 546 nm; r₁：R₂: sample 100: 100: 20, i.e. after addition of 100. mu.l of R1, 20. mu.l of the sample was added, incubated at 37 ℃ for 5 minutes, and then 100. mu. l R was added₂Reading the light absorption values of 17 points and 34 points, and calculating the light absorption difference values of 34 th points and 17 th points: Δ a ═ a34-a 17. Dripping deviceThe results of the assay are shown in the following table (one), and in FIG. 1, the titers selected according to the monoclonal antibody of table (one) are 1: 64, selected titers of multiple antibodies were 1: 128.

TABLE (I) anti-human albumin monoclonal or polyclonal antibody titer determination

Numbering	HSA monoclonal antibody	△	HAS polyclonal antibody	△
					1	1：8	7270	1：8	3689
2	1：16	7084	1：16	4589
					3	1：32	12468	1：32	7895
4	1：64	14326	1：64	14567
					5	1:128	8452	1:128	15665
6	1:256	2431	1:256	8674
					7	0	145	0	567

Example 3

3.1 preparation of a human urinary albumin latex enhanced secondary antibody competitive immunoturbidimetric assay kit:

the following formula is adopted to prepare the detection reagent for the competitive immunoturbidimetry of the second antibody

Kit A:

r1: pH7.0,0.02M phosphate buffer

Rabbit anti-mouse IgG secondary antibody-latex (0.01-0.05%)

Glycine (0.002M)

Tween20(0.01％)

Proclin 300(0.1％)

R2: pH7.5,0.02M phosphate buffer

Mouse anti-human albumin monoclonal antibody (1:200)

NaCl(0.15M)

Casein (0.01%)

Tween20(0.001％)

Proclin 300(0.1％)

Kit B

R1: pH7.0,0.02M phosphate buffer

Goat anti-rabbit IgG secondary antibody-latex (0.01-0.05%)

Glycine (0.002M)

Tween20(0.01％)

Proclin 300(0.1％)

R2: pH7.5,0.02M phosphate buffer

Rabbit anti-human albumin (1: 500)

NaCl(0.15M)

Casein (0.01%)

Tween20(0.001％)

Proclin 300(0.1％)

3.2 measuring parameters

Setting a measuring program on a Hitachi 7170S full-automatic biochemical analyzer according to the following parameters; measuring wavelength: 546 nm; r₁：R₂: sample 100: 100: 5, i.e. after adding 100. mu.l of R1, add 5. mu.l of sample, incubate at 37 ℃ for 5 minutes, add 100. mu. l R₂Reading the light absorption values of 17 points and 34 points, and calculating the light absorption difference values of 34 th points and 17 th points: Δ a ═ a34-a 17. The measurement parameters of the commercial kit are set according to the kit instruction. 3.3 calibration Curve, sample determination and comparison

Diluting standard human albumin antigen with water to 0, 2.5, 5, 20, 50, 100 and 200mg/L, respectively establishing each calibration curve according to the measurement parameters and the measurement parameters of a commercially available kit, as shown in a figure (2), collecting 100 clinical urine samples, respectively comparing and analyzing the urine samples with the commercially available kit by using the kit method (A and B) of the invention, determining each sample twice by using the same calibrator, determining in reverse direction for the second time, calculating the average value of each kit determination, and calculating the correlation coefficient and a linear equation. Shown in Table (II), FIGS. 3-1, (3-2) and (3-3).

TABLE (II) two-antibody Competition methods A, B and kit for 100-part urine test results comparison

Example 4

Samples containing albumin at high concentration were prepared from low concentration urine at concentrations of 50, 100, 200, 1000, 2000, 3000mg/L, and the high concentration samples were measured by the above three methods according to the above measurement procedures, each sample was measured in triplicate, and the results were expressed as mean ± sd. As shown in table (iii), the second antibody competition method has a large difference from the theoretical value when measuring a high concentration sample, but does not show a low value lower than the highest concentration sample, and if necessary, the sample can be diluted and then measured to obtain an accurate value, while the commercially available kit shows a low value lower than the highest calibrator concentration when measuring a high concentration.

Specifically, when the haze in the sample is more than 1000mg/L as shown in Table (III), the measured value of the comparative sample commercially available for the comparison of the present method is decreased, that is, the hook effect is exhibited, at a comparative sample concentration of 200mg/L (when the sample reagent concentration is 1000mg/L, the result is less than 100 mg/g). Although the detection value of the method is larger than the actual value, the detection value still rises (when the sample is 1000mg/L, the detection value is still larger than 200mg/L, namely, the detection result still increases along with the increase of the sample concentration), namely, the error result that the sample concentration is lower than the pathological threshold value is not given. If a high-concentration sample needs to be detected, the detection result indicates that the concentration is higher than the accurate detection threshold of the method (for example, the accurate detection threshold of the method is 200mg/L, and when the detection result is a concentration greater than 200mg/L, the concentration of the sample exceeds the accurate detection threshold), at this time, the sample is diluted by multiple times, and then the accurate concentration of the high-concentration sample can be detected through detection.

TABLE (III) determination of high concentration urine

All of the following schemes are also within the scope of the present invention:

1. reagent type of ligation reaction

There are at least three types of commercially available latex, including carboxyl, hydroxyl, thiol, etc., and the methods employed to attach the secondary antibody are different, but the basic design is to attach the secondary antibody to the latex particles, and none of them is used, and the most obtained result is to prepare a suitable secondary antibody-latex attachment substance to facilitate the secondary antibody-enhanced competitive immunoturbidimetry assay. For carboxyl latex, various activating reagents including EDC, EDC sodium salt, TBE, etc. can be used to prepare the secondary antibody-latex conjugate under certain conditions of suitable pH, temperature, etc., and are within the scope of the present invention.

2. Type of particle

Commercially available latexes include those of various particle sizes, e.g., carboxy latexes of 40, 80, 100, 150, 200, 300, 500, 1000 nanometers, etc., and no latexes of any size are optionally available, and it is within the scope of the invention that secondary antibody-latex conjugates can be prepared under conditions to accomplish the above process.

3. Name, competitive immunoturbidimetric reaction

The method for enhancing competitive turbidimetry established based on the secondary antibody-linker is not unified in professional presentation at present, and is called competitive immunoturbidimetry or inhibition immunoturbidimetry, and the like, although the names are different, the actual principles are completely the same, so the names are different, but the reagents and the steps are the same and are also in the protection scope of the invention.

4. The kind of antibody and the kind of antigen

The secondary antibodies attached to the latex particles can be various, including monoclonal antibodies including murine monoclonal antibodies and rabbit monoclonal antibodies, and polyclonal antibodies including rabbit polyclonal antibodies, sheep polyclonal antibodies, and the like, and are within the scope of the present invention.

5. Types of instruments

At present, there are at least two types of instruments for detecting immunoturbidimetric reactions, a biochemical analyzer based on transmission turbidimetric assay and a so-called specific protein analyzer based on nephelometric turbidimetric assay. The automatic control system comprises a semi-automatic type and a full-automatic type, and a plurality of manufacturers are available, relatively famous foreign manufacturers comprise Hitachi, Siemens, Roche, Beckman and the like, and domestic manufacturers comprise Mirui, KuBi Er, Kowa, Youlite and the like. Various instrument types can be used for antibody screening and titer determination, kit method establishment and clinical application. It is therefore within the scope of the invention to use various instruments for detection.

While embodiments of the invention have been disclosed above, it is not limited to the applications set forth in the specification and the embodiments, which are fully applicable to various fields of endeavor for which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

Claims

1. A human urinary albumin latex enhanced secondary antibody competitive immunoturbidimetric assay kit is characterized by comprising a reagent 1 and a reagent 2; the reagent 1 comprises a support on which an antibody to be used with a primary antibody, i.e., a secondary antibody, is supported; the secondary antibody is rabbit anti-mouse IgG or goat anti-rabbit IgG; the reagent 2 comprises a primary antibody to be used, wherein the primary antibody to be used is a mouse anti-human albumin monoclonal antibody or a rabbit anti-human albumin polyclonal antibody;

preparation of reagent 1: taking 0.2ml of 10% carboxyl nano latex particles with the particle size of 100nm, adding 0.8ml of pH 4.6 and 0.02M phosphate buffer, adding 20 mul of dicyclohexylcarbodiimide with the concentration of 20mg/M l, carrying out heat preservation reaction on a shaker at 37 ℃ for 20 minutes, activating, adding 0.5ml of pH7.5 and 0.02M phosphate buffer and 0.02ml of purified rabbit anti-mouse IgG antibody or 0.05ml of goat anti-rabbit IgG, carrying out heat preservation reaction on the shaker at 37 ℃ for 60 minutes, after the connection reaction is finished, adding 0.7.5 and 0.02M phosphate buffer to ensure that the volume of the total solution is 10ml, adding 0.1ml of 0.2M glycine and 200.1 ml of 1% Tween, carrying out heat preservation reaction at 37 ℃ for 30 minutes, sealing and stopping the reaction, and adding proclin300 according to 0.1%, thus preparing a reagent 1 containing rabbit anti-mouse IgG or goat anti-rabbit IgG-latex connectors;

preparation of reagent 2: diluting the primary antibody by water fold ratio, taking phosphate buffer solution with 0.02M, pH7.5 and containing NaCl 0.15M as R1 ', taking rabbit anti-mouse IgG or goat anti-rabbit IgG secondary antibody-latex conjugate as R2', and respectively determining the diluted primary antibody by each fold ratio on a biochemical analyzer according to the following parameter setting measuring programs; measuring wavelength: 546 nm; r₁’：R₂': sample to be tested = 100: 100: 20, i.e. addition of R₁After the sample is 100 mu l, adding 20 mu l of the sample to be detected, keeping the temperature at 37 ℃ for 5 minutes, and adding R₂' 100 μ l, reading light absorption values at 17 and 34 points, and calculating light absorption difference values at 34 and 17 points: Δ a = a34-a 17; absorption difference of lightThe dilution with the highest value was the primary antibody dilution, which was diluted with 0.02M, pH7.0 and NaCl 0.15M phosphate buffer to 0.1% proclin300, 0.01% tween20 to give reagent 2.

2. The use method of the human urinary albumin latex-enhanced secondary antibody competitive immunoturbidimetric assay kit as claimed in claim 1, comprising the following steps:

establishing a calibration curve: setting a measuring program according to the following parameters on a biochemical analyzer by using a reagent 1 and a reagent 2, namely R2, and establishing a calibration curve by using the concentration ranges of the human albumin calibrator as 0, 2.5, 5, 20, 50, 100 and 200mg/L respectively; measuring wavelength: 546 nm; reagent 1: reagent 2: calibrator = 100: 100: 5, after adding 1100 mul of reagent, adding 5 mul of calibration material, keeping the temperature at 37 ℃ for 5 minutes, adding 2100 mul of reagent, reading the light absorption values of 17 points and 34 points, and calculating the light absorption difference values of the 34 th point and the 17 th point: the delta A = A34-A17, and a calibration curve is established according to the concentration of each calibrator and the corresponding delta A by multipoint spline fitting; the reagent 1 contains a load on which an antibody to be used as a primary antibody, i.e., a secondary antibody, is loaded; the reagent 2 contains a primary antibody to be used;

and (3) sample determination: setting a measuring program on a biochemical analyzer by using a reagent 1 and a reagent 2 according to the following parameters, and taking human urine as a measuring sample; measuring wavelength: 546 nm; r₁：R₂: sample = 100: 100: 5, i.e. addition of R₁After 100 mul, adding 5 mul of a sample, preserving heat at 37 ℃ for 5 minutes, and adding R₂And (5) reading light absorption values of 17 points and 34 points by 100 mu l, and calculating light absorption difference values of 34 th points and 17 th points: Δ A = A34-A17, from which the albumin concentration in the urine sample is calculated according to the calibration curve established above.