CN109298181A - A kind of ELISA kit and its preparation process with recombinant antigen detection human cytomegalovirus IgM and IgA antibody - Google Patents

A kind of ELISA kit and its preparation process with recombinant antigen detection human cytomegalovirus IgM and IgA antibody Download PDF

Info

Publication number
CN109298181A
CN109298181A CN201810968554.9A CN201810968554A CN109298181A CN 109298181 A CN109298181 A CN 109298181A CN 201810968554 A CN201810968554 A CN 201810968554A CN 109298181 A CN109298181 A CN 109298181A
Authority
CN
China
Prior art keywords
hcmv
igm
iga
recombinant antigen
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
CN201810968554.9A
Other languages
Chinese (zh)
Inventor
杨致亭
唐玉蓉
马金环
朱元祺
王婷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
WEIFANG KANGHUA BIOTECH CO Ltd
Original Assignee
WEIFANG KANGHUA BIOTECH CO Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by WEIFANG KANGHUA BIOTECH CO Ltd filed Critical WEIFANG KANGHUA BIOTECH CO Ltd
Priority to CN201810968554.9A priority Critical patent/CN109298181A/en
Publication of CN109298181A publication Critical patent/CN109298181A/en
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a kind of ELISA kit and its preparation process with recombinant antigen detection human cytomegalovirus IgM and IgA antibody, the present invention is HCMV recombinant antigen S (pp150862‑1048+gp52297‑433+pUL57545‑601) for detecting the ELISA kit of human cytomegalovirus IgM and IgA, including coating mouse it is anti-human-ELISA Plate and the HCMV recombinant antigen S of horseradish peroxidase-labeled and the anti-human-IgA monoclonal antibody of the mouse of label of the ELISA Plate of IgM monoclonal antibody and coating HCMV recombinant antigen S.Wherein, coating mouse it is anti-human-ELISA Plate of IgM monoclonal antibody for detecting HCMV IgM antibody, the ELISA Plate of coating HCMV recombinant antigen S is for detecting HCMV IgA antibody.Reagent of the present invention has preferable sensitivity, specificity, stability and repeatability, and operating method is easy, and standard is easily controllable.

Description

A kind of ELISA examination detecting human cytomegalovirus IgM and IgA antibody with recombinant antigen Agent box and its preparation process
Technical field
The present invention relates to it is a kind of with recombinant antigen detect human cytomegalovirus IgM and IgA antibody ELISA kit and its Preparation process.
Background technique
Human cytomegalovirus (HCMV) is β-herpesviral of infection mankind extensively a kind of.Although general adult infection It is asymptomatic or symptoms are mild after HCMV, but it is serious miscarriage, stillborn foetus, fetal anomaly and hypoevolutism to be often resulted in after infection of pregnant women etc. Consequence, and in the individual (such as organ transplant recipients) that immune function is suppressed, HCMV often causes lethal infection.Therefore early Phase, quickly and accurately diagnosis HCMV Active infection is of great significance.
Human immune system can generate Multiple Antibodies, such as IgA, IgM, IgG after HCMV infection.HCMV-IgM and HCMV- IgA type antibody is the antibody that occurs earliest after viral antigen stimulation body, is the mark of HCMV acute HIV infection, therefore exploit person The detection reagent of cytomegalovirus IgA, IgM antibody help to early diagnose and find HCMV infection, to the treatment in later period and pre- It is most important afterwards.
The clinical diagnosis of HCM V early infection at present mainly detects specific antibody IgM in serum with ELISA, but common ELISA kit mostly using the totivirus of complicated component as antigen, there are cross reactions with the virus of other Herpesvirus, lead It causes the specific and sensitive gender gap of detection kit larger, easily causes false positive and false negative result, and then influence difference The HCMV in clinical infection stage is diagnosed, and easily causes to fail to pinpoint a disease in diagnosis and mistaken diagnosis.Using gene engineering method construct HCMV recombinant antigen with Replace totivirus antigen, is greatly improved the specificity and sensibility of detection；Therefore the ELISA-IgM of recombinant antigen HCMV is researched and developed Sensitivity and the specificity of clinical detection can be improved in detection kit, in addition, auxiliary detection IgA can further improve the spirit of diagnosis Quick property reduces rate of missed diagnosis.
Pp150 belongs to HCMV envelope protein, and with other protein herpesvirus without homology, at least there are two antigens for the albumen Determinant, positioned at the 862-1048 amino acid and 495-691 amino acid of C-terminal；Gp52 and pUL57 is DNA binding protein, wherein Gp52 can identify that IgM antigenic determinant is in 297-433 amino acid by most HCMV positive serums；The antigen of pUL57 is determined Determine the 1144-1196 amino acid that cluster is predominantly located at 545-601 amino acid and C-terminal, compared with pp150 and gp52, pUL57 can be earlier Identification IgM antibody, therefore the detection of acute stage IgM is more advantageous to, however, the recombinant antigen that detection HCMV IgM is used at present Mainly pp150 and gp52, sensibility and specificity are all to be improved.Based on this, we are by pp150, gp52 and pUL57 Dominant antigen epitope Combined expression (pp150862-1048+gp52297-433+pUL57545-601), recombinant protein is obtained, is made with the albumen IgM antibody is detected for prize law；In addition, detecting IgA antibody using indirect method, the diagnosis of HCMV acute stage can further improve Rate.
Summary of the invention
The purpose of the present invention is to provide a kind of ELISA with recombinant antigen detection human cytomegalovirus IgM and IgA antibody Kit and its preparation process, to solve the problems mentioned in the above background technology.
To achieve the above object, the invention provides the following technical scheme: a kind of detect human cytomegalovirus with recombinant antigen The ELISA kit and its preparation process of IgM and IgA antibody, it is characterized in that: include coating mouse it is anti-human-IgM monoclonal antibody The HCMV recombinant antigen S and label of the ELISA Plate and horseradish peroxidase-labeled of ELISA Plate and coating HCMV recombinant antigen S Anti-human-IgA the monoclonal antibody of mouse；In addition, further include sample diluent, cleaning solution, developing solution, terminate liquid, negative control and Positive control.
A kind of preparation process with recombinant antigen detection human cytomegalovirus IgM and IgA antibody, the coating mouse is anti-human- The preparation method of the ELISA Plate of IgM monoclonal antibody is: being resisted mouse with pH9.0,0.05M carbonate buffer solution (coating buffer) People-IgM monoclonal antibody is diluted to suitable concentration, is then coated with again on the ELISA Plate of blank, reaches optimal coating effect；
The preparation method of the recombinant protein S of the horseradish peroxidase-labeled is:
The acquisition of A.HCMV recombinant protein S
(1) according to the HCMV gene order announced in NCBI, determine that HCMV (pp150+gp52+pUL57) gene encodes Sequence, the aa545-601 sequence of the aa297-433 and pUL57 of aa862-1048, gp52 of synthetic gene pp150, three G is used between kind gene4S link son connection, is cloned on PET28a carrier, and convert e. coli bl21, lures through IPTG After leading, mycoprotein is identified through SDS-PAGE；
(2) the Escherichia coli mass propgation of HCMV recombinant protein S will be expressed, thallus is collected in induction, and ultrasonication is simultaneously centrifuged Supernatant is collected, obtains a large amount of HCMV recombinant protein S after nickel column chromatographic purifying；
B. horseradish peroxidase-labeled HCMV recombinant antigen S:
(1) 5mg HRP is weighed to be dissolved in 1ml distilled water；
(2) the 0.1M sodium periodate (NaIO that 0.2ml newly matches is added4) solution, it is protected from light stirring 20min at room temperature, then fills Enter in bag filter, to 1mM, the sodium-acetate buffer of pH4.4 is dialysed, and 4 DEG C overnight；
(3) add 20 μ l 0.2M, pH9.5 carbonate buffer solution, pH is made to be increased to 9.0-9.5,10mg weight is added immediately after Group antigen S, room temperature, which is protected from light, is gently mixed 2h；
(4) the 4mg/ml sodium borohydride (NaBH for adding 0.1ml newly to match4) solution, it mixes, places 4 DEG C of 2h, be then charged into dialysis It in bag, dialyses to 0.15M pH7.4PBS, 4 DEG C overnight；
(5) isometric saturated ammonium sulfate is added dropwise under stiring, sets 4 DEG C of 1h；
3000r/min is centrifuged 30min, abandons supernatant, and precipitating is washed twice with semi-saturation ammonium sulfate, and last sediment is dissolved on a small quantity The PBS buffer solution of 0.15M, pH 7.4 is dialysed, and (is detected with Nai Shi reagent) after removing ammonium ion, 10000r/min is centrifuged 30min Removal precipitating, supernatant is enzyme conjugates, after packing, stored frozen；
It is described coating recombinant antigen S ELISA Plate preparation method be with coating mouse it is anti-human-IgM monoclonal antibody method；
The preparation method of the anti-human-IgA monoclonal antibody of the mouse of the horseradish peroxidase-labeled is: using sodium periodate Antibody and HRP are coupled by method, and specific method is the same as recombinant antigen S labeling method.
As a further improvement of the present invention, the positive control that the detection HCMV IgM is used is IgM antibody containing HCMV Human serum；Negative control is the serum for being clinically diagnosed as HCMV IgM feminine gender；The positive that the detection HCMV IgA is used Control is the human serum of the IgA antibody containing HCMV；Negative control is the serum for being clinically diagnosed as HCMV IgA feminine gender；Sample is dilute Release the phosphate buffer that liquid is the 0.01M pH7.4 containing 10% fetal calf serum；Cleaning solution is the phosphoric acid containing 0.05%Tween20 Salt buffer；Developing solution is TMB-H2O2Urea liquid；Terminate liquid is the sulfuric acid solution of 2M.
As a further improvement of the present invention, the amino acid sequence of the recombinant protein S is as shown in SEQ No.1.
As a further improvement of the present invention, the nucleotide sequence of the recombinant protein S is as shown in SEQ No.2.
Compared with prior art, the beneficial effects of the present invention are: the present invention can accurate quick diagnosis go out sample whether be HCMV IgM is positive；The present invention is compared with domestic and international similar kit, and specificity is 100%, sensibility 99.3%.Due to this Invention combines detection IgM and IgA, and specificity and sensibility are above domestic certain company production HCMV IgM kit (respectively It is 97% and 89.1%), especially sensibility greatly improves.
Detailed description of the invention
Gene electrophoretogram that Fig. 1 is recombinant protein S (swimming lane 1 is marker, swimming lane 2,3 attach most importance to histone gene)；
(swimming lane 1 is marker to the SDS-PAGE electrophoresis that Fig. 2 is recombinant protein S, and swimming lane 2,3 is that expressing in series recombinates egg White BL21).
Specific embodiment
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment Only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects It encloses.
It please refers to shown in Fig. 1-2:
Embodiment 1
Human cytomegalovirus IgM antibody, cytomegalovirus IgM antibody detection examination of the present invention are detected using prize law Agent box, including coating mouse it is anti-human-ELISA Plate of IgM monoclonal antibody, HRP-S, cleaning solution, sample diluent, developing solution, termination Liquid, negative control and positive control.
One, the preparation of HCMV recombinant antigen S
1, dominant antigen epitope is determined
According to the HCMV gene order announced in NCBI, HCMV (pp150+gp52+pUL57) gene code sequence is determined Column are analyzed by software and determine genetic superiority epitope, then aa862-1048, gp52 of synthetic gene pp150 The aa545-601 sequence of aa297-433 and pUL57 uses G between three kinds of genes4S link son connection.
2. target DNA fragment is inserted into expression vector
By PET28 carrier and target DNA double digestion, digestion products run gel extraction after electrophoresis, connect through T4DNA ligase After be transferred to e. coli bl21, be coated on the LB plate containing ammonia benzyl antibiotic, 37 DEG C of inversion overnight incubations, next day picking is flat Single colonie on plate, which is set in LB Liquid Culture bacterium, to be grown, and plasmid, digestion identification are then extracted.
3. recombinant protein S inducing expression
Strain inoculated containing recombinant plasmid is cultivated into the LB liquid medium containing ammonia benzyl antibiotic, it is raw to bacterial strain It grows to OD value and adds IPTG inducing expression 5h for 0.5 or so, collect thallus, ultrasonication, after centrifugation, supernatant chromatographs pure through nickel column Change, then SDS-PAGE is identified.
Two, horseradish peroxidase-labeled recombinant antigen S
1. weighing 5mg HRP to be dissolved in 1ml distilled water；
2. the 0.1M sodium periodate (NaIO that 0.2ml newly matches is added4) solution, it is protected from light stirring 20min at room temperature, is then charged into In bag filter, to 1mM, the sodium-acetate buffer of pH4.4 is dialysed, and 4 DEG C overnight；
3. adding 20 μ l, 0.2M, pH9.5 carbonate buffer solution, pH is made to be increased to 9.0-9.5,10mg weight is added immediately after Group antigen S, room temperature, which is protected from light, is gently mixed 2h；
4. the 4mg/ml sodium borohydride (NaBH for adding 0.1ml newly to match4) solution, it mixes, places 4 DEG C of 2h, be then charged into dialysis It in bag, dialyses to 0.15M pH7.4PBS, 4 DEG C overnight；
5. isometric saturated ammonium sulfate is added dropwise under stiring, 4 DEG C of 1h are set；
6. 3000r/min is centrifuged 30min, supernatant is abandoned, precipitating is washed twice with semi-saturation ammonium sulfate, and last sediment is dissolved in The PBS buffer solution dialysis of a small amount of 0.15M, pH 7.4 (is detected), 10000r/min centrifugation after removing ammonium ion with Nai Shi reagent 30min removal precipitating, supernatant is enzyme conjugates, after packing, stored frozen.
Three, measurement mouse it is anti-human-the most suitable peridium concentration of IgM monoclonal antibody
Utilize the optimum concentration of chessboard method measurement coated antibody
1. antibody stoste (0.05 μ g/ μ l) is diluted to 0.04 μ g/ml, 0.03 μ g/ml, 0.02 μ g/ respectively with coating buffer ml、0.01μg/ml、0.009μg/ml、0.008μg/ml、0.007μg/ml、0.006μg/ml、0.005μg/ml、0.004μg/ Ml, every hole are added 100 μ l and are coated with, are washed；
2. strong positive serum, weak positive serum and negative serum dilution are diluted by 10 times of ascending series and are loaded, Washing；
3. the HRP-S of optimum concentration is added, 37 DEG C are incubated for and wash；
4. adding developing solution colour developing, enzyme standard liquid after terminate liquid is added to read OD450 value；
5. select the OD value of strong positive serum between 0.8-1.0, coated antibody of the OD value less than 0.1 of negative serum Dilution is 0.007 μ g/ml as optimum concentration.
Four, mouse it is anti-human-IgM monoclonal antibody enzyme reaction plate coating
With pH9.0,0.05M carbonate buffer solution (coating buffer) by mouse it is anti-human-IgM monoclonal antibody is diluted to albumen Matter content is 0.007 μ g/ml, and 100 μ l are added in ELISA Plate hole, sets 4 DEG C overnight, discards liquid in hole, clap after PBST board-washing 5 times It is dry；Then it is closed with the PBS containing 10% fetal calf serum, 37 DEG C of placement 1h (every 100 μ l of hole), PBST board-washing 5 times.
Five, HRP-S optimum concentration measures
1. be coated with mouse it is anti-human-IgM monoclonal antibody (0.007 μ g/ml)；
2. strong positive serum, weak positive serum and negative serum dilution are diluted by 10 times of ascending series and are loaded, Washing；
3. being added in coating hole after recombinant antigen S (2.4 μ g/ μ l) is made a series of dilutions, it is incubated for washing；
4. adding developing solution colour developing, terminate liquid terminates reaction, measures OD450 value, selecting the OD value of strong positive serum is 0.8- Between 1.0, the dilution of HRP-S of the OD value less than 0.1 of negative serum is 0.01 μ g/ml as optimum concentration.
Six, the specific operation method is as follows for HCMV-IgM detection kit:
1) the reagent equilibrium at room temperature 15min or more that will be stored in cold storage environment；
2) concentrated cleaning solution press 1:40 dilution proportion, shake up it is spare, as apply cleaning solution；
3) take 100 μ l of sample diluent (2 drop) in the reacting hole of each ELISA Plate, blank and yin, yang control wells are not added；
4) it goes 10 μ l samples to be measured to be added in reacting hole, is blown and beaten repeatedly with pipettor, liquid becomes blue, yin, yang in hole Property control wells respectively add 100 μ l positive and negative to compare, blank well is vacant；
5) it mixes well sample reaction plate oscillation 30s, sets 37 DEG C of reaction 20min；
6) ELISA Plate is taken out, is washed 5 times, is patted dry with using cleaning solution；
7) 50 μ l of enzyme conjugates (1 drop) is added in every hole, and blank well is not added, and gently oscillation mixes, and sets 37 DEG C of reaction 20min；
8) ELISA Plate is taken out, is washed 5 times, is patted dry with using cleaning solution；
9) 50 μ l of substrate solution A, 50 μ l of substrate solution B (each 1 drop) is added, blank well is not added, and gently oscillation mixes, and sets 37 DEG C React 10min；
10) add 50 μ l (1 drop) terminate liquid after withdrawing plate, blank well is not added, in microplate reader, at wavelength 450nm, with sky Each hole OD value is surveyed after white control wells zeroing, it is as positive if more than 2.1 multiples of defined negative control OD value.
Embodiment 2
Human cytomegalovirus IgA antibody is detected using indirect method.Cytomegalovirus IgA antibody detection examination of the present invention Agent box, ELISA Plate, the anti-human-IgA monoclonal antibody of HRP- mouse, concentrated cleaning solution, sample including being coated with HCMV recombinant antigen S are dilute Release liquid, substrate solution A, substrate solution B, terminate liquid, negative control and positive control.One, the preparation of HCMV recombinant antigen S
Method is same as above.
Two, the anti-human-IgA monoclonal antibody of horseradish peroxidase-labeled mouse
1. weighing 5mg HRP to be dissolved in 1ml distilled water；
2. the 0.1M sodium periodate (NaIO that 0.2ml newly matches is added4) solution, it is protected from light stirring 20min at room temperature, is then charged into In bag filter, to 1mM, the sodium-acetate buffer of pH4.4 is dialysed, and 4 DEG C overnight；
3. adding 20 μ l 0.2M, pH9.5 carbonate buffer solution, pH is made to be increased to 9.0-9.5, it is anti-that 10mg is added immediately after Body, room temperature, which is protected from light, is gently mixed 2h；
4. the 4mg/ml sodium borohydride (NaBH for adding 0.1ml newly to match4) solution, it mixes, places 4 DEG C of 2h, be then charged into dialysis It in bag, dialyses to 0.15M pH7.4PBS, 4 DEG C overnight；
5. isometric saturated ammonium sulfate is added dropwise under stiring, 4 DEG C of 1h are set；
6. 3000r/min is centrifuged 30min, supernatant is abandoned, precipitating is washed twice with semi-saturation ammonium sulfate, and last sediment is dissolved in The PBS buffer solution dialysis of a small amount of 0.15M, pH 7.4 (is detected), 10000r/min centrifugation after removing ammonium ion with Nai Shi reagent 30min removal precipitating, supernatant is enzyme conjugates, after packing, stored frozen.
Three, the most suitable peridium concentration of recombinant antigen S is measured
Utilize the optimum concentration of chessboard method measurement coated antibody
1. being added in coating hole after recombinant antigen S (2.4 μ g/ μ l) is made a series of dilutions, it is incubated for washing；
2. strong positive serum, weak positive serum and negative serum dilution are diluted by 10 times of ascending series and are loaded, Washing；
3. anti-human-IgA the monoclonal antibody of mouse of the horseradish peroxidase-labeled of optimum concentration is added, 37 DEG C are incubated for and wash It washs；
6. adding developing solution colour developing, enzyme standard liquid after terminate liquid is added to read OD450 value；
7. selecting the OD value of strong positive serum between 0.8-1.0, coating recombination of the OD value of negative serum less than 0.1 is anti- The dilution of former S is 0.01 μ g/ml as optimum concentration.
Four, recombinant antigen S enzyme reaction plate is coated with
It is 0.01 that recombinant antigen, which is diluted to protein content, with pH9.0,0.05M carbonate buffer solution (coating buffer) μ g/ml adds 100 μ l in ELISA Plate hole, sets 4 DEG C overnight, discards liquid in hole, pat dry after PBST board-washing 5 times；Then with containing The PBS of 10% fetal calf serum is closed, 37 DEG C of placement 1h (every 100 μ l of hole), and PBST board-washing 5 times.
Five, the anti-human-IgA monoclonal antibody optimum concentration measurement of the mouse of horseradish peroxidase-labeled
1. being coated with recombinant antigen S (0.01 μ g/ml)；
2. strong positive serum, weak positive serum and negative serum dilution are diluted by 10 times of ascending series and are loaded, Washing；
3. after the anti-human-IgA monoclonal antibody of the mouse of horseradish peroxidase-labeled (1.5 μ g/ μ l) is made a series of dilutions It is added in coating hole, is incubated for washing；
4. adding developing solution colour developing, terminate liquid terminates reaction, measures OD450 value, selecting the OD value of strong positive serum is 0.8- Between 1.0, the dilution of the OD value of negative serum less than the anti-human-IgA monoclonal antibody of the mouse of 0.1 horseradish peroxidase-labeled Degree is used as optimum concentration, is 0.008 μ g/ml.
Six, the specific operation method is as follows for HCMV-IgA detection kit:
1) the reagent equilibrium at room temperature 15min or more that will be stored in cold storage environment；
2) concentrated cleaning solution press 1:40 dilution proportion, shake up it is spare, as apply cleaning solution；
3) take 100 μ l of sample diluent (2 drop) in the reacting hole of each ELISA Plate, blank and yin, yang control wells are not added；
4) it takes 10 μ l samples to be measured to be added in reacting hole, is blown and beaten repeatedly with pipettor, liquid becomes blue, yin, yang in hole Property control wells respectively add 100 μ l positive and negative to compare, blank well is vacant；
5) it mixes well sample reaction plate oscillation 30s, sets 37 DEG C of reaction 20min；ELISA Plate is taken out, is washed with application Liquid washs 5 times, pats dry；
6) 50 μ l of enzyme conjugates (1 drop) is added in every hole, and blank well is not added, and gently oscillation mixes, and sets 37 DEG C of reaction 20min；
7) ELISA Plate is taken out, is washed 5 times, is patted dry with using cleaning solution；
8) 50 μ l of substrate solution A, 50 μ l of substrate solution B (each 1 drop) is added, blank well is not added, and gently oscillation mixes, and sets 37 DEG C React 10min；
9) add 50 μ l (1 drop) terminate liquid after withdrawing plate, blank well is not added, in microplate reader, at wavelength 450nm, with sky Each hole OD value is surveyed after white control wells zeroing, it is as positive if more than 2.1 multiples of defined negative control OD value.
The amino acid sequence (SEQ No.1) of heretofore described recombinant protein:
DDVVSPATSPLSMLSSASPSPAKSAPPSPVKGRGSRVGVPSLKPTLGGKAVVGRPPSVPVSGSAPGRLS GSSRAASTTPTYPAVTTVYPPSSTAKSSVSNAPPVASPSILKPGASAALQSRRSTGIAAVGSPVKSTTGMKTVAFDL SSPQKSGTGPQPGSAGMGGAKTPSDAVQNILQKIEKIKNTEGGGGSGSLSSLANAGGLHDDGPGLDNDIMNEPMGLG GLGGGGGGGGKKHDRGGGGGSGTRKMSSGGGGGDHDHGLSSKEKYEQHKITSYLTSKGGSGGGGGGGGGGLDRNSGN YFNDAKEESDSEDSVTFEFVPNTKKQKCGGGGGSGVPGGGAGGGGGRDVSGGPSDGLGGGRGGGGGGDSGGMMGRGG RMLGASVDRTYRLN
The DNA nucleotide sequence (SEQ No.2) of heretofore described recombinant protein
gatgatgtcgtgtcccctgccacatcgccgctgtccatgctttcgtcagcctctccgtccccggccaa gagtgcccccccgtctccggtgaaaggccggggcagccgcgtcggtgttccttccttgaaacctactttgggcggc aaggcggtggtaggtcgaccgccctcggtccccgtgagcggtagcgcgccgggtcgcctgtccggcagcagccggg ccgcctcgaccacgccgacgtatcccgcggtaaccaccgtttacccaccgtcgtctacggccaaaagcagcgtatc gaatgcgccgcctgtggcctccccctccatcctgaaaccgggggcgagcgc ggctttgcaa
tcacgccgctcgacggggatcgccgccgtaggttcccccgtcaagagcacgacgggcatgaaaacggt ggctttcgacttatcgtcgccccagaagagcggtacggggccgcaaccgggttctgccggcatggggggcgccaaa acgccgtcggacgccgtgcagaacatcctccaaaagatcgagaagattaagaacacggagggaggcggaggtagtg gcagcctctcttcgctggctaatgccggcggtctgcacgacgacggcccgggtctggataacgatattatgaacga gcccatgggtctcggcggtctgggaggaggtggcggcggtggcggcaagaagcacgaccgcggtggcggcggtggt tccggtacgcggaaaatgagcagcggtggcggcggcggtgatcacgaccacggtctttcctccaaggaaaaatacg agcagcacaagatcaccagctacctgacgtccaaaggtggatcgggcggcggcggcggaggaggaggcggcggttt ggatcgcaactccggcaattacttcaacgacgcgaaagaggagagcgacagcgaggattctgtaacgttcgagttc gtccctaacaccaagaagcaaaagtgcggcggaggcggaggtagt
ggggttccgggcggcggtgctggcgggggtggtggacgggacgtgagcgggggcccgagcgacggtct gggcggcggtcgtggtggtgggggtggcggggattccgggggaatgatggggcgcggcggtcgcatgctgggcgct agcgtggaccgtacctatcggctcaat
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding And modification, the scope of the present invention is defined by the appended.

Claims (5)

1. a kind of ELISA kit with recombinant antigen detection human cytomegalovirus IgM and IgA antibody, it is characterized in that: including packet By mouse it is anti-human-ELISA Plate, the horseradish peroxidase-labeled of the ELISA Plate of IgM monoclonal antibody and coating HCMV recombinant antigen S Anti-human-IgA the monoclonal antibody of mouse of HCMV recombinant antigen S and label, sample diluent, cleaning solution, developing solution, terminate liquid, Negative control and positive control.
2. a kind of preparation process with recombinant antigen detection human cytomegalovirus IgM and IgA antibody, it is characterized in that: the coating Mouse is anti-human-preparation method of the ELISA Plate of IgM monoclonal antibody is: with pH9.0,0.05M carbonate buffer solution by mouse it is anti-human- IgM monoclonal antibody is diluted to suitable concentration, is then coated with again on the ELISA Plate of blank, reaches optimal coating effect；
The preparation method of the recombinant protein S of the horseradish peroxidase-labeled is:
The acquisition of A.HCMV recombinant protein S
(1) according to the HCMV gene order announced in NCBI, HCMV (pp150+gp52+pUL57) gene coded sequence is determined, The aa545-601 sequence of the aa297-433 and pUL57 of aa862-1048, gp52 of synthetic gene pp150, three kinds of bases G is used because between4S link son connection, is cloned on PET28a carrier, and convert e. coli bl21, after IPTG is induced, Mycoprotein is identified through SDS-PAGE；
(2) the Escherichia coli mass propgation of HCMV recombinant protein S will be expressed, thallus is collected in induction, and ultrasonication is simultaneously collected by centrifugation Supernatant obtains a large amount of HCMV recombinant protein S after nickel column chromatographic purifying；
B. horseradish peroxidase-labeled HCMV recombinant antigen S:
(1) 5mg HRP is weighed to be dissolved in 1ml distilled water；
(2) the 0.1M sodium periodate (NaIO that 0.2ml newly matches is added4) solution, it is protected from light stirring 20min at room temperature, is then charged into dialysis In bag, to 1mM, the sodium-acetate buffer of pH4.4 is dialysed, and 4 DEG C overnight；
(3) add 20 μ l 0.2M, pH9.5 carbonate buffer solution, pH is made to be increased to 9.0-9.5, it is anti-that 10mg recombination is added immediately after Former S, room temperature, which is protected from light, is gently mixed 2h；
(4) the 4mg/ml sodium borohydride NaBH for adding 0.1ml newly to match4Solution mixes, places 4 DEG C of 2h, be then charged into bag filter, right 0.15M pH7.4PBS dialysis, 4 DEG C overnight；
(5) isometric saturated ammonium sulfate is added dropwise under stiring, sets 4 DEG C of 1h；
3000r/min is centrifuged 30min, abandons supernatant, and precipitating is washed twice with semi-saturation ammonium sulfate, and last sediment is dissolved on a small quantity The PBS buffer solution of 0.15M, pH 7.4 is dialysed, and (is detected with Nai Shi reagent) after removing ammonium ion, 10000r/min is centrifuged 30min Removal precipitating, supernatant is enzyme conjugates, after packing, stored frozen；
It is described coating recombinant antigen S ELISA Plate preparation method be with coating mouse it is anti-human-IgM monoclonal antibody method.
The preparation method of the anti-human-IgA monoclonal antibody of the mouse of the horseradish peroxidase-labeled is: using Over-voltage protection will Antibody and HRP are coupled, and specific method is the same as recombinant antigen S labeling method.
3. a kind of ELISA for detecting human cytomegalovirus IgM and IgA antibody with recombinant antigen according to claim 1 is tried Agent box, it is characterised in that: the positive control that the detection HCMV IgM is used is the human serum of the IgM antibody containing HCMV；It is negative right According to the serum to be clinically diagnosed as HCMV IgM feminine gender；The positive control that the detection HCMV IgA is used is IgA containing HCMV The human serum of antibody；Negative control is the serum for being clinically diagnosed as HCMV IgA feminine gender；Sample diluent is containing 10% tire ox The phosphate buffer of the 0.01M pH7.4 of serum；Cleaning solution is the phosphate buffer containing 0.05%Tween20；Developing solution For TMB-H2O2Urea liquid；Terminate liquid is the sulfuric acid solution of 2M.
4. a kind of ELISA for detecting human cytomegalovirus IgM and IgA antibody with recombinant antigen according to claim 1 is tried Agent box and its preparation process, it is characterised in that: the amino acid sequence of the recombinant protein S is as shown in SEQ No.1.
5. a kind of ELISA for detecting human cytomegalovirus IgM and IgA antibody with recombinant antigen according to claim 1 is tried Agent box and its preparation process, it is characterised in that: the nucleotide sequence of the recombinant protein S is as shown in SEQ No.2.
CN201810968554.9A 2018-08-23 2018-08-23 A kind of ELISA kit and its preparation process with recombinant antigen detection human cytomegalovirus IgM and IgA antibody Withdrawn CN109298181A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810968554.9A CN109298181A (en) 2018-08-23 2018-08-23 A kind of ELISA kit and its preparation process with recombinant antigen detection human cytomegalovirus IgM and IgA antibody

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810968554.9A CN109298181A (en) 2018-08-23 2018-08-23 A kind of ELISA kit and its preparation process with recombinant antigen detection human cytomegalovirus IgM and IgA antibody

Publications (1)

Publication Number Publication Date
CN109298181A true CN109298181A (en) 2019-02-01

Family

ID=65165621

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810968554.9A Withdrawn CN109298181A (en) 2018-08-23 2018-08-23 A kind of ELISA kit and its preparation process with recombinant antigen detection human cytomegalovirus IgM and IgA antibody

Country Status (1)

Country Link
CN (1) CN109298181A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111413496A (en) * 2020-05-18 2020-07-14 博奥赛斯（天津）生物科技有限公司 Novel coronavirus IgM/IgG antibody chemiluminescence method detection kit
CN113788880A (en) * 2021-09-12 2021-12-14 南京珀尔泰生物技术有限公司 Purification method of human cytomegalovirus recombinant antigen

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111413496A (en) * 2020-05-18 2020-07-14 博奥赛斯（天津）生物科技有限公司 Novel coronavirus IgM/IgG antibody chemiluminescence method detection kit
CN111413496B (en) * 2020-05-18 2023-05-05 天津博奥赛斯生物科技股份有限公司 Novel coronavirus IgM/IgG antibody chemiluminescence method detection kit
CN113788880A (en) * 2021-09-12 2021-12-14 南京珀尔泰生物技术有限公司 Purification method of human cytomegalovirus recombinant antigen
CN113788880B (en) * 2021-09-12 2023-11-28 南京珀尔泰生物技术有限公司 Purification method of human cytomegalovirus recombinant antigen

Similar Documents

Publication Publication Date Title
Ndifuna et al. Recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus
CN103172752B (en) Mycoplasma bovis diagnosis reagent and its application
CN110221065B (en) Poultry bursa of sliding mycoplasma indirect ELISA detection kit
CN111381032A (en) Indirect ELISA detection method for detecting porcine epidemic diarrhea virus antibody and kit thereof
CN1307423C (en) Method for making recombinant antigen pp 65 envelope enzyme-linked reaction plate and ELISA test kit
CN113336845B (en) Porcine single B cell antibody of PRRSV nucleocapsid protein and competitive ELISA antibody detection kit
US20230358757A1 (en) Antigen for 2019 novel coronavirus and detection use thereof
CN113265006B (en) Fusion protein 3AN for capturing PRRSV nucleocapsid protein antibody and application thereof
CN108680744A (en) It is a kind of detection novel duck reovirus antibody indirect ELISA testing kit and application
CN111751553A (en) Peste des petits ruminants virus H protein antibody iELISA detection method and application
CN109298181A (en) A kind of ELISA kit and its preparation process with recombinant antigen detection human cytomegalovirus IgM and IgA antibody
CN102221616B (en) Indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic kit of mycoplasma gallisepticum
CN106518989B (en) Polypeptide for detecting porcine Delta coronavirus antibody, preparation method and application thereof
CN113943354B (en) Recombinant feline herpesvirus gB protein antigen and application thereof in antibody diagnosis and vaccine preparation
CN108918869B (en) Application of fiber2 protein and recombinant protein thereof in detecting serum type 4 avian adenovirus antibody
CN103499693A (en) Competitive Alpha LISA (linked immuno sorbent assay) detection kit for classical swine fever virus (CSFV) antibody and detection method thereof
CN105061602A (en) Fusion protein for detecting anti-ETEC (enterotoxigenic escherichia coil) antibody of pigs, as well as preparation method and application thereof
CN103499689A (en) Competitive Alpha LISA (linked immuno sorbent assay) detection kit for porcine circovirus (PCV) 2 antibody and detection method thereof
CN109374886B (en) Infectious bovine rhinotracheitis virus antibody detection kit and application thereof
CN107576791A (en) One boar lawsonia intracellularis ELISA detection kit
CN104569387B (en) A kind of ELISA detects human serum HCMV pp150 IgM antibody kits
CN104502605A (en) ELISA kit for detecting canine toxoplasmosis IgM antibody and preparation method of ELISA kit
CN107219365A (en) A kind of chemiluminescence detection kit based on foot and mouth disease virus 3B neoepitope Westerns
CN103293311A (en) Indirectly competitive ELISA (Enzyme Linked Immunosorbent Assay) immune kit for detecting porcine reproductive and respiratory syndrome virus
CN101303349A (en) Cysticercosis cellulosae indirect ELISA testing kit and preparation method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WW01 Invention patent application withdrawn after publication

Application publication date: 20190201

WW01 Invention patent application withdrawn after publication