CN111103281A

CN111103281A - Detection method of insulin-resistant autoantibody

Info

Publication number: CN111103281A
Application number: CN201911363086.3A
Authority: CN
Inventors: 李彬
Original assignee: Hebei Bio High Technology Deve Co ltd
Current assignee: Hebei Bio High Technology Deve Co ltd
Priority date: 2019-12-26
Filing date: 2019-12-26
Publication date: 2020-05-05

Abstract

The invention relates to the field of immunodetection, and discloses a method for detecting an anti-insulin autoantibody, which comprises the following steps: taking a human serum sample to be detected, adding the human serum sample into the sample pad of the test strip, and judging the result according to the following conditions: the color development is negative only on the quality control line; the color development is positive on both the quality control line and the detection line; and the shade of the color line is directly proportional to the antibody titer, i.e., the higher the antibody titer, the deeper the color line. The detection method of the insulin resistance autoantibody is simple and convenient to operate; the specificity is strong, and the cross reaction with other autoantibodies is avoided; sensitivity and batch-to-batch precision detection, stable property of result display, good reproducibility and good application prospect.

Description

Detection method of insulin-resistant autoantibody

Technical Field

The invention relates to the field of immunodetection, in particular to a method for detecting an anti-insulin autoantibody.

Background

Diabetes is a common disease caused by the interaction of genetic and environmental factors, hyperglycemia is taken as a main clinical sign, common symptoms comprise polydipsia, diuresis, polyphagia, emaciation and the like, and the diabetes is divided into type 1 diabetes and type 2 diabetes.

Type 1 diabetes has some familial aggregation. Research reports that parents have a history of diabetes, and the incidence rate of the children type 1 diabetes is 4-11%; the incidence of family aggregation of type 1 diabetes between brothers and sisters is 6-11%; the concordance of the onset of type 1 diabetes in the same ovum is less than 50%.

Human Leukocyte Antigen (HLA) genes are located on the 6 th chromosome short arm, and form a group of closely linked genes, and HLA is coded by I, II and III 3 genes. The class I gene region comprises HLA-A, HLA-B, HLA-C and other genes with unknown functions and pseudogenes, and antigen molecules coded by the gene are present on the surfaces of all nucleated cells and are responsible for presenting foreign antigens to T lymphocytes of CD 8; the II-class gene region mainly comprises 3 subregions of HLA-DR, HLA-DQ and HLA-DP, respectively encodes DR, DQ and DP antigens, is present on the surfaces of mature B lymphocytes and antigen presenting cells and is responsible for presenting the antigens to CD4 cells; the class III gene region encodes some soluble proteins including some complement components, such as C2C4A, C4B, Tumor Necrosis Factor (TNF), and Heat Shock Protein (HSP). HLA is restricted by Major Histocompatibility Complex (MHC), is involved in the interaction of T lymphocytes in recognizing antigens and other immune cells, and in the formation and maintenance of self tolerance, and plays an important role in recognizing self and heterosis, inducing and regulating immune responses, and the like. As can be seen, HLA is associated with the development of many autoimmune diseases, including type 1 diabetes.

Type 1 diabetes often occurs in association with or following certain infections. Common infection is with mumps virus, rubella virus, cytomegalovirus, measles virus, influenza virus, encephalitis virus, poliovirus, coxsackie virus, Epstein-Barr virus, and the like.

It is well established that type 1 diabetes is caused by immune-mediated islet β cell destruction, and it has been demonstrated that during the development of type 1 diabetes, a variety of autoantibodies (IAAs) against islet β cells can be detected, the positive rate of insulin autoantibodies IAA is about 50% in diagnosed type 1 diabetes patients.

Disclosure of Invention

The invention aims to provide a method for detecting an anti-insulin autoantibody, which comprises the steps of adding a human serum sample to be detected into a sample pad of the test strip, and judging the result according to the following conditions:

negative: only the color is developed on the quality control line, and the color is not developed on the detection line;

positive: color development is carried out on the quality control line and the detection line;

the test strip comprises a PVC base plate, a sample pad, a combination release pad, a nitrocellulose membrane and an absorption pad, wherein the sample pad, the combination release pad, the nitrocellulose membrane and the absorption pad are sequentially lapped on the PVC base plate, the combination release pad is coated with anti-human IgG containing colloidal gold marks, and the nitrocellulose membrane is provided with a quality control line coated by a second antibody of the anti-human IgG and a detection line coated by insulin antigen.

Preferably, the anti-human IgG is of murine origin. More preferably, the second anti-human IgG antibody is a goat anti-mouse secondary antibody.

The invention applies the antigen-antibody reaction and the immunochromatography one-step principle, namely, an anti-insulin autoantibody (IAA) in Human serum is combined with anti-Human IgG marked by colloidal gold in a detection reagent and then combined with insulin antigen fixed on a nitrocellulose membrane, so that a detection color band is formed by deposition, and the detection color band is displayed according to a Test-line (T line) in the middle section of a Test paper and a Control-line (C line) above the Test-line, and is used as a rapid detection method for the auxiliary diagnosis of insulin-dependent diabetes (type 1 diabetes).

The detection method of the insulin resistance autoantibody is simple and convenient to operate; the specificity is strong, and the cross reaction with other autoantibodies is avoided; sensitivity and batch-to-batch precision detection, and stable property and good reproducibility of result display.

Description of the drawings:

FIG. 1 is a schematic structural diagram of the test strip of the present invention.

1-sample pad

2-binding release pad containing colloidal gold labeled anti-human IgG

3-nitrocellulose membrane coated with insulin antigen and goat anti-mouse IgG antibody

4-absorbent pad

5-PVC soleplate

Detailed Description

The invention discloses a detection method of an anti-insulin autoantibody, which can be realized by appropriately improving process parameters by a person skilled in the art with reference to the content in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Example 1:

1) intermediate solution preparation

1. Preparation of 0.2M Potassium carbonate solution

2. Preparation of 1% sodium citrate solution

3. Preparation of 1% chloroauric acid solution

4. Preparation of 10% bovine serum albumin solution

5. Method for preparing 0.1Mtris-HCL solution with pH of 8.0-8.5

6. Method for preparing 0.1M phosphate buffer solution

1. Preparation of colloidal gold solution

1.1 preparation of the device:

electronic temperature-control magnetic stirring electric heating sleeve pipette gun measuring cylinder

1.2 formula:

1ml of 1% chloroauric acid solution was added to 100ml of water, and 1.0-1.8ml of 1% sodium citrate solution was added after boiling.

1.3, operation steps:

1.3.1 measuring and preparing laboratory tertiary water with a certain volume;

1.3.2 completely dissolving 1% chloroauric acid solution in a certain proportion in laboratory tertiary water, uniformly mixing and heating to boil;

1.3.3 adding 1% citric acid solution, stirring to change the color of the solution until the color does not change, stopping stirring and heating, and naturally cooling to room temperature.

2. Mouse anti-human IgG antibody labeling and centrifugation

2.1 preparation of the device:

liquid-transfering gun, flask, electronic temp. -controlled magnetic stirring electric heating jacket and centrifugal machine

2.2 formula:

adding 2-6 microliters of 0.2M potassium carbonate solution into 1 milliliter of colloidal gold solution, shaking and uniformly mixing, and adding 10-15 microliters of 1.0mg/ml potassium carbonate solution

The mouse anti-human IgG antibody is mixed evenly, 10% BSA 100ul is added into 1ml colloidal gold solution of the labeled protein as a stabilizing agent, and the solution is dissolved by one tenth of the volume before redissolution after centrifugation.

2.3, operation steps:

2.3.1 taking colloidal gold solution which is qualified in inspection.

2.3.2 adding a calculated and determined amount of 0.2M potassium carbonate solution under stirring, then adding 1.0mg/ml of mouse anti-human IgG antibody and uniformly mixing; rest for 20 minutes.

2.3.3 adding 10% bovine serum albumin solution according to the calculated amount, uniformly mixing and standing for 20 minutes;

2.3.4 then put into a centrifuge tube, centrifuge at 10000rpm for 10min, and discard the supernatant.

2.3.5 the precipitate was dissolved with a calculated amount of tris-HCL pH8.0-8.5 redissolution.

3. IAA metal spraying

3.1 required appliances:

three-dimensional plane film-drawing instrument reagent bottle liquid-transferring gun

3.2 Process conditions:

drying is carried out in a drying box with the relative humidity of less than or equal to 40 percent at the temperature of 35-40 ℃.

3.3, operation steps:

3.3.1 put the gold spraying pipette of the three-dimensional plane film scribing instrument into the colloidal gold solution of the labeled protein.

3.3.2 spreading the glass fiber on the instrument, setting the spraying amount of 8-12 mul/cm, and spraying gold.

3.3.3 placing the sprayed glass fiber on a test tube rack, placing the test tube rack in a drying box, drying for 5-8 hours at the temperature of 35-40 ℃ and the relative humidity of less than or equal to 40%.

4. IAA coating

4.1 required appliances:

continuous film-drawing instrument reagent bottle liquid-transferring gun

4.2 Process conditions

4.3, operation steps:

4.3.1 pipette amounts of insulin antigen and goat anti-mouse secondary antibody calculated according to the production amount were measured in reagent bottles, respectively.

4.3.2 insulin antigen was diluted to 0.8-2.0mg/ml and goat anti-mouse secondary antibody was diluted to 0.8-2.0mg/ml with 0.1M phosphate buffer, respectively.

4.3.3 tear off the end lining paper of PVC plate, align underline and paste the nitrocellulose membrane of the equivalent width, then lay it on the continuous film-drawing instrument and draw line, set the concentration of drawing line to be 0.8-1.5 μ l/cm, the first pipeline is coated with insulin antigen, the second pipeline is coated with goat anti-mouse second antibody.

4.3.4 placing the sprayed PVC board on a test tube rack, placing the test tube rack in a drying oven, drying for 5-8 hours at 35-40 ℃ and with the relative humidity less than or equal to 40%.

5. IAA large panel assembly

5.1 required instruments:

paper cutter

5.2, operation steps:

6.2.11 the large test paper is prepared by sticking adhesive sticker to the hand-held end and sticking adhesive sticker marked with MAX line to the sample-adding end, and cutting into test paper strips with specified width. Before cutting into strips in batch, 10 strips are cut, and 3 strips are randomly spot-checked and measured by production personnel, wherein the width error of a normal test strip is not more than +/-0.04 mm. Card type: and (4) placing the test paper into a clamping and molding part, and pressing the shell by using a shell pressing machine.

6.2.2 bagging

6.2.2.1 each test paper is put into a drying agent and one test paper strip in an aluminum plastic bag. The card type adds the straw one.

6.2.2.2 the aluminum plastic bag is sealed by heat sealing on a multifunctional automatic film sealing machine at the temperature of 180-240 ℃.

Example 2: quality control standard

1) Raw material quality standard

1.1 appearance

The antibody is clear and transparent liquid observed by naked eyes, and does not contain foreign matters or turbid or non-scattering sediment.

1.2 purity and molecular weight:

the molecular weight of the mouse anti-human IgG antibody protein is mainly concentrated on about 25KD, and the purity is more than or equal to 95 percent

The purity of the goat anti-mouse IgG secondary antibody is more than or equal to 90 percent

1.3 protein concentration

The content of insulin antigen is more than or equal to 27U/ml

The mouse anti-human IgG antibody is more than or equal to 1.0mg/ml, and the goat anti-mouse IgG secondary antibody is more than or equal to 1.0 mg/ml.

1.4 titer:

murine anti-human IgG antibody titers 1: 64

Goat anti-mouse IgG secondary antibody 1: 64

1.5 biological Activity

The general method of preparing colloidal gold is to coat the detection line with insulin antigen, coat the quality control line with goat anti-mouse IgG antibody, label the colloidal gold with mouse anti-human IgG antibody, prepare a reagent sample, and meet the requirements of detection sensitivity, specificity and stability.

2) Standard of acceptance of auxiliary materials

1 nitrocellulose membrane

Appearance: the membrane strip is flat, has no bending wrinkles and has no stains on the surface.

Thickness: 215 +/-20 um.

Capillary migration rate: the chromatography time of water per 4cm was 135. + -.30 s.

2. Glass cellulose membrane

Appearance: the surface is smooth, no stain is generated, and the fiber structure is uniform and compact.

Thickness: and (3) gold pad gluing: 0.5 +/-0.05 mm.

Liquid moving speed: the moving speed is more than or equal to 5mm multiplied by 70mm/min

3. Other auxiliary materials

PVC board, absorbent filter paper, MAX line label, color leather label, etc. are purchased from qualified manufacturers, and corresponding acceptance standard is established.

1. Quality standard of colloidal gold solution

Appearance: rose transparent liquid.

Absorbance: the absorbance of A395/A405 is more than or equal to 1.

2. Marking, centrifugation quality standards

Appearance after gold spraying and drying: smooth and clean, no damage and no pollution. The original rose color of the gold-labeled mouse anti-human IgG antibody is kept, and the color change cannot occur.

And (3) performance detection:

and (3) sensitivity detection: blank control solution: the phosphate buffer solution containing bovine serum albumin was not detected to develop color. Detecting a sample solution containing 50mU/mLIAA standard: only the quality control line is colored, and the detection line is not colored. Detecting a sample solution containing 150mU/mLIAA standard: the quality control line and the detection line are colored. Detecting a sample solution containing 300mU/mLIAA standard: the quality control line and the detection line are colored.

And (3) specific detection: does not cross-react with other autoantibodies.

And (3) detecting the precision in the batch: detecting a sample solution containing 50mU/mLIAA standard: the reaction results are consistent, and the color development is uniform. Detecting a sample solution containing 150mU/mLIAA standard: the reaction results are consistent, and the color development is uniform. Detecting a sample solution containing 300mU/mLIAA standard: the reaction results are consistent, and the color development is uniform.

3. Quality standard of semi-finished product

Appearance: the adhesive is flat and clean, has no damage and pollution, has complete components, correct position, firm adhesion and no falling.

And (3) performance detection:

And (3) specific detection: does not cross-react with other autoantibodies.

4. Quality standard of finished product

1.2 absorbance: 1ml of the sample to be detected is taken by a sample injector and put into a cuvette, the absorption value at the position of 395nm and 405nm of the wavelength is measured by a spectrophotometer, and the ratio of A395 to A405 is calculated to meet the requirement.

Example 3:

1.2 functional assays

And sticking the qualified large plate with the scratched film and the colloidal gold pad to be detected to prepare a test strip for functional detection.

1.2.1 sensitivity detection

1.2.1.1 reagents: blank control solution: 0.01mol/L Phosphate Buffer Solution (PBS) containing bovine serum albumin (BSA for short, the same applies below), pH7.2-pH7.4.

And (3) measuring a sample solution: sample solutions containing 50mU/mL, 150mU/mL, and 300mU/mL IAA standards.

The preparation method comprises the step of diluting IAA standard substance with BSA-containing phosphate buffer solution to obtain 50mU/mL, 150mU/mL and 300mU/mL respectively.

1.2.1.2 instruments: stopwatch

1.2.1.3 operating procedure:

1.2.1.3.1 extracting 3 products of the same batch, detecting blank control liquid, taking out the strip-shaped sample pad after one end is immersed in the sample liquid for at least 20 seconds, adding 3 drops of sample liquid into the sample adding hole if the strip-shaped sample pad is in a card shape, starting timing, reading the result within 10-15 minutes, judging the product to be qualified if the quality control line is uniformly colored and the detection line is not colored, otherwise judging the product to be unqualified;

1.2.1.3.250 mU/mL sample liquid detection amount and detection method are the same, the results of 3 product detection lines only develop color on the quality control line, the detection line does not develop color, the product is judged to be qualified, otherwise, the product is judged to be unqualified;

1.2.1.3.3150 mU/mL sample liquid detection amount and detection method are the same as those of 1.2.1.3.1, and if the result quality control line and the detection line of 3 product detection lines are colored, the product is judged to be qualified, otherwise, the product is judged to be unqualified;

1.2.1.3.3300 mU/mL sample liquid detection amount and detection method are the same as those of 1.2.1.3.1, and the result homogenization control line and detection line of 3 product detection lines are colored, so that the product is judged to be qualified, otherwise, the product is judged to be unqualified.

1.2.2 specific assay

Does not cross-react with other autoantibodies.

1.2.3 determination of in-batch precision

1.2.3.1 reagents

The preparation method comprises the steps of preparing 50mU/mL, 150mU/mL and 300mU/mL IAA samples by using a phosphate buffer solution containing BSA

1.2.3.2 instruments: stopwatch

1.2.3.3 working procedure

1.2.3.3.1 extracting 10 products of the same batch, detecting 50mU/mL IAA standard sample liquid, immersing one end of the strip-shaped sample pad into the sample liquid for at least 20 seconds, taking out, adding 3 drops of sample liquid into the sample adding hole if the sample pad is card-shaped, laying flat on a plane, starting timing, reading the result within 10-15 minutes, wherein the reaction result should be consistent, and the color should be uniform.

1.2.3.3.2150 mU/mL IAA standards were assayed as above.

1.2.3.3.3300 mU/mL IAA standards were assayed as above.

3) Method and control for inspecting gold pad

1.1 appearance: the appearance of the product is required to be observed by naked eyes.

1.2 functional assays

1.2.1 sensitivity detection

1.2.1.2 instruments: stopwatch

1.2.1.3 operating procedure:

1.2.1.3.3300 mU/mL sample liquid detection amount and detection method are the same as those of 1.2.1.3.1, and the result quality control line and detection line of 3 product detection lines are colored, so the product is judged to be qualified, otherwise, the product is judged to be unqualified.

1.2.2 specific assay

Does not cross-react with other autoantibodies.

1.2.3 determination of in-batch precision

1.2.3.1 reagents

1.2.3.2 instruments: stopwatch

1.2.3.3 working procedure

1.2.3.3.1 extracting 10 products of the same batch, detecting sample liquid of 50mU/mL IAA standard, immersing one end of the strip-shaped sample pad into the sample liquid for at least 20 seconds, taking out, adding 3 drops of sample liquid into the sample adding hole if the sample pad is card-shaped, laying flat on a plane, starting timing, reading the result within 10-15 minutes, and obtaining consistent reaction result and uniform color development.

1.2.3.3.2150 mU/mL IAA standards were assayed as above.

1.2.3.3.3300 mU/mL IAA standards were assayed as above.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.

Claims

1. A method for detecting an anti-insulin autoantibody, comprising the steps of:

taking a human serum sample to be detected, adding the human serum sample into the sample pad of the test strip, and judging the result according to the following conditions:

2. The detection method according to claim 1, wherein the anti-human IgG is of murine origin.

3. The detection method according to claim 2, wherein the second anti-human IgG antibody is a goat anti-mouse secondary antibody.