WO2018001214A1 - Rapid mouse antibody subtype typing kit and preparation method therefor - Google Patents

Abstract

A rapid mouse antibody subtype typing kit and preparation method therefor. The kit comprises a first test strip for typing detection of IgG1, Kappa and Lambda, a second test strip for typing detection of IgG1, IgG2a, IgG2b and IgG3, and a third test strip for typing detection of IgA, IgD, IgE and IgM; the test strips are formed by sequentially connecting a sample pad, a gold colloid bonding pad, a nitrocellulose membrane and a water absorption pad to a PVC base plate; the gold colloid bonding pad is coated by a gold colloid-labeled anti-mouse IgG (H+L) antibody; a detection area and a quality control area on the nitrocellulose membrane comprise a specific capture antibody and a quality control mouse monoclonal antibody which are arranged sequentially in parallel and spaced apart from each other. The kit is comprehensive in subtype detection, may be used to conduct typing detection on all heavy chains and light chains, is convenient and easy to use, has a short operation time, and may be used to complete subtype detection on a sample in 30 minutes.

Description

Mouse antibody subtype rapid typing kit and preparation method thereof Technical field

The invention belongs to the technical field of biological testing, and more particularly, to a mouse antibody subtype rapid typing kit and a preparation method thereof.

Background technique

According to physicochemical properties and biological functions, antibodies can be divided into five categories: IgM, IgG, IgA, IgE, and IgD. IgM antibodies are the first antibodies secreted in the immune response. They bind to the antigen and initiate a cascade of complements. They also connect the invaders to each other and form a bunch to facilitate the phagocytosis of macrophages. IgG antibodies activate complement, neutralize A variety of toxins, IgG lasts for a long time, is the only antibody that can pass through the placenta to protect the fetus during pregnancy. They are also secreted from the mammary gland into the colostrum, which protects the newborn. According to the different heavy chain structure, IgG antibodies can be divided. There are 4 subclasses, namely IgG1, IgG2a, IgG2b, IgG3. These four subclasses have different biological properties, so they play different roles in the development and progression of the disease. In an immune response, different IgGs can be produced because of the type of antigen (including the type of vector, the structure and nature of the antigenic determinant), the difference in dose and entry into the body, and the genetic predisposition of the host;; IgA antibodies enter the body. Mucosal surface, including mucous membranes of respiratory, digestive, reproductive and other pipelines, neutralizes infectious agents, and can also deliver this antibody to the digestive tract mucosa of newborns through the colostrum of breast milk, which is the most important in breast milk, the most important A type of antibody; the tail of the IgE antibody binds to the cell membrane of basophils and mast cells. When the antibody binds to the antigen, basophils and mast cells release tissue-like substances to promote the development of inflammation, which is also a rapid allergic reaction. The role of IgD antibodies is not well understood. They are mainly found on the surface of mature B lymphocytes and may be involved in the differentiation of B cells.

Since the antibody has a binding site (antigen-binding cluster) corresponding to the antigenic determinant, binding of the antibody to the antigen is specific. On the other hand, the antibody itself is a protein with its own amino acid composition, arrangement and steric structure, which is an antigen for xenobiotics. All types of Ig have antigen specificity that can be detected by serological methods, and they exhibit different serological types. There are three specificity of Ig antigen: 1. Isotype: the antigen specificity shared by all individuals of the same genus. 2. Allotypic: The difference in antigenicity of one or several amino acids (genetic markers) on the same type of Ig in the same genus. 3. Unique type: refers to the Ig V area and T produced by different B cell clones. An antigen-specific marker possessed by the B cell surface antigen receptor V region. The number of unique determinants of Ig is large, and under certain conditions can stimulate the body to produce anti-idiotypic antibodies, which is of great significance for immune regulation.

The existing mouse antibody subtype rapid typing kit generally uses a double antibody sandwich ELISA method to identify monoclonal antibodies or specific affinity purified subclasses. The secondary antibody coated with the consensus site is coated with the microplate, and the antibody in the culture supernatant is added, and then the HRP marker subtype antibody is added to react separately, and finally the color is detected by the TMB substrate system and terminated with dilute sulfuric acid. The antibody subtype was determined by measuring the absorbance by a microplate reader. The ELISA kit has an extremely high resolution and is a reliable tool for identifying antibody subtypes. The number of samples tested is relatively large, relatively economical; relatively time-consuming (about 3 hours), and requires an ELISA instrument system; can detect subtypes (IgG: IgG1, IgG2a, IgG2b, IgG3) and other antibodies Type (IgM, IgA), heavy chain subtypes are incompletely detected, and few products contain a classification of light chain subtypes.

The existing colloidal gold detection products in the market can also detect subtypes (IgG: IgG1, IgG2a, IgG2b, IgG3) and other antibody subtypes (IgM, IgA), heavy chain subtypes are incompletely detected, and few products are available. A classification containing light chain subtypes. Currently, there are similar colloidal gold detection products on the market, but generally, due to the limited antibody materials developed, it is usually possible to detect conventional subtypes (IgG: IgG1, IgG2a, IgG2b, IgG3) and other antibody subtypes (IgM, IgA). Heavy chain subtypes are incompletely detected, and few products contain a classification of light chain subtypes.

Summary of the invention

Based on this, in order to overcome the above drawbacks of the prior art, the present invention provides a mouse antibody subtype rapid typing kit and a preparation method thereof.

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

A mouse antibody subtype rapid typing kit, comprising:

Test strip 1 for IgG1, Kappa and Lambda typing, strip 2 for IgG1, IgG2a, IgG2b and IgG3 typing, and test strips for IgA, IgD, IgE and IgM typing Article 3, the test strip 1, the test strip 2 and the test strip 3 are each composed of a sample pad, a colloidal gold bond pad, a nitrocellulose film, and an absorbent pad which are sequentially overlapped on a PVC substrate, and the colloidal gold is combined. The pad is coated with a colloidal gold-labeled anti-mouse IgG (H+L) antibody; the nitrocellulose membrane comprises a detection zone coated with anti-mouse typing antibodies and coated mice arranged in parallel and spaced apart from each other The control region of the monoclonal antibody control antibody.

In some of the embodiments, in the test strip 1, the concentration of the anti-mouse IgG1 antibody coated in the detection zone is 0.7-1.0 mg/mL, and the coating amount in the detection zone is 1-1.2 ul/ Cm; the concentration of anti-mouse Kappa antibody is 0.5-0.8 mg/mL, the coating amount in the detection zone is 0.7 ul/cm; the concentration of anti-mouse Lambda antibody is 0.5-0.8 mg/mL, and the package in the detection zone The dose was 1-1.2 ul/cm; the concentration of the mouse monoclonal antibody-controlled antibody coated in the QC region was 1 mg/mL, and the coating amount in the QC region was 1.0 ul/cm.

In some of the embodiments, in the test strip 2, the detection zone is coated with an anti-mouse IgG1 antibody The concentration of the anti-mouse IgG2a antibody, the anti-mouse IgG2b antibody, and the anti-mouse IgG3 antibody are all 0.7-1.0 mg/mL, and the coating amount in the detection zone is 1-1.2 ul/cm; the quality control zone The concentration of the coated mouse monoclonal antibody control antibody was 0.7-1.0 mg/mL, and the coating amount in the QC region was 1-1.2 ul/cm.

In some of the embodiments, in the test strip 3, the concentration of the anti-mouse IgA antibody, the anti-mouse IgD antibody, the anti-mouse IgE antibody, and the anti-mouse IgM antibody coated in the detection zone is 1.0. -1.5mg/mL, the coating amount in the detection area is 1-1.2ul / cm; the concentration of the mouse monoclonal antibody control antibody coated in the quality control area is 0.7-1.0mg / mL, the package in the quality control area The amount was 1-1.2 ul/cm.

In some of the embodiments, the colloidal gold-labeled anti-mouse IgG (H+L) antibody has a concentration of 20-30 ug/mL colloid in the test strip 1, the test strip 2, and the test strip 3. Gold is used in an amount of 1.5-2.5 μg/cm ² on the colloidal gold bond pad.

The invention also provides a preparation method of a mouse antibody subtype rapid typing kit, comprising the following steps:

A. Anti-mouse IgG1 antibody, anti-mouse Kappa antibody, anti-mouse Lambda antibody working solution, and mouse monoclonal antibody control antibody working solution were coated on nitrocellulose membrane, and dried at 36-38 ° C for 4-6 After hours, the nitrocellulose membrane 1 is prepared;

B. Apply anti-mouse IgG1 antibody, anti-mouse IgG2a antibody, anti-mouse IgG2b antibody, anti-mouse IgG3 antibody working solution and mouse monoclonal antibody control antibody working solution on nitrocellulose membrane, 36-38 ° C Drying for 4-6 hours, preparing a nitrocellulose membrane 2;

C. Apply anti-mouse IgA antibody, anti-mouse IgD antibody, anti-mouse IgE antibody, anti-mouse IgM antibody working solution and mouse monoclonal antibody control antibody working solution on nitrocellulose membrane, 36-38 ° C Drying for 4-6 hours, preparing a nitrocellulose membrane 3;

D. Applying a colloidal gold-labeled anti-mouse IgG (H+L) antibody to the gold-labeled pad material, and drying it at 25-35% for more than 6 hours to prepare a colloidal gold binding pad;

E. The sample pad, the colloidal gold bond pad, the nitrocellulose membrane 1, and the absorbent pad are sequentially overlapped on the PVC bottom plate to obtain a test strip 1 for IgG1, Kappa and Lambda typing detection; the sample pad and the colloidal gold are used. The bonding pad, the nitrocellulose membrane 2, and the absorbent pad are sequentially overlapped on the PVC substrate to obtain a test strip 2 for IgG1, IgG2a, IgG2b and IgG3 typing detection, a sample pad, a colloidal gold binding pad, and a nitrocellulose membrane. 3. The absorbent pad is sequentially overlapped on the PVC bottom plate to obtain the test strip 3 for IgA, IgD, IgE and IgM typing detection; the test strip 1, the test strip 2 and the test strip 3 are combined. Kit of the invention.

The mouse antibody subtype rapid typing kit of the present invention utilizes the principle of lateral chromatography for rapidly and accurately determining the subtypes and light chain types of mouse monoclonal antibodies. The kit consists of three sets of test strips, the first group being mouse antibody IgG1 and light chain (IgG1/Kappa/Lambda) typing test strips, and the second group being mouse antibody IgG (IgG1/IgG2a/ The IgG2b/IgG3) typing test strips, and the third group were mouse antibody IgA/D/E/M typing test strips. The specific capture antibodies against the mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, IgD, IgE, κ and λ light chains were coated on solid phase nitrocellulose membranes of three different test strips, respectively. The anti-mouse IgG (H+L) antibody was detected by broad-spectrum labeling with colloidal gold. When using, a small amount of sample (hybridoma cell culture supernatant or purified monoclonal antibody dilution) is placed at the sample pad at the leading end of the test strip arrow or directly inserted into the cell culture solution by the test strip arrow indicating the front end of the sample pad. In the lateral chromatography, the test antibody in the sample is combined with the colloidal gold-labeled detection antibody to form a complex. When the complex moves forward along the test strip by chromatography, it forms a "capture antibody-mouse monoclonal antibody-colloidal gold-labeled anti-mouse IgG (H+L) antibody with the corresponding capture antibody immobilized in different detection lines. "Double antibody sandwich complex and agglutination in the corresponding subtype position, so as to achieve the purpose of typing detection of mouse monoclonal antibody; if it is a negative specimen, the double antibody sandwich complex cannot be formed in the detection line region, so Color development. Regardless of whether the mouse monoclonal antibody is present in the test sample, a red band will appear in the control line region. The red band appearing in the control line area is a criterion for determining whether there is enough specimen, whether the chromatographic process is normal, and also as an internal control standard for the reagent.

When using, the first group of mouse antibody IgG1 and light chain typing test strips can be used to determine whether the mouse antibody is an IgG1 subtype and distinguish between the κ and λ light chains; if it is not possible to determine the subtype of the detection antibody, The second group of mouse antibody IgG typing test strips determine whether the mouse antibody is IgG1, IgG2a, IgG2b, IgG3 subtype; and the third group of mouse antibody IgA / D / E / M typing test strip determination Whether the antibody is of the IgA, IgM, IgD, IgE subtype. The three sets of test strips were used in combination to determine IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, IgD, IgE and kappa and lambda light chains in mice.

Compared with the prior art, the present invention has the following significant advantages:

1. The inventors of the present invention performed a large number of experimental verifications on the coating parameters of the test strip of the mouse antibody subtype rapid typing kit on the test strip, and obtained the optimal coating parameters. Therefore, the test strip of the present invention can sensitively perform subtype detection of the sample, and the kit of the present invention can detect all kinds of heavy chain IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, IgD, IgE. Typing detection and typing detection of κ and λ light chains;

2. The test strip of the mouse antibody subtype rapid typing kit of the present invention has a minimum detection concentration of 100 ng/mL for the sample, and there is no cross between the subtypes;

3. The mouse antibody subtype rapid typing kit of the invention is ingeniously designed, and most of the mouse hybridoma antibody subtypes are considered to be IgG1 subtypes, and the user can use the kit first according to the product specification during the subtype detection process. IgG1/Kappa/Lambda typing test strip 1 preliminarily determines whether the sample is IgG1 subtype and carries out preliminary screening of the light chain, and then decides whether it is necessary to use the other two types of test strips to reduce the workload and save the cost of testing. ;

4. The mouse antibody subtype rapid typing kit of the invention is convenient and simple to use, does not require auxiliary instruments, and the operator does not need professional training; the operation time is short, and the subtype detection of the sample can be completed in 30 minutes.

DRAWINGS

1 is a schematic structural view of a test strip 1 according to Embodiment 1 of the present invention, wherein reference numerals are: 11, a PVC bottom plate; 12, a sample pad; 13, a colloidal gold bond pad; 14, a nitrocellulose film; Pad; 16, IgG1 detection line; 17, Kappa detection line; 18, Lambda detection line; 19, quality control line;

2 is a schematic structural view of a test strip 2 according to Embodiment 1 of the present invention, wherein reference numerals are: 21, a PVC bottom plate; 22, a sample pad; 23, a colloidal gold bond pad; 24, a nitrocellulose film; Pad; 26, IgG1 detection line; 27, IgG2a detection line; 28, IgG2b detection line; 29, IgG3 detection line; 20, quality control line;

3 is a schematic structural view of a test strip 3 according to Embodiment 1 of the present invention, wherein reference numerals are: 31, a PVC bottom plate; 32, a sample pad; 33, a colloidal gold bond pad; 34, a nitrocellulose film; Pad; 36, IgA test line; 37, IgD test line; 38, IgE test line; 39, IgM test line; 30, quality control line.

Detailed ways

The technical solutions of the present invention are further illustrated by the following specific examples, which are not intended to limit the scope of the present invention. Some non-essential modifications and adaptations made by others in accordance with the teachings of the present invention are still within the scope of the present invention.

The steps in the examples are routinely operated in the art unless otherwise specified, and the starting materials used in the following examples are all commercially available.

Example 1 A mouse antibody subtype rapid typing kit

Please refer to FIG. 1-3, which is a mouse antibody subtype rapid typing kit of the present invention, comprising:

1. Test strip 1 (Fig. 1) for IgG1, Kappa and Lambda typing detection, the test strip is sequentially overlapped by sample pad 12, colloidal gold bond pad 13, nitrocellulose membrane 14, and absorbent pad 15 Constructed on a PVC substrate 1, the colloidal gold bond pad 13 is coated with a colloidal gold-labeled broad-spectrum detection anti-mouse IgG (H+L) antibody (Jackson Immuno Research cat #715-545-151); The nitrocellulose membrane 14 includes a detection line 16 coated with anti-mouse IgG1 antibody, a detection line 17 against mouse Kappa antibody, and a detection line 18 against anti-mouse Lambda antibody, and a small control coating, which are arranged in parallel and arranged in parallel. Quality control line 19 of mouse monoclonal antibody (Jackson Immuno Research cat #315-155-006);

The concentration of the colloidal gold-labeled antibody was 25 ug/mL colloidal gold, and the amount on the binding pad was 2 μg/cm ² ; the concentration of the anti-mouse IgG1 antibody coated in the detection zone was 1.0 mg/mL, in the detection zone. The coating amount was 1.0 μL/cm; the concentration of the anti-mouse Kappa antibody was 0.8 mg/mL, the coating amount in the detection zone was 1.0 μL/cm; and the concentration of the anti-mouse Lambda antibody was 0.8 mg/mL. The coating amount of the region was 1.0 μL/cm; the concentration of the control antibody coated in the QC region was 1.0 mg/mL, and the coating amount in the QC region was 1.0 μL/cm.

2. Test strip 2 (Fig. 2) for IgG1, IgG2a, IgG2b and IgG3 typing detection, the test strip consisting of sample pad 22, colloidal gold bond pad 23, nitrocellulose membrane 24, and absorbent pad 25 in sequence Laminated on the PVC substrate 21, the colloidal gold bond pad 23 is coated with a broad-spectrum detection anti-mouse IgG (H+L) antibody labeled with colloidal gold; the detection zone of the nitrocellulose membrane 24 includes parallel The detection line 26 of the anti-mouse IgG1 antibody, the detection line 27 of the anti-mouse IgG2a antibody, the detection line 28 of the detection line of the anti-mouse IgG2b antibody, and the detection line of the anti-mouse IgG3 antibody are provided and spaced apart from each other. 29 and a control line of coated mouse monoclonal antibody control antibody 20;

The concentration of the colloidal gold-labeled antibody was 25 ug/mL, and the amount on the binding pad was 2 μg/cm ² ; the concentration of the anti-mouse IgG1 antibody coated in the detection zone was 1.0 mg/mL, and the coating in the detection zone was The amount is 1.0 μL/cm; the concentration of the anti-mouse IgG2a antibody is 1.0 mg/mL, the coating amount in the detection zone is 1.0 μL/cm; the concentration of the anti-mouse IgG2b antibody is 1.0 mg/mL, in the detection zone. The coating amount was 1.0 μL/cm; the concentration of the anti-mouse IgG3 antibody was 1.0 mg/mL, and the coating amount in the detection zone was 1.0 μL/cm; the concentration of the control antibody coated in the QC region was 1.0 mg/mL. , the coating amount in the quality control area is 1.0 μL / cm;

as well as

3. Test strip 3 (Fig. 3) for IgA, IgD, IgE and IgM typing detection, the test strip is sampled The product pad 32, the colloidal gold bonding pad 33, the nitrocellulose membrane 34, and the absorbent pad 35 are sequentially laminated on the PVC substrate 31, and the colloidal gold bonding pad 33 is coated with a colloidal gold-labeled broad-spectrum detection anti-mouse. An IgG (H+L) antibody; the detection zone on the nitrocellulose membrane comprises a detection line 36 coated with anti-mouse IgA antibody, a detection line 37 against the mouse IgD antibody, and a small anti-small detection line a detection line 38 of a murine IgE antibody and a detection line 39 against an anti-mouse IgM antibody and a quality control line 30 coated with a mouse monoclonal antibody control antibody;

The concentration of the colloidal gold-labeled antibody was 25 ug/mL, and the amount on the binding pad was 2 μg/cm ² ; the concentration of the anti-mouse IgA antibody coated in the detection zone was 1.5 mg/mL, and the coating in the detection zone was The amount is 1.0 μL/cm; the concentration of the anti-mouse IgD antibody is 1.5 mg/mL, the coating amount in the detection zone is 1.0 μL/cm; the concentration of the anti-mouse IgE antibody is 1.5 mg/mL, in the detection zone. The coating amount was 1.0 μL/cm; the concentration of the anti-mouse IgM antibody was 1.5 mg/mL, and the coating amount in the detection zone was 1.0 μL/cm; the concentration of the control antibody coated in the QC region was 1.0 mg/mL. The amount of coating in the quality control zone was 1.0 μL/cm.

The invention also provides a preparation method of a mouse antibody subtype rapid typing kit, comprising the following steps:

A. Anti-mouse IgG1 antibody, anti-mouse Kappa antibody, anti-mouse Lambda antibody working solution, and mouse monoclonal antibody control antibody working solution were coated on nitrocellulose membrane, and dried at 36-38 ° C for 4-6 After hours, the nitrocellulose membrane 1 is prepared;

B. Apply anti-mouse IgG1 antibody, anti-mouse IgG2a antibody, anti-mouse IgG2b antibody, anti-mouse IgG3 antibody working solution and mouse monoclonal antibody control antibody working solution on nitrocellulose membrane, 36-38 ° C Drying for 4-6 hours, preparing a nitrocellulose membrane 2;

C. Apply anti-mouse IgA antibody, anti-mouse IgD antibody, anti-mouse IgE antibody, anti-mouse IgM antibody working solution and mouse monoclonal antibody control antibody working solution on nitrocellulose membrane, 36-38 ° C Drying for 4-6 hours, preparing a nitrocellulose membrane 3;

D. Applying a colloidal gold-labeled broad-spectrum detection anti-mouse IgG (H+L) antibody on the gold-labeled pad material, and drying it at 25-35% for more than 6 hours to prepare a colloidal gold binding pad;

E, 1) The components that need to be cut (sample pad, colloidal gold bond pad, absorbent pad) are cut according to the specifications of the product according to the required specifications;

2) Gently uncover the protective film on the absorbing pad of the nitrocellulose membrane, adhere the cut absorbent pad to it, and push it evenly and gently to enhance the adhesion and prevent bubbles. The absorption pad is covered on the NC film by 1 mm;

3) Gently uncover the protective film on the lower side of the PVC substrate with the colloidal gold bond pad, and attach the cut colloidal gold bond pad to it. The method is the same as the nitrocellulose film and the colloidal gold bond pad covers the NC film. 1mm;

4) Gently uncover the protective film at the lowermost end of the PVC substrate, and attach the sample pad to the colloidal gold bonding pad. The method is the same as the absorbent pad, and the sample pad is covered on the colloidal gold bonding pad by 3 mm;

5) The sample pad, the colloidal gold bonding pad, the nitrocellulose membrane 1, the absorbent pad, and the PVC substrate are firmly pressed, and cut according to the required specifications to obtain a test strip 1 for IgG1, Kappa and Lambda typing detection; The sample pad, colloidal gold bond pad, nitrocellulose membrane 2, absorbent pad, PVC bottom plate are firmly pressed, cut according to the required specifications, and the test strip 2 for IgG1, IgG2a, IgG2b and IgG3 typing detection will be obtained. Sample pad, colloidal gold bond pad, nitrocellulose film 3, absorbent pad, PVC bottom plate are firmly pressed, cut according to the required specifications, and obtained test strip 3 for IgA, IgD, IgE and IgM typing test; test paper The kit of the present invention is obtained by combining the strip 1, the test strip 2 and the test strip 3.

The method for using the mouse antibody subtype rapid typing kit of the present embodiment is as follows:

1. Restore the test strips 1-3 in the kit to room temperature before use;

2. Add 60 ul of the test sample solution to the sample pad in the test strip according to the direction of the arrow, or directly insert the test strip into the cell culture medium sample according to the arrow direction (note that the position of the inserted liquid cannot exceed the arrow) The front end position is taken out and placed on a clean and flat surface after 10 minutes; when tested, the test antibody in the sample binds to the colloidal gold-labeled detection antibody to form a complex. When the complex moves forward along the test strip by chromatography, it forms a "capture antibody-mouse monoclonal antibody-colloidal gold-labeled anti-mouse IgG (H+L) antibody with the corresponding capture antibody immobilized in different detection lines. "Double antibody sandwich complex and agglutination in the corresponding subtype position, so as to achieve the purpose of typing detection of mouse monoclonal antibody; if it is a negative specimen, the double antibody sandwich complex cannot be formed in the detection line region, so Color development. Regardless of whether the mouse monoclonal antibody is present in the test sample, a red band will appear in the control line region. The red band appearing in the control line area is a criterion for determining whether there is enough specimen, whether the chromatographic process is normal, and also as an internal control standard for the reagent.

3. Read the test results in 20-30 minutes. If the sample contains the antibody to be tested, a red band will appear in the test area (T), indicating a positive result, and the antibody subtype is determined according to the colorimetric image; Only one red band appears, that is, there is a colored band in the control line area, and the detection line area is colorless and negative; regardless of whether the antibody is present in the sample, the mixture will continue to be chromatographed to the quality control area (C), the quality control area The corresponding antibody reacts directly with the colloidal gold-labeled antibody conjugate to produce a red band. Red appearing in the quality control area (C) The color strip is the standard for judging whether the chromatographic process is normal. It is also used as the internal control standard of the reagent. When there is no colored band in the control line area, it indicates that the test failed or the reagent failed, which is invalid and needs to be repeatedly tested.

Test Example 1 Determination of the minimum detection limit of the rapid typing kit of Example 1.

1. Determination of the minimum detection limit of test strip 1 for IgG1, Kappa and Lambda typing

Test strip 1 was tested by diluting the above three monoclonal antibodies with 400 ng/ml, 200 ng/ml, 100 ng/ml, 50 ng/ml, 25 ng/ml, and 12.5 ng/ml using mouse IgG1, Kappa, and Lambda mAb. The lowest detection limit of each detection index can be detected by the lowest concentration capable of detecting the color of the color. After detection, the mouse IgG1 can detect 100 ng/ml, and the Kappa and Lambda monoclonal antibodies can detect 25 ng/ml.

2. Determination of the minimum detection limit of test strip 2 for IgG1, IgG2a, IgG2b and IgG3 typing

Using the mouse IgG1, IgG2a, IgG2b and IgG3 mAb, the above four monoclonal antibodies were diluted at 400 ng/ml, 200 ng/ml, 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml to test strip 2 The detection is performed at the lowest concentration that can detect the color of the color, and the minimum detection limit of each detection index is detected. After detection, the mouse IgG1 can detect 100 ng/ml, the IgG2a monoclonal antibody can detect 50 ng/ml, and the lowest of IgG2b and IgG3. The detection limits were 25 ng/ml and 100 ng/ml, respectively;

3. Determination of the minimum detection limit of test strip 3 for IgA, IgD, IgE and IgM typing

Using the mouse IgA, IgD, IgE and IgM mAb, the above four monoclonal antibodies were diluted at 400 ng/ml, 200 ng/ml, 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml to test strip 3 The detection is performed at the lowest concentration that can detect the color of the color, and the minimum detection limit of each detection index is detected. After detection, the mouse IgA can detect 100 ng/ml, the IgD monoclonal antibody can detect 50 ng/ml, and the lowest IgE and IgM. The test was 25 ng/ml.

Test Example 2 Precision of the rapid typing kit of Example 1

1. Test strip 1 for precision testing of IgG1, Kappa and Lambda typing

Dilute to 200 ng/ml with mouse Kappa monoclonal antibody, randomly test 10 test strips, and the test strips have the same color development; indicating that the precision performance is good.

2. Test strip 2 for precision detection of IgG1, IgG2a, IgG2b and IgG3 typing

Dilute to 200 ng/ml with mouse IgG1 monoclonal antibody, and randomly test 10 test strips, and the test strips have the same color development; indicating that the precision performance is good.

3. Test strip 3 precision performance test for IgA, IgD, IgE and IgM typing

Dilute to 200 ng/ml with mouse IgM monoclonal antibody, and randomly test 10 test strips, and the test strips have the same color development; indicating that the precision performance is good.

The above-described embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims (6)

1. A mouse antibody subtype rapid typing kit, characterized in that it comprises:

   Test strip 1 for IgG1, Kappa and Lambda typing, strip 2 for IgG1, IgG2a, IgG2b and IgG3 typing, and test strips for IgA, IgD, IgE and IgM typing Article 3, the test strip 1, the test strip 2 and the test strip 3 are each composed of a sample pad, a colloidal gold bond pad, a nitrocellulose film, and an absorbent pad which are sequentially overlapped on a PVC substrate, and the colloidal gold is combined. The pad is coated with a colloidal gold-labeled anti-mouse IgG (H+L) antibody; the nitrocellulose membrane comprises a detection zone coated with anti-mouse typing antibodies and coated mice arranged in parallel and spaced apart from each other The control region of the monoclonal antibody control antibody.
2. The mouse antibody subtype rapid typing kit according to claim 1, wherein in the test strip 1, the concentration of the anti-mouse IgG1 antibody coated in the detection zone is 0.7-1.0 mg/ mL, the amount of coating in the detection zone is 1-1.2 ul / cm; the concentration of anti-mouse Kappa antibody is 0.5-0.8 mg / mL, the coating amount in the detection zone is 0.7 ul / cm; anti-mouse Lambda antibody The concentration of the test is 0.5-0.8 mg/mL, the coating amount in the detection area is 1-1.2 ul/cm; the concentration of the mouse monoclonal antibody-controlled antibody in the quality control area is 1 mg/mL, and the package in the quality control area The amount was 1.0 ul/cm.
3. The mouse antibody subtype rapid typing kit according to claim 1, wherein in the test strip 2, the detection zone is coated with an anti-mouse IgG1 antibody, an anti-mouse IgG2a antibody, and an anti-mouse The concentration of mouse IgG2b antibody and anti-mouse IgG3 antibody was 0.7-1.0 mg/mL, and the coating amount in the detection area was 1-1.2 ul/cm; the control group coated mouse monoclonal antibody control antibody The concentration was 0.7-1.0 mg/mL, and the coating amount in the quality control zone was 1-1.2 ul/cm.
4. The mouse antibody subtype rapid typing kit according to claim 1, wherein in the test strip 3, the detection zone is coated with an anti-mouse IgA antibody, an anti-mouse IgD antibody, and an anti-mouse The concentration of mouse IgE antibody and anti-mouse IgM antibody was 1.0-1.5 mg/mL, and the coating amount in the detection area was 1-1.2 ul/cm; the control group coated mouse monoclonal antibody control antibody The concentration was 0.7-1.0 mg/mL, and the coating amount in the quality control zone was 1-1.2 ul/cm.
5. The mouse antibody subtype rapid typing kit according to any one of claims 1 to 4, wherein the test strip 1, the test strip 2, and the test strip 3, the colloidal gold mark The concentration of the anti-mouse IgG (H+L) antibody was 20-30 ug/mL colloidal gold, and the amount on the colloidal gold binding pad was 1.5-2.5 μg/cm ² .
6. The method for preparing a mouse antibody subtype rapid typing kit according to any one of claims 1 to 5, comprising the steps of:

   A. Anti-mouse IgG1 antibody, anti-mouse Kappa antibody, anti-mouse Lambda antibody working solution, and mouse monoclonal antibody control antibody working solution were coated on nitrocellulose membrane, and dried at 36-38 ° C for 4-6 After hours, the nitrocellulose membrane 1 is prepared;

   B. Apply anti-mouse IgG1 antibody, anti-mouse IgG2a antibody, anti-mouse IgG2b antibody, anti-mouse IgG3 antibody working solution and mouse monoclonal antibody control antibody working solution on nitrocellulose membrane, 36-38 ° C Drying for 4-6 hours, preparing a nitrocellulose membrane 2;

   C. Apply anti-mouse IgA antibody, anti-mouse IgD antibody, anti-mouse IgE antibody, anti-mouse IgM antibody working solution and mouse monoclonal antibody control antibody working solution on nitrocellulose membrane, 36-38 ° C Drying for 4-6 hours, preparing a nitrocellulose membrane 3;

   D. Applying a colloidal gold-labeled anti-mouse IgG (H+L) antibody to the gold-labeled pad material, and drying it at 25-35% for more than 6 hours to prepare a colloidal gold binding pad;

   E. The sample pad, the colloidal gold bond pad, the nitrocellulose membrane 1, and the absorbent pad are sequentially overlapped on the PVC bottom plate to obtain a test strip 1 for IgG1, Kappa and Lambda typing detection; the sample pad and the colloidal gold are used. The bonding pad, the nitrocellulose membrane 2, and the absorbent pad are sequentially overlapped on the PVC substrate to obtain a test strip 2 for IgG1, IgG2a, IgG2b and IgG3 typing detection, a sample pad, a colloidal gold binding pad, and a nitrocellulose membrane. 3. The absorbent pad is sequentially overlapped on the PVC bottom plate to obtain the test strip 3 for IgA, IgD, IgE and IgM typing detection; the test strip 1, the test strip 2 and the test strip 3 are combined. .

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