CN109738631A - A kind of preparation method of human cytomegalovirus IgM antibody Test paper - Google Patents
A kind of preparation method of human cytomegalovirus IgM antibody Test paper Download PDFInfo
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- CN109738631A CN109738631A CN201910051438.5A CN201910051438A CN109738631A CN 109738631 A CN109738631 A CN 109738631A CN 201910051438 A CN201910051438 A CN 201910051438A CN 109738631 A CN109738631 A CN 109738631A
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Abstract
The present invention provides a kind of preparation methods of human cytomegalovirus IgM antibody Test paper, the specific steps are as follows: (1) first manufactures test paper loading plate and test paper；Test paper is made of drop sample pad, gold conjugation pad, nitrocellulose filter and sample suction pad；(2) drop sample pad is pasted on to one end of test paper loading plate, one end of drop sample pad is bonded gold conjugation pad, and the other end of gold conjugation pad is bonded nitrocellulose filter, and the other end of nitrocellulose filter is bonded sample suction pad；Nitrocellulose filter is coated with the detection T line being separated from each other and Quality Control C line, and detection T line is anti-IgM μ chain monoclonal antibody, and Quality Control C line is people's CMV pp150-gB-pp65 fusion protein IgM antibody；Antigen pp150-gB-pp65 fusion protein containing label human cytomegalovirus-specific in gold conjugation pad.The technical problem to be solved in the present invention is to provide a kind of verification and measurement ratio is higher, effect is more preferable, the preparation method for the human cytomegalovirus IgM antibody Test paper that required time is short, expense is low.
Description
Technical field
The present invention relates to a kind of preparation methods of test paper, the especially preparation of human cytomegalovirus IgM antibody Test paper
Method.
Background technique
Human cytomegalovirus detection in clinical practice is generally used in addition to using the method for hospital's blood sampling oil (gas) filling device analysis
The human cytomegalovirus IgM antigen Test paper of monoclonal antibody production, but protein-specific used in this method is lower, therefore
Recall rate is low, and the time used is long, and effect is poor, it is impossible to be used in accurately diagnosis.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of verification and measurement ratio is higher, effect is more preferable, and required time is short, expense is low
Human cytomegalovirus IgM antibody Test paper preparation method.
In order to solve the above technical problems, the present invention provides the preparation method of human cytomegalovirus IgM antibody Test paper,
Specific step is as follows: (1) first manufacturing test paper loading plate and test paper；The test paper loading plate is strip PVC board, the examination
Paper is made of drop sample pad, gold conjugation pad, nitrocellulose filter and sample suction pad；The drop sample pad is by glass fibre membrane or nothing
Woven fabric is made, and the sample suction pad is made of absorbent filter；(2) the drop sample pad is pasted on to the test paper loading plate
One end, one end bonding of drop sample pad gold conjugation pad, the other end bonding of the gold conjugation pad
The nitrocellulose filter, the other end connection of the nitrocellulose filter sample suction pad；The cellulose nitrate
Plain film is coated with the detection T line being separated from each other and Quality Control C line, and the detection T line is anti-IgM μ chain monoclonal antibody, described
Quality Control C line is people's CMV pp150-gB-pp65 fusion protein IgM antibody；Contain mark in the gold conjugation pad
Remember the antigen pp150-gB-pp65 fusion protein of human cytomegalovirus-specific.
This programme institute sortilin is the high albumen of high specificity, binding force, it is contemplated that Percentage bound up to 95% or more, if by
Cause Percentage bound below standard in certain unknowable reasons, then can be solved by replacement albumen, pp28 (UL99) or gH can be used
(gpUL75) one of pp150-gB-pp65 fusion protein is substituted；Pp28 can be increased in pp150-gB-pp65 fusion protein
(UL99) and the one or two of gH (gpUL75).
Preferably, as further Optimal improvements, the preparation method of the test paper is following steps:
(1) recombination building and fusion protein separation and Extraction, 1. chain reaction primer of polymerase, according to target fragment
DNA sequence dna, designs three pairs of primers (P1, P2), (P3, P4) and (P5, P6), P1 and P2 for expanding pp150 genetic fragment, P3 and
P4 has I enzyme of Nde I and Xho for expanding pp65 genetic fragment, P1 and P6 primer for expanding gB genetic fragment, P5 and P6
Enzyme site, P2 and P3, the sequence of P4 and P5 primer are complementary, and P2, P3 correspond to jointly on the 3 ' ends and gB of the above-mentioned segment of pp150
5 ' end sequences of segment are stated, P4, P5 correspond to 3 ' ends of the above-mentioned segment of gB and 5 ' end sequences of the above-mentioned segment of pp65 jointly
Column；2. the clone of target gene takes 1 μ l of virus-culturing fluid, 10 × Pfu high fidelity enzyme buffer, 5 μ l, concentration 2mmol/L is added
Each 0.5 μ l of 2 μ l, Pfu high fidelity enzyme of 5 μ l, P1, P2 primer of dNTPS adds sterilizing distilled water to 50 μ l and mixes well；React item
Part is 94 DEG C of 30s of denaturation, and anneal 65 DEG C of 30s, 72 DEG C of 60s of renaturation, carries out 30 circulations, then 72 DEG C of extension 10min；P3, P4 and
P5, P6 primer cloning process are same as above；Then the pp150 segment that is obtained with first round PCR amplification, gB (UL55) are template, are added
Primer P1 and P4 expand pp150-gB segment, and reaction condition is pp150 segment, gB is respectively 1 μ l, and 10 × Pfu high-fidelity is added
Each 0.5 μ l of 2 μ l, Pfu high fidelity enzyme of dNTPS5 μ l, P1, P4 primer of enzyme buffer liquid 5 μ l, concentration 2mmol/L adds sterilizing is double to steam
Water is mixed well to 50 μ l, and reaction condition is 94 DEG C of 30s of denaturation, and anneal 65 DEG C of 30s, 72 DEG C of 90s of renaturation, carries out 30 circulations,
72 DEG C of extension 10min again obtain concatenated target gene recombinant fragment pp150-gB；It is obtained later with the first and second wheel PCR amplification
Pp150-gB segment, pp65 segment be template, primer P1 and P6 is added, expands pp150-gB-pp65 segment, reaction condition is same
On, obtain concatenated target gene recombinant fragment pp150-gB-pp65；
3. the building and identification of pET28a-pp150-gB-pp65 recombinant plasmid, by the PCR product pp150-gB- of purifying
Pp65 NdeI and XhoI digestion, and connect with by the pET28a prokaryotic expression carrier equally handled, it is transformed into Escherichia coli
In TOP10, Plasmid DNA is extracted, carries out digestion identification, and be transformed into e. coli bl21；
4. pET28a-pp150-gB-pp65 is recombinated matter by the inducing expression of pET28a-pp150-gB-pp65 fusion protein
The BL21 bacterium solution of grain is added in L.B culture solution with the ratio of 1:100, and 37 DEG C IPTG is added after shaken cultivation 2 hours to dense eventually
Degree be 1mmol/L, 37 DEG C induction 3 hours after thalline were collected by centrifugation, be resuspended in 2X sample buffer, 100 DEG C are boiled, carry out
SDS-PAGE electrophoretic analysis；
5. expressing the purifying of albumen, thalline were collected by centrifugation for the bacterial culture fluid after taking induction, and Ni is utilized after ultrasonication
Protein purification column is purified, and collects eluting peak albumen respectively, and carry out PAGE gel electrophoretic analysis；
6. the antigenicity analysis of recombinant protein cuts glue progress by the mycoprotein of recombination after PAGE gel electrophoresis
Protein electrotransfer goes to destination protein electricity on pvdf membrane, closes pvdf membrane with the PBS of the defatted milk of 50g/L containing mass concentration,
4 DEG C overnight, and PBS is washed film 3 times, dilutes positive serum with confining liquid 1:20, with pvdf membrane after 37 DEG C of common incubation 2h, uses PBS
Film is washed, the Goat anti-Human IgG of horseradish peroxidase-labeled is added, 37 DEG C of incubation 1h, TMB develops the color after PBS washes film, until purpose item
Reaction is terminated when band develops the color clear；
(2) manufacture of gold conjugation pad prepares diameter with gold chloride-trisodium citrate reduction method as the glue of 30-50nm
Body gold solution takes 100ml colloidal gold liquid to be placed in beaker, pH8.0 is adjusted to 0.2MK2CO3, by 100ml colloid after the completion of preparation
0.5-2mg human cytomegalovirus pp150-gB-pp65 antigen is added in gold solution, is stirred at room temperature 2 hours, and it is pure that 1% ox blood is added
Protein B SA, 1% polyethylene glycol PEG20000 close 20min, and 12000r/m is centrifuged 30 minutes, supernatant is abandoned, with colloidal gold working solution
It redissolves to 100ml, is layered on glass fibre membrane or non-woven fabrics with spreading the ratio uniform of 20cm2 by 1ml solution, then set drying room,
20-25 DEG C of temperature, humidity is 2-5 hours dry less than 30%, and gold conjugation pad is made；
(3) the detection T line of nitrocellulose filter and the manufacture of Quality Control C line, by anti-IgM μ chain McAb 50mmol/L Tris-
HCl solution (pH 7.8) is diluted to 4 μ g/ml, is carried out with spray film instrument in nitrocellulose filter lower part with the parameter of 1-1.5 μ l/cm
Scribing line, coating detection T line, while in nitrocellulose filter top coating pp150-gB-pp65 fusion protein IgM antibody as matter
C line is controlled, by nitrocellulose filter at 20-25 DEG C of drying temperature after scribing line, humidity is 2-5 hours dry less than 30%；
(4) assembly of Test paper, in hothouse, 20-25 DEG C of temperature, humidity takes test paper loading plate less than 40%, will
The middle part that coated nitrocellulose filter has been placed on test paper loading plate is pasted, and is overlapped on the detection tip side of nitrocellulose filter
It is pasted at the 1/3 of gold conjugation pad, is overlapped in the gold conjugation pad other side and paste drop sample pad, take the 1/ of gold conjugation pad
5；Sample suction pad is overlapped in nitrocellulose filter Quality Control C line side, takes the 1/10 of sample suction pad；Eventually form test paper.
Compared with prior art, the invention has the advantages that colloidal gold immune chromatography test method of the present invention compared with
Original method has stronger amplification, and gold is marked particle marker on pp150-gB-pp65 fusion protein by the present invention, in human body
HCMVpp150-gB-pp65 fusion protein antibody is combined with pp150-gB-pp65 fusion protein, thus indirectly with gold mark
Burl closes, and the antigenic determinant on fusion protein is more, therefore increases the Percentage bound of antibody and antigen, keeps verification and measurement ratio higher,
Effect is more preferable.
The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
Detailed description of the invention
Fig. 1 is the schematic diagram of human cytomegalovirus IgM antibody Test paper of the invention.
Fig. 2 is recombination building and fusion protein separation schematic diagram of the invention.
Fig. 3 is test paper disassembled schematic of the invention.
Fig. 4 is test paper testing principle schematic diagram of the invention.
Specific embodiment
To enable the above objects, features and advantages of the present invention to be clearer and more comprehensible, human cytomegalovirus of the invention hereafter is sick
The preparation method and its manufacturing method of malicious IgM antibody Test paper will cooperate appended relevant drawings with preferred embodiment, make detailed
It describes in detail bright.
In the description of the present invention, it should be noted that term " on ", "lower", "vertical", "horizontal", "inner", "outside" etc.
The orientation or positional relationship of instruction is to be based on the orientation or positional relationship shown in the drawings, and is merely for convenience of the description present invention and letter
Change description, rather than the device or element of indication or suggestion meaning must have a particular orientation, with specific orientation construct and
Operation, therefore be not considered as limiting the invention.
The preparation method of the human cytomegalovirus IgM antibody Test paper of the present embodiment, the specific steps are as follows: (1) first make
Make test paper loading plate 1 and test paper；Test paper loading plate 1 is strip PVC board, and test paper is by drop sample pad 2, gold conjugation pad 3, nitric acid
Cellulose membrane 4 and sample suction pad 5 form；Drop sample pad 2 is made of glass fibre membrane or non-woven fabrics, and sample suction pad 5 is made of absorbent filter；
(2) drop sample pad 2 is pasted on to one end of test paper loading plate 1, one end of drop sample pad 2 is bonded gold conjugation pad 3, and colloidal gold combines
The other end of pad 3 is bonded nitrocellulose filter 4, and the other end of nitrocellulose filter 4 is bonded sample suction pad 5；Nitrocellulose filter 4 wraps
There are the detection T line 6 and Quality Control C line 7 being separated from each other, detection T line 6 is anti-IgM μ chain monoclonal antibody, and Quality Control C line 7 is that people is huge
Cell virus pp150-gB-pp65 fusion protein IgM antibody；It is special containing label human cytomegalovirus in gold conjugation pad 3
The antigen pp150-gB-pp65 fusion protein of property.
Two kinds or a kind of in pp28 (UL99) and/or gH (gpUL75) substitution pp150, gB and pp65 can be used.
The application method of human cytomegalovirus IgM antibody Test paper: it by tested serum or plasma equilibrium to room temperature, will examine
Test paper is laid flat, and 5-20 μ l test sample is added in sample pad, adds the sample diluting liquid of 50-100 μ l, keeps colloidal gold molten
It solves and is chromatographed on nitrocellulose filter, the appearance situation then with the naked eye directly observed Quality Control C line in 10 minutes, detect T line,
And determine testing result.
In conclusion gold is marked particle marker on pp150-gB-pp65 fusion protein by the present invention, in human body
HCMVpp150-gB-pp65 fusion protein antibody is combined with pp150-gB-pp65 fusion protein, thus indirectly with gold mark
Burl closes, and the antigenic determinant on fusion protein is more, therefore increases the Percentage bound of antibody and antigen, keeps verification and measurement ratio higher,
Effect is more preferable.Therefore, the present invention has substantive distinguishing features outstanding and significant progress.
The above description is only a preferred embodiment of the present invention, is not intended to restrict the invention, for those skilled in the art
For member, the invention may be variously modified and varied.All within the spirits and principles of the present invention, it is made it is any modification,
Equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (4)
1. a kind of preparation method of human cytomegalovirus IgM antibody Test paper, it is characterised in that: specific step is as follows: (1) first
Manufacture test paper loading plate and test paper；The test paper loading plate is strip PVC board, and the test paper is by drop sample pad, colloidal gold
Bonding pad, nitrocellulose filter and sample suction pad composition；The drop sample pad is made of glass fibre membrane or non-woven fabrics, the suction
Sample pad is made of absorbent filter；(2) the drop sample pad is pasted on to one end of the test paper loading plate, the drop sample pad
One end bonding gold conjugation pad, the other end bonding of the gold conjugation pad nitrocellulose
Film, the other end bonding of the nitrocellulose filter sample suction pad；The nitrocellulose filter, which is coated with, mutually to be divided
From detection T line and Quality Control C line, the detection T line be anti-IgM μ chain monoclonal antibody, the Quality Control C line be people it is big and small
Cellular virus pp150-gB-pp65 fusion protein IgM antibody；It is special containing label human cytomegalovirus in the gold conjugation pad
Anisotropic antigen pp150-gB-pp65 fusion protein.
2. the preparation method of human cytomegalovirus IgM antibody Test paper according to claim 1, it is characterised in that: can
With one of pp28 (UL99) or gH (gpUL75) substitution pp150-gB-pp65 fusion protein.
3. the preparation method of human cytomegalovirus IgM antibody Test paper according to claim 2, it is characterised in that: can
Increase the one or two of pp28 (UL99) and gH (gpUL75) in pp150-gB-pp65 fusion protein.
4. the preparation method of human cytomegalovirus IgM antibody Test paper according to claim 3, it is characterised in that: institute
The preparation method for the test paper stated is following steps:
(1) recombination building and fusion protein separation and Extraction, 1. chain reaction primer of polymerase, according to the DNA sequence of target fragment
Column design three pairs of primers (P1, P2), (P3, P4) and (P5, P6), P1 and P2 and use for expanding pp150 genetic fragment, P3 and P4
In amplification gB genetic fragment, P5 and P6 are used to expand pp65 genetic fragment, P1 and P6 primer has I restriction enzyme site of Nde I and Xho,
P2 and P3, the sequence of P4 and P5 primer are complementary, and P2, P3 correspond to 3 ' ends and the above-mentioned segment of gB of the above-mentioned segment of pp150 jointly
5 ' end sequences, P4, P5 correspond to 3 ' ends of the above-mentioned segment of gB and 5 ' end sequences of the above-mentioned segment of pp65 jointly；
2. the clone of target gene takes 1 μ l of virus-culturing fluid, 10 × Pfu high fidelity enzyme buffer, 5 μ l, concentration 2mmol/L is added
Each 0.5 μ l of 2 μ l, Pfu high fidelity enzyme of 5 μ l, P1, P2 primer of dNTPS adds sterilizing distilled water to 50 μ l and mixes well；React item
Part is 94 DEG C of 30s of denaturation, and anneal 65 DEG C of 30s, 72 DEG C of 60s of renaturation, carries out 30 circulations, then 72 DEG C of extension 10min；P3, P4 and
P5, P6 primer cloning process are same as above；Then the pp150 segment that is obtained with first round PCR amplification, gB (UL55) are template, are added
Primer P1 and P4 expand pp150-gB segment, and reaction condition is pp150 segment, gB is respectively 1 μ l, and 10 × Pfu high-fidelity is added
Each 0.5 μ l of 2 μ l, Pfu high fidelity enzyme of dNTPS5 μ l, P1, P4 primer of enzyme buffer liquid 5 μ l, concentration 2mmol/L adds sterilizing is double to steam
Water is mixed well to 50 μ l, and reaction condition is 94 DEG C of 30s of denaturation, and anneal 65 DEG C of 30s, 72 DEG C of 90s of renaturation, carries out 30 circulations,
72 DEG C of extension 10min again obtain concatenated target gene recombinant fragment pp150-gB；It is obtained later with the first and second wheel PCR amplification
Pp150-gB segment, pp65 segment be template, primer P1 and P6 is added, expands pp150-gB-pp65 segment, reaction condition is same
On, obtain concatenated target gene recombinant fragment pp150-gB-pp65；
3. the PCR product pp150-gB-pp65 of purifying is used in the building and identification of pET28a-pp150-gB-pp65 recombinant plasmid
NdeI and XhoI digestion, and connect with by the pET28a prokaryotic expression carrier equally handled, it is transformed into Escherichia coli TOP10
In, Plasmid DNA is extracted, carries out digestion identification, and be transformed into e. coli bl21；
4. the inducing expression of pET28a-pp150-gB-pp65 fusion protein will contain pET28a-pp150-gB-pp65 recombinant plasmid
BL21 bacterium solution be added in L.B culture solution with the ratio of 1:100,37 DEG C IPTG is added after shaken cultivation 2 hours to final concentration
For 1mmol/L, 37 DEG C thalline were collected by centrifugation after induction 3 hours, is resuspended in 2X sample buffer, 100 DEG C are boiled, and SDS- is carried out
PAGE electrophoretic analysis；
5. expressing the purifying of albumen, thalline were collected by centrifugation for the bacterial culture fluid after taking induction, and Ni albumen is utilized after ultrasonication
Purification column is purified, and collects eluting peak albumen respectively, and carry out PAGE gel electrophoretic analysis；
6. the antigenicity analysis of recombinant protein cuts glue and carries out albumen by the mycoprotein of recombination after PAGE gel electrophoresis
Matter electrotransfer goes to destination protein electricity on pvdf membrane, closes pvdf membrane with the PBS of the defatted milk of 50g/L containing mass concentration, and 4 DEG C
Overnight, PBS is washed film 3 times, dilutes positive serum with confining liquid 1:20, with pvdf membrane after 37 DEG C of common incubation 2h, is washed with PBS
The Goat anti-Human IgG of horseradish peroxidase-labeled is added in film, and 37 DEG C of incubation 1h, TMB develops the color after PBS washes film, until purpose band
Reaction is terminated when developing the color clear；
(2) manufacture of gold conjugation pad prepares diameter with gold chloride-trisodium citrate reduction method as the colloidal gold of 30-50nm
Solution takes 100ml colloidal gold liquid to be placed in beaker, is adjusted to pH8.0 with 0.2MK2CO3 after the completion of preparation, molten by 100ml colloidal gold
0.5-2mg human cytomegalovirus pp150-gB-pp65 antigen is added in liquid, is stirred at room temperature 2 hours, and 1% bovine serum albumin(BSA) is added
BSA, 1% polyethylene glycol PEG20000 close 20min, and 12000r/m is centrifuged 30 minutes, abandons supernatant, are redissolved with colloidal gold working solution
It to 100ml, is layered on glass fibre membrane or non-woven fabrics with spreading the ratio uniform of 20cm2 by 1ml solution, then sets drying room, temperature
20-25 DEG C, humidity is 2-5 hours dry less than 30%, and gold conjugation pad is made；
(3) the detection T line of nitrocellulose filter and the manufacture of Quality Control C line, anti-IgM μ chain McAb is molten with 50mmol/L Tris-HCl
Liquid (pH 7.8) is diluted to 4 μ g/ml, is crossed with spray film instrument in nitrocellulose filter lower part with the parameter of 1-1.5 μ l/cm,
Coating detection T line, while in nitrocellulose filter top coating pp150-gB-pp65 fusion protein IgM antibody as Quality Control C
Line, by nitrocellulose filter at 20-25 DEG C of drying temperature after scribing line, humidity is 2-5 hours dry less than 30%；
(4) assembly of test paper, in hothouse, 20-25 DEG C of temperature, humidity takes test paper loading plate less than 40%, will be coated
The middle part that nitrocellulose filter is placed on test paper loading plate is pasted, and overlaps colloidal gold knot on the detection tip side of nitrocellulose filter
It closes and is pasted at the 1/3 of pad, overlapped in the gold conjugation pad other side and paste drop sample pad, take the 1/5 of gold conjugation pad；In nitric acid
Cellulose membrane Quality Control C line side overlaps sample suction pad, takes the 1/10 of sample suction pad；Eventually form test paper.
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