Reagents, kits, methods and devices for whole blood sample five-item immune assay

Abstract

The present application discloses a reagent, a kit, a method and a device for the five-item immune assay of a whole blood sample. The reagent used in the five-item immune assay of a whole blood sample in the present application includes a buffer, an osmotic pressure regulator, a surfactant, a polymerization promoter, a blocking agent and a preservative; wherein the surfactant is a combination of a cationic surfactant and a non-ionic surfactant. The reagent used in the five-item immune assay of a whole blood sample in the present application can directly hemolyze the whole blood sample, and after hemolysis, it can be used as a reaction buffer for immune reaction, and antiserum reagents are directly added thereto to perform the five-item immune assay. The reagent of the present application can not only directly perform the five-item immune assay on the whole blood sample, omitting many pre-treatment steps; but also has good sensitivity, strong specificity, and fast detection speed, and can meet the requirements of rapid detection; it provides a new formula and scheme for the five-item immune assay of a whole blood sample.

Description

Technical Field

[0001] The present application relates to the field of five immune test items, and in particular to a reagent, a kit, a method and a device for five immune test items of whole blood samples. Background Art

[0002] The five immune items refer to IgA, IgG, IgM, complement C3 and C4, of which IgG, IgA and IgM are three types of immunoglobulins (Immune Globulin, Ig), and C3 and C4 are two types of complements, and their concentrations vary in different age groups. In certain disease situations, the concentrations of these indicators will increase or decrease, thus having a reference value for disease diagnosis.

[0003] The existing five-item immunoassay technology can only detect serum or plasma. After each venous blood is drawn, sample pretreatment is required, such as pre-dilution and / or centrifugation to separate serum / plasma before subsequent testing. This not only requires centrifugal equipment, which is costly, but also requires a large amount of blood. Generally, 3-5mL of anticoagulated venous blood needs to be drawn and centrifuged to obtain serum and plasma before testing. The large amount of blood drawn is extremely unfavorable for collecting peripheral blood from infants and young children. In addition, the process is cumbersome and the detection speed is slow.

[0004] Currently, there is no immunological five-item test kit that can directly detect whole blood samples, and there are no related studies or reports. Summary of the invention

[0005] The purpose of the present application is to provide a new reagent, kit, method and device for five immune assays of whole blood samples.

[0006] In order to achieve the above objectives, this application adopts the following technical solutions:

[0007] The first aspect of the present application discloses a reagent for five-item immunoassay of whole blood samples, the reagent comprising a buffer, an osmotic pressure regulator, a surfactant, an aggregation promoter, a blocking agent and a preservative; wherein the surfactant is a combination of a cationic surfactant and a nonionic surfactant.

[0008] It should be noted that the reagent of the present application can not only effectively hemolyze the whole blood sample through the interaction of buffer, osmotic pressure regulator, surfactant, aggregation promoter, blocking agent and preservative, and play the role of hemolytic agent; but also can maintain the stability of the reaction system of the subsequent five immune tests, avoid the influence on the subsequent five immune tests, and can be directly used as a reaction buffer for immune reaction. Therefore, the reagent of the present application can directly detect the five immune tests of the whole blood sample, and there is no need to pre-treat the whole blood sample.

[0009] It can be understood that in the reagents of the present application, the main function of the buffer is to provide a basic reaction environment for the immune response of the antigen and antibody; the role of the osmotic pressure regulator is to adjust the ionic strength of the solution, maintain the osmotic pressure, and avoid causing changes in the osmotic pressure of the cells; the role of the surfactant is hemolysis and reduction of nonspecific adsorption; the role of the coagulation promoter is to increase the reactivity, promote the reaction, and increase the reaction speed; the role of the blocking agent is to protect the protein activity in the antiserum reagent and avoid the destruction of the hydration layer; the role of the preservative is antiseptic and improves the service life of the reagent. In principle, the above components can be selected according to their functions with reference to the prior art. The key to this application lies in the organic combination of the above components, rather than the specific selection of each component; therefore, the specific selection of buffer, osmotic pressure regulator, surfactant, coagulation promoter, coagulation promoter and preservative is not specifically limited here. However, in order to ensure the hemolytic effect and the quality of the subsequent five immune determinations, the preferred technical scheme of this application defines the buffer, osmotic pressure regulator, surfactant, coagulation promoter, coagulation promoter and preservative in detail, see the following technical scheme for details.

[0010] In one implementation of the present application, the reagent of the present application contains 100-200 mmol / L of buffer, 6-15 g / L of osmotic pressure regulator, 30-60 g / L of surfactant, 30-50 g / L of aggregation promoter, 30-100 g / L of sealing agent and 0.5-1.5 g / L of preservative.

[0011] It can be understood that the above specific formula is only a reference formula in one implementation method of the present application. Under the concept of the present application, appropriate adjustments can be made based on the above formula according to needs, and no specific limitation is made here.

[0012] In one implementation of the present application, the cationic surfactant is tetradecyltrimethylammonium bromide (TTAB), and the nonionic surfactant is selected from at least one of alkyl polyglucoside (alkyl polyglycoside APG), Tween 20, Tween 80, Triton X100, saponin and Emulgen B-66.

[0013] Preferably, the surfactant consists of a cationic surfactant and a nonionic surfactant at the same weight volume concentration.

[0014] Preferably, the surfactant consists of tetradecyltrimethylammonium bromide and alkyl polyglucoside at the same weight volume concentration.

[0015] It should be noted that the reagents of the present application are not only used to hemolyze the whole blood sample, but also as a buffer for the subsequent immune reaction, which is particularly important for the selection of surfactants. If the surfactant strength is insufficient, the hemolysis speed will be slow and time-consuming or the hemolysis will be insufficient, which will affect the test results. Excessive surfactant will destroy the hydration layer of the subsequent immune five-item antiserum due to excessive hemolysis, thereby destroying the stability of the reaction system and affecting the specificity of the reaction. Therefore, the present application adopts a combination of cationic surfactants and non-ionic surfactants; and preferably uses tetradecyl trimethylammonium bromide as the cationic surfactant of the present application, and at least one of alkyl polyglucosides (alkyl polyglycosides APG), Tween20, Tween80, TritonX100, saponin and EmulgenB-66 as the non-ionic surfactant of the present application; the combination of cationic surfactants and non-ionic surfactants has better stability while ensuring the hemolysis effect. As for the surfactant composed of tetradecyltrimethylammonium bromide and alkyl polyglucoside of the same weight volume concentration, this is only a surfactant combination used in a specific implementation of the present application. On the basis of the above inventive concept, other cationic surfactants and non-ionic surfactants with the same performance can also be used for replacement; for example, Tween20, Tween80, TritonX100, saponin or EmulgenB-66 can be used to replace alkyl polyglucoside, which is not specifically limited here.

[0016] In one implementation of the present application, the buffer is a phosphate buffer or a Tris buffer; preferably a phosphate buffer.

[0017] It should be noted that the optimum pH value of the antigen-antibody reaction solution is 6.5-8.5. If it exceeds this limit, it is not easy to form a complex, and it may even cause the complex to dissociate. Within a certain range, the ionic strength is large and the complex forms quickly. If the ionic strength is too low or there is no electrolyte, it is not easy to see a precipitation reaction. The type of ions can also affect the formation of immune complexes. Therefore, the buffer can use a conventional phosphate buffer or Tris buffer. In one implementation of the present application, the immunoturbidimetry method is specifically used for the five immune tests, so it is preferred to use a phosphate buffer.

[0018] In one implementation of the present application, the osmotic pressure regulator is at least one of sodium chloride, potassium chloride, magnesium chloride and calcium chloride.

[0019] In one implementation of the present application, the aggregation promoter is at least one of polyethylene glycol 6K-2W, polyvinyl pyrrolidone (PVP), and dextran 2W-7W; preferably polyethylene glycol 8000 (PEG8000).

[0020] It should be noted that the role of the polymerization promoter is to promote the immune response. Small molecular weight polyethylene glycol has a small polymerization effect on the immune response, and even if the amount used is increased, the ideal state cannot be achieved; and if the molecular weight is too large, the initial photoelectric value of the low concentration or even blank sample will become higher, and it cannot be distinguished from the high concentration sample. Therefore, this application preferably uses polyethylene glycol with a molecular weight of 6K-2W, as well as PVP, dextran 2W-7W, etc. with similar performance. Taking into account the polymerization effect on the immune response and the initial photoelectric value of the blank sample, this application preferably uses polyethylene glycol 8000.

[0021] In one implementation of the present application, the pH value of the reagent of the present application is 6-9, preferably 7-8.

[0022] It should be noted that the reagent of the present application is used as a buffer solution for immune reaction, and the preferred pH value is 6-9. This pH range can meet both the hemolysis requirements and the basic requirements of subsequent immune response; especially considering that the optimal pH value of the antigen-antibody reaction solution is 6.5-8.5, the more preferred pH value of the reagent of the present application is 7-8.

[0023] In one implementation of the present application, the blocking agent is at least one of bovine serum albumin (BSA), gelatin, casein and trehalose.

[0024] Preferably, the blocking agent consists of trehalose and bovine serum albumin at the same weight volume concentration.

[0025] It should be noted that the blocking agent of the present application mainly protects the protein activity in the antiserum reagent, can protect the reaction system from being destroyed, avoid the destruction of the hydration layer, and avoid drastic changes in the reaction system; therefore, the present application preferably uses at least one of bovine serum albumin, gelatin, casein and trehalose as the blocking agent of the present application. Moreover, the blocking agent composed of trehalose and bovine serum albumin of the same weight volume concentration has the best effect. It can be understood that the combination of trehalose and bovine serum albumin is only a blocking agent combination used in a specific implementation of the present application, and it is not excluded that the same or similar performance blocking agents can be used to replace trehalose and bovine serum albumin respectively, which is not specifically limited here.

[0026] In one implementation of the present application, the preservative is at least one of Proclin 300, sodium azide, Krovin series and Kasson (i.e., Kasson).

[0027] It should be noted that the preservative of the present application mainly has a preservative effect and increases the shelf life of the reagent. Its mechanism of action is: (1) interfering with the enzyme system of microorganisms, destroying their normal metabolism, and inhibiting the activity of enzymes; (2) causing the protein of microorganisms to solidify and denature, interfering with their survival and reproduction; (3) changing the permeability of the cell plasma membrane, causing the enzymes and metabolites in the body to escape and inactivate them. As long as the reagent can play a preservative role, it can be used in the present application. However, considering safety and harmlessness, not reacting with other active ingredients in the reagent, and being able to effectively preserve the hemolysis buffer and extend the shelf life; the present application preferably uses at least one of Proclin300, sodium azide, Krovin series and Kaisong as the preservative of the present application; Proclin300 is particularly preferred.

[0028] The second aspect of the present application discloses a kit for five-item immunoassay of a whole blood sample, wherein the kit contains the reagents for five-item immunoassay of a whole blood sample of the present application.

[0029] In one implementation of the present application, the kit further contains antiserum reagents, which include IgA antiserum reagents, IgG antiserum reagents, IgM antiserum reagents, C3 antiserum reagents and C4 antiserum reagents.

[0030] The third aspect of the present application discloses a method for five-item immunoassay of a whole blood sample, comprising the following steps:

[0031] The hemolysis step includes directly contacting and mixing the whole blood sample with the reagent for whole blood sample five-item immune determination of the present application to lyse the red blood cells; in one implementation of the present application, specifically, only the two need to be mixed evenly, and the hemolysis can be completed within 10 seconds, for example, 3-5 seconds, and the hemolysis speed is fast and the effect is good;

[0032] The immune reaction step includes directly adding an antiserum reagent for the five-item immune assay to the product of the hemolysis step to perform an immune reaction; in one implementation of the present application, after 3-5 seconds of hemolysis, the antiserum reagent is directly added thereto, for example, an IgA antiserum reagent, an IgG antiserum reagent, an IgM antiserum reagent, a C3 antiserum reagent or a C4 antiserum reagent is added;

[0033] The steps of determining the five immune items include determining the concentration of the five immune items by turbidimetry after adding the antiserum reagent for the five immune items determination; in one implementation of the present application, a protein analyzer is specifically used to read the photoelectric values ​​at the 5th second and the 20th second after adding the antiserum reagent, and the content of the five immune items in the whole blood sample is calculated by comparing with the standard curve.

[0034] In one implementation of the present application, the method of the present application further includes a correction step, which includes correcting the concentration determined in the five immune determination steps according to the hematocrit, and the specific correction formula is shown in Formula 1;

[0035] Formula-C mod =C×100 / (100-Hct%)

[0036] Among them, C mod represents the corrected concentration, C represents the concentration measured by the five-item immunoassay procedure, and Hct% represents the hematocrit.

[0037] It should be noted that the method of the five-item immune assay of whole blood samples of the present application can directly detect whole blood samples because the reagents used for the five-item immune assay of whole blood samples of the present application are used; moreover, the reagents of the present application can be used for hemolysis and as an immune reaction buffer, which reduces the process flow of the five-item immune assay of whole blood samples, saves the cost of centrifugal equipment, shortens the reaction time, and improves the detection speed and quality. The method of the present application can directly detect whole blood samples, thereby reducing the amount of blood collected, and peripheral blood can be taken for testing of subjects such as infants and young children. In a preferred embodiment, the results of the five immune assays determined by the present application can be corrected according to the hematocrit, thereby improving accuracy.

[0038] The fourth aspect of the present application discloses a device for whole blood sample five-item immune assay, comprising a hemolysis and immune response module, a five-item immune assay module and a correction module;

[0039] Hemolysis and immune response module, including a module for directly contacting and mixing a whole blood sample with the reagent for five immune assays of the whole blood sample of the present application to lyse red blood cells, and directly adding antiserum reagent for five immune assays thereto after hemolysis is completed to carry out immune reaction;

[0040] An immuno-five-item assay module, comprising a module for determining the concentration of the immuno-five-item assay by a turbidimetric method after adding an antiserum reagent for the immuno-five-item assay;

[0041] A correction module, including a correction module for correcting the concentration determined in the five immune determination steps according to the hematocrit, wherein the specific correction formula is shown in Formula 1;

[0042] Formula-C mod =C×100 / (100-Hct%)

[0043] Among them, C mod represents the corrected concentration, C represents the concentration measured by the five-item immunoassay procedure, and Hct% represents the hematocrit.

[0044] It should be noted that the device of the present application can realize the automated detection of the five-item immune determination method of whole blood samples of the present application and the automated output and correction of the results through various modules; it greatly facilitates the five-item immune determination of whole blood samples.

[0045] Due to the adoption of the above technical solution, the beneficial effects of this application are:

[0046] The reagent used in the present application for the five-item immune assay of whole blood samples can directly hemolyze the whole blood sample, and after hemolysis, it can be used as a reaction buffer for immune reaction, and antiserum reagents can be directly added thereto to perform the five-item immune assay. The reagent of the present application can not only directly perform the five-item immune assay on the whole blood sample, omitting many pre-treatment steps; but also has good sensitivity, strong specificity, and fast detection speed, and can meet the requirements of rapid detection; it provides a new formula and scheme for the five-item immune assay. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is the standard curve drawn by the IgA standard in the examples of the present application;

[0048] Figure 2 is the standard curve drawn by the IgG standard in the examples of this application;

[0049] Figure 3 is the standard curve drawn by the IgM standard in the example of the present application;

[0050] Figure 4 is the standard curve drawn by the C3 standard in the example of the present application;

[0051] Figure 5 is the standard curve drawn by the C4 standard in the example of the present application;

[0052] Figure 6 This is a correlation analysis diagram of IgA measured by reagent 1 and hospital values ​​in the embodiment of the present application;

[0053] Figure 7 This is a correlation analysis chart of IgG measured by reagent 1 and hospital values ​​in the example of the present application;

[0054] Figure 8 This is a correlation analysis chart of IgM measured by reagent 1 and hospital values ​​in the embodiment of the present application;

[0055] Fig. 9 This is a correlation analysis diagram of C3 measured by reagent 1 and hospital value in the embodiment of the present application;

[0056] Fig.10 This is a correlation analysis chart of C4 measured by reagent 1 and hospital value in the example of this application. DETAILED DESCRIPTION

[0057] The applicant has found that the main reason why the existing technical solutions cannot directly perform five-item immune tests on whole blood samples is that the existing technical solutions of turbidimetric immune five-item reagents do not contain hemolytic agents that can effectively hemolyze whole blood, so whole blood samples cannot be directly tested, and only serum or plasma can be tested.

[0058] Hemolysis of whole blood samples is a relatively simple process in itself, and only requires the selection of a surfactant that can destroy blood cells. However, the present application has found that for the five-item immune test, if the surfactant strength is insufficient, the hemolysis rate will be slow and time-consuming or the hemolysis will be insufficient, which will affect the test results. Excessive surfactant will destroy the hydration layer of the subsequent five-item immune antiserum due to excessive hemolysis, thereby destroying the stability of the reaction system and affecting the specificity of the reaction. Therefore, for conventional projects, the existing hemolytic agents can meet the hemolysis requirements, but due to the particularity of the antiserum of the five-item immune project, the existing commercially available hemolytic agents cannot fully meet the requirements of the five-item immune test for whole blood samples. In addition, the inventors of the present application have found through a large number of experiments that most surfactants will change the turbidity of the subsequent immune reaction and affect the results, so they cannot be used for turbidimetric five-item immune tests.

[0059] After a lot of research and experiments, the inventors of this application creatively proposed that the use of a surfactant of appropriate strength, in combination with a blocking agent, can stabilize the hemolyzed mixture and avoid the destruction of the hydration layer, so that it can be directly used as a buffer for subsequent immune reactions; in particular, the combination of cationic surfactants and non-ionic surfactants has the best effect. Furthermore, in combination with an aggregation promoter, the immune response can be promoted; in combination with an osmotic pressure regulator and a conventional buffer, rapid, efficient, sensitive, and specific detection of whole blood samples can be achieved directly. Furthermore, in combination with a preservative, the hemolysis buffer can be effectively preserved and the shelf life can be extended.

[0060] Based on the above research and understanding, the present application creatively developed a new reagent for five-item immunoassay of whole blood samples, which includes a buffer, an osmotic pressure regulator, a surfactant, a polymerization promoter, a blocking agent and a preservative; wherein the surfactant is a combination of a cationic surfactant and a non-ionic surfactant.

[0061] It should be noted that the existing routine project detection of whole blood samples usually uses a hemolytic agent to hemolyze the whole blood sample, and then the hemolyzed sample is added to the reaction buffer for detection. The reagent of the present application can be hemolyzed by directly adding the whole blood sample to the reagent of the present application for mixing for 3-10 seconds; then, the antiserum reagent is directly added thereto to perform turbidimetric determination. Therefore, the reagent of the present application can be used as a hemolytic agent and as a reaction buffer for immune response. The reagent of the present application is simple and convenient to use, shortens the reaction time, and improves the detection speed. It can be implemented simultaneously with other biological detection projects that do not require centrifugation, such as routine blood detection projects, etc., and can also be carried out on the same equipment, thereby meeting the requirements of rapid detection.

[0062] It should also be noted that the reagent of the present application can be used not only for whole blood samples, but also for serum and plasma samples. It is understandable that when applied to serum and plasma samples, since hemolysis is not required, the reagent of the present application mainly plays its role as an immune response buffer.

[0063] In general, the reagents of the present application have the following advantages:

[0064] 1. It can be used as a reaction buffer for immune reactions and as a hemolytic agent for whole blood samples;

[0065] 2. It can detect whole blood samples, serum or plasma at the same time, and is suitable for a variety of equipment, making the test more convenient and quick;

[0066] 3. Does not affect subsequent immune response, good sensitivity and strong specificity;

[0067] 4. Whole blood samples can be directly tested, thus reducing the amount of blood collected. Peripheral blood can be taken for testing of infants and other subjects;

[0068] 5. The combined use of cationic surfactants and nonionic surfactants can exert a highly efficient hemolytic effect, hemolyze whole blood samples, and lyse blood cells;

[0069] 6. The addition of blocking agent can protect the reaction system from being destroyed and avoid drastic changes in the reaction system;

[0070] 7. The pH of the reagent of the present application is 6-9, especially when the pH is 7-8, so that the target in the whole blood sample can remain in a natural state, so the results of the subsequent immune response determination can more truly reflect the target information of the sample;

[0071] 8. The reagent of the present application can directly detect whole blood samples, omitting the process of centrifuging the whole blood samples, saving the cost of centrifugal equipment, shortening the reaction time, and can be implemented simultaneously with other biological detection projects that do not require centrifugation, such as routine blood tests, and can be performed on the same equipment, thereby meeting the requirements of rapid detection;

[0072] 9. The reagent of the present application is used for immune five-item nephelometry, and the measurement results can be corrected according to the hematocrit, which can further improve the accuracy.

[0073] The present invention is further described in detail below by specific examples and accompanying drawings. The following examples are only further described herein and should not be construed as limiting the present invention. In the following examples, unless otherwise specified, all reagents, materials and equipment can be purchased from the market.

[0074] Embodiment 1

[0075] The reagents used for the five-item immunoassay of whole blood samples in this example consist of 100-200 mmol / L buffer, 6-15 g / L osmotic pressure regulator, 30-60 g / L surfactant, 30-50 g / L aggregation promoter, 30-100 g / L blocking agent and 0.5-1.5 g / L preservative. Among them, the surfactant is selected from at least one of tetradecyl trimethylammonium bromide, alkyl polyglucosides, sodium dodecyl sulfate, Tween 20, Tween 80, Triton X100, sodium deoxycholate, Emulgen B-66 and saponin; the buffer is phosphate buffer or Tris buffer; the osmotic pressure regulator is at least one of sodium chloride, potassium chloride, magnesium chloride and calcium chloride; the polymerization promoter is at least one of polyethylene glycol 6K-2W, PVP, dextran 2W-7W; the blocking agent is at least one of bovine serum albumin, gelatin, casein and trehalose; the preservative is at least one of Proclin 300, sodium azide, Krovin series and Kaisong. In this example, five different specific selections and dosages of reagents were prepared according to the above scheme, as follows:

[0076] Reagent 1

[0077] Prepare hemolysis buffer according to the following composition:

[0078]

[0079] Take purified water and add the above reagents in sequence, stir to dissolve and then make up to 1L, adjust the pH to 7.4, filter and package, and obtain reagent 1 in this example.

[0080] Reagent 2

[0081] Prepare hemolysis buffer according to the following composition:

[0082]

[0083]

[0084] Take purified water and add the above reagents in sequence, stir to dissolve and then make up to 1L, adjust the pH to 7.4, filter and package, and obtain reagent 2 in this example.

[0085] Reagent 3

[0086] Prepare hemolysis buffer according to the following composition:

[0087]

[0088] Take purified water and add the above reagents in sequence, stir to dissolve and then make up to 1L, adjust the pH to 7.4, filter and package, and obtain reagent 3 in this example.

[0089] Reagent 4

[0090] Prepare hemolysis buffer according to the following composition:

[0091]

[0092] Take purified water and add the above reagents in sequence, stir to dissolve and then make up to 1L, adjust the pH to 7.4, filter and package, and obtain reagent 4 in this example.

[0093] Reagent 5

[0094] Prepare hemolysis buffer according to the following composition:

[0095]

[0096]

[0097] Take purified water and add the above reagents in sequence, stir to dissolve and then make up to 1L, adjust the pH to 7.4, filter and package, and obtain reagent 5 in this example.

[0098] The five reagents prepared in this example were used in combination with the five immune antiserum reagents and calibrators of Guosai Biological to perform five immune tests on whole blood samples. The details are as follows:

[0099] Test 1 Hemolytic effect verification

[0100] After calibration with reagents 1-5 and Guosai Biological Immune Five Antiserum Reagents and Calibrators, 5 homologous whole blood samples and 5 serum samples from Fuyong People's Hospital were randomly selected for testing. The test results of reagents 1-5 with different formulations on whole blood samples and serum samples were compared. The sample detection method is consistent with the calibration method. The calibration is to test different concentrations of calibrators covering the detection range of the immune five items. The specific method of the immune five-item determination in this case is as follows:

[0101] IgA:

[0102] A. Take 400 μL of the reagent prepared in this example and add it to the reaction cup. Add 2 μL of whole blood or serum sample and mix thoroughly for 3-5 seconds to dissolve red blood cells.

[0103] B. Add 50 μL of IgA antiserum and stir in the Guosai Bio-specific protein analyzer to allow immune reaction to occur, resulting in turbidity changes;

[0104] C. Read the photoelectric value at 5s and 35s after adding the antiserum reagent, and calculate the content of the five immune items in the whole blood sample by comparing with the standard curve and hematocrit;

[0105] D. The correction formula for correcting the five immune concentrations using hematocrit is as follows:

[0106] C mod =C×100 / (100-Hct%)

[0107] Among them, C mod represents the corrected concentration, C represents the initial concentration obtained by immunoreaction detection, and Hct% represents the hematocrit.

[0108] IgG:

[0109] A. Take 400 μL of hemolysis buffer and add it to the reaction cup. Add 2 μL of whole blood / serum sample and mix thoroughly for 3-5 seconds to lyse red blood cells.

[0110] B. Add 50 μL of IgG antiserum reagent and stir in the Guosai Bio-specific protein analyzer to generate immune reaction and turbidity change;

[0111] C. Read the photoelectric value at 5s and 20s after adding the antiserum reagent, and calculate the content of the five immune items in the whole blood sample by comparing with the standard curve and hematocrit;

[0112] D. The correction formula for correcting the five immune concentrations using hematocrit is as follows:

[0113] C mod =C×100 / (100-Hct%)

[0114] Among them, C mod represents the corrected concentration, C represents the initial concentration obtained by immunoreaction detection, and Hct% represents the hematocrit.

[0115] IgM:

[0116] A. Take 400 μL of hemolysis buffer and add it to the reaction cup. Add 2 μL of whole blood / serum sample and mix thoroughly for 3-5 seconds to lyse red blood cells.

[0117] B. Add 35 μL of IgM antiserum reagent and stir in the Guosai Bio-specific protein analyzer to generate immune reaction and turbidity change;

[0118] C. Read the photoelectric value at 5s and 125s after adding the antiserum reagent, and calculate the content of the five immune items in the whole blood sample by comparing with the standard curve and hematocrit;

[0119] D. The correction formula for correcting the five immune concentrations using hematocrit is as follows:

[0120] C mod =C×100 / (100-Hct%)

[0121] Among them, C mod represents the corrected concentration, C represents the initial concentration obtained by immunoreaction detection, and Hct% represents the hematocrit.

[0122] C3:

[0123] A. Take 400 μL of hemolysis buffer and add it to the reaction cup. Add 2 μL of whole blood / serum sample and mix thoroughly for 3-5 seconds to lyse red blood cells.

[0124] B. Add 35 μL of C3 antiserum reagent and stir in the Guosai Bio-specific protein analyzer to generate immune reaction and turbidity change;

[0125] C. Read the photoelectric value at 10s and 40s after adding the antiserum reagent, and calculate the content of the five immune items in the whole blood sample by comparing with the standard curve and hematocrit;

[0126] D. The correction formula for correcting the five immune concentrations using hematocrit is as follows:

[0127] C mod =C×100 / (100-Hct%)

[0128] Among them, C mod represents the corrected concentration, C represents the initial concentration obtained by immunoreaction detection, and Hct% represents the hematocrit.

[0129] C4:

[0130] A. Take 400 μL of hemolysis buffer and add it to the reaction cup. Add 4 μL of whole blood / serum sample and mix thoroughly for 3-5 seconds to lyse red blood cells.

[0131] B. Add 45 μL of C4 antiserum reagent and stir in the Guosai Bio-specific protein analyzer to generate immune reaction and turbidity change;

[0132] C. Read the photoelectric value at 10s and 70s after adding the antiserum reagent, and calculate the content of the five immune items in the whole blood sample by comparing with the standard curve and hematocrit;

[0133] D. The correction formula for correcting the five immune concentrations using hematocrit is as follows:

[0134] C mod =C×100 / (100-Hct%)

[0135] Among them, C mod represents the corrected concentration, C represents the initial concentration obtained by immunoreaction detection, and Hct% represents the hematocrit.

[0136] The above five immune test results of reagents 1 to 5 are shown in Tables 1 to 5. Among them, Table 1 is the result of the five immune tests using reagent 1, Table 2 is the result of the five immune tests using reagent 2, Table 3 is the result of the five immune tests using reagent 3, Table 4 is the result of the five immune tests using reagent 4, and Table 5 is the result of the five immune tests using reagent 5.

[0137] The standard curves of IgA, IgG, IgM, C3 and C4 were prepared using the calibration products provided by the Guosai Bio-immune five-item antiserum reagent. The test method was the same as above. The standard curve results are shown in the figure below. Figures 1 to 5 As shown. Among them, Figure 1 The standard curve drawn for the IgA standard, Figure 2 The standard curve drawn for the IgG standard, Figure 3 The standard curve drawn for the IgM standard, Figure 4 The standard curve drawn for the C3 standard, Figure 5 Standard curve plotted for C4 standards. Figures 1 to 5 The standard curve is obtained by fitting with concentration as the horizontal axis and photoelectric value as the vertical axis.

[0138] Table 1 Verification results of hemolytic effect of reagent 1

[0139]

[0140] Table 2 Verification results of hemolytic effect of reagent 2

[0141]

[0142] Table 3 Verification results of hemolytic effect of reagent 3

[0143]

[0144]

[0145] Table 4 Hemolytic effect verification results of reagent 4

[0146]

[0147] Table 5 Hemolytic effect verification results of reagent 5

[0148]

[0149] In Tables 1 to 5, "Control" is the test value of Fuyong People's Hospital, whole blood 1 to whole blood 5 are the test results of five whole blood samples, serum 1 to serum 5 are the test results of five serum samples, and T1 and T2 are two repeated test results of the same sample.

[0150] The results of Tables 1 to 5 show that the reagents 1 to 5 of this example can effectively hemolyze within 3-5 seconds after adding the whole blood sample, and will not affect the subsequent immune response. The results of Table 1 show that the deviations of the whole blood sample and serum sample test results of reagent 1 from the hospital values ​​are less than 10%; the results of Table 2 show that only the cationic surfactant tetradecyltrimethylammonium bromide (TTAB) is added to reagent 2 alone. Therefore, although reagent 2 can also perform hemolysis and subsequent five immune tests, its hemolysis is not sufficient, and the test results of the whole blood sample have a large deviation; the results of Table 3 show that only the non-ionic surfactant alkyl polyglucoside (alkyl polyglycoside APG) is added to reagent 3 alone. Therefore, although reagent 3 can also perform hemolysis and subsequent five immune tests, the same Its hemolysis is insufficient, and the test results of whole blood samples have large deviations. The results in Table 4 show that only the blocking agent trehalose is added to reagent 4. Therefore, although reagent 4 can also perform hemolysis and subsequent five-item immune assays, the blocking agent effect is poor, and the subsequent reaction system is destroyed, resulting in large deviations in sample testing and poor stability. The results in Table 5 show that only the blocking agent bovine serum albumin is added to reagent 5. Therefore, although reagent 5 can also perform hemolysis and subsequent five-item immune assays, the blocking agent effect is also poor, and the subsequent reaction system is destroyed, resulting in large deviations in sample testing and poor stability.

[0151] According to the above results and analysis, it can be seen that reagents 1 to 5 in this example can all detect whole blood samples and serum samples; among them, reagent 1, which uses a cationic surfactant and a non-ionic surfactant in combination with two blocking agents, namely trehalose and bovine serum albumin, has the smallest deviation in the test results and the best stability.

[0152] Test 2 Accuracy Verification

[0153] This test selected 10 fresh whole blood samples and 10 fresh serum samples of different concentrations from Fuyong People's Hospital for five immune tests, and used reagent 1 to perform five immune tests. The test results of the 10 whole blood samples and 10 serum samples were statistically analyzed to analyze the accuracy of reagent 1. The specific test method is as follows:

[0154] IgA:

[0155] A. Take 400 μL of hemolysis buffer and add it to the reaction cup. Add 2 μL of whole blood / serum sample and mix thoroughly for 3-5 seconds to lyse red blood cells.

[0156] B. Add 50 μL of IgA antiserum and stir in the Guosai Bio-specific protein analyzer to generate immune reaction and turbidity change;

[0157] C. Read the photoelectric value at 5s and 35s after adding the antiserum reagent, and calculate the content of the five immune items in the whole blood sample by comparing with the standard curve and hematocrit. The standard curve is shown in Figure 1 , the result data are shown in Table 6, and the consistency of the results is shown in Figure 6 ;

[0158] D. The correction formula for correcting the five immune concentrations using hematocrit is as follows:

[0159] C mod =C×100 / (100-Hct%)

[0160] Among them, C mod represents the corrected concentration, C represents the initial concentration obtained by immunoreaction detection, and Hct% represents the hematocrit.

[0161] IgG:

[0162] A. Take 400 μL of hemolysis buffer and add it to the reaction cup. Add 2 μL of whole blood / serum sample and mix thoroughly for 3-5 seconds to lyse red blood cells.

[0163] B. Add 50 μL of IgG antiserum reagent and stir in the Guosai Bio-specific protein analyzer to generate immune reaction and turbidity change;

[0164] C. Read the photoelectric value at 5s and 20s after adding the antiserum reagent, and calculate the content of the five immune items in the whole blood sample by comparing with the standard curve and hematocrit. The standard curve is shown in Figure 2 , the result data are shown in Table 6, and the consistency of the results is shown in Figure 7 ;

[0165] D. The correction formula for correcting the five immune concentrations using hematocrit is as follows:

[0166] C mod =C×100 / (100-Hct%)

[0167] Among them, C mod represents the corrected concentration, C represents the initial concentration obtained by immunoreaction detection, and Hct% represents the hematocrit.

[0168] IgM:

[0169] A. Take 400 μL of hemolysis buffer and add it to the reaction cup. Add 2 μL of whole blood / serum sample and mix thoroughly for 3-5 seconds to lyse red blood cells.

[0170] B. Add 35 μL of IgM antiserum reagent and stir in the Guosai Bio-specific protein analyzer to generate immune reaction and turbidity change;

[0171] C. Read the photoelectric value at 5s and 125s after adding the antiserum reagent, and calculate the content of the five immune items in the whole blood sample by comparing with the standard curve and hematocrit. The standard curve is shown in Figure 3 , the result data are shown in Table 6, and the consistency of the results is shown in Figure 8 ;

[0172] D. The correction formula for correcting the five immune concentrations using hematocrit is as follows:

[0173] C mod =C×100 / (100-Hct%)

[0174] Among them, C mod represents the corrected concentration, C represents the initial concentration obtained by immunoreaction detection, and Hct% represents the hematocrit.

[0175] C3:

[0176] A. Take 400 μL of hemolysis buffer and add it to the reaction cup. Add 2 μL of whole blood / serum sample and mix thoroughly for 3-5 seconds to lyse red blood cells.

[0177] B. Add 35 μL of C3 antiserum reagent and stir in the Guosai Bio-specific protein analyzer to generate immune reaction and turbidity change;

[0178] C. Read the photoelectric value at 10s and 40s after adding the antiserum reagent, and calculate the content of the five immune items in the whole blood sample by comparing with the standard curve and hematocrit. The standard curve is shown in Figure 4 , the result data are shown in Table 6, and the consistency of the results is shown in Fig. 9 ;

[0179] D. The correction formula for correcting the five immune concentrations using hematocrit is as follows:

[0180] C mod =C×100 / (100-Hct%)

[0181] Among them, C mod represents the corrected concentration, C represents the initial concentration obtained by immunoreaction detection, and Hct% represents the hematocrit.

[0182] C4:

[0183] A. Take 400 μL of hemolysis buffer and add it to the reaction cup. Add 4 μL of whole blood / serum sample and mix thoroughly for 3-5 seconds to lyse red blood cells.

[0184] B. Add 45 μL of C4 antiserum reagent and stir in the Guosai Bio-specific protein analyzer to generate immune reaction and turbidity change;

[0185] C. Read the photoelectric value at 10s and 70s after adding the antiserum reagent, and calculate the content of the five immune items in the whole blood sample by comparing with the standard curve and hematocrit. The standard curve is shown in Figure 5 , the result data are shown in Table 6, and the consistency of the results is shown in Fig.10 ;

[0186] D. The correction formula for correcting the five immune concentrations using hematocrit is as follows:

[0187] C mod =C×100 / (100-Hct%)

[0188] Among them, C mod represents the corrected concentration, C represents the initial concentration obtained by immunoreaction detection, and Hct% represents the hematocrit.

[0189] Table 6 Accuracy verification results of reagent 1

[0190]

[0191] In Table 6, "Control" is the test value of Fuyong People's Hospital, whole blood 1 to whole blood 10 are the test results of ten whole blood samples, serum 1 to serum 10 are the test results of ten serum samples, T1 is the result of comparison with the standard curve and hematocrit calculated according to the above method, and "Deviation" is the deviation between the test result of this experiment and the test value of the hospital.

[0192] The results in Table 6 show that the test results of whole blood samples and serum samples using reagent 1 in this experiment, except for the individual low concentrations of C4 items, the deviations of the other test results from the hospital test values ​​are all less than 10%, indicating that reagent 1 has high accuracy. Among them, because the concentration of individual samples of C4 items is low, even if the test results differ by only 0.01 or 0.02, the relative deviation is also large. For example, the relative deviation between 0.12 and 0.14 is 16.7%, and the relative deviation with 0.13 is 8.33%. Therefore, in the low concentration range, the relative deviation calculated in a very small range of concentration deviation may exceed 10%; generally, this situation can be expressed by absolute deviation. Although this example uniformly uses relative deviation expression, the absolute deviation of C4 items is relatively small; so it can still be said that it has high accuracy.

[0193] This test Figures 6 to 10 , is the correlation obtained by linear regression of the accuracy test results of reagent 1 and the hospital value. Figures 6 to 10 The results showed that the accuracy and stability of reagent 1 were good, and the test results were consistent with the hospital values, R>0.99; this indicated that the five-item whole blood sample determination using reagent 1 in this example had a high correlation with the hospital serum test results, was highly consistent, and the stability of multiple groups of test results was good.

[0194] Comparative test 1 CRP hemolytic test five immune items

[0195] This experiment uses the existing five-item immune test kit to perform five-item immune tests on the same five whole blood samples and five serum samples as in Experiment 1. Specifically, the whole blood samples are pre-hemolyzed using Shenzhen Youdi Biotech CRP hemolytic reagent; then the Guosai scattering turbidimetry five-item immune test kit is used for detection. Different from the operation in Example 7, the buffer used is the buffer corresponding to the five reagents of Guosai Biotech, and the Youdi CRP hemolytic reagent needs to be used for hemolysis and dilution before detection. The specific method is as follows:

[0196] The CRP hemolytic agent is Shenzhen Youdi Biological CRP hemolytic agent, and the specific preparation method is as follows:

[0197] 1. Preparation of surfactant mother solution: Weigh 3.4 g of betaine, add 100 mL of purified water, stir to fully dissolve it, and adjust the pH to 6.0 with 1 mol / L hydrochloric acid.

[0198] 2. Preparation of polyethylene glycol mother solution: Weigh 10.0 g of polyethylene glycol 10000, add 100 mL of purified water, and stir to fully dissolve.

[0199] 3. Preparation of sodium ethylenediaminetetraacetate (EDTA) salt buffer: weigh 0.11 g of disodium ethylenediaminetetraacetate, add 100 mL of purified water, stir to fully dissolve it, and adjust the pH to 6.0 with 1 mol / L hydrochloric acid.

[0200] 4. Thoroughly mix the surfactant mother solution, polyethylene glycol mother solution, and sodium ethylenediaminetetraacetic acid buffer solution at a ratio of 1:3:30 to obtain the hemolytic agent for whole blood C-reactive protein (CRP) detection in this test, namely, the CRP hemolytic agent.

[0201] For whole blood samples, you need to first mix 20μL of whole blood sample with 200μL of CRP hemolytic agent, perform hemolysis and dilution, and then use the hemolysis and dilution mixture for subsequent tests. For consistency, this example also mixes and dilutes 20μL of serum sample with 200μL of CRP hemolytic agent for subsequent tests. Hemolyzed whole blood samples and hemolytic agent diluted serum samples are collectively referred to as hemolysis and dilution samples.

[0202] IgA:

[0203] A. Take 400 μL of Guosai IgA buffer and add it to the reaction cup. Add 20 μL of the hemolyzed diluted sample and mix thoroughly for 3-5 seconds to dissolve the red blood cells.

[0204] B. Add 50 μL of IgA antiserum and stir in the Guosai Bio-specific protein analyzer to allow immune reaction to occur, resulting in turbidity changes;

[0205] C. Read the photoelectric value at 5s and 35s after adding the antiserum reagent, and calculate the content of the five immune items in the whole blood sample by comparing with the standard curve and hematocrit. The results are shown in Table 7;

[0206] D. The correction formula for correcting the five immune concentrations using hematocrit is as follows:

[0207] C mod =C×100 / (100-Hct%)

[0208] Among them, C mod represents the corrected concentration, C represents the initial concentration obtained by immunoreaction detection, and Hct% represents the hematocrit.

[0209] IgG:

[0210] A. Take 400 μL of Guosai IgG buffer and add it to the reaction cup. Add 10 μL of the hemolysis diluted sample and mix thoroughly for 3-5 seconds to dissolve the red blood cells.

[0211] B. Add 20 μL of IgG antiserum reagent and stir in the Guosai Bio-specific protein analyzer to generate immune reaction and turbidity change;

[0212] C. Read the photoelectric value at 5s and 20s after adding the antiserum reagent, and calculate the content of the five immune items in the whole blood sample by comparing with the standard curve and hematocrit. The results are shown in Table 7;

[0213] D. The correction formula for correcting the five immune concentrations using hematocrit is as follows:

[0214] C mod =C×100 / (100-Hct%)

[0215] Among them, C mod represents the corrected concentration, C represents the initial concentration obtained by immunoreaction detection, and Hct% represents the hematocrit.

[0216] IgM:

[0217] A. Take 400 μL of Guosai IgM buffer and add it to the reaction cup. Add 20 μL of the hemolyzed diluted sample and mix thoroughly for 3-5 seconds to dissolve the red blood cells.

[0218] B. Add 30 μL of IgM antiserum reagent and stir in the Guosai Bio-specific protein analyzer to generate immune reaction and turbidity change;

[0219] C. Read the photoelectric value at 5s and 125s after adding the antiserum reagent, and calculate the content of the five immune items in the whole blood sample by comparing with the standard curve and hematocrit. The results are shown in Table 7;

[0220] D. The correction formula for correcting the five immune concentrations using hematocrit is as follows:

[0221] C mod =C×100 / (100-Hct%)

[0222] Among them, C mod represents the corrected concentration, C represents the initial concentration obtained by immunoreaction detection, and Hct% represents the hematocrit.

[0223] C3:

[0224] A. Take 400 μL of Guosai C3 buffer and add it to the reaction cup. Add 25 μL of the hemolyzed diluted sample and mix thoroughly for 3-5 seconds to dissolve the red blood cells.

[0225] B. Add 30 μL of C3 antiserum reagent and stir in the Guosai Bio-specific protein analyzer to generate immune reaction and turbidity change;

[0226] C. Read the photoelectric value at 10s and 40s after adding the antiserum reagent, and calculate the content of the five immune items in the whole blood sample by comparing with the standard curve and hematocrit. The results are shown in Table 7;

[0227] D. The correction formula for correcting the five immune concentrations using hematocrit is as follows:

[0228] C mod =C×100 / (100-Hct%)

[0229] Among them, C mod represents the corrected concentration, C represents the initial concentration obtained by immunoreaction detection, and Hct% represents the hematocrit.

[0230] C4:

[0231] A. Take 400 μL of Guosai C4 buffer and add it to the reaction cup. Add 40 μL of the hemolyzed diluted sample and mix thoroughly for 3-5 seconds to dissolve the red blood cells.

[0232] B. Add 40 μL of C4 antiserum reagent and stir in the Guosai Bio-specific protein analyzer to generate immune reaction and turbidity change;

[0233] C. Read the photoelectric value at 10s and 70s after adding the antiserum reagent, and calculate the content of the five immune items in the whole blood sample by comparing with the standard curve and hematocrit. The results are shown in Table 7;

[0234] D. The correction formula for correcting the five immune concentrations using hematocrit is as follows:

[0235] C mod =C×100 / (100-Hct%)

[0236] Among them, C mod represents the corrected concentration, C represents the initial concentration obtained by immunoreaction detection, and Hct% represents the hematocrit.

[0237] Table 7 Results of validation of the effect of CRP hemolytic agent

[0238]

[0239] In Table 7, “Control” is the test value of Fuyong People’s Hospital, whole blood 1 to whole blood 5 are the test results of five whole blood samples, serum 1 to serum 5 are the test results of five serum samples, and T1 and T2 are two repeated test results of the same sample.

[0240] The results in Table 7 show that when the whole blood sample is hemolyzed with a CRP hemolytic agent and then the five immune tests are performed using the existing kit, the serum test results are highly correlated with the hospital test values, but the whole blood test results have large deviations and poor stability. The above results show that for serum samples, since hemolysis is not required, the test results are highly correlated with the hospital test values; and although the existing hemolytic agents can have a hemolytic effect, hemolysis is not conducive to the subsequent five immune tests, and the test results have large deviations and poor stability.

[0241] Comparative test 2 HbA1C hemolytic test immune five items

[0242] The HbA1c hemolytic agent was prepared using the formula of Shenzhen Dima Biotechnology's HbA1C hemolytic agent, and the same 5 whole blood samples and 5 serum samples were subjected to five immune tests using the Guosai turbidimetric immunoassay five-item kit. The difference from comparative experiment 1 is that the hemolytic agent used was the Dima HbA1c hemolytic agent.

[0243] The HbA1c hemolytic agent is prepared by the formula of the HbA1c hemolytic agent of Dima, and the specific preparation method is as follows:

[0244] Weigh 1.0 g of sodium dodecyl sulfate, 5.0 g of Tween 20, 4.0 g of dodecyl dimethyl betaine, and 0.2 g of sodium hydroxide, and dilute to 1 L with purified water to obtain the hemolytic agent for this test.

[0245] The test methods for the remaining items are the same as those for comparative test 1. The test results are shown in Table 8.

[0246] Table 8 Results of validation of the effect of HbA1c hemolytic agent

[0247]

[0248] In Table 8, “Control” is the test value of Fuyong People’s Hospital, whole blood 1 to whole blood 5 are the test results of five whole blood samples, serum 1 to serum 5 are the test results of five serum samples, and T1 and T2 are two repeated test results of the same sample.

[0249] The results in Table 8 show that when the whole blood sample is hemolyzed using the HbA1c hemolytic agent, and then the five immune tests are performed using the existing kit, the serum test results are highly correlated with the hospital test values, but the whole blood test results have large deviations and poor stability. The above results show that for serum samples, since hemolysis is not required, the test results are highly correlated with the hospital test values; and although the existing HbA1c hemolytic agent can have a hemolytic effect, it is not conducive to the subsequent five immune tests after hemolysis, and the test results have large deviations and poor stability.

[0250] Embodiment 2

[0251] In this example, based on the reagent 1 of Example 1, the concentrations of the buffer, osmotic pressure regulator, surfactant, polymerization promoter, blocking agent, preservative, etc. were tested. The specific formula is shown in Table 9.

[0252] Table 9 Reagents with different formulations

[0253]

[0254]

[0255] In this example, 11 reagents for five immune tests of whole blood samples were prepared according to the formula in Table 9. The preparation method of the reagents is the same as that in Example 1, wherein Formula 1 is Reagent 1 in Example 1. Using 11 reagents, the five immune tests of whole blood samples were performed according to the method of "Test 1 Hemolytic Effect Verification" in Example 1. The test results are shown in Tables 10 to 14, which are the IgA test results, IgG test results, IgM test results, C3 test results, and C4 test results of each formula, respectively.

[0256] Table 10 IgA test results of each formulation

[0257]

[0258]

[0259] Table 11 IgG test results of each formulation

[0260]

[0261] Table 12 IgM test results of each formulation

[0262]

[0263] Table 13 C3 test results of each formulation

[0264]

[0265] Table 14 C4 test results of each formulation

[0266]

[0267]

[0268] In Tables 10 to 14, the values ​​in the "row" where "Control" is located are the hospital test values, whole blood 1 to whole blood 5 are five whole blood samples. For example, the hospital test value of "Whole Blood 1" is "1.56", and the "column" corresponding to "1.56" of "Whole Blood 1" is the test values ​​of 11 formulas of "Whole Blood 1", and the "column" corresponding to "Deviation" of "Whole Blood 1" refers to the deviation between the test values ​​of the 11 formulas and the hospital test value "1.56"; the structure of the rest of the table is similar.

[0269] The results in Tables 10 to 14 show that the 11 reagents prepared in this example can stably measure the five immune items after hemolysis, and the deviation between the whole blood sample test results of all reagents and the hospital values ​​is less than 10%. The test results show that the concentration of the buffer is 100-200mmol / L, the concentration of the osmotic pressure regulator is 6-15g / L, the concentration of the surfactant is 30-60g / L, the concentration of the aggregation promoter is 30-50g / L, the concentration of the blocking agent is 30-100g / L, and the concentration of the preservative is 0.5-1.5g / L. The prepared reagents can meet the use requirements of the five immune items determination of whole blood samples.

[0270] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A reagent for whole blood sample five-item immune assay, characterized in that: It includes a buffer, an osmotic pressure regulator, a surfactant, a polymerization promoter, a blocking agent and a preservative; the surfactant is a combination of a cationic surfactant and a nonionic surfactant; The cationic surfactant is tetradecyltrimethylammonium bromide, and the nonionic surfactant is selected from at least one of alkyl polyglucoside, Tween 20, Tween 80, Triton X100, saponin and Emulgen B-66; The polymerization promoter is at least one of polyethylene glycol 6K-2W, polyvinyl pyrrolidone, and dextran 2W-7W; The blocking agent is at least one of bovine serum albumin, gelatin, casein and trehalose.

2. The reagent according to claim 1, characterized in that: The reagent contains 100-200 mmol / L of buffer, 6-15 g / L of osmotic pressure regulator, 30-60 g / L of surfactant, 30-50 g / L of polymerization promoter, 30-100 g / L of sealing agent and 0.5-1.5 g / L of preservative.

3. The reagent according to claim 1 or 2, characterized in that: The surfactant consists of a cationic surfactant and a nonionic surfactant at the same weight volume concentration.

4. The reagent according to claim 3, characterized in that: The surfactant consists of tetradecyltrimethylammonium bromide and alkyl polyglucoside at the same weight volume concentration.

5. The reagent according to claim 1 or 2, characterized in that: The buffer is phosphate buffer or Tris buffer.

6. The reagent according to claim 1 or 2, characterized in that: The osmotic pressure regulator is at least one of sodium chloride, potassium chloride, magnesium chloride and calcium chloride.

7. The reagent according to claim 1 or 2, characterized in that: The pH value of the reagent is 6-9.

8. The reagent according to claim 1 or 2, characterized in that: The blocking agent consists of trehalose and bovine serum albumin with the same weight volume concentration.

9. The reagent according to claim 1 or 2, characterized in that: The preservative is at least one of Proclin 300, sodium azide, Krovin series and Kesong.

10. A kit for whole blood sample five-item immune assay, characterized in that: The kit contains the reagent according to any one of claims 1 to 9.

11. The kit according to claim 10, characterized in that: The kit also contains antiserum reagents, which include IgA antiserum reagents, IgG antiserum reagents, IgM antiserum reagents, C3 antiserum reagents and C4 antiserum reagents.

12. A method for using a whole blood sample five-item immune assay kit, characterized in that: The following steps are included: A hemolysis step, comprising directly contacting and mixing the whole blood sample with the reagent according to any one of claims 1 to 9 to perform red blood cell lysis; An immune reaction step, comprising directly adding an antiserum reagent for five-item immune assay to the product of the hemolysis step to carry out an immune reaction; The immunopentagram assay procedure includes determining the concentration of the immunopentagrams by turbidimetry after adding the immunopentagram assay antiserum reagent.

13. The method according to claim 12, characterized in that: It also includes a correction step, which includes correcting the concentration determined in the five immune determination steps according to the hematocrit, and the specific correction formula is shown in Formula 1; Formula-C mod =C×100 / (100-Hct%) Among them, C mod represents the corrected concentration, C represents the concentration measured by the five-item immune assay, and Hct% represents the hematocrit.

14. A device for whole blood sample five-item immune assay, characterized in that: It includes hemolysis and immune response module, five immune determination module and correction module; The hemolysis and immune response module includes a module for directly contacting and mixing a whole blood sample with the reagent according to any one of claims 1 to 9 to perform red blood cell lysis, and after the hemolysis is completed, directly adding the antiserum reagent for the five immune assays thereto to perform an immune response; The immune five-item assay module includes a module for determining the concentration of the immune five-item assay by turbidimetry after adding the antiserum reagent for the immune five-item assay; The correction module includes a module for correcting the concentration determined in the five immune determination steps according to the hematocrit, and the specific correction formula is shown in Formula 1; Formula-C mod =C×100 / (100-Hct%) Among them, C mod represents the corrected concentration, C represents the concentration measured by the five-item immune assay, and Hct% represents the hematocrit.

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