CN117890576A - A reagent, method and application of detecting CD89-IgA immune complex - Google Patents

Abstract

The invention belongs to the field of immunodetection, and particularly discloses a reagent and a method for detecting a CD89-IgA immune complex and application thereof. The detection reagent of the CD89-IgA immune complex comprises a capture antibody and a detection secondary antibody, wherein the capture antibody comprises an anti-CD 89 antibody, and the detection secondary antibody comprises an anti-IgA antibody. The invention also provides diagnostic products for IgA nephropathy, allergic purpura, methods of detecting CD89-IgA immune complexes and uses thereof. The invention can detect CD89-IgA-IC with high specificity and high sensitivity, and combines detection of IgA immune complex combined with non-CD 89 to make the detection of the whole IgA immune complex tend to perfect, thereby improving the sensitivity of using serological immune complex as biomarker in the auxiliary diagnosis process, and having important significance for auxiliary diagnosis, disease tracking, drug administration assessment and the like of IgA related diseases.

Description

A reagent, method and application of detecting CD89-IgA immune complex

Technical Field

The present invention relates to the field of immune detection, and in particular to a reagent, method and application thereof for detecting CD89-IgA immune complex.

Background technique

During the human body's autoimmune response, human immunoglobulin A (IgA) binds to pathogens to initiate an immune response. With the participation of downstream antibodies IgG, IgM, C3 complement of the complement pathway and other molecules, IgA forms IgA-IgG, IgA-IgM, IgA-C3, IgA-IgG-IgM and other complexes, collectively referred to as IgA complexes. Abnormal clearance of IgA complexes in the immune system may lead to several autologous diseases: the deposition of IgA complexes that are not effectively cleared in the kidneys leads to common IgA nephropathy (IgANehphropathy, IgAN), which often occurs in young adults; the deposition of IgA complexes that are not effectively cleared in capillaries leads to IgA vasculitis (IgAV), also known as purpura or Henoch-Schonlein purpura (HSP), which often occurs in children. Therefore, circulatory system IgA complexes can serve as important biomarkers for these diseases.

CD89 is the Fc receptor for IgA and is a type I transmembrane glycoprotein expressed on the surface of myeloid cells (monocytes/macrophages, dendritic cells, Kupffer cells, neutrophils and eosinophils). CD89 binds to IgA1 and IgA2 molecules by interacting with the Cα2/Cα3 attachment site of IgA through its amino-terminal end. The immune system utilizes CD89 as a typical function of the IgA Fc receptor on monocytes, responsible for clearing circulating IgA pathogens or polymeric IgA complexes. CD89 has a higher affinity for polymeric IgA than for monomeric IgA, enabling phagocytes to selectively capture and clear polymeric IgA complexes. Studies have shown that, on the one hand, in addition to attaching to immune cells, some cells can secrete soluble CD89 (sCD89) in the circulatory system. These sCD89 can bind to IgA complexes before they trigger the clearance of immune cells to form CD89-IgA immune complexes, denoted as CD89-IgA-IC. For people susceptible to IgA nephropathy, CD89-IgA-IC can be deposited in the kidneys, and together with other IgA immune complexes that can bind to CD89, they are called inducing factors of IgA nephropathy; on the other hand, those IgA immune complexes captured by the transmembrane protein CD89 of immune cells (including circulating immune cells and immune cells infiltrating renal tissue) may be detached during the immune response, thereby entering the circulatory system in the form of CD89-IgA-IC, and recirculating and depositing in the kidneys, becoming inducing factors of IgA nephropathy together with other IgA immune complexes that can bind to CD89.

In the patent "Application of Reagents for Detecting IgA Immune Complexes" (patent number: 2022102944568), the reagents and applications for detecting IgA complexes using CD89 as a capture probe are described in detail. However, due to the use of CD89 as a capture probe, the scheme cannot capture and detect IgA or IgA complexes that have bound to CD89, that is, it cannot capture and detect CD89-IgA-IC. Therefore, in addition to detecting IgA or IgA complexes that are not bound by CD89, a method for detecting CD89-IgA-IC is also urgently needed as a complementary method to improve the overall IgA immune complex detection, thereby improving the sensitivity of using serological immune complexes as biomarkers in the auxiliary diagnosis of IgA nephropathy and purpura.

Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a detection reagent for CD89-IgA immune complexes. The CD89-IgA immune complex (CD89-IgA-IC) detection reagent provided by the present invention can detect CD89-IgA-IC with high specificity and high sensitivity. The use of the CD89-IgA immune complex detection reagent of the present invention, combined with the detection of non-CD89-bound IgA immune complexes, can make the overall IgA immune complex detection more perfect, thereby improving the sensitivity of using serological immune complexes as biomarkers in the auxiliary diagnosis process of diseases such as IgA nephropathy and purpura, and has a high application value.

The present invention also provides a diagnostic product for IgA nephropathy, Henoch-Schonlein purpura and/or IgA immune complex-related diseases.

The invention also provides a method for detecting CD89-IgA immune complex.

The present invention also provides the application of a CD89-IgA immune complex detection reagent and a method for detecting the CD89-IgA immune complex.

In a first aspect of the present invention, a detection reagent for CD89-IgA immune complex is provided, the reagent comprising a capture antibody and a detection secondary antibody;

Wherein, the capture antibody comprises an anti-CD89 antibody, and the detection secondary antibody comprises an anti-IgA antibody.

The capture antibody of the detection reagent of the present invention can bind to and capture CD89-IgA-IC, and the detection secondary antibody further binds to the CD89-IgA immune complex, thereby specifically detecting CD89-IgA-IC in the sample.

In some embodiments of the present invention, the secondary detection antibody is labeled with a detectable marker.

Detectable markers refer to a class of substances that have properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

Through the capture of the capture antibody and the combination of the detection secondary antibody, the CD89-IgA-IC in the sample can be specifically detected. A detectable marker is connected, coupled or labeled to the detection secondary antibody, and the marker is processed in a targeted manner, thereby characterizing the CD89-IgA-IC content information of the sample and realizing CD89-IgA-IC detection.

In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

In some embodiments of the present invention, the detectable marker is selected from an enzyme that catalyzes the color development of a substrate, a chemiluminescent reagent, a fluorescent microsphere, a radioactive isotope or a nanoparticle marker.

In some preferred embodiments of the present invention, the enzyme that catalyzes the color development of the substrate is selected from horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase or 6-phosphoglucose deoxidase.

In some more preferred embodiments of the present invention, the enzyme that catalyzes the color development of the substrate comprises horseradish peroxidase.

In some preferred embodiments of the present invention, the chemiluminescent agent is selected from acridinium esters and their derivatives, luminol and its derivatives, bipyridine ruthenium and its derivatives, lucigenin, crustacean fluorescein and its derivatives, dioxetane and its derivatives, lophanine and its derivatives or peroxyoxalate and its derivatives.

In some more preferred embodiments of the present invention, the chemiluminescent agent includes acridinium ester and its derivatives.

In some preferred embodiments of the present invention, the fluorescent substance on the fluorescent microsphere is selected from fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) or protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), prephycocyanin-chlorophyll protein (preCP), etc.).

In some embodiments of the present invention, the detection reagent also includes a calibrator.

In some preferred embodiments of the present invention, the calibrator includes at least one of a CD89-IgA complex formed by the combination of recombinant CD89 and recombinant polymeric IgA or polymeric IgA purified from commercial human serum IgA, a CD89-IgA complex formed by directional coupling of CD89 and polymeric IgA, and a natural CD89-IgA complex in commercial human serum IgA.

In some embodiments of the present invention, the samples detected by the reagent include serum, plasma, and urine.

The second aspect of the present invention provides a diagnostic product for IgA nephropathy, Henoch-Schonlein purpura and/or IgA immune complex-related diseases, the product comprising the CD89-IgA immune complex detection reagent described in the first aspect of the present invention.

The diagnostic product provided by the present invention can be used as a complementary method and in combination with the diagnostic product of the patent "Application of Reagents for Detecting IgA Immune Complexes" (Patent No.: 2022102944568), so that the overall IgA immune complex detection is perfected, and the sensitivity of using serological immune complexes as biomarkers in the auxiliary diagnosis of IgA nephropathy and purpura is improved; the detection indicators of the two diagnostic products are used for linear regression to establish a comprehensive index of IgA immune complexes, which is of great significance for auxiliary diagnosis, disease tracking, and medication evaluation of IgA nephropathy and purpura. In addition, the statistical characteristics, clinical characteristics, and treatment effects of different populations can also be established, so as to carry out personalized precision medical treatment and disease management of IgA nephropathy.

In some embodiments of the present invention, the reagents of the diagnostic product also include ELISA plates, coating buffer, calibrators, colorimetric solution, stop solution, washing solution, sample diluent, blocking buffer, sealing film and ziplock bag, positive control, negative control, and standard curve card.

The third aspect of the present invention provides a method for detecting CD89-IgA immune complexes for non-diagnostic purposes, wherein the method uses the CD89-IgA immune complex detection reagent described in the first aspect of the present invention or the diagnostic product described in the second aspect of the present invention.

In some embodiments of the invention, the method comprises the following steps:

S1, capture CD89-IgA immune complexes using capture antibodies;

S2, using the detection secondary antibody to specifically bind to the CD89-IgA immune complex captured in step S1;

S3, detecting the luminescence value or fluorescence value after processing the detectable marker marked by the secondary antibody;

S4. Calculate the content of CD89-IgA immune complex in the sample according to the standard curve.

In some preferred embodiments of the present invention, the method comprises the following steps:

S1, dissolving the capture antibody in the coating buffer, adding it to the ELISA plate for coating, washing the ELISA plate with a washing solution after coating, adding a blocking solution for blocking, adding the sample after blocking, and capturing the CD89-IgA-IC in the sample with the capture antibody;

S2, wash the ELISA plate with washing solution, add the detection secondary antibody to bind to the CD89-IgA-IC captured in step S1;

S3, wash the ELISA plate with washing solution, add color developing solution, protect from light, add stop solution after color development, and use ELISA reader, chemiluminescence detector or fluorescence detector to detect luminescence value or fluorescence value;

S4. Calculate the content of CD89-IgA immune complex in the sample according to the standard curve.

In actual use, technicians in this field can select a fully automatic detector to implement the above-mentioned method for detecting CD89-IgA-IC according to the detection conditions or actual needs. If its detection principle is consistent with the principle of the above-mentioned detection method or the detection method is obtained by technicians in this field without creative work based on the principle of the above-mentioned detection method, it belongs to the protection scope of the present invention.

The fourth aspect of the present invention provides the use of the detection reagent for CD89-IgA immune complexes described in the first aspect of the present invention in the preparation of diagnostic products for IgA nephropathy, Henoch-Schonlein purpura and/or IgA immune complex-related diseases.

Since the application provided by the present invention uses the detection reagent for the CD89-IgA immune complex described in the first aspect of the present invention, it at least has all the beneficial effects of the above detection reagent.

In some embodiments of the present invention, the product includes any one of a kit, a chip or a test paper.

In some embodiments of the present invention, the kit includes a capture antibody, a detection secondary antibody, an ELISA plate, a coating buffer, a calibrator, a colorimetric solution, a stop solution, a washing solution, a sample diluent, a blocking buffer, a sealing film and a ziplock bag, a positive control, a negative control, and a standard curve card.

In some embodiments of the present invention, the capture antibody is coated on the chip.

In some embodiments of the present invention, the test paper includes a test card and an antibody binding pad.

In some embodiments of the present invention, the antibody binding pad contains the secondary detection antibody.

In some embodiments of the present invention, the test card has a test line and a quality control line arranged in parallel, and the capture antibody is coated on the test line.

In some preferred embodiments of the present invention, the kit includes an ELISA detection kit, a chemiluminescence detection kit, a time-resolved immunochromatography detection kit, an immunocolloidal gold detection kit, and an immunoturbidimetry detection kit.

In some embodiments of the present invention, the diagnostic product further comprises a non-CD89-binding IgA complex detection reagent.

Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

FIG1 is a schematic diagram of the detection process of the ELISA detection kit for CD89-IgA-IC according to Example 1 of the present invention;

FIG. 2 is a graph showing the ROC results corresponding to the single indicators of two IgA complexes in the detection example of the present invention;

FIG. 3 is a graph showing ROC results corresponding to the linear regression model of two indicators in the detection example of the present invention.

Detailed ways

The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

If the specific conditions are not specified in the specific implementation mode, the conventional conditions or the conditions recommended by the manufacturer shall be followed. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased from the market.

In a specific embodiment, the calibrator is a CD89-IgA complex formed by the combination of recombinant CD89 (rCD9) and recombinant poly IgA (rPolyIgA), specifically recorded as rCD89-rPolyIgA. The preparation method of recombinant poly IgA refers to the patent "A recombinant poly IgA protein and its preparation method and use" (publication number: CN116789806A), wherein the preparation of the rCD89-rPolyIgA complex is as follows:

1. Use 0.01M PBS (pH 7.4) to prepare a mixed solution with rCD89 and rPolyIgA at a molar ratio of 1:1 to 1:100; preferably, the concentration of rCD89 is 10nM, and the concentration of rPolyIgA is in the range of 10nM to 10000nM. The preferred ratio is a molar ratio of rCD89 to rPolyIgA of 1:20;

2. Place the rCD89-rPolyIgA mixture at 18-25°C for 3 hours and mix thoroughly;

3. After the reaction is completed, store the samples in aliquots at -20℃ to avoid repeated freezing and thawing.

In a specific embodiment, the recombinant CD89 may be commercial CD89, and the polymeric IgA may be commercial human serum IgA. The CD89-IgA complex is obtained by binding CD89 to commercial human serum IgA.

In a specific embodiment, another calibrator is a natural CD89-IgA complex in commercial human serum IgA. The commercial human serum IgA calibrator was purchased from Shanghai Yuduo Biological, item number LY210-1mg, and the initial gradient of the calibration curve was 1600ng/mL, and the calibrator titer curve containing 1600, 800, 400, 200, 100, 50, and 25ng/mL was obtained in sequence according to the dilution multiple of 1:2.

In a specific embodiment, rabbit anti-human CD89 antibody was purchased from Beijing Sino Biological Co., Ltd. (SinoBiological), Catalog No. Cat: 10414-R002; commercial human serum IgA calibrator was purchased from Shanghai Yuduo Biotechnology, Catalog No. LY210-1mg; HRP-labeled mouse anti-human IgA antibody was purchased from Wuhan Boster Biological Technology Co., Ltd. (BosterBiological Technology); streptavidin-modified magnetic particles were purchased from: JSR Company, Catalog No. MS300, Streptavidin; colorimetric solution A and colorimetric solution B were both purchased from Beijing Sino Biological Co., Ltd. (SinoBiological), Catalog No. SEKCR01; CD89 protein standard was purchased from Beijing Sino Biological Co., Ltd.

The biotinylated rabbit anti-human CD89 antibody used in the examples was produced in-house. The preparation method is as follows:

1. Materials:

Biotin-N-succinimidyl ester, rabbit anti-human CD89 antibody, buffer solution is 0.1 mol/L sodium bicarbonate buffer solution (pH 8.0) or 0.5 mol/L boric acid buffer solution (pH 8.6), 1 mol/L NH ₄ Cl, organic solvent DMSO, glycerol.

2. Steps:

A. Dilute the protein to be biotinylated to 1 mg/mL with 0.1 mol/L sodium bicarbonate buffer (pH 8.0) or 0.5 mol/L boric acid buffer (pH 8.6). The biotinylation volume used in general laboratories is 1 to 2.5 mL.

B. Use 0.1 mol/L sodium bicarbonate buffer (pH 8.0) or 0.5 mol/L boric acid buffer (pH 8.6) alternately to fully dialyze the protein;

C. Dissolve 1 mg of biotin succinimidyl ester (NHSB) in 1 mL of DMSO;

D. Add 120 μl of NHSB solution (containing 120 μg of NHSB) to 1 mL of protein solution (containing 1 mg of protein);

E. Continue stirring at room temperature and keep warm for 0.5 to 4 hours;

F. Add 9.6 μL 1 mol/L NH ₄ Cl (1 μL for every 25 μg NHSB) and stir at room temperature for 10 to 60 minutes;

G. Dialyze the solution thoroughly against PBS at 4°C to remove free biotin;

H. Place the sample on a 1 mL molecular sieve column and slowly elute with PBS, collecting 1 mL/tube. The protein should elute in 1 to 3 mL.

I. Finally, add 0.05% sodium azide or PC300 preservative to the sample. Store the combined product at 4°C for a short term and away from light, or add 50% glycerol and store at -20°C for a long term.

In a specific embodiment, the term "antibody" includes polyclonal antibodies and monoclonal antibodies; "rabbit anti-human CD89 antibody" can also be selected from anti-human CD89 specific antibodies from other animals; "mouse anti-human IgA antibody" can also be selected from anti-human IgA antibodies from other animals; "goat anti-chicken IgY antibody" can also be selected from anti-chicken IgY antibodies from other animals.

Example 1

This embodiment provides an ELISA detection kit and detection method for human serum or plasma CD89-IgA-IC:

1. ELISA detection kit for CD89-IgA-IC, the components of the kit include:

ELISA plate: 8×12 well standard plate;

Molecular probe: rabbit anti-human CD89 antibody;

Coating buffer: 0.05 M carbonate buffer, pH 9.6;

Calibrator: Commercial human serum IgA;

Secondary antibody: HRP-labeled mouse anti-human IgA antibody;

Color development solution A: jujube phosphate + citric acid + TMB + H ₂ O ₂ ;

Color development solution B: jujube phosphate + citric acid + ABTS + H ₂ O ₂ ;

Stop solution: sulfuric acid with a mass concentration of 10%;

Washing solution: 0.15 M phosphate buffer containing 0.05% Tween-20;

Sample diluent: a solution containing 1‰ BSA prepared with washing solution;

Blocking buffer: a solution containing 5% BSA prepared with washing buffer;

Sealing film and ziplock bags.

2. The detection method of the ELISA detection kit for CD89-IgA-IC specifically comprises the following steps:

(1) Sample preparation: Use disposable test tubes to collect peripheral blood, extract serum (or plasma), and use it as the sample to be tested for subsequent testing (or it can be packaged and frozen for use at an appropriate time);

(2) Detection: The schematic diagram of the detection process is shown in Figure 1, which specifically includes the following steps:

A) Coating of molecular probes: Add 100 μL of 2 ng/mL rabbit anti-human CD89 antibody coating buffer to each well of the ELISA plate, seal the plate and store at 4°C for 16 h (overnight coating), shake off the solution in the well, wash the plate gently 3 times with washing solution, let stand for 60 s each time and then discard the washing solution, pat the ELISA plate dry on clean absorbent paper after washing, add 200 μL of blocking solution to each well, seal the plate and store for 2 h;

B) Sample addition: Pour off the blocking solution, wash once with washing solution, dilute the sample to be tested 50 times with sample diluent, and do not dilute the calibrator, negative control (sample diluent) and positive control (CD89 protein standard, 1 mg/mL). Add 100 μL of each diluted sample, calibrator, negative control and positive control to different wells of the ELISA plate, seal and store for 2 hours;

C) Adding secondary antibody: Wash three times with washing solution, add 100 μL of 2 ng/mL HRP-labeled mouse anti-human IgA antibody, seal the membrane and store for 60 min;

D) Color development: Wash three times with washing solution, take 50 μL of color development solution A and color development solution B into each well, mix well and add to the ELISA plate, and color develop for 10 min at room temperature in the dark;

E) Stop: Add 50 μL of stop solution to each well.

F) Determination of luminescence by microplate reader: After gently shaking the microplate, place it on a microplate reader to determine the test results. Use dual wavelengths to measure the OD value, with the first wavelength being 450nm and the second wavelength being 630nm;

G) Data processing: Use a four-parameter model (a quadratic polynomial model or a log-log linear model can also be used to fit the calibration curve), and then use the calibration curve to calculate the CD89-IgA-IC concentration of the sample.

Example 2

This embodiment provides an ELISA detection kit for CD89-IgA-IC in human urine and a detection method thereof: the difference between this embodiment and Example 1 is only that "collecting peripheral blood and extracting serum (plasma may also be used)" in the detection method of Example 1 is replaced by "collecting urine".

Example 3

This embodiment provides a chemiluminescent detection kit and detection method for human serum or plasma CD89-IgA-IC:

1. Chemiluminescence detection kit for CD89-IgA-IC, the components of the kit include:

Reagent M: 0.2 mg/mL, 300 nm in diameter, streptavidin-modified magnetic particles, preservative PC300;

R1 reagent: Molecular probe: 1.0 mg/mL biotinylated rabbit anti-human CD89 antibody;

R1 buffer: pH 7.5, 0.05 M phosphate buffer, preservative PC300;

Reagent R2: mouse anti-human IgA antibody labeled with acridinium ester;

R2 buffer: pH 7.5, 0.05 M phosphate buffer;

Pre-excitation solution A: a solution containing HNO ₃ with a molar concentration of 0.1 M and hydrogen peroxide with a mass concentration of 1%;

Excitation solution B: a solution containing 0.5 mM sodium hydroxide and 7 mM CTAC;

Calibrator: Commercial human serum IgA;

Washing solution: 0.15 M phosphate buffer containing 0.05% Tween-20;

Sample diluent: a solution containing 1‰ BSA prepared with washing solution;

2. The detection method of the chemiluminescence detection kit for CD89-IgA-IC specifically comprises the following steps:

(1) Sample preparation: Use disposable test tubes to collect peripheral blood, extract serum (plasma can also be used), collect using standard or vacuum tubes with separation gel (can also be packaged and frozen at -20°C and then thawed at 37°C for use), and dilute 50 times with sample diluent to obtain diluted samples;

(2) Detection: Specifically includes the following steps:

Add the components of the kit and the diluted samples to the corresponding reagent positions of the fully automatic chemiluminescence immunoassay analyzer, and the analyzer automatically performs the following steps:

A) Add 20 μL of calibrator and diluted sample into the reaction cup respectively;

B) Add 50 μL of M reagent and 50 μL of R1 reagent to each reaction cup, incubate at 37°C for 10 min, and wash the instrument three times;

C) Add 100 μL of R2 reagent to each reaction cup, incubate at 37°C for 10 min, and wash the instrument three times;

D) adding pre-excitation solution A and excitation solution B successively to cause chemiluminescence reaction;

E) Output the test results. There is a positive relationship between the total amount of CD89-IgA-IC in the sample and the value of the relative light unit (RLUs) detected by the system. Use a four-parameter model (a quadratic polynomial model or a log-log linear model can also be used to fit the calibration curve), and then use the calibration curve to calculate the CD89-IgA-IC concentration of the sample.

The fully automatic chemiluminescence immunoassay analyzer used in this embodiment can be Shine i900 or Shine i2000 fully automatic chemiluminescence immunoassay analyzer.

Example 4

This embodiment provides a chemiluminescence detection kit for CD89-IgA-IC in human urine and a detection method thereof: the difference between this embodiment and Example 3 is only that "collecting peripheral blood and extracting serum (plasma may also be used)" in the detection method of Example 3 is replaced by "collecting urine".

Example 5

This embodiment provides a human serum or plasma CD89-IgA-IC time-resolved immunochromatography (TRFIA) detection kit and detection method thereof:

1. CD89-IgA-IC time-resolved immunochromatographic detection kit, the components of the kit include:

Test card: Two parallel test lines are arranged on the test card, namely, the test line T line and the quality control line C line. The T line is coated with rabbit anti-human CD89 antibody, and the C line is coated with chicken IgY. The front end of the test card is provided with a sample hole and an antibody binding pad. The antibody binding pad is located between the sample hole and the test card. The antibody binding pad contains mouse anti-human IgA antibody labeled with fluorescent microspheres and goat anti-chicken IgY antibody coated with fluorescent microspheres.

Sample diluent: PBS buffer containing 1% BSA;

IC card (standard curve card): provides standard curve;

Quality control: PBS buffer containing different CD89-IgA-IC concentrations;

2. The detection method of the POCT fluorescent immunochromatography detection kit for CD89-IgA-IC specifically comprises the following steps:

(1) Sample preparation: Use disposable test tubes to collect venous blood, extract serum (or plasma), dilute 50 times with sample diluent to obtain a diluted sample, and store the remaining sample at -80°C;

(2) Detection: Specifically includes the following steps:

A) Take 100 μL of the diluted sample and add it to the sample well;

B) react at room temperature for 15 min;

C) placing the test card in a fluorescence detector, using the fluorescence detector to detect the test card, detecting the fluorescence values of the T line and the C line respectively, and calculating the ratio of the T line signal to the C signal;

D) Calculate the concentration of CD89-IgA-IC in the sample according to the standard curve provided by the IC card.

Example 6

This embodiment provides a chemiluminescence detection kit for CD89-IgA-IC in human urine and a detection method thereof: the difference between this embodiment and Example 5 is only that "collecting peripheral blood and extracting serum (plasma may also be used)" in the detection method of Example 5 is replaced by "collecting urine".

Example 7

This embodiment provides a method for detecting all IgA immune complexes in a sample:

The two IgA immune complexes respectively use the patent "Application of Reagents for Detecting IgA Immune Complexes" (Patent No.: 2022102944568) based on the method of using CD89 as a capture molecule to detect non-CD89-bound IgA complexes, and use the scheme of Example 1 of the present invention (Example 2, Example 3, Example 4, Example 5 or Example 6 can also be used) based on the specific antibody against human CD89 as a capture molecule to detect CD89-IgA-IC immune complexes.

After the above tests, the samples were divided into four types according to the test results:

Non-CD89-bound IgA complex (+) & CD89-IgA-IC (+);

Non-CD89-bound IgA complex (+) & CD89-IgA-IC (-);

Non-CD89-bound IgA complex (-) & CD89-IgA-IC (+);

Non-CD89-binding IgA complex (-) & CD89-IgA-IC (-);

Among them, + represents positive and - represents negative.

Through the detection of all IgA immune complexes in this embodiment, the indicators of the two detection schemes can be used for linear regression to establish a comprehensive index of IgA immune complexes, which is of great significance for auxiliary diagnosis, disease tracking, and medication evaluation of IgA nephropathy and purpura. In addition, statistical characteristics, clinical characteristics, treatment effects, etc. of different populations can also be established, so as to carry out personalized precision medicine and disease management of IgA nephropathy.

Test example

In this test example, the kit and detection method disclosed in the patent "Application of Reagents for Detecting IgA Immune Complexes" (Patent No.: 2022102944568) are used to detect non-CD89-bound IgA complexes, and the ELISA detection kit and detection method of CD89-IgA-IC of the present invention are used to detect CD89-IgA-IC, so that all IgA immune complexes in the sample can be detected, thereby improving the clinical effect of a single test of the two, and then more accurately used to assist in the diagnosis of diseases such as IgA nephropathy (IgAN) or HSP.

(1) Detection method:

This test case used 9 positive samples, including 7 IgA nephropathy patients diagnosed by renal puncture and 2 plasma samples of purpura patients; 18 negative samples were also used, including 4 urine samples of diabetic nephropathy (DN), 5 membranous nephropathy (MN) and 6 minimal change nephropathy (MCD) patients. The samples were captured using CD89 and anti-human CD89 antibodies, respectively, and the corresponding subsequent detection steps were performed to obtain the titer of the non-CD89-bound IgA complex and the optical density (OD) of CD89-IgA-IC. Since the calibrator of the ELISA detection kit of CD89-IgA-IC in the scheme of the present invention is still being improved, the measured luminescence value, i.e., the OD value, was directly used for analysis. Generally speaking, the model for converting the OD value to the titer through the calibrator is formed by a logarithmic linear model, so two linear regression methods are used here: one is to directly perform linear regression using two indicators; the other is to first take the logarithm of the titer of the non-CD89-bound IgA immune complex, and then perform linear regression with the latter OD value.

(2) Analysis of test results:

Receiver operating characteristic (ROC) curves were used for analysis and comparison. The ROC results of the two IgA immune complexes and their linear combination for the diagnosis of IgA nephropathy and purpura are shown in Table 1, where auc represents the area under the curve; fpr represents the false positive rate; tpr represents the true positive rate; cutoff represents the positive judgment value; and acc represents the accuracy.

Table 1 ROC results of two IgA immune complexes and their linear combination for the diagnosis of IgA nephropathy and purpura

index auc fpr tpr cutoff acc CD89-IgA-IC (OD value) 0.8 0.22 0.78 0.5 0.78 IgA immune activity indicator (non-CD89 binding) 0.81 0.33 0.78 7.51 0.7 Linear model: OD+IgA immune activity index (non-CD89 connection) 0.83 0.28 0.89 0.24 0.78 Linear model: OD + log (IgA immune activity index (non-CD89 connection)) 0.82 0.33 1 0.24 0.74

The parameters of the two bivariate linear regression models are shown in Table 2, where Intercept represents the model constant; OD_avg represents the mean of repeated OD of CD89-IgA-IC samples; IgAN_ImmunoIndex_avg represents the mean of detection of CD89-captured IgA complex samples retested; IgAN_ImmunoIndex_log represents the logarithm (log2) of detection of CD89-captured IgA complex samples retested. In the linear regression model parameters: estimate represents the weight of the linear model, stderr is the standard deviation, tv is the t-statistic, pv is the corresponding p-value, and rsq is the R square of the model.

Table 2 Parameters of two bivariate linear regression models

The ROCs corresponding to the two IgA complex single indicators are plotted as shown in Figure 2. The ROCs corresponding to the two bivariate linear regression models are shown in Figure 3. The OD+IgA immune activity indicator linear model is: Score=0.22×CD89_IgA_OD+0.04×NoCD89_IgA_IC-0.15 (left figure in Figure 3), and the OD+log ₂ (IgA immune activity indicator) linear model is: Score=0.33×CD89_IgA_OD+0.22×log ₂ (NoCD89_IgA_IC)-0.75 (right figure in Figure 3), where NoCD89_IgA_IC represents the titer of non-CD89-bound IgA immune complexes.

From the above results, it can be seen that CD89-IgA-IC has a similar specificity (1-fpr) to the non-CD89-bound IgA immune activity index: 78% vs 67%. The relatively better CD89-IgA-IC here may be due to the bias in the selection of test samples; the two linear models combining CD89-IgA-IC and non-CD89-bound IgA immune activity index have better sensitivity than each single index. The two linear models: OD+IgA immune activity index and OD+log (IgA immune activity index), the true positive rate of the former, that is, the sensitivity is 89%, and the latter is 100%, while the sensitivity of a single index is only 78%.

As for the detection method of CD89-IgA-IC in this scheme, the OD value of CD89-IgA-IC in the IgA nephropathy population and other non-IgA nephropathy populations was compared by ROC (Table 1, first row), auc was 0.8, specificity (1-false positive rate) was 78%, sensitivity was 78%, and accuracy was 78%. In contrast, the kit and detection method disclosed in the patent "Application of Reagents for Detecting IgA Immune Complexes" (Patent No.: 2022102944568) were used to detect non-CD89-bound IgA complex indicators. The ROC results (Table 1, second row) showed that auc was 0.81, specificity (1-false positive rate) was 67%, sensitivity was 78%, and accuracy was 70%. Therefore, the detection of CD89-IgA-IC in this scheme has similar clinical utility and detection effect as the kit and detection method disclosed in the patent "Application of Reagents for Detecting IgA Immune Complexes" (Patent No.: 2022102944568) for detecting non-CD89-bound IgA complexes. In addition, the linear combination of the two increases the sensitivity to 89% (the third row of Table 1) while ensuring specificity (72%), while the linear combination of the former OD and the logarithm of the latter increases the sensitivity to 100% (the fourth row of Table 1) while ensuring specificity (63%). This shows that the method of detecting CD89-IgA-IC in this scheme has independent and similar detection effects and clinical value on the one hand, and can be used as a complementary method for the detection of non-CD89-bound IgA complexes with the kit and detection method disclosed in the patent "Application of Reagents for Detecting IgA Immune Complexes" (Patent No.: 2022102944568), covering those cases with CD89-IgA-IC as the main source of immune complex pathogenicity of IgA nephropathy, thereby improving sensitivity and comprehensiveness of detection.

Since CD89-IgA-IC and non-CD89 bound IgA immune complexes may be complementary to the patient population, different IgA nephropathy patients, or the same patient at different stages of the disease, may show different expression levels of the two pathogenic IgA complexes. If a single test is performed, for patients with only one IgA complex expressed at a high level, the other test method will result in a false negative. Therefore, simultaneous detection of both pathogenic IgA complexes can improve detection sensitivity.

In summary, the CD89-IgA-IC detection kit and detection method of the present invention have the advantages of high specificity and high sensitivity for the detection of CD89-IgA-IC. Combined with the detection of non-CD89-bound IgA immune complexes, the overall IgA immune complex detection can be improved, thereby improving the sensitivity of using serological immune complexes as biomarkers in the auxiliary diagnosis process of diseases such as IgA nephropathy and purpura, and has a high application value.

The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims (10)

1. A detection reagent for CD89-IgA immune complex, characterized in that the reagent comprises a capture antibody and a detection secondary antibody; Wherein, the capture antibody comprises an anti-CD89 antibody, and the detection secondary antibody comprises an anti-IgA antibody. 2. The CD89-IgA immune complex detection reagent according to claim 1, characterized in that the secondary detection antibody is labeled with a detectable marker. 3. The detection reagent for CD89-IgA immune complex according to claim 2, characterized in that the detectable marker is selected from an enzyme that catalyzes the color development of a substrate, a chemiluminescent reagent, a fluorescent microsphere, a radioactive isotope or a nanoparticle marker; Preferably, the enzyme catalyzing the color development of the substrate is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase or 6-phosphoglucose deoxidase; Preferably, the chemiluminescent agent is selected from acridinium esters and their derivatives, luminol and their derivatives, bipyridyl ruthenium and their derivatives, lucigenin, crustacean fluorescein and their derivatives, dioxetane and their derivatives, lophanine and their derivatives, or peroxyoxalate and their derivatives; Preferably, the fluorescent substance on the fluorescent microspheres is selected from fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, or protein dyes and their derivatives. 4. The detection reagent for CD89-IgA immune complex according to claim 1, characterized in that the detection reagent also includes a calibrator; Preferably, the calibrator comprises at least one of a CD89-IgA complex formed by the combination of recombinant CD89 and recombinant polymeric IgA or polymeric IgA purified from commercial human serum IgA, a CD89-IgA complex formed by directional coupling of CD89 and polymeric IgA, and a natural CD89-IgA complex in commercial human serum IgA. 5. A diagnostic product for IgA nephropathy, Henoch-Schonlein purpura and/or IgA immune complex-related diseases, characterized in that the product comprises a detection reagent for the CD89-IgA immune complex according to any one of claims 1 to 4. 6. A method for detecting CD89-IgA immune complexes for non-diagnostic purposes, characterized in that the method uses the CD89-IgA immune complex detection reagent according to any one of claims 1 to 4 or the diagnostic product according to claim 5. 7. The method for detecting CD89-IgA immune complex according to claim 6, characterized in that the method comprises the following steps: S1, capture CD89-IgA immune complexes using capture antibodies; S2, using the detection secondary antibody to specifically bind to the CD89-IgA immune complex captured in step S1; S3, detecting the luminescence value or fluorescence value after processing the detectable marker marked by the secondary antibody; S4. Calculate the content of CD89-IgA immune complex in the sample according to the standard curve. 8. Use of the CD89-IgA immune complex detection reagent according to any one of claims 1 to 4 in the preparation of diagnostic products for IgA nephropathy, Henoch-Schonlein purpura and/or IgA immune complex-related diseases. 9. The use according to claim 8, characterized in that the diagnostic product comprises any one of a kit, a chip or a test paper. 10. The use according to claim 8, characterized in that the diagnostic product further comprises a non-CD89-binding IgA complex detection reagent.