A p200 pemphigus diagnostic kit
By using immunoblotting with human dermal extract and LMγ1 recombinant protein strips, combined with enzyme-labeled antibodies, the problem of misdiagnosis and missed diagnosis of P200 pemphigus was solved, and the simultaneous detection of IgG and IgA antibodies was achieved, improving the comprehensiveness and accuracy of diagnosis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- DALIAN UNIV
- Filing Date
- 2022-12-14
- Publication Date
- 2026-06-19
AI Technical Summary
The lack of effective methods for detecting LMγ1 autoantibodies in existing technologies has led to serious misdiagnosis and missed diagnosis of P200 pemphigoid, making it difficult to make a diagnosis based on clinical manifestations. Furthermore, most laboratories only perform IgG antibody testing while ignoring IgA antibodies.
An immunoblotting method combining human dermal extract protein strips and multiple LMγ1 recombinant protein strips, along with enzyme-labeled antibodies, can simultaneously detect IgG and IgA type LMγ1 autoantibodies, simplifying the detection process and improving diagnostic accuracy.
This approach achieves comprehensive and accurate serological diagnosis of P200 pemphigus, reduces the rate of misdiagnosis and missed diagnosis, and ensures the correct treatment plan.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of reagent detection technology, specifically relating to a P200 type pemphigus diagnostic kit. Background Technology
[0002] P200 pemphigoid is an important subtype of pemphigoid, a complex, difficult-to-diagnose, and difficult-to-treat autoimmune disease. Clinically, it primarily manifests as skin lesions, with some patients also experiencing mucosal lesions. P200 pemphigoid was first reported in 1996, and it was discovered that patients with this condition all possessed an autoantibody in their serum that recognizes a 200 kDa protein (P200), hence the name P200 pemphigoid. [1,2] In 2009, this 200kDa protein was identified as lamininγ1 (LMγ1), and P200 pemphigoid was thus also called anti-LMγ1 pemphigoid. [3] P200 pemphigoid cannot be diagnosed based on clinical manifestations alone; a definitive diagnosis requires the detection of LMγ1 autoantibodies in the patient's serum.
[0003] Currently, there are no clinically available test kits for LMγ1 autoantibodies. Detection primarily relies on laboratory immunoblotting using human dermal extracts and / or various recombinant LMγ1 proteins. These methods are mastered by only a very few laboratories both internationally and domestically. Furthermore, although some patients only have IgA type LMγ1 autoantibodies, most laboratories only perform IgG antibody testing. Therefore, misdiagnosis and missed diagnosis of P200 pemphigoid are very serious problems, leading to inadequate treatment. Summary of the Invention
[0004] To overcome the shortcomings of existing technologies, this invention provides a diagnostic kit for pemphigus type P200. Based on years of practical experience, this invention has developed a detection kit for pemphigus type P200, which uses immunoblotting with two types of antigens to simultaneously detect anti-LMγ1 autoantibodies of IgG and IgA. Therefore, it can solve the problem of the difficulty in diagnosing pemphigus type P200 in clinical practice both domestically and internationally in one go.
[0005] The above-mentioned objective of this invention is achieved through the following technical solution: a P200 pemphigus diagnostic kit, the kit comprising human dermal extract protein strips, LM521 / LM421 / LM411 / LM111 recombinant protein strips, a positive control, a negative control, enzyme-labeled IgG antibody, enzyme-labeled IgA antibody, reaction buffer, washing buffer, and enzyme matrix solution; wherein the positive control component is: LMγ1 IgG positive antibody serum was diluted in reaction buffer containing 0.05% sodium azide, with a concentration of 1 ml. The negative control consisted of: normal human serum diluted in reaction buffer containing 0.05% sodium azide, with a concentration of 1 ml; enzyme-labeled IgG antibody was horseradish peroxidase-labeled rabbit anti-human IgG antibody, with a concentration of 3.0 ml; enzyme-labeled IgA antibody was horseradish peroxidase-labeled rabbit anti-human IgA antibody, with a concentration of 1.0 ml; the reaction buffer was 1xPBS containing 0.05% v / v Tween 20 and 0.05% w / v NaN3, with a concentration of 2.0 ml; the washing buffer was 10xPBS containing 0.5% v / v Tween 20, with a concentration of 10 ml; and the enzyme matrix solution was a mixed solution of TMB and H2O2, with a concentration of 5.0 ml, wherein TMB was dissolved in anhydrous ethanol at a concentration of 2.5 mg / ml and H2O2 concentration was 0.03% by volume.
[0006] Furthermore, the LM521 recombinant protein, LM421 recombinant protein, LM411 recombinant protein, and LM111 recombinant protein used in the LM521 / LM421 / LM411 / LM111 recombinant protein strips were purchased from Biolamina, with catalog numbers LN521-05, LN421-02, LN411-02, and LN111-0501, respectively.
[0007] Furthermore, the preparation method of the LM521 / LM421 / LM411 / LM111 recombinant protein strips is as follows: Take 0.5 μg each of LM521 recombinant protein, LM421 recombinant protein, LM411 recombinant protein and LM111 recombinant protein and mix with 300 μl of 4-fold concentrated protein loading buffer, boil at 100 degrees for 5 minutes, stand on ice for 1 minute, and then collect the recombinant protein sample to the bottom of the tube. Using a 7.5% PAGE gel (one-well gel), all recombinant protein samples were added and electrophoresed at 20 mA for 3 hours. After electrophoresis, the protein was transferred using an iBlot2 NC regular transfer stack (Thermofisher) on an iBlot2 device (Thermofisher) for 7 minutes. The NC membrane containing the transferred protein was removed and longitudinally cut into 5 mm wide protein strips. The protein strips were blocked at room temperature for 1 hour with 5% skim milk (diluted with reaction buffer), then washed three times with washing buffer (3 minutes each time), and dried at room temperature. These are the protein strips used in this invention. The 4x concentrated protein loading buffer consists of 2.4 wt% Tris, 8 wt% SDS, 0.1 wt% bromophenol blue, 1.5 wt% DTT, 40 v / v glycerol, and the balance being water. The preparation method for human dermal extract protein strips in this invention is the same as above.
[0008] Furthermore, the P200 pemphigus diagnostic kit has 8 staining chambers, of which 4 staining chambers contain human dermal extract protein strips and the other 4 staining chambers contain LM521 / LM421 / LM411 / LM111 recombinant protein strips.
[0009] Furthermore, before adding the protein strips, the staining tank is filled with the above-mentioned reaction buffer diluted to 5% skim milk and sealed at room temperature for 1 hour. Then, it is washed with the above-mentioned washing buffer and air-dried.
[0010] Taking a mixture of recombinant proteins LM521 / LM421 / LM411 / LM111 as an example, this method uses a mixture of four LMγ1 recombinant proteins as the antigen and utilizes Western blotting to detect LMγ1 autoantibodies in human serum. During detection, positive and negative control sera, as well as sera from suspected patients, are added to their respective protein strips. LMγ1 IgG / IgA antibodies in the serum bind to the antigen. After washing, unbound serum proteins are removed. Then, the human IgG / IgA antibodies bound to the protein strips bind to horseradish peroxidase-labeled anti-human IgG / IgA antibodies. After another wash, the horseradish peroxidase bound to the protein strips reacts with the substrate to form a visible pink band, which indicates a positive result; the absence of a band indicates a negative result.
[0011] The advantages of this invention compared to existing technologies are: it simplifies the complex immunoblotting technique, uses human dermal extracts and mixtures of various LMγ1 recombinant proteins as antigens, and simultaneously analyzes IgG and IgA autoantibodies. This provides a comprehensive and one-time solution to the serological diagnostic problem of P200 pemphigus. Attached Figure Description
[0012] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.
[0013] Figure 1 This is a schematic diagram showing the results of testing suspected patients using the P200 pemphigus diagnostic kit of the present invention. Detailed Implementation
[0014] The present invention is described in detail below through specific embodiments, but this does not limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained commercially.
[0015] Example 1
[0016] A diagnostic kit for mucosal pemphigoid, the kit comprising:
[0017] (1) Dermal extract protein strips (4 strips, placed in 4 staining tanks respectively)
[0018] (2) LM521 / LM421 / LM411 / LM111 recombinant protein strips (4 strips, placed in 4 staining tanks respectively)
[0019] (3) Positive control (serum from patients with positive LMγ1IgG autoantibody diluted in 1 ml of reaction buffer containing 0.05% sodium azide)
[0020] (4) Negative control (normal human serum diluted in 1 ml of reaction buffer containing 0.05% sodium azide)
[0021] (5) Enzyme-labeled IgG antibody (horseradish peroxidase-labeled rabbit anti-human IgG antibody, 3.0 ml)
[0022] (6) Enzyme-labeled IgA antibody (horseradish peroxidase-labeled rabbit anti-human IgA antibody, 1.0 ml)
[0023] (7) Reaction buffer (2 ml of 1xPBS containing 0.05% v / v Tween 20 and 0.05% w / v NaN3)
[0024] (8) Washing buffer (10 ml of 10x PBS containing 0.5% v / v Tween 20)
[0025] (9) Enzyme substrate solution (3,3',5,5'-tetramethylbenzidine dihydrochloride / hydrogen peroxide (TMB / H2O2) solution, TMB concentration 2.5 mg / ml (dissolved in anhydrous ethanol), H2O2 concentration 0.03% (V / V), total 5.0 ml)
[0026] Preparation method of protein strips:
[0027] The dermal extract involved in this patent was prepared in-house, and the specific method is described in the literature. [4] The recombinant proteins LM521, LM421, LM411, and LM111 were purchased from Biolamina (catalog numbers LN521-05, LN421-02, LN411-02, and LN111-0501, respectively).
[0028] The preparation method of LM521 / LM421 / LM411 / LM111 recombinant protein strips is as follows:
[0029] Take 2 μg of total protein (a mixture of four recombinant proteins in a 1:1:1:1 ratio) and mix with 300 μl of 4-fold concentrated protein loading buffer. Boil at 100°C for 5 minutes, let stand on ice for 1 minute, and then briefly collect the protein sample to the bottom of the tube. Use a 7.5% PAGE gel (one-well gel), add all the sample, and perform electrophoresis (conditions: 20 mA, 3 hours). After electrophoresis, use an iBlot2NCregulartransferstack (Thermofisher) and an iBlot2 device (Thermofisher) for membrane transfer (transfer time: 7 minutes). Remove the NC membrane containing the transferred protein, cut it longitudinally into 5 mm wide protein strips, and block the protein strips with 5% milk (reaction buffer) at room temperature for 1 hour. Then wash three times with washing buffer (3 minutes each time) and dry at room temperature to obtain the protein strips used in this invention.
[0030] The staining tank is sealed at room temperature for 1 hour (1 ml / tank) after being diluted with 5% milk (reaction buffer), then thoroughly washed with washing buffer and air-dried before being prepared to add protein strips. Therefore, this staining tank does not non-specifically bind serum proteins when staining protein strips, thereby improving the sensitivity of detecting antibodies in serum.
[0031] Methods for detection using the P200 pemphigus diagnostic kit:
[0032] (1) Reagent preparation
[0033] Before the experiment, place all test materials at room temperature (20-30 degrees Celsius); dilute an appropriate amount of washing buffer (1:10) with distilled water as needed. Additionally, prepare 100ml of tap water and absorbent paper.
[0034] (2) Serum sample preparation
[0035] Dilute patient serum 1:20: Add 70 μl of serum to 1400 μl of reaction buffer and mix well.
[0036] (3) Detection steps (taking LM521 / LM421 / LM411 / LM111 protein strips as an example; the same applies to dermal extract protein strips)
[0037] 1) Take out four staining wells containing LM521 / LM421 / LM411 / LM111 protein strips, and add 300 μl of positive control, negative control, and diluted patient serum ×2 to each well. Incubate at room temperature (20-30 degrees Celsius) for 120 minutes;
[0038] 2) Washing protein strips: Add 500 μl of diluted washing buffer to each staining tank and wash 3 times (3 minutes each time);
[0039] 3) Add 300 μl of enzyme-labeled antibody solution to each staining tank (for positive control, negative control, and one patient serum tank, add enzyme-labeled IgG antibody solution, and for another patient serum tank, add enzyme-labeled IgA antibody solution), and incubate at room temperature (20-30 degrees Celsius) for 60 minutes;
[0040] 4) Washing protein strips: Add 500 μl of diluted washing buffer to each staining tank and wash 3 times (3 minutes each time);
[0041] 5) Add 300 μl of enzyme substrate solution to each staining tank and incubate at room temperature (20-30 degrees Celsius) for 10 minutes.
[0042] 6) Washing the protein strips: Add 500 μl of tap water to each staining tank and wash 3 times (1 minute each time);
[0043] 7) Remove the protein strips from the staining bath and lay them flat on absorbent paper to dry.
[0044] 8) Visually inspect the results and make a judgment.
[0045] The result is determined as follows: if a visible pink band forms on the protein strip and its position is consistent with that of the positive band in the positive control, it is considered positive; if no band appears or the position of the band is inconsistent with that of the positive control, it is considered negative.
[0046] Figure 1The results of serological diagnosis using the kit of this invention for a patient suspected of having P200 pemphigus are shown. 1 is a human dermal extract protein strip, and 2 is a recombinant protein strip of LM521 / LM421 / LM411 / LM111. 1+ / 2+ represents the positive control result; 1- / 2- represents the negative control result; 1G / 2G represents the result of IgG autoantibodies in the patient's serum; and 1A / 2A represents the result of IgA autoantibodies in the patient's serum. Based on the results, it can be determined that the patient is positive for 1G and 2G (red arrows) and negative for 1A and 2A, meaning the patient's serum is positive for IgG anti-LMγ1 autoantibodies. The serological diagnosis indicates that the patient has P200 pemphigus and is positive for IgG autoantibodies.
[0047] Experimental results:
[0048] Specificity and sensitivity:
[0049]
[0050] Repeatability:
[0051] The five samples were repeated six times, and the results of each repetition were consistent.
[0052] Interfering substances:
[0053] It is necessary to avoid using highly hemolyzed samples, as well as high-concentration serum diluted too low for testing.
[0054] The embodiments described above are merely preferred embodiments of the present invention, and not all feasible embodiments of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.
[0055] References
[0056] [1]Hua Qian,Zhijun Zhou,Luhuai Shi,Huicheng Li,Weijun Liu,Yong Ai,Yangmin Gao,Suying Feng,Takashi Hashimoto,Xiaoguang Li.Case report:Variety oftarget antigens during one year’s follow up of a patient initially diagnosedwith bullous pemphigoid.Front Immunol.2022 Jan 13;12:825226.
[0057] [2]Li X,Qian H,Ishii N,Yamaya M,Fukuda H,Mukai H,Hirako Y,HashimotoT.A case of concurrent anti-lamininγ1pemphigoid and anti-laminin 332-typemucous membrane pemphigoid.Br J Dermatol.2014;171(5):1257-9.
[0058] [3]Liu W,Sun X,Gao Y,Li H,Shi L,Cheng L,Zhou Z,Li X,Qian H.A Chinesecase of concurrent anti-lamininγ1pemphigoid and anti-laminin 332-type mucousmembrane pemphigoid.J Dermatol.2022 Jul 11
[0059] [4]Hashimoto T,Ogawa MM,Konohana A,Nishikawa T.Detection of pemphigusvulgaris and pemphigus foliaceus antigens by immunoblot analysis usingdifferent antigen sources.J Invest Dermatol 1990;94:327-31.
Claims
1. A diagnostic kit for pemphigus type P200, characterized in that, The kit comprises human dermal extract protein strips, recombinant protein strips, a positive control, a negative control, enzyme-labeled IgG antibody, enzyme-labeled IgA antibody, reaction buffer, washing buffer, and enzyme matrix solution. The recombinant protein strips are composed of a mixture of LM521, LM421, LM411, and LM111 recombinant proteins. The positive control consists of 1 ml of LMγ1 IgG positive antibody serum diluted in a reaction buffer containing 0.05% sodium azide. The negative control consists of 1 ml of normal human serum diluted in a reaction buffer containing 0.05% sodium azide. The enzyme-labeled IgG antibody is horseradish peroxidase-labeled rabbit anti-human IgG antibody, with a content of 3.0 ml. The enzyme-labeled IgA antibody is horseradish peroxidase-labeled rabbit anti-human IgA antibody, with a content of 1.0 ml. The reaction buffer contains 0.05% v / v... The recombinant protein strips were prepared as follows: 2.0 ml of 1xPBS containing Tween 20 and 0.05% w / v NaN3; 10 ml of 10xPBS containing 0.5% v / v Tween 20; and 5.0 ml of a mixture of TMB and H2O2, with TMB dissolved in anhydrous ethanol at a concentration of 2.5 mg / ml and H2O2 at a concentration of 0.03% v / v. The recombinant protein strips were prepared as follows: 0.5 µg each of LM521, LM421, LM411, and LM111 recombinant proteins were mixed with 300 µl of 4-fold concentrated protein loading buffer, boiled at 100°C for 5 minutes, incubated on ice for 1 minute, and the recombinant protein sample was collected to the bottom of the tube. All recombinant protein samples were added to a 7.5% PAGE gel and electrophoresed at 20 mA for 3 hours. After electrophoresis, the samples were analyzed using Thermofisher's iBlot2 NC regular transfer instrument. The protein was transferred using a Thermofisher iBlot2 device for 7 minutes. The NC membrane containing the transferred protein was removed and longitudinally cut into 5 mm wide protein strips. The protein strips were blocked at room temperature for 1 hour with 5% skim milk diluted with reaction buffer, followed by washing three times with washing buffer for 3 minutes each time, and then dried at room temperature to obtain LM521 / LM421 / LM411 / LM111 recombinant protein strips.
2. The P200 pemphigus diagnostic kit according to claim 1, characterized by, The LM521, LM421, LM411, and LM111 recombinant proteins used in the recombinant protein strips were purchased from Biolamina, with catalog numbers LN521-05, LN421-02, LN411-02, and LN111-0501, respectively.
3. The P200 pemphigus diagnostic kit according to claim 1, characterized in that, The P200 pemphigus diagnostic kit has 8 staining chambers, of which 4 contain human dermal extract protein strips and the other 4 contain recombinant protein strips.
4. The P200 pemphigus diagnostic kit according to claim 3, characterized by, Before adding the protein strips, the staining tank is filled with the above reaction buffer diluted to 5% skim milk and sealed at room temperature for 1 hour. Then, it is washed with the above washing buffer and air-dried.
5. The P200 pemphigus diagnostic kit according to claim 4, characterized in that, The 4x concentrated protein loading buffer consists of 2.4wt% Tris, 8wt% SDS, 0.1wt% bromophenol blue, 1.5wt% DTT, 40v / v% glycerol, and the balance being water.