CN109142723A - Human immunodeficiency virus quickly detects test card and its application - Google Patents
Human immunodeficiency virus quickly detects test card and its application Download PDFInfo
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- CN109142723A CN109142723A CN201810953554.1A CN201810953554A CN109142723A CN 109142723 A CN109142723 A CN 109142723A CN 201810953554 A CN201810953554 A CN 201810953554A CN 109142723 A CN109142723 A CN 109142723A
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- fluorescent microsphere
- antibody
- antigen
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- hiv
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- 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
- G01N33/56988—HIV or HTLV
Abstract
The present invention provides HIV human antibodies and HIV antigen rapid detection testing card, it includes bottom plate, sample pad, nitrocellulose filter and water absorption pad and fluorescent microsphere pad, wherein, fluorescent microsphere pad adsorbs the fluorescent microsphere of the fluorescent microsphere of I type HIV antigenic mark, the fluorescent microsphere of II type HIV antigenic mark, the fluorescent microsphere of p24 antibody label and Quality Control protein labeling, successively adsorbs I type HIV antigen, II type HIV antigen, p24 antibody and the antibody for resisting the Quality Control albumen on nitrocellulose filter.In addition, the present invention also provides the preparation methods of the test card and quick detection HIV human antibody and the application of antigen etc..
Description
Technical field
The invention belongs to technical field of immunoassay, and in particular to quickly detect human immunodeficiency virus (including I type
With II type) test card of antibody and human immunodeficiency virus p24 antigen and its application etc..
Background technique
AIDS, acquired immunodeficiency syndrome are a kind of reversions by human immunodeficiency virus (abbreviation HIV)
After recording virus infection, because immune system is destroyed, it is increasingly becoming the target of attack of many waiting for an opportunity property diseases, facilitates a variety of face
Bed symptom, is referred to as syndrome, rather than a kind of simple disease, and this syndrome can be by directly contacting mucous membrane tissue
(mucosa) oral cavity, genitals, anus etc. or with virulent blood, sperm, vaginal secretion, milk and infect.
Due to the highly infective of AIDS, high spread scope and high mortality are actually just built from finding AIDS
Corresponding laboratory diagnostic method is found；Since the purpose of detection is different, different detection methods is also respectively set up.Detection
Method is roughly divided into two classes:
1. pathogeny detection
1) pathogen separation: strict demand carries out in the laboratory P3, by highly concentrated to patients serum, in electronic display
Micro- microscopic observation separates inhibition of HIV.
2) in situ hybridization: the inhibition of HIV cDNA segment cloned with isotope labelling carries out core with the histocyte of patient
Acid hybridization, by autoradiograph, to determine the cell of virus infection.
3) PCR: amplification HIV cDNA segment, by the specific dna probe of fluorescent marker, come check whether by
HIV infection.
4) measurement of reverse transcriptase: by measurement patients serum in reverse transcriptase activity, come diagnose whether by
HIV infection.
5) lymphocyte function measures: the function by measuring lymphocyte, mainly to T4Helper lymphocyte comes
Diagnosis whether infected by HIV.
6) detection of p24 antigen: by immune response, detecting the p24 antigen in blood, as HIV infection
Early diagnosis.
2.HIV antibody test
There are many method of HIV antibody detection, such as enzyme-linked immunosorbent assay (Enzyme linked immunosorbent
Assay, ELISA), gelatin particle agglutination experiment (Gelatine particle agglutination assay, PA), latex
Agglutination experiment (Latex agglutination assay, LA), various quick test experiences (Rapid tests) etc..These
Test the pyrolysis product that the antigen that uses is intact virus in early days, this antigen usually due to the ingredient containing host cell and
There is false positive reaction, while the preparation and purification of this antigen are all relatively difficult, risk is big.Hereafter reagent uses recombination
Or synthesis polypeptide HIV antigen, antigenic determinant relevant to immune response is selected, sensibility and specificity increases, this
Class antigen prepare it is safer, easy, reproducible, but recombinant antigen sometimes due to the component containing carrier and there is vacation
Positive findings, especially inhibition of HIV itself are easy mutation, and polypeptide antigen due to lack suitable three-dimensional conformation can also make it is quick
So as to cause false negative, this will be rated as catastrophic for popularization and application for perception decline.
For example, Chinese patent application CN105842462A discloses a kind of immune chromatograph testing strip for detecting HIV antibody,
Using HIV-I the and HIV-2 antibody in fluorescence immune chromatography technology and dual-antigen sandwich method detection human serum；Chinese patent Shen
Please CN1858594A disclose multiple index quick detecting method for human immune defect virus antibody, using including p24, gp41
Fixation with the antigen proteins segment such as gp36 is detected；Chinese patent application CN102445538A discloses AIDS
(HIV-1/2) urine quick detection reagent detects HIV recombinant antigen p41, p42 and p36 based on colloidal gold technique.
However, the above-mentioned prior art is not related to the selection to corresponding antigens or antigene fragment, therefore it is easy to dash forward in detection
When the inhibition of HIV of change, it is difficult to avoid false negative result, for this purpose, the present inventor studies intensively, have developed a kind of new HIV
Human antibody and HIV antigen rapid detection testing card, not only can quickly, highly sensitive, the low coefficient of variation detect simultaneously I type and
The human antibody of II type HIV and the p24 antigen of HIV, and accuracy rate is high, is avoided that the appearance of false negative result, is particularly suitable for using
In the primary dcreening operation of HIV.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of HIV human antibody and HIV antigen rapid detection testing card,
It is detected in which can be carried out quick, highly sensitive, the low coefficient of variation, convenient for the primary dcreening operations such as field condition detection and bedside detection, simultaneously
Accuracy with height, especially avoids false negative result, is suitble to promote the use of.In addition, the present invention also provides the tests
The preparation method and application etc. of card.
Specifically, in a first aspect, the present invention provides HIV human antibody and HIV antigen rapid detection testing card,
Sample pad (2), nitrocellulose filter (8) and the water absorption pad (9) including bottom plate (1) and being successively fitted on bottom plate, cellulose nitrate
Plain film (8) connect with water absorption pad (9), wherein is connected with and is fitted on bottom plate between sample pad (2) and nitrocellulose filter (7)
Fluorescent microsphere pad (3), wherein fluorescent microsphere pad (3) absorption I type HIV antigenic mark fluorescent microsphere, II type HIV antigen mark
The fluorescent microsphere of the fluorescent microsphere of note, the fluorescent microsphere of p24 antibody label and Quality Control protein labeling, on nitrocellulose filter (8)
Have on the direction of sample pad (2) to water absorption pad (9) and successively adsorbs I type HIV antigen, II type HIV antigen, p24 antibody and resist
Three of the antibody of Quality Control albumen separation perpendicular to sample pad (2) to the line in the direction of water absorption pad (9).
It is preferred that in the test card of first aspect present invention, the amino acid sequence such as SEQ ID of the I type HIV antigen
Shown in NO:1.The antigen is that the present inventor develops from the albumen of a large amount of HIV, test to the first aspect of the present invention
The high accuracy of card has played key effect.
It is also preferred that in the test card of first aspect present invention, the amino acid sequence such as SEQ ID of the II type HIV antigen
Shown in NO:2.The antigen is that the present inventor develops from the albumen of a large amount of HIV, test to the first aspect of the present invention
The high accuracy of card has played key effect.
Fluorescent microsphere shiploads of merchandise, however the inventors discovered that, their effects in the present invention are irregular not
Together, it has been investigated that, have absorption peak at wavelength 480nm (is such as purchased from Ocean Nanotech with carboxylic group fluorescent microsphere
) particularly suitable in the present invention.It is preferred, therefore, that in the test card of first aspect present invention, the fluorescent microsphere be
Have absorption peak at wavelength 480nm has carboxylic group fluorescent microsphere.
It is preferred that the fluorescent microsphere of the label is prepared via a method which in the test card of first aspect present invention: will
It after the fluorescent microsphere is mixed with EDC oscillation, is washed with PBS buffer solution, adds the antigen, antibody or Quality Control albumen, shaken
Mixing is swung, is then added to vibrate containing the PBS buffer solution of glycine and Β SA and close, finally with SA's containing Β and Proclin300
PBS buffer solution is washed and is diluted.Fluorescent microsphere, the institute of the I type HIV antigenic mark can be prepared respectively by the above method
State the glimmering of the fluorescent microsphere of II type HIV antigenic mark, the fluorescent microsphere of p24 antibody label and the Quality Control protein labeling
Light microballoon.
The present inventor studies the mutation of the upper p24 antigen of discovery HIV usually not in its epitope, therefore the existing p24 of use
Antigen prepares its antibody and can effectively identify.It is preferred that in the test card of first aspect present invention, the antigen of the p24 antibody
Amino acid sequence as shown in SEQ ID NO:3.
The inventors discovered that Quality Control albumen also has certain influence to effect of the invention, in order to avoid the object in proper manners sheet
The influence of confrontation nature controlling line, it is preferred to use haptens and its antibody.It is preferred that in the test card of first aspect present invention, Quality Control
Albumen is DNP-BSA (dinitrofluorobenzene-bovine serum albumin(BSA)), that is, the BSA of DNP (haptens) has been conjugated；Corresponding antibodies are preferred
It is the antibody for resisting the haptens, i.e., anti-DNP antibody.Haptens using DNP as Quality Control albumen can be effectively prevented from people
Influence of the sample to nature controlling line.
In second aspect, the present invention provides the methods of the test card of preparation first aspect present invention comprising: (a) make
The standby fluorescent microsphere of the I type HIV antigenic mark, the fluorescent microsphere of the II type HIV antigenic mark, p24 antibody label
Fluorescent microsphere and the Quality Control protein labeling fluorescent microsphere, then mixing and absorption is on fluorescent microsphere pad (3)；
(b) by the I type HIV antigen, the II type HIV antigen, the p24 antibody and described resist the Quality Control albumen
Antibody lined on nitrocellulose filter (8) respectively；With,
(c) nitrocellulose filter (8) same bottom plate that the fluorescent microsphere pad (3) and step (b) obtained step (a) obtains
(1), sample pad (2) and water absorption pad (9) are assembled into the test card.
Wherein, the method for preparing the fluorescent microsphere of the HIV antigenic mark and the fluorescent microsphere of the Quality Control protein labeling
It can be following method:
After the fluorescent microsphere and EDC oscillation are mixed, washed with PBS buffer solution, add the antigen, antibody or
Quality Control albumen, oscillation mixing, be then added containing the PBS buffer solution of glycine and Β SA vibrate close, finally with SA containing Β with
The PBS buffer solution of Proclin300 is washed and is diluted.The I type HIV antigenic mark can be prepared respectively by the above method
The fluorescent microsphere and the Quality Control egg that fluorescent microsphere, the fluorescent microsphere of the II type HIV antigenic mark, the p24 antibody mark
The fluorescent microsphere of white marker.
In the third aspect, the present invention provides the test cards of first aspect present invention to prepare low coefficient of variation detection HIV
Application in human antibody and the testing product of HIV antigen.
The test card accuracy of first aspect present invention is good, and the coefficient of variation is low.It is preferred that in the application of third aspect present invention
In, the low coefficient of variation is that the coefficient of variation is no more than 15%.
On the other hand the, the present invention provides the amino acid sequences I type HIV antigen as shown in SEQ ID NO:1 and/
Or application of the amino acid sequence II type HIV antigen as shown in SEQ ID NO:2 in the testing product of preparation HIV antibody.
The application of this aspect can be being used alone for amino acid sequence I type HIV antigen as shown in SEQ ID NO:1
Or amino acid sequence II type HIV antigen as shown in SEQ ID NO:2 is used alone, and is also possible to answer their combine
With.
In addition, this aspect is also possible to them and is directed to amino acid sequence p24 antigen as shown in SEQ ID NO:3
The use in conjunction of p24.Preferably wherein, the testing product of the HIV antibody is the testing product of HIV human antibody and HIV antigen,
The more preferably test card of first aspect present invention.
The beneficial effects of the present invention are detect with can be carried out quick, highly sensitive, the low coefficient of variation HIV human antibody and
Antigen convenient for the primary dcreening operations such as field condition detection and bedside detection, while having the accuracy of height, especially avoids false negative
As a result, being suitble to promote the use of.
The present invention refers to open source literature, these documents are their entire contents in order to more clearly describe the present invention
It is included in and is referred to herein, just look like that repeated description herein has been excessively for their full text.
In order to make it easy to understand, the present invention will be described in detail by specific embodiment and attached drawing below.It needs
It is emphasized that these descriptions are only exemplary description, and it is not meant to limit the scope of the invention.According to this theory
The discussion of bright book, many variations of the invention, change are all obviously for those skilled in the art.
Detailed description of the invention
Fig. 1 shows that HIV of the invention quickly detects the structural schematic diagram of test card, wherein appended drawing reference distinguishes table
Show: 1:PVC bottom plate；2: sample pad；3: fluorescent microsphere pad；4:I type antigen test line；5:II type antigen test line；6:p24 is anti-
Body p-wire；7:DNP antibody test line；8:NC film；9: water absorption pad；10: sample.
Specific embodiment
It will describe to invent by specific embodiment herein below.It, can be according to this field skill such as not specified place
" Molecular Cloning:A Laboratory guide " (third edition) known to art personnel (Cold Spring Harbor laboratory
Press), " cell experiment guide " (Science Press, Beijing, China, 2001), " RNA experimental technique handbook " (science goes out
Version society, Beijing, China, 2004), " immunoassay technology " (Science Press, Beijing, China, 1991) etc. laboratory manuals with
And listed method is implemented in bibliography cited herein；Reagent therein can be obtained by commercial channel.
The determination of embodiment 1 HIV I type, II type recombinant antigen, p24 antibody
The design of people according to the present invention is devised from the antigen of a large amount of inhibition of HIV as shown in SEQ ID NO:1
The antigen of amino acid sequence detects antigen (abbreviation I type antigen), the amino as shown in SEQ ID NO:2 as I type inhibition of HIV
The antigen of acid sequence detects antigen (abbreviation II type antigen) as II type inhibition of HIV.In addition, the present inventor uses such as SEQ
The HIV p24 antigen of amino acid sequence shown in ID NO:3 entrusts Bioisystech Co., Ltd of Beijing Zhong Shan Golden Bridge according to normal
Rule method is prepared for corresponding p24 monoclonal antibody and resists (rabbit-anti p24 is mostly anti-) more.
2 HIV antigen of embodiment, antibody draw film on nitrocellulose
It is carried out respectively with the recombinant antigen of embodiment 1 and antibody in nitrocellulose filter (NC film) with nitrocellulose filter
Film is drawn, Sartori μ s (Sai Duolisi) CN104NC film, 15 μm of aperture, creep speed 150S/4cm are specifically used.Specifically draw film
Process is as follows:
1) it draws the preparation of film buffer: preparing 0.01MPBS (phosphate buffer), pH7.0, contain 1% sucrose, with 0.22 μm
It is filtered.
2) it takes and draws film buffer, dilute antigen, antibody respectively according to a certain concentration, wherein the I type HIV of embodiment 1 is anti-
Former, II type HIV antigen diluent to 1mg/ml, for the II type antigen diluent of embodiment 1 to 1mg/ml, the p24 monoclonal antibody of embodiment 1 is dense
Degree is diluted to 1.8mg/ml, and DNP antibody (the be purchased from DAKO company) concentration dilution as Quality Control is to 1.5mg/ml.
3) with film instrument (being purchased from Hangzhou Feng Hang Science and Technology Ltd.) stroke film on NC film respectively is drawn, according to 1 μ L/cm
Speed draws film, and the I type antigen of embodiment 1 draws film and draws film at 0.5cm, and the II type antigen of embodiment 1 draws film at 0.9cm, real
The p24 monoclonal antibody for applying example 1 draws film at 1.3cm, and DNP antibody draws film at 1.8cm, and 37 DEG C 3-4 hours dry, is sealed 1
~30 DEG C.
The determination of 3 fluorescent microsphere of embodiment and coupling with antigen, antibody
The carboxylic group fluorescent microsphere for taking separate sources is diluted with 0.01M PBS with 1:10；It is returned to zero with 0.01M PBS,
It is scanned within the scope of wavelength 400nm~600nm, whether see has narrow absorption peak (to illustrate fluorescent microsphere grain at wavelength 480nm
Diameter is uniform)；With 0.01M PBS dilution fluorescent microsphere concentration to 2mg/ml, the 10 μ l of point sample on NC film, detection sees whether its is glimmering
Light value >=104.After tested, purchased from the best with carboxylic group fluorescent microsphere effect of Ocean Nanotech, therefore selecting should
Fluorescent microsphere.Specific antigen, the process of antibody coupling are as follows:
1) I type antigenic mark fluorescent microsphere: use activation buffer (0.05M MES (2- (N- morpholino) ethanesulfonic acid)) will
Fluorescent microsphere is diluted to 1mg/ml, is during which cleaned twice with activation buffer, be centrifuged each time after washing (10000 turns, 30
Minute).By 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and fluorescent microsphere according to mass ratio 1:20
Ratio be added, in 25 DEG C of oscillation 30min, with coupling liquid (0.01M P Β S, pH7.4) washing 2 times, according to every milligram of microballoon
The concentration of 10 μ g albumen is added, the I type antigen of embodiment 1,2-8 DEG C of shaken overnight is added.With it is additional be added 40mM glycine,
The coupling liquid of 1% (w/v) Β SA is closed as confining liquid, 25 DEG C of oscillation 60min.(contained in 0.01M P Β S with liquid is saved
0.5% (w/v) Β SA (bovine serum albumin(BSA)), 0.5% (v/v) Proclin300 (are purchased from Shanghai and dodge brilliant molecular biosciences science and technology
Co., Ltd)) washing 2 times.Adding preservation liquid to concentration is 1mg/ml, places 2~8 DEG C of temperature preservations.
2) fluorescent microsphere II type antigenic mark fluorescent microsphere: is diluted to 1mg/ with activation buffer (0.05M MES)
During which ml is cleaned twice with activation buffer, be centrifuged (10000 turns, 30 minutes) each time after washing.By 1- (3- diformazan ammonia
Base propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and fluorescent microsphere be added according to the ratio of mass ratio 1:20, shake at 25 DEG C
30min is swung, is washed 2 times with coupling liquid (0.01M P Β S, pH7.4), the concentration of 10 μ g albumen is added according to every milligram of microballoon,
The II type antigen of embodiment 1,2-8 DEG C of shaken overnight is added.With the additional coupling that 40mM glycine, 1% (w/v) Β SA is added
Liquid is closed as confining liquid, 25 DEG C of oscillation 60min.(contain 0.5% (w/v) Β SA (cow's serum in 0.01M P Β S with liquid is saved
Albumin), 0.5% (v/v) Proclin300) washing 2 times.Adding preservation liquid to concentration is 1mg/ml, places 2~8 DEG C of temperature
It saves.
3) the how anti-mark fluorescent microballoon of p24: being diluted to 1mg/ml for fluorescent microsphere with activation buffer (0.05M MES),
Period is cleaned twice with activation buffer, is centrifuged (10000 turns, 30 minutes) each time after washing.By 1- (3- dimethylamino
Propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and fluorescent microsphere be added according to the ratio of mass ratio 1:20, vibrate at 25 DEG C
30min is washed 2 times with coupling liquid (0.01M P Β S, pH7.4), and the concentration of 5 μ g albumen is added according to every milligram of microballoon, is added
The p24 of embodiment 1 is mostly anti-, 2-8 DEG C of shaken overnight.Use it is additional be added 40mM glycine, 1% (w/v) Β SA coupling liquid as
Confining liquid closing, 25 DEG C of oscillation 60min.(contain 0.5% (w/v) Β SA (bovine serum albumin in 0.01M P Β S with liquid is saved
It is white), 0.5% (v/v) Proclin300) washing 2 times.Adding preservation liquid to concentration is 1mg/ml, places 2~8 DEG C of temperature preservations.
4) DNP-BSA mark fluorescent microballoon: being diluted to 1mg/ml for fluorescent microsphere with activation buffer (0.05M MES),
Period is cleaned twice with activation buffer, is centrifuged (10000 turns, 30 minutes) each time after washing.By 1- (3- dimethylamino
Propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and fluorescent microsphere be added according to the ratio of mass ratio 1:20, vibrate at 25 DEG C
30min is washed 2 times with coupling liquid (0.01M P Β S, pH7.4), and the concentration of 15 μ g albumen is added according to every milligram of microballoon, adds
Enter DNP-BSA (being purchased from DAKO company), 2-8 DEG C of shaken overnight.40mM glycine, 1% (w/v) Β SA is added with additional
It is coupled liquid to close as confining liquid, 25 DEG C of oscillation 60min.(contain 0.5% (w/v) Β SA (ox in 0.01M P Β S with liquid is saved
Seralbumin), 0.5% (v/v) Proclin300) washing 2 times.Adding preservation liquid to concentration is 1mg/ml, places temperature 2~8
DEG C save.
5) fluorescent microsphere of above-mentioned I type antigen, II type antigen, p24 antibody, DNP-BSA label is mixed in equal volume, is mixed
The fluorescent microsphere of conjunction is added on glass fibre, and every milliliter of fluorescent microsphere spreads about 8 square centimeters of fiberglass packing, 37 DEG C of dryings 4
Hour.
4 HIV of embodiment quickly detects the assembling of test card
By the NC film prepared in example 2,3, fluorescent microsphere fiberglass packing (fluorescent microsphere pad) and sample pad, water absorption pad
Etc. sticking on backing, slitting, assembling.It is specific as follows:
Above-mentioned sample pad is cut into length 30cm and width 28mm, fluorescent glass fibre pad is cut into length 30cm and width
10mm, reaction film are cut into length 30cm and width 25mm, and water absorption layer cuts into length 30cm and width 27mm, then successively will
It is attached on lining card, after forming big plate, is cut into the single person-portion test strips of length 8cm and width 4mm, is packed into test and gets stuck
In, by every single part equipped in the hermetic bag of desiccant, deposit in room temperature.The test card structure being prepared into is as shown in Figure 1.
The application of 5 HIV of embodiment detection test card
HIV antibody, the detection method of antigen in test sample, detailed process is as follows:
1) test card prepared by Example 4.
2) serum, plasma sample are drawn 75 μ L with pipettor, are added in test card sample pad.
3) test card after five minutes, is inserted into luminoscope immediately and (is purchased from Hangzhou and steps the limited public affairs of science and technology by test card sample-adding
Department) in jack, detect each band fluorescent value, in which:
I type antigen bands fluorescent value is greater than 500 or more, and Quality Control band fluorescent value >=1000 or more can determine that as I type HIV
Virus antibody positive；
II type antigen bands fluorescent value is greater than 500 or more, and Quality Control band fluorescent value >=1000 or more can determine that as II type
HIV antibody is positive；
P24 antibody band fluorescent value is greater than 500 or more, and Quality Control band fluorescent value >=1000 or more can determine that as HIV
P24 antigen positive；
When Quality Control band fluorescent value < 1000, repeat the 1) step re-measure.
The HIV national standard that detection state food drug assay research institute provides is detected using the above method, as a result such as
The following table 1.
1 HIV national standard testing result of table
Above-mentioned standard product sample is separately repeated 10 times detection respectively, the coefficient of variation (CV) is respectively 8.32%, 9.87%,
10.25% ,≤15%, illustrate that this method accuracy is good.In addition, test card of the invention, which obtains sensitivity, can reach 0.05pg,
The range of linearity can reach 200-1000 times.
Separately accelerate the failure to test card of the invention, places 30 days, be measured, testing result is still in 37 DEG C
As shown above, and its CV≤15%, accuracy is still good, shows that test card stability of the invention is good, can be used for storing
The detection of the quick HIV sample in the bad place of condition.
In addition, detected using the above method to 107 parts of common samples, as a result with clinical Multiple detection (including disease
Poison culture) testing result made a definite diagnosis is consistent, do not find false negative result；And it using the colloidal gold of conventional antigen or is immunized glimmering
Light technology is to above-mentioned sample detection, it may appear that 1~3% false negative result.
<110>Suzhou Hua Yimei Biotechnology Co., Ltd
<120>human immunodeficiency virus quickly detects test card and its application
<130> CN
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 118
<212> PRT
<213> Artificial Sequence
<220>
<223> HIV-1
<400> 1
Gln Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly
1 5 10 15
Ile Lys Gln Leu Gln Ala Arg Gly Gly Gly Ala Val Glu Asn Tyr Leu
20 25 30
Lys Asp Gln Gln Leu Ala Gly Ile Trp Gly Cys Ser Gly Lys His Ile
35 40 45
Cys Thr Ser Ala Val Pro Trp Asn Ser Ser Trp Ser Asn Lys Gly Ser
50 55 60
Asp Glu Ile Trp Asn Asn Met Thr Trp Met Glu Trp Glu Ala Glu Ile
65 70 75 80
Asp Lys Tyr Thr Ser Glu Ile Tyr Ser Leu Ile Glu Ile Ser Gln Thr
85 90 95
Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Asn Trp Ala
100 105 110
Ser Leu Trp Asn Trp Phe
115
<210> 2
<211> 133
<212> PRT
<213> Artificial Sequence
<220>
<223> HIV-II
<400> 2
Gly Thr Lys Asn Leu Gln Ala Arg Val Thr Ala Ile Glu Lys Tyr Leu
1 5 10 15
Lys Asp Gln Ala Leu Leu Asn Ser Trp Gly Cys Ala Phe Arg Gln Val
20 25 30
Cys His Thr Ala Val Gln Trp Pro Asn Glu Gly Pro Asn Trp Asp Asn
35 40 45
Met Thr Trp Gln Gln Trp Glu Lys Gln Val Arg Phe Leu Asp Glu Asn
50 55 60
Ile Thr Val Leu Ser Glu Gln Ala Gln Ile Gln Gln Glu Lys Asn Met
65 70 75 80
Tyr Glu Leu Gln Lys Leu Ser His Trp Asp Ile Phe Ser Asn Trp Phe
85 90 95
Asp Phe Thr Ser Trp Ile Ala Tyr Ile Arg Ile Gly Leu Tyr Ile Val
100 105 110
Ile Leu Ala Val Leu Arg Ile Ala Ile Tyr Ile Leu Gln Met Leu Ala
115 120 125
Arg Leu Arg Lys Gly
130
<210> 3
<211> 103
<212> PRT
<213> Human immunodeficiency virus
<400> 3
Pro Ile Val Gln Asn Ala Gln Gly Gln Met Ile His Gln Ser Leu Ser
1 5 10 15
Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys Ala Phe
20 25 30
Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr
35 40 45
Pro Phe Asp Leu Asn Met Met Leu Asn Ile Val Gly Gly His Gln Ala
50 55 60
Ala Met Leu Met Leu Lys Asp Thr Ile Asn Glu Glu Ala Ala Glu Trp
65 70 75 80
Asp Arg Leu His Pro Val His Ala Gly Pro Val Ala Pro Gly Gln Met
85 90 95
Arg Glu Pro Arg Gly Ser Asp
100
Claims (10)
1.HIV human antibody and HIV antigen rapid detection testing card comprising bottom plate (1) and the sample that is successively fitted on bottom plate
Pad (2), nitrocellulose filter (8) and water absorption pad (9), nitrocellulose filter (8) are connect with water absorption pad (9), which is characterized in that sample
The fluorescent microsphere pad (3) being fitted on bottom plate is connected between product pad (2) and nitrocellulose filter (7), wherein fluorescent microsphere pad
(3) it is micro- that the fluorescent microsphere of I type HIV antigenic mark, the fluorescent microsphere of II type HIV antigenic mark, the fluorescence of p24 antibody label are adsorbed
The fluorescent microsphere of ball and Quality Control protein labeling has on the direction of sample pad (2) to water absorption pad (9) on nitrocellulose filter (8)
Successively adsorb I type HIV antigen, II type HIV antigen, p24 antibody and resist the Quality Control albumen three of antibody separation it is vertical
In sample pad (2) to the line in the direction of water absorption pad (9).
2. test card described in any one of aforementioned claim, wherein the amino acid sequence such as SEQ ID of the I type HIV antigen
Shown in NO:1；And/or the amino acid sequence of the II type HIV antigen is as shown in SEQ ID NO:2.
3. test card described in any one of aforementioned claim, wherein the antigen of the Quality Control albumen is haptens；It is preferred that described
Quality Control albumen is DNP-BSA, and the antibody of the Quality Control albumen is anti-DNP antibody.
4. test card described in any one of aforementioned claim, wherein the fluorescent microsphere is that have absorption peak at wavelength 480nm
Have carboxylic group fluorescent microsphere.
5. test card described in any one of aforementioned claim, wherein the fluorescent microsphere of the label is prepared via a method which:
After the fluorescent microsphere is mixed with EDC oscillation, is washed with PBS buffer solution, adds the antigen, antibody or Quality Control albumen,
Then oscillation mixing is added to vibrate containing the PBS buffer solution of glycine and Β SA and close, finally with SA's containing Β and Proclin300
PBS buffer solution is washed and is diluted.
6. test card described in any one of aforementioned claim, wherein the amino acid sequence such as SEQ of the antigen of the p24 antibody
Shown in ID NO:3.
7. the method for preparing test card described in any one of aforementioned claim comprising:
(a) fluorescent microsphere of the I type HIV antigenic mark, fluorescent microsphere, the p24 of the II type HIV antigenic mark are prepared
The fluorescent microsphere of antibody label and the fluorescent microsphere of the Quality Control protein labeling, then mixing and absorption is on fluorescent microsphere pad (3)；
(b) by the I type HIV antigen, the II type HIV antigen, the p24 antibody and the antibody for resisting the Quality Control albumen
It is lined on nitrocellulose filter (8) respectively；With,
(c) nitrocellulose filter (8) same to bottom plate (1), the sample that the fluorescent microsphere pad (3) and step (b) obtained step (a) obtains
Product pad (2) and water absorption pad (9) are assembled into the test card.
8. described in any item test cards of claim 1-6 are preparing low coefficient of variation detection HIV human antibody and HIV antigen
Application in testing product.
9. application according to any one of claims 8, wherein the low coefficient of variation is that the coefficient of variation is no more than 15%.
10. amino acid sequence I type HIV antigen and/or amino acid sequence as shown in SEQ ID NO:1 such as SEQ ID NO:2 institute
Application of the II type HIV antigen shown in the testing product of preparation HIV antibody.
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| CN113912677A (en) * | 2021-11-04 | 2022-01-11 | 苏州华益美生物科技有限公司 | Hepatitis C virus detection related peptide and visible time-resolved fluorescent microsphere test strip thereof |
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