CN114371294A - Use of crystallin alpha A for detecting or diagnosing pemphigoid - Google Patents

Abstract

The invention discloses the use of phacoelenterazine alpha A for the detection or diagnosis of pemphigoid. The present inventors have discovered and identified a novel pemphigoid autoantigen-phaa (CRYAA). The accuracy of the detection of the pemphigoid can be improved by detecting the autoantibody of the protein, and the occurrence of false negative can be avoided. Based on the discovery of the autoantigen, the invention also provides an ELISA kit for detecting the anti-human CRYAAIgG antibody and a detection method thereof, wherein the kit contains a microporous band coated by CRYAA recombinant protein, and the kit can realize the detection of the CRYAAIgG autoantibody in the serum of the pemphigoid patient. The invention provides a new technical means for detecting and diagnosing the pemphigoid.

Description

Use of crystallin alpha A for detecting or diagnosing pemphigoid

Technical Field

The present invention relates to a novel pemphigoid autoantigen and its use, and more particularly to a novel pemphigoid autoantigen-phaa (CRYAA) and its use for detecting or diagnosing pemphigoid. The invention belongs to the technical field of medicines.

Background

Pemphigoid is a group of autoimmune blistering diseases of the skin that affects mainly the elderly, mainly manifested by lesions of the skin and the oral, ocular and other mucous membranes. Diagnosis cannot be made simply by clinical manifestations, and diagnosis must be confirmed by detection of autoantibodies in the serum of the patient. Known autoantigens of pemphigoid include BP180, laminin gamma 1, collagen VII, laminin-332, integrin alpha 6 beta 4. Currently, approximately 20% of pemphigoid cannot be diagnosed with precision due to the presence of unknown autoantigens.

Recently, the present inventors have discovered and identified a novel pemphigoid autoantigen, crystallin α a (CRYAA). The inventor of the invention finds that CRYAA is mainly distributed in a basement membrane zone (a linear strong fluorescence staining zone between epidermis and dermis) through immunofluorescence staining of normal human skin tissue sections, and the zone is also a zone in which known autoantigens of pemphigoid, including BP180, laminin-332, integrin alpha 6 beta 4, laminin gamma 1, collagen VII and the like exist. Thus, CRYAA may also be an autoantigen for pemphigoid, in other words, autoantibodies against CRYAA may be present in patients with pemphigoid. The inventor constructs an expression plasmid of human CRYAA, expresses and purifies CRYAA recombinant protein, thereby establishing an ELISA kit for detecting anti-human CRYAAIgG antibody and a method thereof, thereby being capable of realizing the detection of CRYAAIgG autoantibody in the serum of a pemphigoid patient, and being capable of improving the diagnosis accuracy of pemphigoid.

Disclosure of Invention

It is an object of the present invention to provide a novel autoantigen useful for detecting or diagnosing pemphigoid and use thereof.

It is another object of the present invention to provide an ELISA kit for detecting or diagnosing pemphigoid.

In order to achieve the purpose, the invention adopts the following technical means:

the invention relates to the use of crystallin alpha A (CRYAA) for producing a reagent for detecting or diagnosing pemphigoid.

Preferably, the reagent is a reagent for detecting human anti-CRYAA antibodies by an ELISA method.

The invention also provides an ELISA kit for detecting or diagnosing pemphigoid, wherein the kit contains a micropore strip coated by CRYAA recombinant protein.

Preferably, the kit further comprises standard serum 1, standard serum 2, an enzyme labeled antibody, a diluent, a reaction buffer solution, a washing buffer solution, an enzyme matrix solution and a stop solution.

Wherein, preferably, the standard serum 1 is a reaction buffer containing 0.05% w/v of sodium azide; the standard serum 2 is a reaction buffer solution containing 100U/ml CRYAAIgG antibody and 0.05% w/v sodium azide; the enzyme-labeled antibody is a rabbit anti-human IgG antibody labeled by horseradish peroxidase; the reaction buffer solution is 1xPBS containing 0.05% v/v Tween20 and 0.05% w/v NaN3, and the washing buffer solution is 10xPBS containing 0.5% v/v Tween 20; the enzyme substrate liquid is 3, 3 ', 5, 5' -tetramethylcascade aniline dihydrochloride/hydrogen peroxide (TMB/H)₂O₂) A solution; the stop solution is 0.5N hydrochloric acid solution.

Preferably, the lenticular protein α a-coated microporous strip is prepared by the following method:

the purified human CRYAA recombinant protein obtained by prokaryotic expression is dissolved by PBS, and the storage concentration is 1 mg/ml. Before coating, PBS is used for 1000-fold dilution to make the final concentration of protein reach 1 mu g/ml, a 96-well plate is taken for coating of protein, 100 mu l of the diluted CRYAA recombinant protein solution with the concentration of 1 mu g/ml is added into each well, and coating is carried out for 12-16 hours at the temperature of 4 ℃.

The invention provides an ELISA kit for detecting or diagnosing pemphigoid, which utilizes an ELISA method to qualitatively determine IgG antibody of CRYAA in serum. During detection, patient serum and standard serum are added into a micropore strip coated with CRYAA recombinant protein, and the IgG antibody of CRYAA is combined with antigen. After washing, removing unbound serum protein, then adding an anti-human IgG antibody marked by horseradish peroxidase into the micropores to be bound with human IgG, after washing, adding a horseradish peroxidase substrate to react with the horseradish peroxidase, and finally adding an acid solution to stop the enzyme reaction. And (5) measuring the absorbance by using a microplate reader, and quantifying the detection result.

Preferably, when the ELISA kit is used for detecting human cryaigg antibody, the following steps are performed:

(1) reagent preparation

Before the experiment, all detection materials are placed at room temperature, and a proper amount of washing buffer solution is diluted by distilled water as required;

(2) sample preparation

1:50 dilution of each patient serum: adding 500 mul reaction buffer solution into 10 mul serum, and mixing evenly;

(3) detection step

1) Adding 100 mul of diluted sample into the microporous strip, and incubating for 60 minutes at room temperature;

2) washing the microporous strip: washing 4 times with 200. mu.l/well diluted washing buffer;

3) adding 100 mul/hole enzyme-labeled antibody liquid, and incubating for 45 minutes at room temperature;

4) washing the microporous strip: washing 4 times with 200. mu.l/well diluted washing buffer;

5) adding 100 mul/hole enzyme substrate liquid, and incubating for 30 minutes at room temperature;

6) adding 100 mul/hole stop solution;

7) reading the absorbance at 450 nm;

8) result calculation and decision

Unit value (U/ml) calculation formula:

(A₄₅₀<sample (I)>-A₄₅₀<Standard serum 1>)*100/(A₄₅₀<Standard serum 2>-A₄₅₀<Standard serum 1>)

And (3) judging standard: negative with a unit value < 20; the unit value is more than or equal to 20 and is positive;

quality control: each test result must satisfy the following condition, otherwise the result is invalid:

standard serum 1 had an OD450 of 0.100 or less

The OD450 of the standard serum 2 was 0.500 or more.

Finally, the invention also provides the application of the ELISA kit in the preparation of a reagent for detecting the human CRYAAIgG antibody.

Compared with the prior art, the invention has the beneficial effects that:

1. the inventor finds and identifies a new pemphigoid autoantigen, namely crystallina alpha A (CRYAA), and can improve the accuracy of pemphigoid detection by detecting the protein and avoid the occurrence of false negative;

2. the invention provides an ELISA kit for detecting an anti-human CRYAAIgG antibody and a method thereof, thereby realizing the detection of the CRYAAIgG autoantibody in the serum of a pemphigoid patient and providing a new technical means for the detection and diagnosis of the pemphigoid.

Drawings

FIG. 1 is SDS-PAGE (left) and western blot (right) analysis of CYRAA cloned in pET-30a (+) and expressed in BL21(DE3) strain;

wherein, lane M1: protein molecular weight marker 1; lane M2: protein molecular weight marker 2; lane PC1: BSA (1. mu.g); lane PC2: BSA (2. mu.g); lane NC: (ii) an uninduced cell lysate; lane 1: inducing cell lysates for 16 hours at 15 ℃; lane 2: cell lysates induced at 37 ℃ for 4 hours; lane NC 1: (ii) an uninduced cell lysate supernatant; lane 3: cell lysate supernatant induced at 15 ℃ for 16 h; lane 4: cell lysate supernatant induced at 37 ℃ for 4 hours; lane NC 2: precipitation of uninduced cell lysate; lane 5: cell lysates were pelleted and induced at 15 ℃ for 16 h. Lane 6: cell lysates were induced at 37 ℃ for 4 hours.

FIG. 2 is an SDS-PAGE result of purified CYRAA;

wherein, lane M1: a protein molecular weight marker; lane 1: BSA; lane 2: purified CYRAA.

Detailed Description

The present invention is further illustrated by the following experiments in conjunction with examples, it being understood that these examples are for illustrative purposes only and in no way limit the scope of the present invention.

Example 1 discovery of Lectinom alpha A (Crystallina alpha A, CRYAA)

By immunofluorescent staining of normal human skin tissue sections (FIG. 1), we found that CRYAA is distributed mainly in the basement membrane zone (the linear strongly fluorescent stained region between the epidermis and the dermis, which is also the region where known autoantigens for pemphigoid, including BP180, lamin-332, integrins α 6 β 4, laminin γ 1, collagenVII, etc. therefore, we suspected that CRYAA may also be an autoantigen for pemphigoid, in other words, autoantibodies against CRYAA may be present in patients with pemphigoid.

Example 2 expression of Lectinoglobulin alpha A (CryaNalphaA, CRYAA)

1. A complete CRYAA gene sequence (NdeI-ATG-CYRAA-His tag-Stop codon-HindIII, shown in SEQ ID NO.1 and the coded amino acid sequence is shown in SEQ ID NO. 2) is designed and synthesized, and is subcloned into a target vector pET-30a (+) and is transformed into escherichia coli for expression.

2. Inducible expression

(1) Three well-separated single colonies were picked and inoculated into 4ml of LB medium containing 50. mu.g/ml kanamycin, respectively.

(2) The cells were placed in a 37 ℃ shake flask and shaken at 200 rpm.

(3) When the OD600 value reaches 0.6-0.8, IPTG with the final concentration of 0.5mM IPTG is added into two of the three test tubes, induction is carried out for 16h at 15 ℃ and 4h at 37 ℃ respectively, and the last test tube is used as a negative control.

(4) Protein expression and solubility were examined using SDS-PAGE and western blot.

3. Sample preparation

Cell pellets were taken from 450. mu.l of culture and lysed with 300. mu.l lysis buffer (50mM Tris, 150mM NaCl, 5% glycerol pH 8.0) for 1 minute using a sonicator.

(1) Whole cell lysate: mu.l of 5-fold loading buffer was mixed with 100. mu.l of cell lysate to obtain a whole cell lysate sample. The sample was heated at 100 ℃ for 10 minutes and centrifuged at 15000rpm for 5 minutes.

(2) Supernatant and cell lysis debris: the remaining 200. mu.l of cell lysate was centrifuged at 15000rpm for 10 minutes, and the supernatant and cell lysis debris were collected, respectively;

mu.l of 5-fold addition buffer was mixed with 180. mu.l of the supernatant as a cell lysate supernatant sample. All pellets were resuspended with 150. mu.l of 5 Xloading buffer and used as cell lysate pellet samples. The samples were heated at 100 ℃ for 10 minutes and centrifuged at 15000rpm for 5 minutes before being loaded into the gel.

4. Purification of

Purification of the protein was performed using high affinity Ni-NTA resin.

5. Results

(1) Whole cell lysates, cell lysate supernatants, and pellet samples were analyzed by SDS-PAGE and Westernblot. The results are shown in FIG. 1.

(2) The protein purification results are shown in FIG. 2.

EXAMPLE 3 preparation of ELISA kit for detecting CRYAAIgG antibody

1. Composition of

The ELISA kit comprises:

(1) CRYAA recombinant protein micropore strip (48 holes)

(2)0U/ml of Standard serum 1 (reaction buffer containing 0.05% sodium azide, 1.5ml)

(3) Standard serum 2(CRYAAIgG autoantibody positive patient serum diluted in 0.05% w/v reaction buffer sodium azide, 1.5ml) at 100U/ml

(4) Enzyme-labeled antibody (horse radish peroxidase-labeled rabbit anti-human IgG antibody, 8ml)

(5)1x reaction buffer (0.01M PBS containing 0.05% v/vTween20 and 0.05% w/vNaN3, 50ml)

(6)10x washing buffer (0.1M PBS containing 0.5% v/vTween20, 100ml)

(7) Enzyme substrate liquid (3, 3 ', 5, 5' -tetramethylbenzidine dihydrochloride/hydrogen peroxide (TMB/H)₂O₂) Solution, 8ml)

(8) Middle-stop liquid (0.5N hydrochloric acid solution, 8ml)

2. Coating of CRYAA recombinant protein microporous band

(1) Coated material

Prokaryotic expression and purification of human CRYAA full-length recombinant protein (containing his tag).

(2) Protein solubilization and coating

CRYAA recombinant protein (prepared in example 2) was solubilized in PBS and stored at a concentration of 1 mg/ml. A1000-fold dilution with PBS was performed before coating to achieve a final protein concentration of 1. mu.g/ml. The protein was coated in a 96-well plate, and 100. mu.l of the diluted CRYAA recombinant protein (1. mu.g/ml) was added to each well and coated overnight (12 to 16 hours) at 4 ℃.

3. Detection method

(4) Reagent preparation

Before the experiment, all detection materials are placed at room temperature (20-30 ℃); an appropriate amount of the washing buffer (1:10) was diluted with distilled water as necessary.

(5) Sample preparation

1:50 dilution of each patient serum: mu.l of serum was added to 500. mu.l of reaction buffer and mixed well.

(6) Detection step

1) Adding 100 μ l of diluted sample (1:50) into the microporous strip, and incubating at room temperature (20-30 deg.C) for 60 min;

2) washing the microporous strip: washing 4 times with 200 mul/well diluted washing buffer solution;

3) adding 100 mul/hole enzyme-labeled antibody liquid for 45 minutes at room temperature (20-30 ℃);

4) washing the microporous strip: washing 4 times with 200 mul/well diluted washing buffer solution;

5) adding 100 μ l/well enzyme matrix solution, and incubating at room temperature (20-30 deg.C) for 30 min

6) Add 100. mu.l/well stop solution

7) Reading absorbance (450nm)

8) Result calculation and decision

Unit value (U/ml) calculation formula:

(A₄₅₀<sample (I)>-A₄₅₀<Standard serum 1>)*100/(A₄₅₀<Standard serum 2>-A₄₅₀<Standard serum 1>)

And (3) judging standard: negative with a unit value < 20; positive results were obtained at a unit value of 20 or more.

Quality control: each test result must satisfy the following condition, otherwise the result is invalid:

(1) standard serum 1 had an OD450 of 0.100 or less

(2) Standard serum 2 had an OD450 of 0.500 or more

Example 4 use of CRYAA in the detection or diagnosis of pemphigoid

(1) Sample source

100 parts of pemphigoid patient serum, 50 parts of pemphigoid patient serum, a part from the university of longtime university of japan dermatology, donated by professor Takashi Hashimoto (collected from patients in japan and all over the world), and another part from the remaining patient serum after serological diagnosis of skin autoimmune blistering disease provided by the dermatology department of the tri hospital or dermatology specialty hospital in china. Normal human serum 300 remaining sera from normal physical examinations in hospitals.

The serum adopts 1: and (5) diluting by 50.

(2) Specificity and sensitivity

By using the kit of example 3 of the present invention to detect 100 sera from pemphigoid patients, 15 cases were found in which CRYAAIgG antibody positivity was confirmed, and only 10 sera from known pemphigoid autoantibodies were detected from the 15 sera, that is, 5 of them were negative to other known pemphigoid autoantibodies but positive to CRYAAIgG antibodies, thus indicating that CRYAA is one of the autoantigens of pemphigoid. The results are shown in table 1 below:

TABLE 1

(2) Repeatability of

For 5 samples, 6 replicates were performed, each with a CV% value of less than 15%.

(3) Detection range

The detection range of the kit is 5-150U/ml.

Sequence listing

<110> Tang Xian Ling

<120> use of Lenticoccun alpha A for detecting or diagnosing pemphigoid

<160> 2

<170> SIPOSequenceListing 1.0

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<211> 552

<212> DNA

<213> Homo sapiens

<400> 1

catatggatg taacaataca acacccctgg tttaaaagga ctctagggcc cttctacccg 60

agccgtctgt tcgatcagtt ctttggcgag ggcctgttcg agtacgacct gctgccgttt 120

ctgagcagca ccatcagccc gtactatcgt cagagcctgt tccgtaccgt gctggatagc 180

ggtatcagcg aggttcgtag cgaccgtgat aagttcgtga tttttctgga cgttaaacac 240

ttcagcccgg aagatctgac cgtgaaggtt caggacgatt ttgtggagat ccacggcaaa 300

cacaacgaac gtcaagacga tcacggttac attagccgtg agttccaccg tcgttatcgt 360

ctgccgagca acgttgatca aagcgcgctg agctgcagcc tgagcgcgga tggtatgctg 420

accttttgcg gcccgaagat ccaaaccggt ctggatgcga cccatgcgga acgtgcgatt 480

ccggttagcc gtgaggaaaa gccgaccagc gcgccgagca gccaccacca ccaccatcat 540

taatgaaagc tt 552

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Met Asp Val Thr Ile Gln His Pro Trp Phe Lys Arg Thr Leu Gly Pro

1 5 10 15

Phe Tyr Pro Ser Arg Leu Phe Asp Gln Phe Phe Gly Glu Gly Leu Phe

20 25 30

Glu Tyr Asp Leu Leu Pro Phe Leu Ser Ser Thr Ile Ser Pro Tyr Tyr

35 40 45

Arg Gln Ser Leu Phe Arg Thr Val Leu Asp Ser Gly Ile Ser Glu Val

50 55 60

Arg Ser Asp Arg Asp Lys Phe Val Ile Phe Leu Asp Val Lys His Phe

65 70 75 80

Ser Pro Glu Asp Leu Thr Val Lys Val Gln Asp Asp Phe Val Glu Ile

85 90 95

His Gly Lys His Asn Glu Arg Gln Asp Asp His Gly Tyr Ile Ser Arg

100 105 110

Glu Phe His Arg Arg Tyr Arg Leu Pro Ser Asn Val Asp Gln Ser Ala

115 120 125

Leu Ser Cys Ser Leu Ser Ala Asp Gly Met Leu Thr Phe Cys Gly Pro

130 135 140

Lys Ile Gln Thr Gly Leu Asp Ala Thr His Ala Glu Arg Ala Ile Pro

145 150 155 160

Val Ser Arg Glu Glu Lys Pro Thr Ser Ala Pro Ser Ser His His His

165 170 175

His His His

Claims (8)

1. Use of Crystallin Alpha a (CRYAA) for the preparation of a reagent for the detection or diagnosis of pemphigoid.

2. The use of claim 1, wherein the agent is an agent for the detection of human anti-CRYAA antibodies by ELISA.

3. An ELISA kit for detecting or diagnosing pemphigoid, wherein the kit contains a microwell band coated with CRYAA recombinant protein.

4. The ELISA kit of claim 3 further comprising standard serum 1, standard serum 2, an enzyme-labeled antibody, a diluent, a reaction buffer, a washing buffer, an enzyme substrate solution, and a stop buffer.

5. The ELISA kit of claim 4 wherein the standard serum 1 is a reaction buffer containing 0.05% w/v sodium azide; the standard serum 2 is a reaction buffer solution containing 100U/ml CRYAA IgG antibody and 0.05% w/v sodium azide; the enzyme-labeled antibody is a rabbit anti-human IgG antibody labeled by horseradish peroxidase; the reaction buffer solution is 1xPBS containing 0.05% v/v Tween20 and 0.05% w/v NaN3, and the washing buffer solution is 10xPBS containing 0.5% v/v Tween 20; the enzyme substrate liquid is 3, 3 ', 5, 5' -tetramethylcascade aniline dihydrochloride/hydrogen peroxide (TMB/H)₂O₂) A solution; the stop solution is 0.5N hydrochloric acid solution.

6. The ELISA kit of claim 3 wherein the coated microwell strip of CRYAA recombinant protein is prepared by:

dissolving the purified human CRYAA recombinant protein obtained by prokaryotic expression by PBS, and storing the protein at the concentration of 1 mg/ml; before coating, PBS is used for 1000-fold dilution to make the final concentration of protein reach 1 mu g/ml, a 96-well plate is taken for coating of protein, 100 mu l of the diluted CRYAA recombinant protein solution with the concentration of 1 mu g/ml is added into each well, and coating is carried out for 12-16 hours at the temperature of 4 ℃.

7. The ELISA kit of any one of claims 3 to 6 wherein the kit is for detecting human CRYAAIgG antibodies by:

(1) reagent preparation

Before the experiment, all detection materials are placed at room temperature, and a proper amount of washing buffer solution is diluted by distilled water as required;

(2) sample preparation

1:50 dilution of each patient serum: adding 500 mul reaction buffer solution into 10 mul serum, and mixing evenly;

(3) detection step

1) Adding 100 mul of diluted sample into the microporous strip, and incubating for 60 minutes at room temperature;

2) washing the microporous strip: washing 4 times with 200. mu.l/well diluted washing buffer;

3) adding 100 mul/hole enzyme-labeled antibody liquid, and incubating for 45 minutes at room temperature;

4) washing the microporous strip: washing 4 times with 200. mu.l/well diluted washing buffer;

5) adding 100 mul/hole enzyme substrate liquid, and incubating for 30 minutes at room temperature;

6) adding 100 mul/hole stop solution;

7) reading the absorbance at 450 nm;

8) result calculation and decision

Unit value (U/ml) calculation formula:

(A₄₅₀<sample (I)>-A₄₅₀<Standard serum 1>)*100/(A₄₅₀<Standard serum 2>-A₄₅₀<Standard serum 1>)

And (3) judging standard: negative with a unit value < 20; the unit value is more than or equal to 20 and is positive;

quality control: each test result must satisfy the following condition, otherwise the result is invalid:

standard serum 1 had an OD450 of 0.100 or less

The OD450 of the standard serum 2 was 0.500 or more.

8. Use of the ELISA kit of any one of claims 3-7 in the preparation of a reagent for detecting human cryaigg antibodies.

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