Reagent combination and its application in eliminating non-specific interference detection of MALB reagent

Abstract

The present application relates to the technical field of clinical in vitro detection reagent, especially relates to reagent combination and its application in eliminating MALB reagent non-specific interference detection. When MALB reagent is calibrated at 0 point with purified water, and normal human urine sample is tested on a biochemical instrument, the test value can be 0 or negative, especially when the concentration of urine is 0 mg / L-5 mg / L, the test value is basically 0. The present application selects suitable Malb antibody and suitable antibody feeding amount to improve the sensitivity of reagent, and selects suitable background solution in the reaction system to reduce the non-specific interference of MALB reagent. Not only the problem of 0 or negative sample value is effectively improved, but also the detection accuracy is improved, which has important significance for improving the accuracy of MALB reagent test sample. The reagent combination provided by the present application has good clinical application and large-scale industrial production prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of clinical in vitro detection reagent technology, and particularly to a reagent combination and application thereof in eliminating non-specific interference detection of MALB reagent. BACKGROUND

[0002] Turbidimetric inhibition immuno assay is a dynamic determination method of antigen-antibody combination. The basic principle is that when the antigen and the antibody react in a special dilution system and the proportion is appropriate (generally, the antibody is in excess), the soluble immune complex formed under the action of the polymerization agent (polyethylene glycol, etc.) in the dilution system is precipitated from the liquid phase to form microparticles, so that the reaction solution appears turbidity. When the antibody concentration is fixed, the amount of immune complex formed increases with the increase of the amount of antigen in the sample, and the turbidity of the reaction solution also increases. By comparing the turbidity of the reaction solution with a series of standard samples, the content of antigen in the sample can be calculated. It is divided into immunoturbidimetry, immunosorbent turbidimetry and immunolatex turbidimetry.

[0003] Albumin is one of the important plasma proteins, under normal circumstances, the molecular weight of albumin is large, can not cross the glomerular basement membrane, therefore, in healthy people urine contains only a very low concentration of albumin, specifically to each liter of urine albumin is not more than 20 mg, so also known as "urinary microalbumin" (MALB). About MALB reagent detection method mainly has two kinds of immune latex turbidity method and immune transmission turbidity method. Non-specific binding refers to the binding of a certain ligand to its corresponding specific structural site outside other irrelevant substances (such as non-specific proteins, containers, separation materials, etc.). Its characteristics are low affinity and large binding capacity. In the past 30 years, there have been many inventions and researches on reducing non-specific binding. There are researches on non-specific reaction inhibitors of two major groups of secondary amines and tertiary amines, mainly suitable for reducing non-specific binding when detecting serum samples; there are also reagents for protein induced vitamin K absence or antagonist-II (PIVKA-II) that use additives such as skim milk, saponin, CaCl2, MgCl2 and sulfobetaine zwitterionic detergent to reduce non-specific binding. However, there is no research on reducing non-specific interference of MALB reagent to test normal urine samples on biochemical analyzer to 0 or negative value. The antibody used in the MALB kit is sheep anti-human albumin antiserum, which has complex components and inevitably introduces some unknown components. In addition, the composition and physicochemical properties of urine itself are also complex, which may cause non-specific binding between the antibody used and the antigen other than MALB in the urine sample, thereby reducing the accuracy of the low-end test. The normal range of microalbumin in normal urine is 0 mg / L to 30 mg / L. From this range, it can be seen that the albumin in normal urine is very small, and some even have none, especially in urine with MALB content in the range of 0 mg / L to 5 mg / L. When using pure water as the 0 point for calibration on the biochemical analyzer, the non-specific antibody / antigen interaction makes the absorbance change of the blank greater than that of the urine sample with very low MALB content when reacting with the reagent, thereby causing the test value of the urine sample to be 0 or negative, and the content of MALB in the normal urine sample cannot be accurately given. Therefore, it is necessary to improve the sensitivity and accuracy of the MALB kit for testing normal urine samples.

[0004] The MALB reagent detection is double reagent, which is divided into reagent 1 and reagent 2, and the reagent 1 is mainly to create a suitable and stable reaction environment for the whole reaction, that is, the reaction of the antibody in the reagent 2 and the antigen in the sample; the method for improving the sensitivity and accuracy of the MALB kit in detecting normal human urine samples is mainly carried out in two aspects, one is to improve the analysis sensitivity of the reagent, mainly by changing the feeding amount of the antibody, but the manufacturing process, purity and composition of the antibody of different manufacturers will be different, and the specific amount needs to be determined according to the actual situation; the other is to provide a suitable background solution for the whole reaction process. The defect is that when the MALB reagent is calibrated at 0 point with purified water on the biochemical instrument, the test value of the normal human urine sample is 0 or negative, especially when the concentration of the urine is 0 mg / L-5 mg / L, the test value is basically 0. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to provide a reagent combination and its application in eliminating the non-specific interference detection of the MALB reagent.

[0006] The present application improves the analysis sensitivity of the reagent by selecting a suitable Malb antibody and a suitable antibody feeding amount, and cooperates with the selection of a suitable background solution in the reaction system to reduce the non-specific interference of the MALB reagent.

[0007] The present application provides a reagent combination, which comprises a reagent combination, characterized by comprising reagent R1 and reagent R2:

[0008] The reagent R1 comprises: phosphate, chloride ion, preservative, PEG6000 and surfactant;

[0009] The mass ratio of each component of the reagent R1 is: phosphate: chloride ion: preservative: PEG6000: surfactant = (1.9-5.7):8:0.01:50:1;

[0010] The reagent R2 comprises: Tris-HCl, chloride ion, preservative, surfactant and sheep anti-human Malb antiserum;

[0011] The mass ratio of each component of the reagent R2 is: Tris-HCl: chloride ion: preservative: surfactant: sheep anti-human Malb antiserum = 12.1:8.77:0.01:1:150.

[0012] In the reagent combination provided by the present application:

[0013] The phosphate buffer solution comprises one of potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium phosphate;

[0014] The surfactant includes one of Triton X-100, Tween 20, Tween 40, Tween 60, Tween 80, SPAN20, SPAN40, SPAN60, SPAN80, Triton X-114, and Triton X-405.

[0015] The preservatives include: Proclin series preservatives, parabens, potassium sorbate, sodium benzoate, sodium nitrite and / or sodium azide;

[0016] The Proclin series of preservatives includes Proclin 150, Proclin 200, Proclin 300 and / or Proclin 5000;

[0017] The paraben preservatives include methylparaben, ethylparaben, propylparaben, and / or butylparaben.

[0018] The chloride ions are derived from potassium chloride or sodium chloride.

[0019] In the reagent combination described in this invention, the mass ratio of reagent R1 to reagent R2 is R1:R2 = 4:1. In the reagent combination, the pH value of both reagent R1 and reagent R2 is 6-8.

[0020] In the reagent combination described in this invention:

[0021] The reagent R1 comprises: 20 mmol / L to 60 mmol / L phosphate buffer, 137 mmol / L sodium chloride, 0.001% Proclin 300, 5% PEG 6000 and 1 g / L Triton X-100;

[0022] The reagent R2 comprises: 100 mmol / L Tris-HCl buffer, 150 mmol / L sodium chloride, 0.001% Proclin 300, 1 g / L Triton X-100 and 15% goat anti-human Malb antiserum.

[0023] The present invention also provides the application of the reagent combination in reducing nonspecific interference in MALB reagent or immunoturbidimetry and / or improving the detection accuracy of MALB reagent.

[0024] In the application described in this invention, improving the detection accuracy of the MALB reagent includes reducing the probability of samples with test concentrations of 0 mg / L to 5 mg / L producing a value of 0 or a negative value.

[0025] In the applications described in this invention, the immunoturbidimetric method includes immunotransmission turbidimetry, immunoscattering turbidimetry, and / or immunolatex turbidimetry.

[0026] The application also provides a method for eliminating non-specific interference in MALB reagent or immune nephelometry, which comprises detecting a sample by using the reagent combination.

[0027] The application adopts the reagent combination of six groups, each group having 38 samples for experiment, and the conclusion obtained is that the components of the reagent support each other and interact with each other to improve the analysis sensitivity of the reagent and reduce the probability of 0 value of the test sample of 0-5 mg / L. However, the greater the analysis sensitivity of the reagent is, the smaller the probability of 0 value is, and the 0 value of the test sample is also related to the background solution. When the reagent combination parameters are as follows: R1 component: phosphate buffer: 20 mmol / L, sodium chloride: 137 mmol / L, Proclin 300: 0.001%, PEG6000: 5%, Triton X-100: 1 g / L, PH = 7.4; R2 component: Tris-HCl buffer: 100 mmol / L, sodium chloride: 150 mmol / L, Proclin 300: 0.001%, Triton X-100: 1 g / L, sheep anti-human Malb antiserum (SHBLS): 15%, PH = 7.4, the analysis sensitivity of the reagent at the low end is the largest, but the number of 0 values of the test urine is not the least.

[0028] Therefore, the effective method for reducing the non-specific interference of the MALB reagent is to jointly reduce the non-specific interference of the MALB reagent and reduce the probability of 0 value of the test sample of 0-5 mg / L by selecting a suitable Malb antibody, the amount of the antibody, and the selection of the reagent components (reagent 1: the best combination of phosphate concentration of 20 mmol / L-60 mmol / L and NaCl concentration of 137 mmol / L; reagent 2: using 100 mmol / L of Tris-Hcl buffer and 250 mmol / L of NaCl). It is found that the parameters of each step support each other and interact with each other.

[0029] In the method, the method comprises using a full-automatic biochemical analyzer to determine by using an end-point method, and the detection main wavelength is 340 nm.

[0030] The application also provides a preparation method of the reagent combination, which comprises:

[0031] The reagent R1 is prepared by adding phosphate buffer, sodium chloride, and Proclin 300 to mix uniformly, adjusting the PH to 7.4, and adding PEG6000 and Triton X-100.

[0032] Reagent R2: Tris-HCl buffer, sodium chloride, Proclin 300, Triton X-100 are added and mixed, the pH is adjusted to 7.4, and goat anti-human Malb antiserum is added.

[0033] The application also provides a method for using the reagent combination, which comprises mixing a sample with the reagents R1 and R2 and performing detection.

[0034] The reagent of the application enables the rapid formation of an antigen-antibody complex between albumin in a sample and the anti-human albumin antibody in the reagent, so that the reaction solution exhibits turbidity. When there is an excess of antibody in the reaction solution, the formed complex increases with the amount of antigen, and the turbidity of the reaction solution also increases, which can be compared with a calibration sample to calculate the content of unknown albumin. The analysis sensitivity of the reagent can be improved by selecting a suitable Malb antibody and a suitable amount of antibody, and the non-specific interference of the MALB reagent can be reduced by selecting a suitable background solution in the reaction system.

[0035] The application can reduce the non-specific interference of the MALB reagent (immunoturbidimetry) in the test of a low-value urine sample by improving the analysis sensitivity of the reagent and appropriately increasing the ion concentration in the entire reaction system, thereby improving the accuracy of the MALB reagent in the test of trace albumin in a low-value urine sample.

[0036] The application provides a reagent combination and its application in eliminating the non-specific interference in the detection of the MALB reagent. In the reagent combination, the combination of a phosphate concentration of 20 mmol / L to 60 mmol / L and a NaCl concentration of 137 mmol / L in reagent R1 is optimal, and the use of a Tris-Hcl buffer of 100 mmol / L and a NaCl of 250 mmol / L in reagent R2 is optimal. The reagent combination not only effectively improves the problem of 0 value or negative value in the test of a normal human urine sample, but also improves the accuracy of the test value of a normal human urine sample and ensures the accuracy of the test of an abnormal urine sample. The reagent combination has important significance for improving the accuracy of the MALB reagent in the test of a normal human sample, and has good clinical application and large-scale industrial production prospects. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor:

[0038] Figure 1 MALB general immunoturbidimetry reagent calibration curve. DETAILED DESCRIPTION

[0039] The present application provides reagent combination and its application in eliminating non-specific interference detection of MALB reagent. Those skilled in the art can refer to the content herein and appropriately improve the process parameters. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0040] The goat anti-human Malb antiserum used in Comparative Example 1 and Comparative Example 2 is from SHYS Biotechnology Co., Ltd.; the goat anti-human Malb antiserum used in Comparative Example 3 and the example is from SHBLS Biotechnology Co., Ltd.

[0041] The embodiments of the present application will be described in detail below in conjunction with the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. The specific conditions not indicated in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be purchased on the market.

[0042] Example 1

[0043] (1) The present application sets three examples and three comparative examples, and the reagent composition of the examples and each comparative example is shown in Table 1.

[0044] Among them, the comparative example is the reagent prepared according to the comparative reagent combination, and the buffer pH, proclin300, PEG6000, TritonX-100 and other components used in the reagents of the examples and the comparative examples are consistent. Reagent 1 is first mixed uniformly with phosphate buffer, sodium chloride and Proclin300 (a preservative), then the pH is adjusted to 7.4, and then PEG6000 (polyethylene glycol) and TritonX-100 (one of the Triton series of surfactants) are added; reagent 2 is first dissolved and mixed uniformly in purified water with phosphate buffer / Tris-HCl buffer, sodium chloride, Proclin300 and TritonX-100, then the pH is adjusted to 7.4, and then the goat anti-human Malb antiserum is added.

[0045] Table 1: Reagent composition table of each comparative example and example

[0046]

[0047]

[0048] Note: The goat anti-human Malb antiserum used in the comparative examples was from the manufacturer SHYS Biotechnology Co., Ltd.; the goat anti-human Malb antiserum used in the examples was from SHBLS Biotechnology Co., Ltd.

[0049] (2) The reagents in the above comparative examples and examples were calibrated on the full-automatic biochemical analyzer TBA120-FR using calibrators, and normal human urine samples were measured. The calibrator used for calibration was a single-level calibrator with a concentration of (1000 mg / L), which needed to be diluted with purified water to 500 mg / L, 250 mg / L, 62.5 mg / L, and 31.25 mg / L. Finally, purified water was used as the 0-point calibrator, and a 6-point calibration was run.

[0050] Table 2: Detection parameters of the full-automatic biochemical analyzer TBA120-FR

[0051]

[0052] (3) The final experimental results were processed and analyzed. The reaction degree (i.e. analysis sensitivity) of the low end of the reagent was mainly analyzed, and the analysis sensitivity of the low end of the reagent, the concentration of ions and the concentration of the buffer were observed, and the influence of the normal human urine measurement value, especially the low value urine sample with a concentration of 0 mg / L to 5 mg / L, was observed.

[0053] The calibration results on the full-automatic biochemical analyzer TBA-120FR are shown in Table 3, and the calibration curve is as follows Figure 1 .

[0054] Table 3: Calibration reaction degree of MALB ordinary immune turbidimetry reagent

[0055]

[0056] Note: Blank reaction degree is the change in blank absorbance; the higher the analysis sensitivity of the reagent, the greater the reaction degree of each calibration point.

[0057] From the calibration results, it can be seen that the reaction degree of each calibration point of the reagent in Example 1 is the largest, followed by Comparative Example 3, Example 2, and Example 3.

[0058] (2) 29 urine samples with a concentration of (0 mg / L to 5 mg / L) and 9 samples with a concentration of (15 mg / L to 40 mg / L) were tested, a total of 38 samples.

[0059] The urine sample measurement results are shown in Table 4:

[0060] Table 4: Urine measurement results

[0061]

[0062]

[0063] From the measured results, 24 cases of 0 value for the reagent of Comparative Example 1, 18 cases of 0 value for the reagent of Comparative Example 2, 9 cases of 0 value for the reagent of Comparative Example 3, and 2 cases of 0 value for the reagents of Examples 1, 2 and 3; the sample with the test concentration of (15 mg / L-40 mg / L) has little difference in the measured results of the three reagents of Comparative Examples and the three reagents of Examples.

[0064] (3) From Table 3 and Table 4, it can be concluded that improving the analysis sensitivity of the reagent can reduce the probability of 0 value of the sample with the test concentration of 0 mg / L-5 mg / L, which can be seen from the calibration reaction rate of the reagents of Comparative Examples 1, 2 and 3, and the number of 0 value of the urine test; however, the greater the analysis sensitivity of the reagent, the smaller the probability of 0 value, and the 0 value of the test sample is also related to the background solution, which can be seen from the reaction rate of each calibration point of the reagents of Comparative Example 3, Examples 1, 2 and 3, and the number of 0 value of the urine sample with the test concentration of 0 mg / L-5 mg / L, the analysis sensitivity of the reagent of Example 1 is the largest at the low value end, but the number of 0 value of the urine test is not the least.

[0065] (4) The effective method for reducing the non-specific interference of the MALB reagent is to select the appropriate Malb antibody and the appropriate antibody dosage on one hand to improve the analysis sensitivity of the reagent; on the other hand, the appropriate background solution and reagent components (reagent 1: the phosphate concentration of 20 mmol / L-60 mmol / L and the NaCl concentration of 137 mmol / L are the best combination; reagent 2: using 100 mmol / L of Tris-Hcl buffer and 150 mmol / L of NaCl) in the reaction system. Both of them work together to reduce the non-specific interference of the MALB reagent and reduce the probability of 0 value of the urine sample with the test concentration of 0 mg / L-5 mg / L.

[0066] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions described in the foregoing examples can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. Application of a reagent combination in reducing non-specific interference of a MALB reagent and improving detection accuracy of the MALB reagent; the improvement of the detection accuracy of the MALB reagent is to reduce the probability of 0 or negative values of samples with a test concentration of 0 mg / L to 5 mg / L. The reagent combination comprises reagent R1 and reagent R2: The reagent R1 is: 20 mmol / L to 60 mmol / L phosphate buffer, 137 mmol / L sodium chloride, 0.001% Proclin 300, 5% PEG6000 and 1 g / L TritonX-100; The reagent R2 is: 100 mmol / L Tris-HCl buffer, 150 mmol / L sodium chloride, 0.001% Proclin 300, 1 g / L TritonX-100 and 15% goat anti-human Malb antiserum.

2. The application of claim 1, wherein: The phosphate buffer comprises one of potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium phosphate.

3. Use according to claim 1, characterized in that, The mass ratio of the reagent R1 and the reagent R2 is R1: R2 = 4:

1.

4. The use according to any one of claims 1 to 3, characterized in that, The pH values of the reagent R1 and the reagent R2 are both 6 to 8.

5. The use according to claim 1, characterized in that, The immunoturbidimetry includes immunotransmission turbidimetry, immunoscattering turbidimetry and / or immunolatex turbidimetry.

6. A method for eliminating non-specific interference of MALB reagent and improving detection accuracy of the MALB reagent, the improvement of the detection accuracy of the MALB reagent being reduction of probability of 0 value or negative value of a sample with a test concentration of 0 mg / L to 5 mg / L, characterized in that, The method comprises detecting a sample using a reagent combination; The reagent combination comprises reagent R1 and reagent R2: The reagent R1 is: 20 mmol / L to 60 mmol / L phosphate buffer, 137 mmol / L sodium chloride, 0.001% Proclin 300, 5% PEG6000 and 1 g / L TritonX-100; The reagent R2 is: 100 mmol / L Tris-HCl buffer, 150 mmol / L sodium chloride, 0.001% Proclin 300, 1 g / L TritonX-100 and 15% goat anti-human Malb antiserum.

7. The method of claim 6, wherein, The method comprises using a full-automatic biochemical analyzer to determine by end-point method, and the detection main wavelength is 340 nm.

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