CN109374899B - ELISA kit for detecting anti-MDA5 antibody and detection method thereof - Google Patents

ELISA kit for detecting anti-MDA5 antibody and detection method thereof Download PDF

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CN109374899B
CN109374899B CN201810973433.3A CN201810973433A CN109374899B CN 109374899 B CN109374899 B CN 109374899B CN 201810973433 A CN201810973433 A CN 201810973433A CN 109374899 B CN109374899 B CN 109374899B
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CN109374899A (en
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王楷文
赵江峰
王忠伟
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Shanghai Jiading District Central Hospital
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6881—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from skin
    • G—PHYSICS
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    • G01N2800/202—Dermatitis

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Abstract

The invention discloses an ELISA kit for detecting anti-MDA5 antibody. The kit is characterized by comprising purified dominant antigen epitope MDA5 protein. The invention also discloses a detection method of the ELISA kit for detecting the anti-MDA5 antibody. The invention has the beneficial effects that: the MDA5 has difficult full-length expression and high technical requirement, and the price of purchasing the finished MDA5 full-length protein is more expensive. The application expresses the synthetic antigen on the basis of searching the MDA5 dominant epitope, thereby greatly reducing the cost. The kit can be used for detecting the anti-MDA5 antibody, fills the blank of the anti-MDA5 antibody detection method, has the characteristics of large detection flux, exact result, high sensitivity and simple and convenient operation, and is easy to popularize and apply.

Description

ELISA kit for detecting anti-MDA5 antibody and detection method thereof
Technical Field
The invention belongs to the field of antibody detection, and particularly relates to an ELISA kit for detecting an anti-MDA5 antibody and a detection method thereof.
Background
Dermatomyositis (DM) is a more common life-threatening inflammatory myopathy in the clinic, mainly involving the skin and muscles. Currently considered to fall into the category of autoimmune diseases. The annual incidence rate of dermatomyositis is about 0.5-1/10 ten thousands of people, and the incidence rate is in an ascending trend. In clinical practice we have observed that patients with DM that involve or have skin as the major organ involved, we call "Dermatomyositis inonotus (CADM)" patients with very poor prognosis. According to the existing report and the clinical data of our family, the reason that DM/CADM has poorer prognosis than PM is that the incidence rate of Interstitial Lung Disease (ILD) is extremely high, and the incidence rate is the leading cause of death of DM/CADM patients.
The anti-MDA5 (melanoma differentiation gene 5) antibody is not only a specific antibody of DM/CADM combined ILD, but also a serological marker of DM/CADM patients accompanied with acute lung interstitial lesion, and the antibody level is highly positively correlated with the occurrence and the severity of the lung interstitial lesion and the occurrence of skin ulcer and vasculitis. Dynamic observation of the antibody level in serum is helpful for predicting disease evolution and guiding clinical treatment.
At present, no marketable anti-MDA5 antibody detection kit exists in the domestic market, so that the development of an anti-MDA5 antibody detection kit which can be widely applied, is efficient and sensitive and a corresponding detection method is particularly important for predicting disease evolution and guiding clinical treatment in view of the importance of detecting an anti-MDA5 antibody.
Disclosure of Invention
In order to overcome the defects in the prior art, one of the purposes of the invention is to provide an ELISA kit for detecting anti-MDA5 antibody, wherein the kit contains purified dominant epitope MDA5 protein.
In a preferred embodiment of the present invention, the amino acid sequence group of the purified dominant antigenic epitope MDA5 protein is:
Asn-Ser-Asn-Met-Gly-Ser-Asp-Ser-Gly-Thr-Met-Gly-Ser-Asp-Ser-AspGlu-Glu-Asn-Val-Ala-Ala-Arg-Ala-Ser-Pro-Glu-Pro-Glu-Leu-Gln-LeuLeu-Leu-Asn-Leu-Glu-Asn-Gly-Glu-Asp-Ala-Gly-Val-Gln-Leu-Ser-AspPhe-Ser-Leu-Ile-Ile-Ile-Asp-Glu-Cys-His-His-Thr-Asn-Lys-Glu-AlaVal-Tyr-Asn-Asn-Ile-Met-Arg-His-Tyr-Leu-Met-Gln-Lys-Leu-Lys-AsnLeu-Gly-Leu-Thr-Ala-Ser
in a preferred embodiment of the invention, the kit is provided with an anti-MDA5 antibody detection strip, and the detection strip is a 96-well enzyme label plate coated with dominant epitope MDA5 protein.
In a preferred embodiment of the invention, the kit is further provided with negative control serum, positive control serum, serum diluent, enzyme-labeled secondary antibody, washing solution, substrate developing solution and stop solution.
In a preferred embodiment of the invention, the negative control serum is a CADM patient immunoblotting negative serum for detecting anti-MDA5 antibody, the positive control serum is a CADM patient immunoblotting positive serum for detecting anti-MDA5 antibody, and the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-human IgG.
The invention also aims to provide a detection method of the ELISA kit for detecting the anti-MDA5 antibody, which comprises the following steps:
(1) coating a 96-well plate with dominant antigen epitope MDA5 protein, and standing overnight at 4 ℃;
(2) washing after coating, removing coating solution, washing with PBST, and repeating for 3-5 times;
(3) adding 5% BSA, incubating at 400 ul/well BSA at room temperature for 2h, washing with PBST, and repeating for 3-5 times;
(4) adding standard (known positive serum) and serum to be detected, 100ul per well, and incubating at normal temperature for 1 h;
(5) dilution of standards and blank control: 1: 40,1: 80,1: 160,1: 320,1: 640,1: 1280,1: 2560, blank: 100ul PBS, test serum 1: 100 mu are added into 100ul of the diluted solution respectively
(6) Washing with PBST for 3-5 times;
(7) adding an enzyme-labeled secondary antibody 1: 2000 (sheep anti-human. HRP), incubation for 30 min at room temperature, washing with PBST, repeating 3-5 times;
(8) adding substrate solution for developing for 15 min;
(9) the reaction was stopped, and read on an 450/630nm microplate reader.
In a preferred embodiment of the invention, in step (1), the dominant epitope MDA5 protein is added at a concentration of 0.5ug/ml to each reaction well, and 100ul is added.
The invention has the following main innovation points:
the MDA5 has difficult full-length expression and high technical requirement, and the price of purchasing the finished MDA5 full-length protein is more expensive. The application expresses the synthetic antigen on the basis of searching the MDA5 dominant epitope, thereby greatly reducing the cost.
The kit can be used for detecting the anti-MDA5 antibody, fills the blank of the anti-MDA5 antibody detection method, has the characteristics of large detection flux, exact result, high sensitivity and simple and convenient operation, and is easy to popularize and apply.
Drawings
FIG. 1 is a diagram of the MDA5 protein structure;
FIG. 2 is a structural diagram of dominant epitope MDA5 protein;
FIG. 3 is a schematic diagram of Western-Blot results of dominant epitope MDA5 protein obtained after protein recombination;
FIG. 4 is a schematic comparison of Anti-MDA5 antibody detection in various groups of humans;
FIG. 5 is a graph showing the comparison of the survival rates of Anti-MDA5 antibody assays.
Detailed Description
The present invention is further illustrated by the following examples, which should not be construed as limiting the invention thereto.
Example 1
1. Because the MDA5 protein has a large structure and a plurality of amino acid sequences, the full-length structure is analyzed by protein dynamics to obtain the dominant antigen structure epitope:
full-length amino acid sequence:
msngystden fryliscfra rvkmyiqvep vldyltflpa evkeqiqrtv atsgnmqave
lllstlekgv whlgwtrefv ealrrtgspl aarymnpelt dlpspsfena hdeylqllnl
lqptlvdkll vrdvldkcme eelltiedrn riaaaenngn esgvrellkr ivqkenwfsa
flnvlrqtgn nelvqeltgs dcsesnaeie nlsqvdgpqv eeqllsttvq pnlekevwgm
ennssessfa dssvvsesdt slaegsvscl deslghnsnm gsdsgtmgsd sdeenvaara
spepelqlrp yqmevaqpal egkni i iclp tgsgktrvav yiakdhldkk kkasepgkvi
vlvnkvllve qlfrkefqpf lkkwyrvigl sgdtqlkisf pevvkscdi i istaqilens
llnlengeda gvqlsdfsli i idechhtnk eavynnimrh ylmqklknnr lkkenkpvip
lpqilgltas pgvggatkqa kaeehilklc anldaftikt vkenldqlkn qiqepckkfa
iadatredpf keklleimtr iqtycqmspm sdfgtqpyeq waiqmekkaa kkgnrkervc
aehlrkynea lqindtirmi daythletfy neekdkkfav ieddsdeggd deycdgdede
ddlkkplkld etdrflmtlf fennkmlkrl aenpeyenek ltklrntime qytrteesar
giiftktrqs ayalsqwite nekfaevgvk ahhligaghs sefkpmtqne qkeviskfrt
gkinlliatt vaeegldike cniviryglv tneiamvqar graradesty vlvahsgsgv
iehetvndfr ekmmykaihc vqnmkpeeya hkilelqmqs imekkmktkr niakhyknnp
slitflcknc svlacsgedi hviekmhhvn mtpefkelyi vrenkalqkk cadyqingei
ickcgqawgt mmvhkgldlp clkirnfvvv fknnstkkqy kkwvelpitf pnldyseccl
fsded
the full-length protein structure is shown in FIG. 1.
After proteome kinetic analysis, the amino acid sequence of the dominant epitope is as follows:
Asn-Ser-Asn-Met-Gly-Ser-Asp-Ser-Gly-Thr-Met-Gly-Ser-Asp-Ser-Asp
Glu-Glu-Asn-Val-Ala-Ala-Arg-Ala-Ser-Pro-Glu-Pro-Glu-Leu-Gln-Leu
Leu-Leu-Asn-Leu-Glu-Asn-Gly-Glu-Asp-Ala-Gly-Val-Gln-Leu-Ser-Asp
Phe-Ser-Leu-Ile-Ile-Ile-Asp-Glu-Cys-His-His-Thr-Asn-Lys-Glu-Ala
Val-Tyr-Asn-Asn-Ile-Met-Arg-His-Tyr-Leu-Met-Gln-Lys-Leu-Lys-Asn
Leu-Gly-Leu-Thr-Ala-Ser
the dominant antigen structure is shown in FIG. 2.
2. After obtaining the dominant antigen epitope amino acid sequence, the dominant antigen epitope protein is obtained by a protein recombination mode, and the method comprises the following steps:
2.1 cloning gene;
2.1.1 obtaining the Gene of interest
The PCR method comprises the following steps: the primers are designed by taking human cDNA containing target genes as a template according to target gene sequences as follows:
F:TCGAATGGGTATTCCACAGACGAGAATTT,R:TCTTGCTGCCACTTCTCTTCTATGAA
the PCR reaction conditions were as follows:
95℃2min；
xm in (2min/kb) at 94 ℃ for 30s,60 ℃ for 30s,68 ℃ for 68 ℃; repeat 35 cycles;
68℃10min；
4℃1min；
and (3) carrying out electrophoresis gel running recovery on the amplification product by using a target fragment recovery kit (QIAGEN QIAqu ickGe l extraction K it) to obtain a purified amplification product.
2.1.2 construction of the cloning plasmid of interest
2.1.2.1 ligation of amplification products
The construction of the connecting body is as follows:
Figure BDA0001776821330000061
Figure BDA0001776821330000071
ligation was performed overnight at 16 ℃ to give a ligation product.
2.1.2.2 performing thermal transformation on the ligation products by using competent cells, then inoculating the ligation products to an LB culture plate with corresponding antibiotics for overnight culture, and obtaining a bacterial colony screening plate through transformation; selecting a single colony, transferring the single colony to an LB culture medium containing corresponding antibiotics again for culture, and then taking 2 mu L for PCR identification; after passing the identification, the sample containing the target fragment is inoculated into LB culture medium for overnight rotary culture.
2.1.2.3 the bacterial liquid obtained by culturing is extracted by plasmid according to the standard steps of the kit by using an Axygen miniprep kit, and the obtained plasmid is sequenced to identify whether the plasmid is the target clone plasmid.
2.2 transfection of cells
The obtained target clone plasmid is transfected into HEK293 cells, the transfected cells containing the target clone plasmid are inoculated into corresponding culture media to be cultured for 48-72h, and then the cultured cells are collected.
And 2.3, purifying the protein.
And (3) cracking the collected cells in an ultrasonic crushing mode, then centrifuging at a high speed to collect cracking supernatant, and purifying the cracking supernatant by an affinity purification method to obtain the purified protein.
2.4Western-Blot identification see FIG. 3.
3. Obtaining a large amount of purified dominant antigen protein, and using the dominant antigen protein as an antigen to perform ELISA test, wherein the ELISA test comprises the following steps:
3.1 antigen concentration 0.5ug/ml MDA5ul coated 96-well plate, adding 100ul in each reaction well, 4 degree overnight;
3.2 washing: after coating, removing coating liquid, washing with PBST, and repeating for 3-5 times;
3.3, sealing: 5% BSA, 400 ul/well, incubation at room temperature for 2h, washing, 3.2;
3.4 adding the standard substance and the serum to be detected, 100ul of each hole, and incubating for 1h at normal temperature;
3.5 dilution standard (known positive serum):
diluting a standard product: blank, 1: 40,1: 80,1: 160,1: 320,1: 640,1: 1280,1: 2560;
blank: 100ul PBS, test serum 1: 100 mu is added into 100 mu after being diluted;
3.6 washing, as in 3.2;
3.7 addition of secondary antibody 1: 2000 (goat anti-human. HRP), incubating for 30 min at room temperature, washing, as same as 3.2;
3.8 adding substrate liquid for developing for 15 min;
3.9 stop the reaction, 450/630nm microplate reader reading.
4. Experimental data:
4.1 comparison of Anti-MDA5 antibody detection among various groups of people, it can be seen that Anti-MDA5 antibody expression is significantly increased in ADM-ILD patients, see FIG. 4.
4.2 comparison of survival rates for Anti-MDA5 antibody assays:
by analyzing 85 IIL-ILD patients, the mortality rate of Anti-MDA5 antibody positive patients is obviously higher than that of Anti-MDA5 antibody negative patients, and the result is shown in figure 5.
Sequence listing
<110> Shanghai Feishan Biotechnology Limited
<120> ELISA kit for detecting anti-MDA5 antibody and detection method thereof
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Pro Glu Leu Thr Asp Leu Pro Ser Pro Ser Phe Glu Asn Ala His Asp
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Glu Tyr Leu Gln Leu Leu Asn Leu Leu Gln Pro Thr Leu Val Asp Lys
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Leu Leu Val Arg Asp Val Leu Asp Lys Cys Met Glu Glu Glu Leu Leu
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Thr Ile Glu Asp Arg Asn Arg Ile Ala Ala Ala Glu Asn Asn Gly Asn
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Glu Ser Gly Val Arg Glu Leu Leu Lys Arg Ile Val Gln Lys Glu Asn
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Trp Phe Ser Ala Phe Leu Asn Val Leu Arg Gln Thr Gly Asn Asn Glu
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Leu Val Gln Glu Leu Thr Gly Ser Asp Cys Ser Glu Ser Asn Ala Glu
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Ile Glu Asn Leu Ser Gln Val Asp Gly Pro Gln Val Glu Glu Gln Leu
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Leu Ser Thr Thr Val Gln Pro Asn Leu Glu Lys Glu Val Trp Gly Met
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Glu Asn Asn Ser Ser Glu Ser Ser Phe Ala Asp Ser Ser Val Val Ser
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Glu Ser Asp Thr Ser Leu Ala Glu Gly Ser Val Ser Cys Leu Asp Glu
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Ser Leu Gly His Asn Ser Asn Met Gly Ser Asp Ser Gly Thr Met Gly
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Ser Asp Ser Asp Glu Glu Asn Val Ala Ala Arg Ala Ser Pro Glu Pro
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Glu Leu Gln Leu Arg Pro Tyr Gln Met Glu Val Ala Gln Pro Ala Leu
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Glu Gly Lys Asn Ile Ile Ile Cys Leu Pro Thr Gly Ser Gly Lys Thr
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Arg Val Ala Val Tyr Ile Ala Lys Asp His Leu Asp Lys Lys Lys Lys
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Ala Ser Glu Pro Gly Lys Val Ile Val Leu Val Asn Lys Val Leu Leu
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Tyr Arg Val Ile Gly Leu Ser Gly Asp Thr Gln Leu Lys Ile Ser Phe
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Pro Glu Val Val Lys Ser Cys Asp Ile Ile Ile Ser Thr Ala Gln Ile
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Leu Glu Asn Ser Leu Leu Asn Leu Glu Asn Gly Glu Asp Ala Gly Val
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Gln Leu Ser Asp Phe Ser Leu Ile Ile Ile Asp Glu Cys His His Thr
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tcgaatgggt attccacaga cgagaatttr tcttgctgcc acttctcttc tatgaa 56

Claims (4)

1. An ELISA kit for detecting anti-MDA5 antibody is characterized in that the kit contains purified dominant antigen epitope MDA5 protein, and the amino acid sequence group of the purified dominant antigen epitope MDA5 protein is as follows:
Asn- Ser- Asn- Met- Gly- Ser-Asp-Ser-Gly-Thr-Met-Gly-Ser-Asp-Ser-Asp
Glu-Glu-Asn-Val-Ala-Ala-Arg-Ala-Ser-Pro-Glu-Pro-Glu-Leu-Gln-Leu
Leu-Leu-Asn-Leu-Glu-Asn-Gly-Glu-Asp-Ala-Gly-Val-Gln-Leu-Ser-Asp
Phe-Ser-Leu-Ile- Ile- Ile-Asp-Glu-Cys-His-His-Thr-Asn-Lys-Glu-Ala
Val-Tyr-Asn-Asn- Ile-Met-Arg-His-Tyr-Leu-Met-Gln-Lys-Leu-Lys-Asn
Leu-Gly-Leu-Thr-Ala-Ser。
2. the ELISA kit of claim 1 for detecting anti-MDA5 antibody, wherein the kit is provided with an anti-MDA5 antibody detection strip, and the detection strip is a 96-well ELISA plate coated with dominant antigen epitope MDA5 protein.
3. The ELISA kit for detecting anti-MDA5 antibody of claim 1, wherein the kit further comprises negative control serum, positive control serum, serum diluent, enzyme-labeled secondary antibody, washing solution, substrate developing solution, and stop solution.
4. The ELISA kit of claim 3 for detecting anti-MDA5 antibody, wherein the negative control serum is CADM patient immunoblotting anti-MDA5 antibody negative serum, the positive control serum is CADM patient immunoblotting anti-MDA5 antibody positive serum, and the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-human IgG.
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