Homogeneous chemiluminescence immunoassay kit integrated with sample pretreatment technology

Abstract

The invention provides a homogeneous chemiluminescence immunodetection kit integrated with a sample pretreatment technology, which comprises a sample pretreatment container filled with an anti-biotin interference reagent filler and a reagent reaction container filled with an immunodetection reagent, the anti-biotin interference reagent filler comprises a molecular sieve filler or an affinity filler. According to the invention, the anti-biotin interference capability of the immunodiagnostic reagent is enhanced, the direct contact between the anti-biotin reagent component and the immunodiagnostic reagent can be effectively avoided, and the influence of the anti-biotin reagent component on the immunoreaction is reduced; by adjusting the filling amount of the filler in the sample cup, the capture capability can be effectively enhanced, and the biotin interference resistance of the kit is improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a homogeneous chemiluminescence immunoassay kit with integrated sample pretreatment technology. Background Art

[0002] Biotin (vitamin H) is an important component of a commonly used biotin-avidin system, which is widely used in biochemistry and clinical diagnosis. However, the presence of high concentrations of biotin in the human body may interfere with immunoassays based on the biotin-avidin system, resulting in inaccurate test results. Therefore, how to improve the ability of test samples to resist biotin interference has become an urgent problem to be solved. Summary of the invention

[0003] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a homogeneous chemiluminescent immunoassay kit integrating sample pretreatment technology.

[0004] To achieve the above-mentioned purpose of the invention, on the one hand, the present invention provides a homogeneous chemiluminescent immunoassay kit integrating sample pretreatment technology.

[0005] A homogeneous chemiluminescent immunoassay kit integrating sample pretreatment technology comprises a sample pretreatment container filled with an anti-biotin interfering reagent filler and a reagent reaction container filled with an immunoassay reagent, wherein the anti-biotin interfering reagent filler comprises a molecular sieve filler or an affinity filler.

[0006] Furthermore, the pore size of the molecular sieve filler is 5 to

[0007] Furthermore, the material of the molecular sieve filler is at least one of silica gel, aluminum oxide, magnetic material, nanocomposite material, organic polymer material, cross-linked dextran or agarose.

[0008] Furthermore, the affinity filler is coupled with one of avidin, streptavidin, neutravidin, and biotin antibody to prepare an affinity filler, wherein the particle size of the affinity filler is between 50 nanometers and 500 micrometers.

[0009] Furthermore, the affinity filler includes at least one of a synthetic material that does not contain natural proteins, a material that has been chemically modified to reduce nonspecific adsorption, a material with high mechanical strength and chemical stability, and a material that does not contain any active components that may interfere with immunoassays. Among them, the synthetic material that does not contain natural proteins includes polymer microspheres, the material that has been chemically modified to reduce nonspecific adsorption includes silica microspheres, the material with high mechanical strength and chemical stability includes magnetic particles, and the material that does not contain any active components that may interfere with immunoassays includes membrane materials or fiber materials.

[0010] In another aspect, the present invention provides a method for preparing the above-mentioned kit, comprising the following steps:

[0011] Prepare a sample pretreatment container and a reagent reaction container, wherein the sample pretreatment container and the reagent reaction container are of an integrated design or a split design;

[0012] Prepare an anti-biotin interference reagent filler, then fill the anti-biotin interference reagent filler into the sample pretreatment container, and fill the immunoassay reagent into the reagent reaction container.

[0013] In another aspect, the present invention provides a method for performing immunoassay using the above kit, comprising the following steps:

[0014] Add the sample to be tested into a sample pretreatment container, and use the sample pretreatment container to pretreat the sample to reduce the interference of biotin;

[0015] The pretreated sample is transferred to a reagent reaction container and mixed with an immunoassay reagent for immunoassay.

[0016] Furthermore, the sample to be tested is added to the sample pretreatment container to allow the sample to contact with the affinity filler so that the biotin in the sample to be tested is captured by the affinity filler. After capture, the remaining sample to be tested enters the reagent reaction container for subsequent immunoassay.

[0017] Furthermore, the sample to be tested is added to the sample pretreatment container to allow the sample to be tested to contact with the molecular sieve filler to separate and remove biotin in the sample, and then the remaining sample to be tested enters the reagent reaction container for subsequent immunoassay.

[0018] On the other hand, the present invention provides an application of the above-mentioned kit in preparing immunoassay samples without biotin interference, as well as its application in the fields of clinical diagnosis, scientific research, and drug development.

[0019] The advantages and beneficial effects of the present invention over the prior art are as follows:

[0020] 1. The existing solutions have weak anti-interference capabilities. The present invention provides a sample pretreatment method for improving the anti-biotin interference capability. Through specific sample pretreatment steps, the interference of biotin in the sample on the immunoassay results is effectively eliminated, thereby improving the accuracy and reliability of the detection.

[0021] 2. In addition to enhancing the anti-biotin interference ability of the immunodiagnostic reagent, the present invention can also effectively avoid direct contact between the anti-biotin reagent components and the immunodiagnostic reagent, reducing the impact of the anti-biotin reagent components on the immune response; by adjusting the filling amount of the filler in the sample cup, the capture ability can be effectively enhanced and the anti-biotin interference ability of the kit can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 This is a diagram showing the deviation of the test results of the experimental group and the control group after adding different concentrations of biotin to the sample 1 in Example 1 of the present invention;

[0024] Figure 2 This is a diagram showing the deviation of the test results of the experimental group and the control group after adding different concentrations of biotin to the sample 2 in Example 1 of the present invention;

[0025] Figure 3 This is a diagram showing the deviation of the test results of the experimental group and the control group after adding different concentrations of biotin to the sample 1 in Example 2 of the present invention;

[0026] Figure 4 This is a diagram showing the deviation of the test results of the experimental group and the control group after adding different concentrations of biotin to the sample 2 in Example 2 of the present invention;

[0027] Figure 5 This is a diagram showing the deviation of the test results of the experimental group and the control group after adding different concentrations of biotin to the sample 1 in Example 3 of the present invention;

[0028] Figure 6 This is a diagram showing the deviation of the test results of the experimental group and the control group after adding different concentrations of biotin to the sample 2 in Example 3 of the present invention;

[0029] Figure 7 This is a diagram showing the deviation of the test results of the experimental group and the control group after adding different concentrations of biotin to the sample 1 in Example 4 of the present invention;

[0030] Figure 8 This is a diagram showing the deviation of the test results of the experimental group and the control group after adding different concentrations of biotin to the sample 2 in Example 4 of the present invention;

[0031] Fig. 9 This is a diagram showing the deviation of the test results of the experimental group and the control group after adding different concentrations of biotin to the sample 1 in Example 5 of the present invention;

[0032] Fig.10 This is a diagram showing the deviation of the test results of the experimental group and the control group after adding different concentrations of biotin to sample 2 in Example 5 of the present invention. DETAILED DESCRIPTION

[0033] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] Example 1: A kit for detecting procalcitonin by using a molecular sieve filler to prevent biotin interference

[0035] Background: Procalcitonin (PCT) is a biomarker that is significantly elevated in inflammatory responses caused by bacterial infections. In clinical diagnosis, PCT testing is important for assessing the extent of infection and guiding antibiotic treatment. However, biotin, as a commonly used marker, may interfere with PCT test results.

[0036] Implementation content:

[0037] This embodiment provides an anti-biotin interference procalcitonin detection kit prepared by molecular sieve filler, which aims to reduce the interference of biotin on PCT detection and improve the accuracy and reliability of the detection results.

[0038] Implementation steps:

[0039] 1. Molecular sieve filler: In this case, Sephadex G-25 Medium filler from Cytiva was used.

[0040] 2. Filling: Fill the prepared molecular sieve filler into the sample pretreatment container in the kit, with a filling mass of 50 mg / kit.

[0041] 3. Sample pretreatment: Add the sample to be tested (the sample to be tested contains biotin) into a sample pretreatment container filled with a molecular sieve filler, and separate and remove the biotin in the sample according to the pore size of the molecular sieve filler.

[0042] 4. Immunoassay: Release the pretreated sample from the sample pretreatment container and add it to the reagent reaction container filled with immunoassay reagent in the kit for PCT detection.

[0043] The above implementation steps are the experimental group. The difference between the control group and the experimental group is that the molecular sieve filler is not contained. The amount of biotin added in the following table is the content of biotin in the sample to be tested. The test data of the control group and the experimental group are as follows:

[0044]

[0045] Through the above implementation steps, the anti-biotin interference procalcitonin detection kit prepared in this embodiment can effectively reduce the interference of biotin on PCT detection and improve the accuracy and reliability of the detection results. Compared with traditional methods, the kit has higher sensitivity, specificity and repeatability. In addition, the kit is simple to operate and is suitable for laboratory and clinical environments.

[0046] Application fields: The anti-biotin interference procalcitonin detection kit of this embodiment is suitable for clinical diagnosis, scientific research, drug development and other fields, and can be used to assess the degree of infection, guide antibiotic treatment and monitor inflammatory response, etc.

[0047] Conclusion: This example provides an anti-biotin interference procalcitonin detection kit prepared by molecular sieve filler, which can effectively reduce biotin interference and improve the accuracy and reliability of PCT detection. The kit is simple to operate, suitable for a variety of application scenarios, and has broad application prospects.

[0048] Example 2: An anti-biotin interference procalcitonin detection kit prepared by a streptavidin-labeled affinity filler

[0049] Background: Procalcitonin (PCT) is a biomarker that is significantly elevated in inflammatory responses caused by bacterial infections. In clinical diagnosis, PCT testing is important for assessing the extent of infection and guiding antibiotic treatment. However, biotin, as a commonly used marker, may interfere with PCT test results.

[0050] Implementation content:

[0051] This embodiment provides an anti-biotin interference procalcitonin detection kit prepared by an affinity filler labeled with streptavidin, which aims to reduce the interference of biotin on PCT detection and improve the accuracy and reliability of the detection results.

[0052] Implementation steps:

[0053] 1. Affinity filler selection: In this case, we selected NHS Activated 4FF.

[0054] 2. Streptavidin labeling: label the streptavidin onto the affinity filler to prepare the affinity filler. The labeling ratios are 10ug / mg filler, 20ug / mg filler, and 50ug / mg filler, respectively.

[0055] 3. Filling with affinity filler: Fill the prepared affinity filler into the sample pretreatment container in the kit, with a filling mass of 50 mg / kit.

[0056] 4. Sample pretreatment: Add the sample to be tested into a sample pretreatment container filled with affinity filler, and separate and remove biotin in the sample through the specific binding between streptavidin and biotin.

[0057] 5. Immunoassay: Release the pretreated sample from the sample pretreatment container and add it to the reagent reaction container filled with immunoassay reagent in the kit for PCT detection.

[0058] The above implementation steps are for the experimental group. The difference between the control group and the control group is that it does not contain affinity fillers.

[0059]

[0060] Through the above implementation steps, the anti-biotin interference procalcitonin detection kit prepared in this embodiment can effectively reduce the interference of biotin on PCT detection and improve the accuracy and reliability of the detection results. Compared with traditional methods, the kit has higher sensitivity, specificity and repeatability. In addition, the kit is simple to operate and is suitable for laboratory and clinical environments.

[0061] Application areas:

[0062] The anti-biotin interference procalcitonin detection kit of this embodiment is suitable for clinical diagnosis, scientific research, drug development and other fields, and can be used to assess the degree of infection, guide antibiotic treatment, monitor inflammatory response, etc.

[0063] in conclusion:

[0064] This embodiment provides an anti-biotin interference procalcitonin detection kit prepared by a streptavidin-labeled affinity filler, which can effectively reduce biotin interference and improve the accuracy and reliability of PCT detection. The kit is simple to operate, suitable for a variety of application scenarios, and has broad application prospects.

[0065] Example 3: Preparation of Anti-biotin Interference Procalcitonin Detection Kit by Biotin Antibody Modification Filler Background technology:

[0067] Biotin is a commonly used marker and is widely used in immunoassays. However, the presence of biotin may interfere with the test results, especially in the case of high concentrations of biotin. Therefore, an effective filler is needed to reduce biotin interference and improve the accuracy and reliability of the test results.

[0068] Implementation content:

[0069] The invention provides an anti-biotin interference procalcitonin detection kit prepared by affinity filling material, and compares the difference in anti-biotin interference ability between streptavidin filling material and biotin antibody filling material.

[0070] Implementation steps:

[0071] 1. Affinity filler selection: In this case, we selected NHS Activated 4FF.

[0072] 2. Streptavidin labeling: Streptavidin and biotinylated antibody were coated onto affinity fillers at a labeling ratio of 50 ug / mg to prepare streptavidin fillers and biotin antibody fillers, respectively.

[0073] 3. Filling of affinity fillers: Fill the prepared streptavidin filler and biotin antibody filler into the sample pretreatment container in the kit respectively, with a filling mass of 50 mg / kit.

[0074] 4. Sample pretreatment: Add the samples to be tested into sample pretreatment containers filled with streptavidin filler and biotin antibody filler respectively to separate and remove biotin in the samples.

[0075] 5. Immunoassay: Release the pretreated samples from the sample pretreatment container and add them to the reagent reaction container filled with immunoassay reagent in the kit to perform PCT detection.

[0076] The above implementation steps are the experimental group. The difference between the control group and the control group is that it does not contain affinity fillers. The amount of biotin added in the following table is the content of biotin in the sample to be tested. The test data of the control group and the experimental group are as follows:

[0077]

[0078]

[0079] Through the above implementation steps, the anti-biotin interference procalcitonin detection kit prepared in this embodiment can effectively reduce the interference of biotin on PCT detection and improve the accuracy and reliability of the detection results. The experimental results show that the anti-biotin interference ability of streptavidin filler is better than that of biotin antibody filler, and has higher sensitivity, specificity and repeatability.

[0080] Application areas:

[0081] The anti-biotin interference procalcitonin detection kit of this embodiment is suitable for clinical diagnosis, scientific research, drug development and other fields, and can be used to assess the degree of infection, guide antibiotic treatment and monitor inflammatory response.

[0082] in conclusion:

[0083] This embodiment provides an anti-biotin interference procalcitonin detection kit prepared by a streptavidin-labeled affinity filler, and compares the difference in anti-biotin interference ability between streptavidin filler and biotin antibody filler. The experimental results show that the streptavidin filler has better anti-biotin interference ability and provides more accurate detection results for PCT detection. The kit is easy to operate, suitable for a variety of application scenarios, and has broad application prospects.

[0084] Example 4: Preparation of anti-biotin interference testosterone detection kit by streptavidin affinity filler Background technology:

[0085] Interleukin 6 (IL-6) is a polypeptide and a useful pro-inflammatory cytokine marker, which is mainly used in the auxiliary diagnosis of inflammation in clinical practice. However, biotin, as a commonly used marker, may interfere with the results of IL-6 detection.

[0086] Implementation content:

[0087] This embodiment provides an anti-biotin interferon 6 detection kit prepared by streptavidin affinity filler, which aims to reduce the interference of biotin on iL-6 detection and improve the accuracy and reliability of the detection results.

[0088] Implementation steps:

[0089] 1. Affinity filler selection: In this case, we selected NHS Activated 4FF.

[0090] 2. Streptavidin labeling: Streptavidin is coated onto the affinity filler at a labeling ratio of 50 ug / mg to prepare an affinity filler.

[0091] 3. Filling with affinity filler: Fill the prepared affinity filler into the sample pretreatment container in the kit, with a filling mass of 50 mg / kit.

[0092] 4. Sample pretreatment: Add the sample to be tested into a sample pretreatment container filled with affinity filler, and separate and remove biotin in the sample through the specific binding between streptavidin and biotin.

[0093] 5. Immunoassay: Release the pretreated sample from the sample pretreatment container and add it to the reagent reaction container filled with immunoassay reagent in the kit for iL-6 detection.

[0094] The above implementation steps are the experimental group. The difference between the control group and the experimental group is that the affinity filler is not contained. The amount of biotin added in the following table is the content of biotin in the sample to be tested. The test data of the control group and the experimental group are as follows:

[0095]

[0096]

[0097] Through the above implementation steps, the anti-biotin interferon 6 detection kit prepared in this embodiment can effectively reduce the interference of biotin on iL-6 detection and improve the accuracy and reliability of the detection results.

[0098] Example 5

[0099] Preparation of anti-biotin interference testosterone detection kit by streptavidin affinity filling, filling mass 50mg Background technology:

[0101] Testosterone (T) is a steroid hormone, also known as testosterone, testosterone or testosterone. Testosterone affects many body systems and functions, including: blood production, calcium balance in the body, bone mineralization, lipid metabolism, sugar metabolism and prostate growth. Testosterone replacement therapy can improve aging-related vascular lesions and improve a variety of cardiovascular risk factors. However, as a commonly used marker, the presence of biotin may interfere with the test results.

[0102] Implementation content:

[0103] This embodiment provides an anti-biotin interference testosterone detection kit prepared by streptavidin affinity filler, which aims to reduce the interference of biotin on testosterone detection and improve the accuracy and reliability of the detection results.

[0104] Implementation steps:

[0105] 1. Affinity filler selection: In this case, we selected NHS Activated 4FF.

[0106] 2. Streptavidin labeling: Streptavidin is coated onto the affinity filler at a labeling ratio of 50 ug / mg to prepare an affinity filler.

[0107] 3. Filling with affinity filler: Fill the prepared affinity filler into the kit with a filling mass of 50 mg / kit.

[0108] 4. Sample pretreatment: Add the sample to be tested into a sample pretreatment container filled with affinity filler, and separate and remove biotin in the sample through the specific binding between streptavidin and biotin.

[0109] 5. Immunoassay: Release the pretreated sample from the sample pretreatment container, add it to the reagent reaction container filled with immunoassay reagent in the kit, and perform Testo detection.

[0110] The above implementation steps are the experimental group. The difference between the control group and the experimental group is that the affinity filler is not contained. The amount of biotin added in the following table is the content of biotin in the sample to be tested. The test data of the control group and the experimental group are as follows:

[0111]

[0112] Through the above implementation steps, the anti-biotin interference testosterone detection kit prepared in this embodiment can effectively reduce the interference of biotin on Testo detection and improve the accuracy and reliability of the detection results.

[0113] Application areas:

[0114] The anti-biotin interference testosterone detection kit of this embodiment is suitable for clinical diagnosis, scientific research, drug development and other fields, and can be used to assess the degree of infection, guide antibiotic treatment and monitor inflammatory response, etc.

[0115] in conclusion:

[0116] This embodiment provides an anti-biotin interference testosterone detection kit prepared by streptavidin affinity filler. The kit is simple to operate, suitable for a variety of application scenarios, and has broad application prospects.

[0117] In the above embodiments, the sample pretreatment container and the reagent reaction container are of an integrated design. The existing solutions have weak anti-interference capabilities. The present invention provides a sample pretreatment method for improving the anti-biotin interference capability. Through specific sample pretreatment steps, the interference of biotin in the sample on the immunoassay results is effectively eliminated, thereby improving the accuracy and reliability of the detection. In addition, in addition to enhancing the anti-biotin interference capability of the immunodiagnostic reagent, the present invention can also effectively avoid direct contact between the anti-biotin reagent component and the immunodiagnostic reagent, reducing the effect of the anti-biotin reagent component on the immune response; by adjusting the filling amount of the filler in the sample cup, the capture capability can be effectively enhanced, and the anti-biotin interference capability of the test kit can be improved.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A homogeneous chemiluminescent immunoassay kit integrating sample pretreatment technology, characterized in that: The kit comprises a sample pretreatment container filled with an anti-biotin interference reagent filler and a reagent reaction container filled with an immunoassay reagent, wherein the anti-biotin interference reagent filler comprises a molecular sieve filler or an affinity filler.

2. The homogeneous chemiluminescent immunoassay kit with integrated sample pretreatment technology according to claim 1, characterized in that: The pore size of the molecular sieve filling is 5 to 3. The homogeneous chemiluminescent immunoassay kit with integrated sample pretreatment technology according to claim 1, characterized in that: The material of the molecular sieve filler is at least one of silica gel, aluminum oxide, magnetic material, nanocomposite material, organic polymer material, cross-linked dextran or agarose.

4. The homogeneous chemiluminescent immunoassay kit with integrated sample pretreatment technology according to claim 1, characterized in that: The affinity filler is coupled with one of avidin, streptavidin, neutravidin and biotin antibody to prepare an affinity filler, wherein the particle size of the affinity filler is between 50 nanometers and 500 micrometers.

5. The homogeneous chemiluminescent immunoassay kit with integrated sample pretreatment technology according to claim 1, characterized in that: The affinity filler includes at least one of a synthetic material that does not contain natural proteins, a material that has been chemically modified to reduce nonspecific adsorption, a material with high mechanical strength and chemical stability, and a material that does not contain any active components that may interfere with immunoassay. Among them, the synthetic material that does not contain natural proteins includes polymer microspheres, the material that has been chemically modified to reduce nonspecific adsorption includes silica microspheres, the material with high mechanical strength and chemical stability includes magnetic particles, and the material that does not contain any active components that may interfere with immunoassay includes membrane materials or fiber materials.

6. A method for preparing the kit according to any one of claims 1 to 5, characterized in that: The following steps are involved: Prepare a sample pretreatment container and a reagent reaction container, wherein the sample pretreatment container and the reagent reaction container are of an integrated design or a split design; Prepare an anti-biotin interference reagent filler, then fill the anti-biotin interference reagent filler into the sample pretreatment container, and fill the immunoassay reagent into the reagent reaction container.

7. A method for performing immunoassay using the kit according to any one of claims 1 to 4, characterized in that: The following steps are involved: Add the sample to be tested into a sample pretreatment container, and use the sample pretreatment container to pretreat the sample to reduce the interference of biotin; The pretreated sample is transferred to a reagent reaction container and mixed with an immunoassay reagent for immunoassay.

8. The method of immunoassay according to claim 7, characterized in that: The sample to be tested is added to the sample pretreatment container, and the sample to be tested is brought into contact with the affinity filler so that the biotin in the sample to be tested is captured by the affinity filler. After capture, the remaining sample to be tested enters the reagent reaction container for subsequent immunoassay.

9. The method of immunoassay according to claim 7, characterized in that: The sample to be tested is added to the sample pretreatment container, and the sample to be tested is brought into contact with the molecular sieve filler to separate and remove biotin in the sample to be tested, and then the remaining sample to be tested is placed in the reagent reaction container for subsequent immunoassay.

10. Use of the kit according to any one of claims 1 to 4 in preparing immunoassay samples without biotin interference, and in the fields of clinical diagnosis, scientific research, and drug development.

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