CN108398554A - A kind of kit of anti-torch-IgM types antibody spectrum chip and preparation method thereof and TORCH detections - Google Patents

A kind of kit of anti-torch-IgM types antibody spectrum chip and preparation method thereof and TORCH detections Download PDF

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Publication number
CN108398554A
CN108398554A CN201810188202.1A CN201810188202A CN108398554A CN 108398554 A CN108398554 A CN 108398554A CN 201810188202 A CN201810188202 A CN 201810188202A CN 108398554 A CN108398554 A CN 108398554A
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China
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torch
igm
chip
quality control
control point
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CN108398554B (en
Inventor
张大准
肖川
张永顶
马伟民
王洪涛
马新民
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SHENZHEN CITY BOLAOTE BIOLOGICAL PRODUCTS CO Ltd
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SHENZHEN CITY BOLAOTE BIOLOGICAL PRODUCTS CO Ltd
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Priority to CN201810188202.1A priority Critical patent/CN108398554B/en
Priority to PCT/CN2018/092768 priority patent/WO2019169792A1/en
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905Protozoa
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76Chemiluminescence; Bioluminescence
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911Bacteria
    • G01N33/56927Chlamydia
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983Viruses
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983Viruses
    • G01N33/56994Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5761Hepatitis B
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08RNA viruses
    • G01N2333/18Togaviridae; Flaviviridae
    • G01N2333/19Rubella virus
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/44Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
    • G01N2333/45Toxoplasma
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00Immunoassays for the detection of microorganisms
    • G01N2469/20Detection of antibodies in sample from host which are directed against antigens from microorganisms

Abstract

The invention belongs to technical field of biological, a kind of anti-torch IgM types antibody spectrum chip and preparation method thereof and the TORCH kits detected and detection method are disclosed.Anti- torch IgM type antibody spectrum chip of the present invention can not only the related pathogenic microorganisms of more kinds of TORCH of simultaneous quantitative detection infection, and coating buffer and coating condition, closed stablity agent by optimizing chip, keep anti-torch IgM types antibody spectrum chip stability more preferable, service life is longer.The kit of TORCH detections of the present invention, can not only effectively reduce the usage amount of related antigen, reduce antigen cost, while keeping its stationary phase longer, and service life is longer, reduces transportation cost, improves the convenience of storage, transport.The infection of more kinds of related pathogenic microorganisms of TORCH detection methods energy simultaneous quantitative detection TORCH of the present invention, high sensitivity, specificity are good.

Description

A kind of anti-torch-IgM types antibody spectrum chip and preparation method thereof and TORCH detections Kit
Technical field
The invention belongs to technical field of biological, and in particular to a kind of anti-torch-IgM types antibody spectrum chip and its system The kit of Preparation Method and TORCH detections.
Background technology
TORCH is the pathogen for leading to congenital intrauterine infection and perinatal infection and causing peri-natal infant deformity, it is one The English name abbreviation of group pathogenic microorganism, wherein T (Toxoplasma) is toxoplasma, and O (Others) is the micro- life of other cause of diseases Object, such as microspironema pallidum, herpes zoster virus, assays for parvovirus B 19, Coxsackie virus, R (Rubella.Virus) is rubella Virus, C (Cytomegalo.Virus) are cytomegaloviruses, and H (Herpes.Virus) is herpe simplex I/II types.TORCH One of an important factor for infection is serious harm Neonatal Health, is known as TORCH syndromes in perinatology, and infection is in generation Criticality is distributed.In population of China, torch infection is widely present, and pregnant woman is after torch infection occurs for gestation without apparent clinical Symptom, but after fetal infection, multiple organs such as fetus or neonatal liver, kidney, the heart, brain may be caused developmental defect and work(occur Energy obstacle, it is possible to cause embryo/fetal abortion, premature labor, intrauterine growth retardation, deformity, stillborn foetus and neonatal death etc. bad Consequence constitutes prenatal and postnatal care and population quality and greatly threatens.Therefore, the birth rate and raising to reduce Disabled children go out stranger Mouthful quality, should pole carry out the serological screening of torch infection to find Disadvantage pregnancy and timely processing early.To newborn TORCH detections should routinely be carried out, neonatal TORCH infection situation is understood, so as to early intervention, early treatment.
Four kinds of toxoplasma, rubella virus, cytomegalovirus, herpe simplex I/II types pathogenic microorganisms in the detection of TORCH Infection account for about 90%, remaining 10% is other pathogenic microorganisms, including assays for parvovirus B 19, Coxsackie virus, treponemal The infection such as body, hepatitis B, Chlamydia.At present to the detection method of TORCH mainly have ELISA, immunofluorescent test (IFT), Colloidal gold and genetic chip etc..In China, most convenient, most common methods for screening are to use ELISA diagnostic techniques. ELISA be detect human serum in specific IgM, IgM antibody, due to IgM be early infection index, on fetus influence it is huge Greatly, so the detection of IgM is concerned, the detection of specific IgM is the reliable basis of diagnosing fetal intrauterine infection in placenta. ELISA reagents are widely used in common lab due to its stabilization, high sensitivity, high specificity, the advantages that at low cost, but one As be used for doing qualitative, cannot quantify.And IFT is generally just for the detection of single index, the method for colloidal gold is generally also only fixed Property or sxemiquantitative, of high cost although biochip technology effect is good, price general charged is expensive.
Invention content
In view of this, the present invention for the defects in the prior art, provides a kind of anti-torch-IgM types antibody spectrum chip And preparation method thereof with TORCH detection kit.
To achieve the purpose of the present invention, the present invention adopts the following technical scheme that.
A kind of anti-torch-IgM types antibody spectrum chip, is coated with the egg that TORCH detects the antigen of relevant pathogenic microorganism White chip.
The present invention uses protein biochip technology, and the multiple pathogens of TORCH are integrated on protein chip and are prepared for height The TORCH associated antibodies IgM chips that flux, more Testing index, performance are stable, reproducible, accuracy is high.
Wherein, preferably, it is toxoplasma (TOX), cytomegalovirus that the TORCH, which detects relevant pathogenic microorganism, (CMV), rubella virus (RV), herpes simplex virus (HSV), Human parvovirus B19, Coxsackie virus, microspironema pallidum, second At least one of hepatovirus (HBV), Chlamydia.
In some embodiments, it is toxoplasma (TOX), giant cell disease that the TORCH, which detects relevant pathogenic microorganism, Malicious (CMV), rubella virus (RV), herpes simplex virus (HSV), Human parvovirus B19, Coxsackie virus, microspironema pallidum, Hepatitis B (HBV) and Chlamydia, totally 9 kinds of the relevant pathogenic microorganisms of TORCH.Wherein herpes simplex virus (HSV) includes single Pure herpesviral-I types, herpes simplex virus-II types.
Anti- torch-IgM types antibody spectrum chip of the present invention, the protein chip also include Quality Control point and/or reference Point.
Wherein, the Quality Control point includes at least one positive quality control point (PC), at least one negative Quality Control point (NC), at least One sample Quality Control point (SC) and/or at least one enzyme mark Quality Control point (EC).The reference point includes that the reference of various concentration is bent Line point (S1-S5) and/or at least one chip position reference point (Loc).
In some embodiments, chip of the present invention includes a positive quality control point (PC), a negative Quality Control point (NC), a sample Quality Control point (SC), an enzyme mark Quality Control point (EC), 5 reference curve points (S1-S5) and a chip position Reference point (Loc).
In some embodiments of the invention, the positive quality control point can be people IgM, the enzyme linked immunological marker used It is the enzyme mark of anti-human IgM.In some other embodiments, the positive quality control point can also be coated with BSA-DNP conjugates, make Enzyme linked immunological marker is exactly the mixed liquor that enzyme marks the enzymic-labelled antibody of anti-human IgM and anti-DNP-BSA.
In some embodiments of the invention, the negative Quality Control point can be less than the micro-concentrations of reaction signal value People IgM.It can also be substituted with other unrelated proteins in some other embodiments.
In some embodiments of the invention, sample Quality Control point can be the anti-human IgM of mouse.In some other embodiments In other anti-human IgM can also be used.
In some embodiments of the invention, the enzyme mark Quality Control point can be people IgM.In some other embodiments The antibody (enzyme mark is rabbit-anti people IgM) of other anti-rabbit, such as goat-anti rabbit IgM antibody can also be used.
In some embodiments of the present invention, the reference curve point is the people IgM of five kinds of various concentrations.Such as 0.5 μ g/ml, 1 The human IgG of μ g/ml, 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml.
In the present invention, it is people's IgM solution of 2 μ g/ml that the position reference point of described chip itself is coated, and main is exactly pair Positioning action when array value.
The present invention also provides the preparation methods of the anti-torch-IgM types antibody spectrum chip, with coating buffer solution dilution TORCH detects the antigen of relevant pathogenic microorganism, Quality Control point albumen and/or reference point albumen, by point sample instrument with lattice array Form is coated in chip matrix.
TORCH is detected antigen and the institute of relevant 10 kinds of pathogenic microorganisms by the point sample instrument of high-precision by the present invention Quality Control point albumen, the reference point albumen needed is coated in the form of 4 × 5 lattice array in chip matrix
Wherein, the coating buffer solution is the buffer solution containing PEG, water soluble Beta-cyclodextrin, trehalose, preservative and glycerine.
In some embodiments, the caching liquid is the Tris buffer solutions or pH7.4- of the CB buffer solutions of pH9.6, pH8.5 7.6 PBS buffer solution.
Contain PEG in coating buffer solution of the present invention.In some embodiments, the PEG is PEG4000.It is described The content of PEG is preferably 0.5%~1%, and more preferably 0.5%.
Contain water soluble Beta-cyclodextrin in coating buffer solution of the present invention so that more stable, uniform, the coated point of coating is more It is regular, more round, CV smallers.Preferably, the water soluble Beta-cyclodextrin is 0.01%~0.02%Captisol, 0.02%- 0.04% 2- hydroxy-beta-cyclodextrins or carboxymethyl-beta-cyclodextrin;
Contain trehalose in coating buffer solution of the present invention.Preferably, the content of the trehalose is 5%~8%;
Contain preservative in coating buffer solution of the present invention.In some embodiments, the preservative is Proclin300.Preferably, the content of the preservative is 0.05%.
Contain glycerine in coating buffer solution of the present invention.Preferably, the content of the glycerine is 15%.
Preferably, coating described in the preparation method of chip of the present invention is 2-8 DEG C, coating 16-24h is stood.
Chip matrix of the present invention includes but not limited to for enzyme reaction plate, sheet glass, chemical films, Bio-sil.Its It is suitble to the carrier of protein attachment to can also be used as chip matrix.
Further, the preparation method of chip of the present invention further includes the steps that being closed with closed stablity agent.
Preferably, the closed stablity agent is mannitol containing 1%-4%, PVP40000,1%-3% of 2%-5% The 0.01M disodium phosphate solns of the pH7.4 of NH2-PEG, 0.05%NaN3.In some embodiments, the closed stablity agent For 1% mannitol, 2% PVP40000,1%NH2The 0.01M disodium phosphate solns of the pH7.4 of-PEG, 0.05%NaN3.
The closing is preferably that room temperature closes 1h.
The present invention also provides a kind of kits of TORCH detections, including the anti-torch-IgM types antibody repertoire core Piece.
Further, the kit of TORCH detection further include enzyme marker, enzyme mark dilution stabilizer, sample diluting liquid, At least one of cleaning solution, developing solution.In some embodiments, the kit of TORCH detection further include enzyme marker, Enzyme mark dilutes stabilizer, sample diluting liquid, cleaning solution and developing solution.
Preferably, the enzyme marker is the anti-human IgM antibodies of horseradish peroxidase-labeled, as goat-anti people IgM is anti- Body.
Preferably, the enzyme mark dilution stabilizer is the cow's serum γ ball eggs of the citric acid comprising 0.05M, 3%-5% In vain, the glycine betaine of the gum arabic 1%-2% of PEG10000,0.05%-0.2% of 2%-4%, 0.05% The Tris solution of the 100mM of the pH7.4 of Proclin3000.It is furthermore preferred that the enzyme mark dilution stabilizer is to include 0.05M's Citric acid, 3% cow's serum gamma Globulin, 2% PEG10000,0.05% gum arabic 1% glycine betaine, 0.05% Proclin3000 pH7.4 100mM Tris solution.
Preferably, the sample diluting liquid is comprising 0.15M NaCl, 0.05%Tween20,0.01% casein The 0.02M Tris solution of pH7.4.
Preferably, the cleaning solution is the 0.02M Tris of the pH7.4 comprising 0.15M NaCl, 0.05%Tween20 Solution.
Preferably, the developing solution is sedimentation type TMB.
The present invention also provides a kind of TORCH detection methods, sample to be tested is added described after being diluted with sample diluting liquid On anti-torch-IgM types antibody spectrum chip, enzyme marker is added, developing solution is added and is protected from light colour developing, fluorescence detection device detection knot Fruit calculates the anti-torch-IgM types antibody signal value in sample to be tested.
Further, cleaning solution is washed after the detection method includes incubating before enzyme marker is added and developing solution is added The step of washing.
Detection method of the present invention detects TORCH in subject's sample by the immunology principle of antigen-antibody reaction The level of the associated antibodies IgM of infection.The signal detection system of detection method of the present invention is chemiluminescent mode, can be used Substrate is the chemiluminescent substrate of such as luminol, and the reading of result is carried out by fluorescence detection device or instrument.
As shown from the above technical solution, the present invention provides a kind of anti-torch-IgM types antibody spectrum chip and its preparation sides The kit and detection method of method and TORCH detections.Anti- torch-IgM types antibody spectrum chip of the present invention, is coated with TORCH Detect the protein chip of the antigen of relevant pathogenic microorganism.Also include further Quality Control point and/or reference point.It is described anti- Torch-IgM type antibody spectrum chips detect the antigen of relevant pathogenic microorganism with coating buffer solution dilution TORCH, egg is ordered in Quality Control White and/or reference point albumen, is coated in the form of lattice array in chip matrix by point sample instrument, further uses closed stablity agent Closing.It can not only determine simultaneously compared to traditional enzyme linked immunological, colloidal gold detection and existing biochip technology, the present invention The infection of amount detection more kinds of related pathogenic microorganisms of TORCH, and coating buffer and coating condition, closing by optimizing chip are steady Determine agent, keeps anti-torch-IgM types antibody spectrum chip stability more preferable (2-8 DEG C refrigerates 2 years, and room temperature can store 8 months), use week Phase is longer.The kit of TORCH detections of the present invention, including the anti-torch-IgM types antibody spectrum chip and enzyme mark Remember at least one of object, enzyme mark dilution stabilizer, sample diluting liquid, cleaning solution, developing solution.The kit can not only effectively drop The usage amount of low related antigen reduces antigen cost, while optimizing kit, keeps its stationary phase longer, 2-8 DEG C can place two Year, room temperature can place 8 months, and the overall performance of kit is more preferable, and service life is longer, can also further decrease transportation cost, Improve the convenience of storage, transport.More kinds of related cause of diseases of TORCH detection methods energy simultaneous quantitative detection TORCH of the present invention The infection of microorganism, high sensitivity, specificity is good, and a kind of efficient, efficiently detection means newly is provided for prenatal and postnatal care.
Specific implementation mode
The invention discloses the reagents that a kind of anti-torch-IgM types antibody spectrum chip and preparation method thereof is detected with TORCH Box.Those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular, it should be pointed out that all Similar replacement and change is apparent to those skilled in the art, they are considered as being included in the present invention.This The method and product of invention are described by preferred embodiment, and related personnel can obviously not depart from the present invention Hold, method described herein is modified or is suitably changed and is combined in spirit and scope, to realize and apply skill of the present invention Art.
For a further understanding of the present invention, below in conjunction with the embodiment of the present invention, to the technical side in the embodiment of the present invention Case is clearly and completely described, it is clear that and described embodiments are only a part of the embodiments of the present invention, rather than all Embodiment.Based on the embodiments of the present invention, those of ordinary skill in the art institute without making creative work The every other embodiment obtained, shall fall within the protection scope of the present invention.
Unless otherwise specified, reagent involved in the embodiment of the present invention is commercial product, can pass through business canal Road purchase obtains.Wherein, the content of BSA is mass volume ratio, based on g/ml, if 5%BSA is to contain 5gBSA in 100ml PBS; The content of Tween-20 is volume ratio, and such as 0.05% Tween-20 is that 1ml PBS contain 0.5 μ l Tween-20s;The content matter of PEG4000 Volume ratio is measured, based on g/ml, if 0.5%PEG is PEG4000 containing 0.5g in 100ml buffer solutions;Trehalose is mass volume ratio, Based on g/ml, if 5% trehalose is trehalose containing 5g in 100ml buffer solutions;Captisol is mass volume ratio, based on g/ml, Captisol such as 0.02% is to contain 0.02gCaptisol in 100ml buffer solutions;Proclin300 is mass volume ratio, by g/ Ml is counted, and the Proclin300 such as 0.05% is Proclin300 containing 0.05g in 100ml buffer solutions.In addition, PVA, glycine betaine, NaN3, P-hydroxybenzoic acid sodium, gum arabic, casein are mass volume ratio, based on g/ml.
The preparation of embodiment 1, anti-torch-IgM types antibody repertoire chip agent box
1, the coating of antigen:
1) the specific distribution of the Array Design of chip and antigen and reference point (is not limited only to following arrangement to set as follows Meter):
2), specifically it is coated with process:
First press the dilution antigen and coherent reference point albumen:
PC, NC, S1, S2, S3, S4, S5, EC point in array coated respectively is 2 μ g/ml, 0.01 μ g/ml, 0.5 μ g/ The people IgM of ml, 1 μ g/ml, 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml, 2.5 μ g/ml, dilution buffer are the CB buffer solutions of pH9.6 (wherein contain 0.5% PEG4000,5% trehalose, 0.02% Captisol, 0.05% Proclin300, and 15% glycerine).
It is the goat-anti IgM antibody of 2 μ g/ml that SC points are coated, and dilution buffer is the CB buffer solutions of pH9.6.
It is the people IgM of 2 μ g/ml that Loc points are coated, and dilution buffer is the CB buffer solutions of pH9.6.
The dilution of envelope antigen:50 μ g/ml, HSV-I recombinant antigen of Tox purified antigens, 20 μ g/ml, HSV-II recombinant antigens 20 μ g/ml, RV recombinant antigen, 15 μ g/ml, CMV recombinant antigen, 10 μ g/ml, HBV antigen, 8 μ g/ml, TP recombinant antigen, 40 μ g/ml, 25 μ g/ml, CV recombinant antigen of B19 recombinant antigens, 30 μ g/ml, CT recombinant antigen, 25 μ g/ml.
By the antigen diluted respectively with 0.22 μm of membrane filtration, then array is carried out with BioDot precisions point sample instrument Coating.After whole arrays complete point sample, chip is placed in 2-8 DEG C, is coated with 16-24h.
2, it closes:
Coated chip is taken out, is cleaned 3 times with the PBST cleaning solutions of pH7.4, the closed stablity of 150 μ l is then added per hole Agent (1% mannitol, 2% PVA40000,1%NH2-PEG, 0.05%NaN3 preservative pH7.4 disodium phosphate soln), Room temperature closes 1h, then pats dry, and in humidity 15% hereinafter, being placed at room temperature for, is sealed after dry 4h, 2-8 DEG C of preservation.
3, enzyme marking reagent is prepared
With enzyme mark dilution stabilizer (Tris of 100mM, wherein the citric acid containing 50mM, 3% cow's serum gamma Globulin, 2% PEG10000,0.05% gum arabic 1% glycine betaine and 0.05% Proclin300) by horseradish peroxide The rabbit-anti human IgM antibody of compound enzyme label is diluted to 6000 times.
4, the foundation of the reaction system of kit
Take out chip reagent, balance to room temperature;
Sample-adding:By negative and positive control serum and with sample diluting liquid (0.02M Tris, 0.15M NaCl, 0.05%Tween20,0.01% casein, pH7.4) 101 times of sample to be tested is diluted, chip to be measured is added per 100 μ l of hole It is reacted in hole.
It incubates:React at room temperature 30min.Add 300 μ l cleaning solutions (0.02M Tris, 0.15M NaCl, 0.05%Tween20, PH7.4), wash 3 times, each 1min.
Add ELIAS secondary antibody (enzyme marker):50 μ l enzyme labelled antibodies are added per hole.
It incubates:React at room temperature 30min.Add 300 μ l cleaning solutions, washs 3 times, each 1min.
Colour developing:50 μ l of TMB color developing agents are added per hole, room temperature is protected from light 30min.
It measures:In 30min, the signal value that each reacting hole corresponds to antibody is read and calculated with detector.
Embodiment 2, anti-torch-IgM types antibody repertoire chip agent box evaluation of the accuracy
1, anti-with the 10 determining resisting toxoplasmosis IgM positive serums and 10 of Diasorin companies ELISA kit screening Toxoplasma Gondi IgM negative serum, the anti-torch-IgM types antibody spectrum chip prepared with embodiment 1 are tested, as a result such as the following table 1 (+indicate is positive ,-indicate negative).
Anti- torch-IgM types antibody repertoire chip testing resisting toxoplasmosis IgM serum results prepared by 1 embodiment 1 of table
2, with 10 determining rubella virus IgM positive serums of Diasorin companies ELISA kit screening and 10 Rubella virus IgM negative serums, the anti-torch-IgM types antibody repertoire chip testing prepared with embodiment 1, as a result such as the following table 2 (+indicate is positive ,-indicate negative).
Anti- torch-IgM types antibody repertoire chip testing rubella virus IgM serum results prepared by 2 embodiment 1 of table
3, with 10 determining anti-cytomegalovirus IgM positive serums and 10 of Diasorin companies ELISA kit screening Example anti-cytomegalovirus IgM negative serums, anti-torch-IgM types antibody spectrum chip prepared by embodiment 1 are tested, as a result such as The following table 3 (+indicate positive ,-indicate negative).
Anti- torch-IgM types antibody repertoire chip testing anti-cytomegalovirus IgM serum results prepared by 3 embodiment 1 of table
4, with 10 determining anti-herpes simplex virus-I IgM positive serums of Diasorin companies ELISA kit screening With 10 herpes simplex virus-I IgM negative serums, the anti-torch-IgM types antibody repertoire chip testing prepared with embodiment 1, As a result such as the following table 4 (+expression is positive, and-expression is negative).
Anti- torch-IgM types antibody repertoire chip testing anti-herpes simplex virus-I IgM serum knots prepared by 4 embodiment 1 of table Fruit
5, with 10 determining anti-herpes simplex virus-II IgM positive bloods of Diasorin companies ELISA kit screening Cleer and peaceful 10 anti-herpes simplex virus-II IgM negative serums, embodiment 1 prepare anti-torch-IgM types antibody spectrum chip into It goes and tests, as a result such as the following table 5 (+expression is positive ,-expression feminine gender).
Anti- torch-IgM types antibody repertoire chip testing anti-herpes simplex virus-II IgM serum prepared by 5 embodiment 1 of table As a result
6, determine that anti-parvovirus B1 IgM sun is infected in infection with 10 of EUROIMMUN companies ELISA kit screening Property serum and 10 anti-parvovirus B1 IgM negative serums, the anti-torch-IgM types antibody spectrum chip prepared with embodiment 1 into It goes and tests, as a result such as the following table 6 (+expression is positive ,-expression feminine gender).
The anti-parvovirus B1IgM serum results of anti-torch-IgM types antibody repertoire chip testing prepared by 6 embodiment 1 of table
7, with the anti-Coxsackie virus IgM positive serums of 10 determinations and 10 of EUROIMMUN companies ELISA kit screening The anti-Coxsackie virus IgM negative serums of example, the anti-torch-IgM types antibody spectrum chip prepared with embodiment 1 are tested, as a result Such as the following table 7 (+expression is positive, and-expression is negative).
The anti-Coxsackie virus IgM serum results of anti-torch-IgM types antibody repertoire chip testing prepared by 7 embodiment 1 of table
8, with anti-microspironema pallidum (TP) the IgM positive serums of 10 determinations of EUROIMMUN companies ELISA kit screening With 10 anti-microspironema pallidum (TP) IgM negative serums, the anti-torch-IgM types antibody spectrum chip prepared with embodiment 1 carries out Test, as a result such as the following table 8 (+expression is positive, and-expression is negative).Anti- torch-IgM types antibody spectrum chip prepared by 8 embodiment 1 of table Test anti-microspironema pallidum IgM serum results
9, with 10 determining anti-hepatitis virus (HBV) core antibody IgM sun of Diasorin companies ELISA kit screening Property serum and 10 anti-HBcIgM negative serums, the anti-torch-IgM types antibody repertoire prepared with embodiment 1 Chip is tested, as a result such as the following table 9 (+expression is positive, and-expression is negative).Anti- torch-IgM types prepared by 9 embodiment 1 of table are anti- Body spectrum chip tests anti-HBcIgM serum result
10, with 10 determining Against Chlamydia Trachomatis (CT) IgM positive serums and 10 of IBL companies ELISA kit screening Example Against Chlamydia Trachomatis (CT) IgM negative serums, the anti-torch-IgM types antibody spectrum chip prepared with embodiment 1 are tested, As a result such as the following table 10 (+expression is positive, and-expression is negative).
Anti- torch-IgM types antibody repertoire chip testing Against Chlamydia Trachomatis IgM serum results prepared by 10 embodiment 1 of table
In conclusion anti-torch-IgM types antibody repertoire chip testing items antipathogen IgM's prepared by the present invention is accurate Property meet the requirements, accuracy is high.
Embodiment 3, anti-torch-IgM types antibody repertoire chip agent box Evaluation on specificity
Anti- torch-IgM types antibody repertoire chip agent box and traditional ELISA kits more of the present invention are special.With bow For shape worm project, 20 resisting toxoplasmosis IgM antibody clinic negative serums are chosen, the anti-torch-IgM types prepared with embodiment 1 Antibody spectrum chip and Diasorin companies ELISA kit are carried out at the same time test, test data such as the following table 11 ("+" indicates the positive, " ± " table is that weak positive "-" indicates negative).
Anti- torch-IgM types antibody spectrum chip specificity comparison result prepared by 11 embodiment 1 of table
The results show that anti-20 negative serums of torch-IgM types antibody repertoire chip testing prepared by embodiment 1 do not occur False sun, and Diasorin companies ELISA kit in the market tests 20 negative serums and 2 official holidays sun occurs.Show the present invention The anti-torch-IgM types antibody spectrum chip specificity is more preferable compared with Diasorin companies ELISA kit.
The estimation of stability of embodiment 4, anti-torch-IgM types antibody repertoire chip agent box
Embodiment 1 is prepared into anti-torch-IgM types antibody repertoire chip agent box and is individually positioned in 2-8 DEG C after 0 month, 6 After month, after 12 months, after 18 months, 24 months, be placed on 18-28 DEG C after 0 month, after 2 months, after 4 months, after 6 months, 8 Test signal Value Data is distinguished with anti-corresponding antigens IgM quality controlled serums after a month, as a result such as table 12 and 13.
12 embodiment 1 of table prepares anti-2-8 DEG C of estimation of stability result of torch-IgM types antibody repertoire chip agent box
13 embodiment 1 of table prepares anti-18-28 DEG C of estimation of stability result of torch-IgM types antibody repertoire chip agent box
The results show that anti-torch-IgM types antibody repertoire chip agent box of the present invention is 2 years in 2-8 DEG C of stationary phase, It it is 1 year in 18-28 DEG C of stationary phase.
Closed stablity agent and enzyme mark dilution are to stability in embodiment 5, anti-torch-IgM types antibody repertoire chip agent box Influence
It is closed with using general in the anti-torch-IgM types antibody repertoire chip agent box prepared using the embodiment of the present invention 1 Liquid (pH=7.4 0.01M PBS contain 5%BSA), (pH=7.4 0.01M PBS are containing 0.05% Tween-20,1% for enzyme mark dilution BSA) the anti-torch-IgM types antibody spectrum chip contrast agent box (kit other methods step is with embodiment 1) formed, respectively Be placed on 2-8 DEG C after 6 months, after 12 months, after 18 months, 24 months, after being placed on 18-28 DEG C, 2 months, after 4 months, 6 Test signal Value Data is distinguished after month, after 8 months with anti-corresponding antigens IgM quality controlled serums, as a result such as table 14 and 15.
The 2-8 DEG C of closed stablity agent of table 14, enzyme mark dilute stabilizer estimation of stability result
The 18-28 DEG C of closed stablity agent of table 15, enzyme mark dilute stabilizer estimation of stability result
The results show that preparing anti-torch-IgM types antibody spectrum chip detection examination using general confining liquid and enzyme mark dilution After 2-8 DEG C of agent box is placed 12 months or 18-28 DEG C place 2 months after using corresponding quality controlled serum test signal intensity it is apparent under Drop, and the anti-torch-IgM types antibody repertoire chip agent box of the present invention places 2-8 DEG C and places placement 2 in 12 months or 18-28 DEG C After month good performance is also shown with the test of corresponding quality controlled serum.Show the anti-torch-IgM types antibody spectrum chip of the present invention Closed stablity agent, enzyme mark dilution stabilizer in detection kit is better than general confining liquid and enzyme mark dilution, can be largely Improve the stability of product.
Embodiment 6, anti-torch-IgM types antibody repertoire chip agent box and antigen cost used in colloidal gold kit Compare
Compared with colloidal gold fast detecting test paper disclosed in the Chinese patent application No. is 201110220541 antigen at This:
Antigen dosage used in fluorescence immune chromatography kit calculates:
By taking toxoplasma antigen is coated with as an example:Toxoplasma antigen peridium concentration 1.5mg/ is mentioned in CN201110220541 patents Ml, by 1 μ l/cm coatings, each wide 0.4cm of test strips, therefore the average dosage per person-portion antigen is about 0.6 μ g.
Antigen dosage used in kit of the present invention calculates:
The present invention uses BioDot precision point sample instruments to carry out the coating of antigen, and the coating volume each put is only 10nL, therefore the amount for being coated with antigen used in every person-portion is:Toxoplasma purified antigen dosage:50 μ g/ml × 10nL × 10-6= 0.005μg。
It follows that the usage amount of toxoplasma antigen will obviously be less than 2 numbers of colloidal gold fast detecting test paper in the present invention Magnitude.Other antigen dosage situations are similarly.In conclusion showing anti-torch-IgM types antibody spectrum chip of the present invention Kit can effectively reduce the usage amount of related antigen, while the sensitivity and specificity for ensureing kit, reduce phase The antigen use cost of pass.

Claims (13)

1. a kind of anti-torch-IgM types antibody spectrum chip, is coated with the albumen that TORCH detects the antigen of relevant pathogenic microorganism Chip.
2. chip according to claim 1, the TORCH detect relevant pathogenic microorganism be toxoplasma (TOX), it is big and small Cellular virus (CMV), rubella virus (RV), herpes simplex virus (HSV), Human parvovirus B19, Coxsackie virus, syphilis spiral shell Revolve at least one of body, hepatitis B (HBV), Chlamydia.
3. chip according to claim 1 or 2, the protein chip also includes Quality Control point and/or reference point;The Quality Control Point comprising at least one positive quality control point (PC), at least one negative Quality Control point (NC), at least one sample Quality Control point (SC) and/ Or at least one enzyme mark Quality Control point (EC);The reference point includes the reference curve point (S1-S5) and/or at least one of various concentration A chip position reference point (Loc).
4. chip according to claim 3, the positive quality control point coating people IgM or coating BSA-DNP conjugates;It is described Negative Quality Control point coating less than the people IgM of the micro-concentrations of reaction signal value or other and TORCH detection pathogenic microorganisms without The albumen of pass;The sample Quality Control point is to be coated with anti-human IgM;The antibody of the enzyme mark Quality Control point coating people IgM or anti-rabbit;It is described Reference curve point is coated with the people IgM of various concentration;People's IgM solution of the chip position reference point coating known concentration.
5. the preparation method of the chip described in claim 1-4 any one, relevant with coating buffer solution dilution TORCH detections Antigen, Quality Control point albumen and/or the reference point albumen of pathogenic microorganism, chip base is coated in by point sample instrument in the form of lattice array In matter.
6. preparation method according to claim 5, the coating buffer solution be containing PEG, water soluble Beta-cyclodextrin, trehalose, The buffer solution of preservative and glycerine;The coating is 2-8 DEG C, stands coating 16-24h;The chip matrix be enzyme reaction plate, Sheet glass, chemical films, Bio-sil.
7. preparation method according to claim 6, the caching liquid is the Tris bufferings of the CB buffer solutions of pH9.6, pH8.5 The PBS buffer solution of liquid or pH7.4-7.6;The PEG is PEG4000;The content of the PEG is 0.5%~1%;It is described water-soluble Property cyclodextrin be 0.01%~0.02%Captisol, the 2- hydroxy-beta-cyclodextrins of 0.02%-0.04% or carboxymethyl-β-ring Dextrin;The content of the trehalose is 5%~8%;The preservative is Proclin300;The content of the preservative is 0.05%;The content of the glycerine is 15%~20%.
Further include the steps that being closed with closed stablity agent 8. according to the preparation method described in claim 5-7 any one.
9. preparation method according to claim 8, the closed stablity agent is mannitol containing 1%-4%, 2%-5%'s PVP40000,1%-3%NH2The 0.01M disodium phosphate solns of the pH7.4 of-PEG, 0.05%NaN3.
10. a kind of kit of TORCH detections, including the anti-torch-IgM types antibody repertoire described in claim 1-4 any one Chip.
11. kit according to claim 10 further includes enzyme marker, enzyme mark dilution stabilizer, sample diluting liquid, washes Wash at least one of liquid, developing solution.
12. kit according to claim 11, the enzyme marker is that the anti-human IgM of horseradish peroxidase-labeled is anti- Body;It is the citric acid comprising 0.05M, the cow's serum gamma Globulin of 3%-5%, 2%-4% that enzyme mark, which dilutes stabilizer, The pH7.4 of the glycine betaine of the gum arabic 1%-2% of PEG10000,0.05%-0.2%, 0.05% Proclin3000 The Tris solution of 100mM;Sample diluting liquid is the pH7.4 comprising 0.15M NaCl, 0.05%Tween20,0.01% casein 0.02M Tris solution;Cleaning solution is the 0.02M Tris solution of the pH7.4 comprising 0.15M NaCl, 0.05%Tween20; The developing solution is sedimentation type TMB.
13. a kind of TORCH detection methods, claim 1-4 any one institute is added after being diluted with sample diluting liquid in sample to be tested On the anti-torch-IgM types antibody spectrum chip stated, enzyme marker is added, developing solution is added and is protected from light colour developing, fluorescence detection device inspection It surveys as a result, calculating the anti-torch-IgM types antibody signal value in sample to be tested.
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