CN105572379A - Kit capable of being used for diagnosis of vivax malaria and/or falciparum malaria - Google Patents
Kit capable of being used for diagnosis of vivax malaria and/or falciparum malaria Download PDFInfo
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- CN105572379A CN105572379A CN201410615078.4A CN201410615078A CN105572379A CN 105572379 A CN105572379 A CN 105572379A CN 201410615078 A CN201410615078 A CN 201410615078A CN 105572379 A CN105572379 A CN 105572379A
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Abstract
The invention relates to a kit capable of being used for diagnosis of vivax malaria and/or falciparum malaria. The kit disclosed by the invention comprises one or more solid carriers, and specific polypeptide sets which are independently connected onto the one or more solid carriers.
Description
Technical field
The present invention relates generally to kit.Specifically, the present invention relates to the kit that can be used for detecting plasmodium vivax infection and/or falciparum infection.
Background technology
Malaria remains global sanitarian vital issue, and annual death reaches 0.7-2.7 1,000,000, and wherein more than 75% is African children.In the past 35 years, the incidence of disease of malaria increased 2-3 doubly.In nineteen fifty-five, the World Health Organization (WHO) starts one and carries out clinical treatment and application DDT(dichloro-diphenyl-trichloroethane by application chloroquine) control the grand plan of mosquito group and eradication malaria.Progressively stop in the later stage sixties 20th century, although in the whole world, many countries cause important and lasting Disease Spectrum to alleviate in this plan.But still have recurrence in many national malaria, it is mainly because the parasitic appearance of resistance and propagation cause.The appearance of resistance to pesticide mosquito, the increase of the density of population is (from 1963, world population has turned over some), global warming (scope that this carrier does not arrive before diffusing to), continue poor, political turbulence, and the loss of the yield-power caused due to communicable disease, the maintenance on the stable public health basis of these all factors disrupt treatment and malaria control.
Malarial parasite belongs to Plasmodium, and the many vertebrate hosts of energy postoperative infection, comprise multiple non-human primates species.Four kinds of Plasmodiums parasitize the mankind: plasmodium falciparum (Plasmodium.fal-
Ciparum), malariae (P.malariae), Plasmodium ovale (P.ovale) and Plasmodium vivax (P.vivax).Wherein, plasmodium falciparum and Plasmodium vivax are relevant to the M & M of most of malaria respectively.
Due to the singularity of malaria treatment medicine, accurately diagnosis fast can not only Timeliness coverage treat malaria patients, greatly reducing case fatality rate, also by getting rid of malaria in early days, saving the life having infected other communicable disease patient in time.With this simultaneously, accurately early diagnosis fast is also the necessary means to the real-time epidemic disease monitor and forecast of malaria.Up to the present, suspicious patient is taken a blood sample and makes the thickness dyed rear microscopy of blood sheet (abbreviation Microscopical Method For Detection) and be considered to " goldstandard " of malaria diagnosis always, its advantage is that expense is low, plasmodium worm kind can be identified, but also there is defect, namely need very professional reviewer, when parasitemia densities is lower, can false negative be there is, cause mistaken diagnosis or fail to pinpoint a disease in diagnosis, also need some utility appliance such as microscope, dyeing liquor simultaneously.Along with Clinical Institutions, the hospital that particularly some up-to-datenesses are high, because reviewer more relies on instrument and equipment, obviously declines to the grasp of microscopy ability; And in some low developed areas, for various reasons, fail to be equipped with the relevant equipment of microscopy and reagent.These factors limit clinically to be diagnosed fast and effectively to malaria.Therefore, the focus that new malaria fast diagnosis method becomes research is at present studied.
Summary of the invention
The object of the present invention is to provide new for Malaria Diagnosis, particularly can be used for Plasmodium Vivax, malignant malaria or mixing malaria, or for distinguishing tertian fever, malignant malaria and mixing the kit of malaria.
Current inventor provides 5 polypeptide.When the biological specimen of originating using experimenter whether in described 5 polypeptide at least two or more has the index of response as judgement experimenter whether by Infected With Plasmodium, can with up to 98% sensitivity (and false positive rate is low to moderate 1.0%) Malaria Diagnosis.
In addition, the present inventor also finds, by using the combination of above-mentioned 5 polypeptide and other 2 polypeptide as detection molecules, can not only Malaria Diagnosis, can also Plasmodium Vivax, malignant malaria or mixing malaria further, or for distinguishing tertian fever, malignant malaria and mixing malaria.
Therefore, the present invention includes:
1. a kit, it comprises one or more solid carrier, and is connected to the following polypeptide set 1 on described one or more solid carrier independently:
Polypeptide shown in SEQIDNO:1;
Polypeptide shown in SEQIDNO:2;
Polypeptide shown in SEQIDNO:3;
Polypeptide shown in SEQIDNO:4; With
Polypeptide shown in SEQIDNO:5.
2. the kit according to item 1, it is for Malaria Diagnosis.
3. the kit according to item 1 or 2, wherein, described polypeptide set 1 also comprises following polypeptide set 2:
Polypeptide shown in SEQIDNO:6; With
Polypeptide shown in SEQIDNO:7.
4. the kit according to item 3, it is for Plasmodium Vivax, malignant malaria or mixing malaria, or for distinguishing tertian fever, malignant malaria and mixing malaria.
5. the kit according to any one of item 1 ~ 4, wherein, whole polypeptide is connected on same solid carrier independently.
6. following polypeptide set 1 is for the preparation of the purposes in the kit of Malaria Diagnosis:
Polypeptide set 1
Polypeptide shown in SEQIDNO:1;
Polypeptide shown in SEQIDNO:2;
Polypeptide shown in SEQIDNO:3;
Polypeptide shown in SEQIDNO:4; With
Polypeptide shown in SEQIDNO:5.
7. the purposes according to item 6, wherein, described Malaria Diagnosis also comprises Plasmodium Vivax, malignant malaria or mixing malaria, or for distinguishing tertian fever, malignant malaria and mixing malaria, and described polypeptide set 1 also comprises following polypeptide set 2:
Polypeptide shown in SEQIDNO:6; With
Polypeptide shown in SEQIDNO:7.
the embodiment of invention
polypeptide set and polypeptide group
In this instructions, polypeptide set 1 is the whole of following polypeptide group 1 ~ 5:
Polypeptide shown in polypeptide group 1:SEQIDNO:1;
Polypeptide shown in polypeptide group 2:SEQIDNO:2;
Polypeptide shown in polypeptide group 3:SEQIDNO:3;
Polypeptide shown in polypeptide group 4:SEQIDNO:4; And
Polypeptide shown in polypeptide group 5:SEQIDNO:5.
In this instructions, sensitivity refers to: in the positive sample confirmed by " goldstandard " method, be determined as the ratio of positive sample by additive method.False positive rate refers to: in the negative sample confirmed by " goldstandard " method, be determined as the ratio of positive sample by additive method." goldstandard " method that the art detects plasmodium vivax infection or falciparum infection is Microscopical Method For Detection.
In this instructions, " malaria " refers to malignant malaria and/or tertian fever.Malignant malaria refers to the malaria caused because of falciparum infection, and tertian fever refers to the malaria caused because of plasmodium vivax infection, and mixing malaria refers to the malaria caused because of plasmodium falciparum and plasmodium vivax infection (also claiming mixed infection).
Preferably, polypeptide set 1 is on whole basis of described polypeptide group 1 ~ 5, add following polypeptide group 6 ~ 7:
Polypeptide shown in polypeptide group 6:SEQIDNO:6; And
Polypeptide shown in polypeptide group 7:SEQIDNO:7.
As shown in the Examples, the serum of described polypeptide set 1 pair of plasmodium falciparum and/or Infected With Plasmodium person is positive, and the serum of healthy normal person or non-malignant plasmodium and/or plasmodium vivax infection person is negative reaction, therefore, described polypeptide set 1 is useful as the testing tool of Infected With Plasmodium.
In this instructions, positive reaction refers to: meet criterion listed in following " diagnostic method " part; Otherwise be negative reaction.
Described polypeptide can be suitable for adopting the known method such as (1) chemical synthesis process or (2) enzyme reaction synthetic method to obtain, and wherein chemosynthesis is more easy.In chemosynthesis polypeptide situation of the present invention, undertaken by using peptide synthesizer synthesis or this polypeptide semi-synthetic.As chemical synthesis process, such as peptide solid-phase synthesis etc. can be listed.The peptide of such synthesis can adopt conventional means such as ion-exchange chromatography, reverse phase high performance liquid chromatogram, affinity chromatography etc. to carry out purifying.Such peptide solid phase synthesis process with and subsequent peptide purification be all well-known in the art.
In addition, when producing polypeptide of the present invention by enzyme reaction, the method such as described in No. WO2004/011653, International Publication pamphlet can be adopted.Namely; can produce like this: by the amino acid of a side or the carboxyl terminal of dipeptides is esterified or amidation and the amino acid that obtains or dipeptides, the amino acid (amino acid of such as carboxy protective) that is in free state with amino acid react under the existence of peptide synthetase, the dipeptides of generation or tripeptides.As peptide synthetase, can list: there is the culture of microorganism of the ability generating peptide, the bacterial disposing thing of the microbial cells be separated by this culture or this microorganism or this microbe-derived peptide synthetase.
Particularly the chemosynthesis of polypeptide achieves commercialization, can easily entrust the Peptide systhesis company of specialty to synthesize described polypeptide.
kit
The invention provides a kind of kit (kit of the present invention), it comprises
One or more solid carrier, and be connected to the aforementioned polypeptides set 1 on described one or more solid carrier independently.
Kit of the present invention can be used for Malaria Diagnosis.Here, " diagnosis " can be make a definite diagnosis, and also can be provide reference information for making a definite diagnosis.
When described polypeptide set 1 is aforementioned polypeptides group 1 ~ 7 whole, kit of the present invention can be used for Plasmodium Vivax, malignant malaria or mixing malaria, or for distinguishing tertian fever, malignant malaria and mixing malaria.
Therefore, described polypeptide set 1 may be used for the kit for the preparation of Malaria Diagnosis.When described polypeptide set 1 is aforementioned polypeptides group 1 ~ 7 whole, described polypeptide set 1 may be used for for the preparation of Plasmodium Vivax, malignant malaria or mixing malaria, or for distinguishing tertian fever, malignant malaria and mixing malaria kit.
Solid carrier in this kit can be one, also can be multiple, but is preferably one, and namely all polypeptide is connected on same solid carrier independently.
In the present invention, solid carrier is not particularly limited, as long as the carrier of solid or insoluble material (be such as can pass through filtration, material that precipitation, magnetic resolution etc. are separated from reaction mixture).
The material forming solid carrier includes but not limited to: silica gel (dimethyl silicone polymer, PDMS), cellulose, teflon
tM, NC Nitroncellulose, agarose, glucosan, shitosan, polystyrene, polyacrylamide, polyester, polycarbonate, polyamide, polypropylene, nylon, polyvinylidene fluoride, latex, silicon dioxide, glass, glass fibre, gold, platinum, silver, copper, iron, stainless steel, ferrite, silicon wafer, tygon, polyethyleneimine, PLA, resin, polysaccharide, albumen (albumin etc.), carbon or their combination etc.
The shape of solid carrier comprises but is not limited to: pearl, magnetic bead, film, microcapillary, filter membrane, plate, micro plate, carbon nano-tube, sensor chip etc.Just as known in the art, the solid carrier that film or plate etc. are smooth can be arranged bottom pit, groove, filter membrane etc.
Magnetic bead can have the sphere diameter of about 25nm ~ about 1mm scope.In a preferred embodiment, magnetic bead has the diameter of about 50nm ~ about 10 μm scope.The size of magnetic bead can be selected according to specific purposes.
The pearl be made up of the contour crosslinked spherical agarose of Sepharose has the diameter of about 24 μm ~ about 165 μm of scopes.Preferably, high crosslinked spherical sepharose 4B has the diameter of about 24 μm ~ about 44 μm of scopes.The size of high crosslinked spherical sepharose 4B can be selected according to specific purposes.
The example with the solid carrier of hydrophobic surface comprises can from Polysciences, Warrington, PA or Spherotech, the polystyrene latex beads such as the goods that Liberville, IL buy.
Silicon dioxide (SiO
2)-process or silicon dioxide (SiO
2) example of solid carrier of base comprises the extraordinary magnetic silica pearl etc. can bought from Polysciences, Warrington, PA, it may be used for catching nucleic acid (such as DNA).Or, the M-280 etc. that can buy from DynalBiotech can also be used.
The magnetic bead with hydrophilic surface can be used for the bacterial cell of seizure proliferation period, nucleic acid and other composition.As the example of this magnetic bead, Polysciences can be listed, Warrington, the pearl (title: Biomag (registered trademark) carboxyl) that PA sells or BangsLaboratory, Inc., the name of Fishers, IN is called the pearl of MC02N/2928.Or, the M-270 etc. that DynalBiotech sells can be used.
In a preferred embodiment, described solid carrier is SJ modified silica-gel, it is the microarray solid support material (iPDMS film, see Chinese patent CN101265329A) of a kind of silicon rubber material of Suzhou Siju Biomaterials Co., Ltd.'s exploitation.This material is based on the conventional PDMS of biological study, add specific initiating agent composition (making this material realize surface-functionalized modification by surface initiated polymerization (SIP)) wherein, obtain through polyethylene glycol methacrylate-styrene polymer (poly (oligo (ethyleneglycol) methacrylate), pOEGMA) finishing again.SJ modified silica-gel has outstanding anti-protein non-specific adsorption (Nonspecificproteinadsorption, NPA) ability, non-specific protein absorption in complicated protein immunization can being detected controls to close to " absolute 0 " level (being near or below the detection limit of instrument), not only can exempt the trouble closed and repeatedly clean, can also by the sensitivity using stronger amplification of signal means to improve protein microarray.And the essence of its silicon rubber imparts the stronger mechanical property of this material and good operability.Suzhou Yvonne gathers the combination assay microarray ELISA kit that SJ modified silica-gel has successfully been applied to 11 tumor markers compositions by company, achieve high flux and high-sensitive detection, demonstrating this material is a kind of outstanding protein microarray solid support material.Meanwhile, this material also has the adjustable characteristic of surface nature, can adjust its surface topography within the specific limits by the controlled modification reaction time.
The connection of polypeptide and solid carrier can adopt the method for attachment that well known to a person skilled in the art polypeptide and solid carrier to carry out.Such as, for the connection on protein/polypeptide and modified silica-gel surface, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides [1-ethyl-3-(3-dimethylami-nopropyl) carbodiimide can be passed through, EDC] and N-hydroxy-succinamide (N-hydroxysuccinimide, NHS) reaction changes the carboxyl (-COOH) group on the macromolecular chain of modified silica-gel surface into activated group, this activated group can with protein/polypeptide on amino (-NH2) react thus realize protein/polypeptide to be fixed on solid carrier surface.
Be not particularly limited for the concentration of polypeptide in the sampling liquid used during point sample, those skilled in the art can conventionally select, and are preferably 1 μ g ~ 1000 μ g/mL, more preferably 10 μ g ~ 500 μ g/mL.In addition, the density distributed on a solid support for polypeptide is not particularly limited, and those skilled in the art can conventionally select, and is preferably 1 ~ 100 points/10mm
2, more preferably 5 ~ 50 points/10mm
2.
This kit can also comprise:
1. the serum dilution prepared or serum dilution component solution: serum dilution, such as, have the Sample dilution (production code member 070021-S2) of Sai Chi bio tech ltd, Beijing, the application of sample variable color Sample dilution (production code member bwj010103) etc. of Bo Weijia bio tech ltd, Zhengzhou.This serum dilution is used for dilute serum, and the serum that kit detects will dilute suitable multiple, such as 2 ~ 200 times, preferably 10 ~ 100 times.
This kit can also comprise:
2. concentrated washing lotion: after solid carrier surface hatches serum and ELIAS secondary antibody, the unconjugated antibody of solid carrier surface and ELIAS secondary antibody need be washed by washing lotion.Concentrated washing lotion is such as the polysorbas20 aqueous solution of 1%, need dilute 2 ~ 40 times, preferably 5 ~ 20 times during use.
This kit can also comprise:
3. ELIAS secondary antibody solution: the plasmodium specific antibody in Infected With Plasmodium (such as malaria) patients serum can be combined by the polypeptide of the present invention on solid carrier (such as SJ modified silica-gel), ELIAS secondary antibody can be combined with antibody, and the label in ELIAS secondary antibody can react with luminous substrate, thus send detectable light.ELIAS secondary antibody can be the goat anti-human igg of such as horseradish peroxidase-labeled.As ELIAS secondary antibody solution, the Goat anti human IgG (H+L) of the horseradish peroxidase-labeled that Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge produces can be listed, production code member ZB-2304.Being not particularly limited the concentration of ELIAS secondary antibody in ELIAS secondary antibody solution, can be such as 1ng ~ 1000ng/mL.
This kit can also comprise:
4. luminescent solution component solution: luminescent solution can react with the horseradish peroxidase that marks in ELIAS secondary antibody, makes to react the chemical light sending instrument and can detect.Luminescent solution is mixed by two kinds of solution, is A liquid-superoxol respectively, and B liquid-luminol solution.Luminol (luminol) only has crosses just meeting luminescence by oxidizer treatment.The mixed aqueous solution of usual use hydrogen peroxide and a kind of hydroxide bases is as exciting agent.Under horseradish peroxidase enzyme catalytic, decomposing hydrogen dioxide solution is oxygen and water:
2H
2O
2→O
2+2H
2O
Luminol and oxyhydroxide generate a pairs of anion when reacting, the dioxygen oxidation that it can be gone out by peroxide decomposition, and product is an organic peroxide.This superoxide is very unstable, decomposites nitrogen immediately, generates the 3-aminophthalic acid of excited state.During excited state to ground state transforms, the energy of release exists with the form of photon, and wavelength is positioned at the blue light components of visible ray.The SuperSignal ELISAFemtoMaximumSensitivitySubstrate of the example such as ThermoSeientific company of luminescent solution component solution, article No. 37074.
This kit can also comprise:
5. one or more reaction cavity (such as Chinese patent Granted publication CN202054829U).
This kit can also comprise:
6. other are for detection molecules (such as polypeptide, protein, nucleic acid etc.) that is qualitative or falciparum infection.
This kit can also comprise:
7. operation instructions.
This kit can also comprise:
8. be connected to described one or more solid carrier or other positive quality control point (such as people's blood plasma gamma globulin (H-I g G) independently on solid carrier, be called for short H-IgG) and/or negative Quality Control point (such as phosphate buffer is called for short PB).
diagnostic method
Whether the present invention also provides a kind of experimenter of diagnosis to be the method for malaria patients, and the method comprises:
Use the kit of the invention described above to detect the polypeptide shown in SEQIDNO:1 ~ 5 and whether have response to the blood sample in experimenter source; Wherein,
When detect have response from least any two or more polypeptide at least two or more polypeptide groups in following polypeptide group 1 ~ 5 to the blood sample that described experimenter originates respectively time (criterion 1), judge that experimenter is malaria patients; Otherwise, judge that experimenter is not malaria patients.
In this manual, " response " refers to: signal to noise ratio (snr) is more than or equal to 2, wherein, and signal to noise ratio (S/N ratio)=(polypeptide point signal value-negative control point signal value)/negative control point signal value.
In addition, when described polypeptide set 1 is aforementioned polypeptides group 1 ~ 7 whole, whether the present invention also provides a kind of experimenter of diagnosis to be the method for malignant malaria patient, and the method comprises:
Use the kit of the invention described above to detect the polypeptide shown in SEQIDNO:1 ~ 7 and whether have response to the blood sample in experimenter source; Wherein,
When detect have response from least any two or more polypeptide at least two or more polypeptide groups in polypeptide group 1 ~ 5 to the blood sample that described experimenter originates respectively and do not detect have response from any polypeptide in polypeptide group 6 ~ 7 to the blood sample that described experimenter originates time (criterion 2), judge that experimenter is malignant malaria patient; Otherwise, judge that experimenter is not malignant malaria patient.
In addition, when described polypeptide set 1 is aforementioned polypeptides group 1 ~ 7 whole, whether the present invention also provides a kind of experimenter of diagnosis to be the method for patients infected with Plasmodium vivax, and the method comprises:
Use the kit of the invention described above to detect the polypeptide shown in SEQIDNO:1 ~ 7 and whether have response to the blood sample in experimenter source; Wherein,
When detect have response from least any two or more polypeptide at least two or more polypeptide groups in polypeptide group 1 ~ 5 to the blood sample that described experimenter originates respectively and detect have response from least any two or more polypeptide in polypeptide group 6 ~ 7 to the blood sample that described experimenter originates respectively time (criterion 3), judge that experimenter is patients infected with Plasmodium vivax; Otherwise, judge that experimenter is not patients infected with Plasmodium vivax.
In addition, when described polypeptide set 1 is aforementioned polypeptides group 1 ~ 7 whole, whether the present invention also provides a kind of experimenter of diagnosis to be the method for mixing malaria patient, and the method comprises:
Use the kit of the invention described above to detect the polypeptide shown in SEQIDNO:1 ~ 7 and whether have response to the blood sample in experimenter source; Wherein,
When detect have response from least any two or more polypeptide at least two or more polypeptide groups in polypeptide group 1 ~ 5 to the blood sample that described experimenter originates respectively and detect have response from least any one or the plural polypeptide in the only polypeptide group in polypeptide group 6 ~ 7 to the blood sample that described experimenter originates time (criterion 4), judge that experimenter is mixing malaria patient; Otherwise, judge that experimenter is not mixing malaria patient.
In addition, when described polypeptide set 1 is aforementioned polypeptides group 1 ~ 7 whole, a kind of method that the present invention also provides that experimenter of differentiation is malignant malaria patient, patients infected with Plasmodium vivax, mixing malaria patient are also non-malaria patients, the method comprises:
Use the kit of the invention described above to detect the polypeptide shown in SEQIDNO:1 ~ 7 and whether have response to the blood sample in experimenter source; Wherein,
When do not detect have response from least any two or more polypeptide at least two or more polypeptide groups in polypeptide group 1 ~ 5 to the blood sample that described experimenter originates respectively time, judge that experimenter is not malaria patients;
When detect have response from least any two or more polypeptide at least two or more polypeptide groups in polypeptide group 1 ~ 5 to the blood sample that described experimenter originates respectively and do not detect have response from any polypeptide in polypeptide group 6 ~ 7 to the blood sample that described experimenter originates time, judge that experimenter is malignant malaria patient;
When detect have response from least any two or more polypeptide at least two or more polypeptide groups in polypeptide group 1 ~ 5 to the blood sample that described experimenter originates respectively and detect have response from least any two or more polypeptide in polypeptide group 6 ~ 7 to the blood sample that described experimenter originates respectively time, judge that experimenter is patients infected with Plasmodium vivax;
When detect have response from least any two or more polypeptide at least two or more polypeptide groups in polypeptide group 1 ~ 5 to the blood sample that described experimenter originates respectively and detect have response from least any one or the plural polypeptide in the only polypeptide group in polypeptide group 6 ~ 7 to the blood sample that described experimenter originates time, judge that experimenter is mixing malaria patient.
It should be noted that, in the detection method of the invention described above, described blood sample can be whole blood, blood plasma or serum, is preferably blood plasma or serum, is more preferably serum.
Embodiment
1. the preparation of polypeptide and confirmation
The polypeptide used in embodiment is synthesized by the biochemical (Shanghai) Co., Ltd. of gill, and is confirmed it by mass spectrum.Wherein,
SEQIDNO:1:HEIVEVEEILPEDEEVKEKEEVKEK; Molecular weight 3035.
SEQIDNO:2:LPEPTVTNEEYGFDDGSAFGGGLPF; Molecular weight 2617.
SEQIDNO:3:CGKNEEFLNDRCDICDEMLDPEASFWG; Molecular weight 3136.
SEQIDNO:4:NFLNDLFKKNNKNDLDDFFKNEKEYDDLCD; Molecular weight 3715.
SEQIDNO:5:KGPEIIIEEVKEEIKKQVEDGIKENDTEGN; Molecular weight is 3413.
SEQIDNO:6:PKDQLDYENDIEKKICKMEK; Molecular weight 2469.
SEQIDNO:7:KAEPKNPRENKLKQPGDRAD; Molecular weight 2291.
2. the preparation of kit
Detection chip be with SJ modified silica-gel (iPDMS film) for solid support material, be prepared from by point sample immobilized polypeptide solution thereon.Modified silica-gel be add in traditional polydimethyl siloxane material band olefin-terminal, the initiating agent of surface initiated polymerization, and be fixed in the three-dimensional structure of dimethyl silicone polymer by heat cross-linking (si-h bond bonding), obtain a kind of new material and SJ modified silica-gel.Its manufacturing process is see International Patent Publication WO2014/044184.
The SJ modified silica-gel film made can be kept in 4 DEG C of refrigerators.
Adopt brilliant core PersonalArrayerTM16 people's point sample instrument on modified silica-gel, prepare polypeptide microarrays (i.e. detection chip), process is:
1) pre-service
By SJ modified silica-gel thin slice (15 × 15mm
2) be immersed in activating solution, take out with deionized water drip washing 3 times after 30min, dry up with nitrogen, be used for point sample at once.
2) point sample
Sampling liquid is diluted and gets well and transfer in the corresponding micropore of 384 orifice plate, 384 orifice plates of band sample are placed on point sample instrument base station, pretreated modified silica-gel thin slice are placed on the base station of point sample instrument simultaneously, carry out point sample at once.Point sample environmental baseline is room temperature (25 DEG C), and humidity set is 50%.Following polypeptide is put into microarray on SJ modified silica-gel thin slice, each polypeptide point sample point, in addition point sample H-IgG point as positive quality control point and a PB point as negative Quality Control point.On the polypeptide microarrays made, the point sample amount of each point is about 0.6nL, and sampling point radius is 200 μm.
Here, on the modified silica-gel thin slice of each kit, the polypeptide of point sample is specific as follows:
Polypeptide shown in kit 1:SEQIDNO:1 ~ 5.
Polypeptide shown in kit 2:SEQIDNO:1 ~ 7.
3) chemistry is fixing
The polypeptide microarrays just made will be placed on fixing at least 6h in climatic chamber (26 DEG C, 60% humidity).Chemistry fixation procedure is see International Patent Publication WO2014/044184.
First by point sample instrument by include catch peptide molecule damping fluid point on modified silica-gel film, then damping fluid starts evaporation, catch peptide molecule and the surface intimate contact interacting of SJ modified silica-gel, by chemical bond, the high molecular end-COOH of ploy (OEGMA) on modified silica-gel surface and peptide molecule--NH
2formed and stablize covalent bond, and then chemically active peptide molecule will be had to be fixed on SJ modified silica-gel surface.
4) assemble
The polypeptide microarrays of fixing 6h must assemble in two days.First by gum, SJ modified silica-gel thin slice is attached on special reaction column, covers reaction cavity.A reactor is made up of two reaction columns and a reaction cavity.
5) preserve
The polypeptide microarrays assembled, needs to vacuumize sealing, is kept in the refrigerator of 4 DEG C, for subsequent use.
3. detect with kit
Checking procedure
(1), before starting to detect, concentrated cleaning solutions is added purified water in the ratio of 1:10 or distilled water dilutes, diluted rear direct use.Use liquid-transfering gun that 2mL cleaning fluid is added to chip surface, soak chip 3 minutes, ensure that chip surface is fully wet out.
(2) test serum sample Sample dilution is mixed according to 1:200 dilution.
(3) discard the cleaning fluid soaking chip, under the state that chip surface is completely moistening, the serum after each serum sample draws 200 μ L dilutions joins in chip reactor.
(4) chip reactor is put into chip holder, be put on shaking table, open shaking table, frequency 150 revs/min, incubated at room 30 minutes.
(5) serum sample in chip reactor is discarded, with 15mL washing lotion cleaning reaction cavity and chip surface 3 times.
(6) after having cleaned, each chip reactor adds 200 μ L enzyme labelled antibody solution respectively, chip reactor is put into chip holder, is put on shaking table, opens shaking table, frequency 150 revs/min, incubated at room 30 minutes.
(7) the enzyme labelled antibody solution in chip reactor is discarded, with 15mL washing lotion cleaning reaction cavity and chip surface 3 times.
(8) after having cleaned, take off reaction cavity, each chip surface adds 15 μ L luminous substrate liquid respectively, makes luminescent solution can be laid on chip surface uniformly.
(9) chip adding luminescent solution is placed in gel imaging instrument chemiluminescence imaging and result of determination, as gel imaging instrument, uses such as sky energy 5500 type Microarray image instrument, the prompt board TN5500 type of Yvonne or Yvonne prompt board CLEAR4000 type etc.
(10) result judges: for every a serum, and whether each polypeptide added up in kit 1 ~ 3 has response (that is, signal to noise ratio (snr) is more than or equal to 2) respectively, and the criterion 1 ~ 4 described in above-mentioned " diagnostic method " part judges.That is,
Criterion 1: when detect have response from least any two or more polypeptide at least two or more polypeptide groups in following polypeptide group 1 ~ 5 to the blood sample that described experimenter originates respectively time, judge that experimenter is malaria patients; Otherwise, judge that experimenter is not malaria patients.
Criterion 2: when detect have response from least any two or more polypeptide at least two or more polypeptide groups in polypeptide group 1 ~ 5 to the blood sample that described experimenter originates respectively and do not detect have response from any polypeptide in polypeptide group 6 ~ 7 to the blood sample that described experimenter originates time, judge that experimenter is malignant malaria patient; Otherwise, judge that experimenter is not malignant malaria patient.
Criterion 3: when detect have response from least any two or more polypeptide at least two or more polypeptide groups in polypeptide group 1 ~ 5 to the blood sample that described experimenter originates respectively and detect have response from least any two or more polypeptide in polypeptide group 6 ~ 7 to the blood sample that described experimenter originates respectively time, judge that experimenter is patients infected with Plasmodium vivax; Otherwise, judge that experimenter is not patients infected with Plasmodium vivax.
Criterion 4: when detect have response from least any two or more polypeptide at least two or more polypeptide groups in polypeptide group 1 ~ 5 to the blood sample that described experimenter originates respectively and detect have response from least any one or the plural polypeptide in the only polypeptide group in polypeptide group 6 ~ 7 to the blood sample that described experimenter originates time, judge that experimenter is mixing malaria patient; Otherwise, judge that experimenter is not mixing malaria patient.
Wherein, signal to noise ratio (S/N ratio)=(polypeptide point signal value-negative control point signal value)/negative control point signal value.Polypeptide point signal value refers to the chemiluminescence intensity value of the polypeptide point that imager software kit reads, and negative control point signal value refers to the chemiluminescence intensity value of the negative control point that imager software kit reads.
For 643 parts of serum samples, adopt respectively the kit 1(of preparation in above-mentioned steps 2 by above-mentioned 3 step) and traditional Microscopical Method For Detection detect, testing result is as shown in table 1.
Table 1
Accuracy rate is=(337+296)/643=98.4%
Sensitivity is=337/344 × 100%=98.0%
Specificity=296/299 × 100%=99.0%
False positive rate=3/299 × 100%=1.0%
For 643 serum samples, to adopt respectively in above-mentioned 2 the kit 2(of preparation by above-mentioned 3 step) and traditional Microscopical Method For Detection detect, testing result is as shown in table 2.
Table 2
Pernicious malaria accuracy rate=(138+99)/239 × 100%=99.2%
Pernicious malaria sensitivity=138/140 × 100%=98.5%
Pernicious malaria specificity=99/99 × 100%=100%
Pernicious malaria false positive rate=0/99 × 100%=0%
Every other day malaria accuracy rate=(148+99)/250 × 100%=98.8%
Every other day malaria sensitivity=148/151 × 100%=98.0%
Every other day malariaspecific=99/99 × 100%=100.0%
Every other day malaria false positive rate=0/99 × 100%=0.0%
Mixed infection accuracy rate=(51+100)/154=98.1%
Mixed infection sensitivity=51/53 × 100%=96.2%
Mixed infection specificity=100/101 × 100%=99%
Mixed infection false positive rate=1/101 × 100%=1%
It can thus be appreciated that, diagnostic data and the clinical microscopy data of mentioned reagent box 1 ~ 2 are basically identical, it meets the requirement as diagnostic kit completely, there is good accuracy rate, sensitivity and specificity, be applicable to the diagnosis of malaria, and can distinguish malignant malaria infection, tertian fever infects, the mixed infection of malignant malaria and tertian fever, is applicable to situation of all-level hospitals and disease control department uses.
Above, by embodiment, more specific description is carried out to the present invention, but be not the restriction to the technology of the present invention scope.By the record of this instructions, those skilled in the art can be easy to modify the present invention/change, and these are included in technical scope of the present invention.
SEQUENCELISTING
<110> Suzhou Siju Biomaterials Co., Ltd.
<120> can be used for the kit of Plasmodium Vivax and/or malignant malaria
<130>sj-1-7
<160>7
<170>PatentInversion3.5
<210>1
<211>25
<212>PRT
<213> artificial sequence
<220>
<221>misc_feature
<223> malaria antigen polypeptide
<400>1
HisGluIleValGluValGluGluIleLeuProGluAspGluGluVal
151015
LysGluLysGluGluValLysGluLys
2025
<210>2
<211>25
<212>PRT
<213> artificial sequence
<220>
<221>misc_feature
<223> malaria antigen polypeptide
<400>2
LeuProGluProThrValThrAsnGluGluTyrGlyPheAspAspGly
151015
SerAlaPheGlyGlyGlyLeuProPhe
2025
<210>3
<211>27
<212>PRT
<213> artificial sequence
<220>
<221>misc_feature
<223> malaria antigen polypeptide
<400>3
CysGlyLysAsnGluGluPheLeuAsnAspArgCysAspIleCysAsp
151015
GluMetLeuAspProGluAlaSerPheTrpGly
2025
<210>4
<211>30
<212>PRT
<213> artificial sequence
<220>
<221>misc_feature
<223> malaria antigen polypeptide
<400>4
AsnPheLeuAsnAspLeuPheLysLysAsnAsnLysAsnAspLeuAsp
151015
AspPhePheLysAsnGluLysGluTyrAspAspLeuCysAsp
202530
<210>5
<211>30
<212>PRT
<213> artificial sequence
<220>
<221>misc_feature
<223> malaria antigen polypeptide
<400>5
LysGlyProGluIleIleIleGluGluValLysGluGluIleLysLys
151015
GlnValGluAspGlyIleLysGluAsnAspThrGluGlyAsn
202530
<210>6
<211>20
<212>PRT
<213> artificial sequence
<220>
<221>misc_feature
<223> malaria antigen polypeptide
<400>6
ProLysAspGlnLeuAspTyrGluAsnAspIleGluLysLysIleCys
151015
LysMetGluLys
20
<210>7
<211>20
<212>PRT
<213> artificial sequence
<220>
<221>misc_feature
<223> malaria antigen polypeptide
<400>7
LysAlaGluProLysAsnProArgGluAsnLysLeuLysGlnProGly
151015
AspArgAlaAsp
20
Claims (7)
1. a kit, it comprises one or more solid carrier, and is connected to the following polypeptide set 1 on described one or more solid carrier independently:
Polypeptide shown in SEQIDNO:1;
Polypeptide shown in SEQIDNO:2;
Polypeptide shown in SEQIDNO:3;
Polypeptide shown in SEQIDNO:4; With
Polypeptide shown in SEQIDNO:5.
2. kit according to claim 1, it is for Malaria Diagnosis.
3. kit according to claim 1 and 2, wherein, described polypeptide set 1 also comprises following polypeptide set 2:
Polypeptide shown in SEQIDNO:6; With
Polypeptide shown in SEQIDNO:7.
4. kit according to claim 3, it is for Plasmodium Vivax, malignant malaria or mixing malaria, or for distinguishing tertian fever, malignant malaria and mixing malaria.
5. the kit according to any one of claim 1 ~ 4, wherein, whole polypeptide is connected on same solid carrier independently.
6. following polypeptide set 1 is for the preparation of the purposes in the kit of Malaria Diagnosis:
Polypeptide set 1
Polypeptide shown in SEQIDNO:1;
Polypeptide shown in SEQIDNO:2;
Polypeptide shown in SEQIDNO:3;
Polypeptide shown in SEQIDNO:4; With
Polypeptide shown in SEQIDNO:5.
7. purposes according to claim 6, wherein, described Malaria Diagnosis also comprises Plasmodium Vivax, malignant malaria or mixing malaria, or for distinguishing tertian fever, malignant malaria and mixing malaria, and described polypeptide set 1 also comprises following polypeptide set 2:
Polypeptide shown in SEQIDNO:6; With
Polypeptide shown in SEQIDNO:7.
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| CN105572379B CN105572379B (en) | 2018-06-05 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002070542A2 (en) * | 2001-03-02 | 2002-09-12 | Caritas St. Elizabeth's Medical Center Of Boston, Inc. | Band 3 antigenic peptides, malaria polypeptides and uses thereof |
| CN1450171A (en) * | 2003-05-09 | 2003-10-22 | 陶开华 | Detection type gene chip for detecting various infectious desease and use thereof |
| CN202054829U (en) * | 2011-05-30 | 2011-11-30 | 苏州偲聚生物材料有限公司 | Incubation reactor for biological chips |
| CN102759621A (en) * | 2011-04-26 | 2012-10-31 | 复旦大学 | High-flux rapid malaria serum detection method based on micro-fluidic chip |
| WO2014044184A1 (en) * | 2012-09-21 | 2014-03-27 | 苏州偲聚生物材料有限公司 | Protein for detecting type 1 diabetes mellitus and partial peptide thereof |
-
2014
- 2014-11-05 CN CN201410615078.4A patent/CN105572379B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002070542A2 (en) * | 2001-03-02 | 2002-09-12 | Caritas St. Elizabeth's Medical Center Of Boston, Inc. | Band 3 antigenic peptides, malaria polypeptides and uses thereof |
| CN1450171A (en) * | 2003-05-09 | 2003-10-22 | 陶开华 | Detection type gene chip for detecting various infectious desease and use thereof |
| CN102759621A (en) * | 2011-04-26 | 2012-10-31 | 复旦大学 | High-flux rapid malaria serum detection method based on micro-fluidic chip |
| CN202054829U (en) * | 2011-05-30 | 2011-11-30 | 苏州偲聚生物材料有限公司 | Incubation reactor for biological chips |
| WO2014044184A1 (en) * | 2012-09-21 | 2014-03-27 | 苏州偲聚生物材料有限公司 | Protein for detecting type 1 diabetes mellitus and partial peptide thereof |
Non-Patent Citations (6)
| Title |
|---|
| ALLAN SAUL ET AL: "Conservation of repeating structures in thePfEMP2/MESA protein of Plasmodium falciparum", 《IMMUNOLOGY AND CELL BIOL》 * |
| DENISEL.DOOLAN ET AL: "Profiling humoral immune responses to P.falciparum infection with protein microarrays", 《PROTEOMICS》 * |
| H.CURTIDOR，ET AL: "Plasmodium falciparum acid basic repeat antigen (ABRA) peptides: erythrocyte binding and biological activity", 《VACCINE》 * |
| JENNIFER K. THOMPSON，ET AL: "A novel ligand from Plasmodium falciparum that binds to asialic acid-containing receptor on the surface of human erythrocytes", 《MOLECULAR MICROBIOLOGY》 * |
| PETERD.CROMPTON ET AL: "A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray", 《PNAS》 * |
| SOCRATES HERR ERA ET AL: "HUMAN RECOGNITION OF T CELL EPITOPES ON THE Plasmodium CIRCUMSPOROZOITE PROTEIN", 《THE JOURNAL OF IMMUNOLOG》 * |
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