CN105759057A - Dry-type immunofluorescence kit for detecting Alzheimer syndrome MS4A6A and preparation method thereof - Google Patents
Dry-type immunofluorescence kit for detecting Alzheimer syndrome MS4A6A and preparation method thereof Download PDFInfo
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- G—PHYSICS
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
The invention relates to a dry-type immunofluorescence kit for detecting Alzheimer syndrome MS4A6A and a preparation method thereof. The kit comprises a PVC (polyvinyl chloride) bottom plate, a fluorescent-binding pad, a nitrocellulose membrane, a sample pad and a water absorbent pad, wherein the sample pad, the nitrocellulose membrane, the fluorescent-binding pad and the water absorbent pad are fixed on the PVC bottom plate horizontally; a coated mouse anti-human MS4A6A monoclonal antibody 1-nano-fluorescence bead compound and a coated chicken IgY-nano-fluorescence bead compound are arranged on the fluorescent-binding pad, and a detection line formed by mouse anti-human MS4A6A monoclonal antibody 2 and a quality control line formed by rabbit anti-chicken IgY antibody are formed on the nitrocellulose membrane. According to the kit, the nano-fluorescence bead is used for detecting MS4A6A, the background value is low, the detection time is short, the specificity and the sensitivity are high, the detection result is accurate, and the kit and the method provide a more reliable basis for clinical diagnosis and Alzheimer syndrome.
Description
Technical field
The present invention relates to a kind of dry type immunofluorescent reagent box detecting alzheimer syndrome MS4A6A and preparation method, the invention belongs to medical product field.
Background technology
Alzheimer syndrome (AD) is the nervous system degenerative disease of the Progressive symmetric erythrokeratodermia development of a kind of onset concealment.AD is one of geratic period most common disease, and the World Health Organization (WHO) estimates that the prevalence of whole world over-65s elderly population AD is 4% ~ 7%.AD prevalence is closely related with the age, and the age the most often increases by 6.1 years old, and prevalence raises 1 times；More than 85 years old in elderly population, AD prevalence may be up to 20% ~ 30%.Calendar year 2001, whole world AD patient was more than 20,000,000, estimated the year two thousand forty, and whole world AD patient will be more than 80,000,000.From 1999, China initially enters aging society, corresponding, and the current Aged in China ill population of crowd AD alreadys more than 6,000,000, expecting the year two thousand fifty ill population will be ill most populous, the country that growth rate is the fastest of AD in the world more than 20,000,000.AD is the most common disease causing old people to lose activity of daily living, is also the 5th cause of disease causing old people dead.AD not only brings huge misery to patient, returns family and society brings heavy stress and medical treatment, Burden of care.Therefore, AD has become the significant problem affecting global public health and social sustainable development.Although countries in the world are a large amount of manpower and materials to the Innovation Input of AD medicine, still fail to develop the medicine delaying or stoping AD to be in progress.Therefore early diagnosis treatment is the critical period of preventing and treating AD.
The diagnostic method that at present alzheimer syndrome clinic is conventional has: 1, neuroimaging inspection, electroencephalogram: as calculated hippocampus neurofibrillary tangles and senile plaque number, with the coincidence rate of clinical diagnosis up to 81%～88%.Also having a number of neurofibrillary tangles and senile plaque due to normal aging people, and have same distribution, accuracy rate is relatively low；2, Five neuropsychological tests: Five neuropsychological tests is the class psychometry method for brain function assessment grown up on the basis of modern psychology is test.Because individual variation is relatively big, accuracy rate is poor.For early stage patient, recall rate is unsatisfactory；3, gene test: by detection alzheimer Comprehensive Criteria sex factor, such as amyloid precursor protein gene (APP), presenilin 1,2 gene (PS1, PS2) etc., there are simplicity, sensitivity, the advantage such as special, but need to detect accordingly equipment, technology requires height, costly, it is difficult to promote.4, hematological examination, cerebrospinal fluid detection: mainly for detection of alzheimer syndrome specific proteins, find the risk factor that the accompanying diseases that exists or complication, discovery are potential.At present in conventional ELISA immune diagnostic technique detection serum or cerebrospinal fluid specific proteins, have conventional program tediously long time-consuming, be not easy to promote, sensitivity is low.Therefore find a kind of quickly, be convenient for carrying, simple to operate, highly sensitive, diagnosis and development of methodology tool to alzheimer syndrome are of great significance.
MS4A6A belongs to MS4A (membrane-spanning 4-domain
Family, subfamily A) family, MS4A family is the coding mankind and the big gene family of more than 20 different proteins of mouse, including CD20, high parent's property IgE receptor β subunit (Fc ε RI β) and transmembrane albumen-IV (HTm4) with hematopoietic cell characteristic.The CD20 of the mankind is designated as MS4A1, Fc ε RI β and is designated as MS4A2, HTm4 and is designated as MS4A3, and remaining gene is then respectively designated MS4A4A etc. 6 kinds；Mouse MS4a gene kinsfolk is designated as MS4al etc. 15 kinds.Each MS4A protein comprises 4 membrane-spanning domains supposed, these membrane-spanning domains have intracellular N-and C-end；All MS4A film sizes are similar, and have a homologous sequence the most similar, and the most conforming region is in front 3 membrane-spanning domains.MS4A protein all has expression in various tissues, especially the most notable in lymphoid tissue.But, even if they are expressed in different cell types, but because in the conservative of gene locus and the concordance in terms of structure and sequence, therefore MS4A family member may participate in some functions the most jointly.MS4A6A, is also 4SPAN3, MS4A4E.Utilizing whole-genome association to find in the astrocyte of Alzheimer's, the up-regulated of MS4A6A, its neurosis caused at alzheimer's disease plays important function.
Fluorescent microsphere nano-particle has tremendous potential in marker detection field.It has the following characteristics that 1, highly sensitive: by extraneous ambient interferences；2, stability is high: avoid sample corrosion or the quenching phenomenon of self decay initiation；3, motility is high: can combine variation spectrum；4, safety is high: be all safe from harm for tester, detection sample and environment；5, convenient simple: sample directly can be detected, it is not necessary to special handling；6, above feature determines that its application prospect in terms of biomarker is broader, especially at the other detection field of bed.By fluorescent microsphere nanoparticle label mouse-anti people's MS4A6A monoclonal antibody, set up a kind of dry type immunofluorescent reagent box detecting alzheimer syndrome MS4A6A and preparation method has important using value in Clinical detection field.
Summary of the invention
For with present on problem, the invention provides a kind of dry type immunofluorescent reagent box detecting alzheimer syndrome MS4A6A and preparation method.The invention have the characteristics that and combine dry type immuno-fluorescence assay people MS4A6A, forming double-antibody sandwich structure by specific monoclonal antibody with MS4A6A antigen in sample, its accuracy and specificity are higher；Utilizing dry type determination of immunofluorescence method, its background value is low, measures convenient, accurate, simple to operate.
It is an object of the invention to provide a kind of dry type immunofluorescent reagent box detecting alzheimer syndrome MS4A6A and preparation method.
Test kit of the present invention includes PVC base plate, fluorescence pad, nitrocellulose filter, sample pad and adsorptive pads；Described sample pad, nitrocellulose filter, fluorescence pad, adsorptive pads are fixed on PVC base plate by horizontal direction.Coated mouse-anti people's MS4A6A monoclonal antibody 1-fluorescent microsphere nano-particle complex, chicken IgY-fluorescent microsphere nano-particle complex is had on described fluorescence pad；Detection line that mouse-anti people's MS4A6A monoclonal antibody 2 constitutes and the nature controlling line that rabbit anti-chicken IgY antibody is constituted is had on described nitrocellulose filter；Described nano-particle footpath is 100 ~ 200nm.
The technical scheme is that
1) preparation of nitrocellulose filter (NC film)-PVC base plate: nitrocellulose filter is cut into 25mm × 300mm, is attached on PVC base plate.Mouse-anti people's MS4A6A monoclonal antibody 2 is rule on nitrocellulose filter and obtains detecting line；Anti-for rabbit chicken IgY is rule on nitrocellulose filter and obtains nature controlling line.Then nitrocellulose filter (NC film)-PVC base plate is placed in drying baker and is dried；
2) utilize nanometer fluorescent microspheres particle marker mouse-anti people's MS4A6A monoclonal antibody 1 and chicken IgY, save backup.
3) preparation of pad: pad is cut into 9mm × 300mm slice, mouse-anti people's MS4A6A monoclonal antibody 1-fluorescent microsphere nano-particle complex, chicken IgY-fluorescent microsphere nano-particle complex are mixed according to a certain percentage, uniform spreading, on pad, puts into stove-drying.
4) sample pad: glass fibre membrane is cut into 17mm × 300mm slice.
5) absorption pad: absorbent paper is cut into 17mm × 300mm slice.
7) assemble: above-mentioned adsorptive pads, sample pad, fluorescence pad are attached on PVC base plate successively, the intermedium cutting machine posted is cut into the reagent strip of one fixed width.
Accompanying drawing explanation
Fig. 1 dry type Immunofluorescence test system structure schematic diagram, wherein 1 is sample pad；2 is fluorescence pad；3 is nitrocellulose filter；4 is adsorptive pads；5 is PVC base plate；6 is target antigen in sample；7 is fluorescent microsphere labelling mouse-anti people's MS4A6A monoclonal antibody 1；8 is fluorescent microsphere labelling chicken IgY；9 is detection line: mouse-anti people's MS4A6A monoclonal antibody 2；10 is nature controlling line: rabbit anti-chicken IgY.
Detailed description of the invention
Embodiment 1 prepares the dry type immunofluorescent reagent box of the alzheimer syndrome MS4A6A of the present invention
Test kit of the present invention includes PVC base plate, fluorescence pad, nitrocellulose filter, sample pad and adsorptive pads；Described sample pad, nitrocellulose filter, fluorescence pad, adsorptive pads are fixed on PVC base plate by horizontal direction.
One, the preparation of nitrocellulose filter (NC film)-PVC base plate
1, draw film: celluloid enzyme action is become 25mm × 300mm, be attached on PVC base plate.Anti-for rabbit chicken IgY PBS is diluted to concentration is 1mg/ml, is coated liquid as C line；With PBS, mouse-anti people's MS4A6A monoclonal antibody 2 is diluted to concentration is 1mg/ml, is coated liquid as T line.By spray film instrument, T line is coated liquid and C line is coated liquid and rule on nitrocellulose filter and obtain detection line and nature controlling line, dry for 37 degree, i.e. can get monoclonal antibody and be coated test strip.
Two, the preparation of pad
1, traget antibody: nanometer fluorescent microspheres granule is to be condensed by schiff base base under mild alkaline conditions with the amino on antibody protein by the aldehyde radical on nanometer fluorescent microspheres with mouse-anti people's MS4A6A monoclonal antibody coupling method, forms nanometer fluorescent microspheres-CH-NH2-MS4A6A antibody complex；The same manner forms nanometer fluorescent microspheres-CH-NH2-chicken IgY antibody complex.Fluorescent microsphere nano-particle and mouse-anti people's MS4A6A monoclonal antibody or chicken IgY are according to 1:1(ul:ug) hybrid reaction 2h, prepare mouse-anti people's MS4A6A monoclonal antibody 1-fluorescent microsphere nano-complex or chicken IgY-fluorescent microsphere nano-particle complex.
2, the preparation of pad: pad is cut into 9mm × 300mm slice, mouse-anti people's MS4A6A monoclonal antibody 1-fluorescent microsphere nano-particle complex or chicken IgY-fluorescent microsphere nano-particle complex mix homogeneously according to a certain percentage are layered on pad, put into and toast 2h under 37 degree of baking oven.
Three, prepared by other auxiliary materials
1, glass fibre membrane is cut into 17mm × 300mm slice.
2, absorption pad: absorbent paper is cut into 17mm × 300mm slice.
Four, assemble
Assemble test strips: paste on PVC base plate successively by nitrocellulose filter, absorbent paper, fluorescence pad, sample pad, be cut into the reagent strip of one fixed width with cutting machine.
The using method of embodiment 2 test kit of the present invention
1, with pipettor pipette 50ul serum, blood plasma, CSF sample are added on the sample-adding pad of test strips；
2, room temperature stands 10min；
3,10min terminates, and test strips is put into reading data in immunofluorescence quantitative analysis instrument；
4, optical signalling is measured and analyzes and processes by immunofluorescence quantitative analysis instrument, quantitatively draws the concentration of measured matter；
5, yin and yang attribute is judged according to reference value.
Embodiment 3 test kit of the present invention compares with ELISA test kit
The test kit of the present invention and ELISA test kit detect alzheimer syndrome patients's serum 185 example of clinical definite simultaneously, CSF sample 28 example, normal human control sera 180 example, and testing result see table 1:
The test kit of table 1 present invention and ELISA test kit testing result
As it can be seen from table 1 the test kit of the present invention to the positive rate of alzheimer syndrome patients apparently higher than ELISA kit, normal person's Virus monitory is feminine gender, it is seen that the test kit of the present invention is more more sensitive than ELISA kit, special, accurate.Illustrate that test kit of the present invention has higher clinical coincidence rate, can be that the detection of current alzheimer syndrome provides more accurately, is worth reliably.
Claims (8)
1. detecting dry type immunofluorescent reagent box and a preparation method of alzheimer syndrome MS4A6A, its special disease is, this test kit includes PVC base plate, fluorescence pad, nitrocellulose filter, sample pad and adsorptive pads；Described sample pad, fluorescence pad, nitrocellulose filter, adsorptive pads are fixed on PVC base plate by horizontal direction；Described knot fluorescence closes has coated mouse-anti people's MS4A6A monoclonal antibody 1-nanometer fluorescent microspheres complex, chicken IgY-nanometer fluorescent microspheres complex on pad；Detection line that mouse-anti people's MS4A6A monoclonal antibody 2 constitutes and the nature controlling line that rabbit anti-chicken IgY antibody is constituted is had on described nitrocellulose filter.
A kind of dry type immunofluorescent reagent box detecting alzheimer syndrome MS4A6A the most as claimed in claim 1 and preparation method, its special disease is, described nanometer fluorescent microspheres is the red fluorescent microsphere of aldehyde radicalization, nano particle diameter is 100 ~ 200nm, excitation wavelength is 365nm, a length of 612 ± the 5nm of transmitted wave, surface has its content of aldehyde groups to be 0.1 ~ 0.5mMol/g.
A kind of dry type immunofluorescent reagent box detecting alzheimer syndrome MS4A6A the most as claimed in claim 1 and preparation method, its special disease is, mouse-anti people's MS4A6A monoclonal antibody 1 and mouse-anti people's MS4A6A monoclonal antibody 2 are MS4A6A specific monoclonal antibody, identify 2 kinds of sites on MS4A6A protein molecular.
A kind of dry type immunofluorescent reagent box detecting alzheimer syndrome MS4A6A the most as claimed in claim 1 and preparation method, its special disease is, celluloid enzyme action is become 25mm × 300mm, is attached on PVC base plate；Anti-for rabbit chicken IgY PBS is diluted to concentration is 1mg/ml, is coated liquid as C line；With PBS, mouse-anti people's MS4A6A monoclonal antibody 2 is diluted to concentration is 1mg/ml, is coated liquid as T line；By spray film instrument, T line is coated liquid and C line is coated liquid and rule on nitrocellulose filter and obtain detection line and nature controlling line, dry for 37 degree, i.e. can get monoclonal antibody and be coated test strip.
5. a kind of dry type immunofluorescent reagent box detecting alzheimer syndrome MS4A6A as described in claim 1 and preparation method, its special disease is that nanometer fluorescent microspheres granule and mouse-anti people's MS4A6A monoclonal antibody or chicken IgY are according to 1:1(ul:ug) mix, 37 degree of reaction 2h, prepare mouse-anti people's MS4A6A monoclonal antibody 1-nanometer fluorescent microspheres complex or chicken IgY-nanometer fluorescent microspheres complex.
6. nanometer fluorescent microspheres granule as claimed in claim 5 is to be condensed by schiff base base under mild alkaline conditions with the amino on antibody protein by the aldehyde radical on nanometer fluorescent microspheres with mouse-anti people's MS4A6A monoclonal antibody coupling method, forms nanometer fluorescent microspheres-CH-NH2-MS4A6A antibody complex；The same manner forms nanometer fluorescent microspheres-CH-NH2-chicken IgY antibody complex.
A kind of dry type immunofluorescent reagent box detecting alzheimer syndrome MS4A6A the most as claimed in claim 1 and preparation method, its special disease is the preparation of pad: pad is cut into 9mm × 300mm slice, mouse-anti people's MS4A6A monoclonal antibody 1-nanometer fluorescent microspheres complex or chicken IgY-nanometer fluorescent microspheres complex mix homogeneously according to a certain percentage are layered on pad, put into and toast 2h under 37 degree of baking oven.
A kind of dry type immunofluorescent reagent box detecting alzheimer syndrome MS4A6A the most as claimed in claim 1 and preparation method, its special disease is that detecting sample is serum, blood plasma, whole blood, cerebrospinal fluid, urine.
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