CN102680702B - Immune-fluorescence test strip component for rapidly detecting C-reactive protein quantitatively, detection card component produced by same and method for preparing same - Google Patents
Immune-fluorescence test strip component for rapidly detecting C-reactive protein quantitatively, detection card component produced by same and method for preparing same Download PDFInfo
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Abstract
The invention discloses an immune-fluorescence test strip component for rapidly detecting C-reactive protein quantitatively, a detection card component produced by the same and a method for preparing the same. The test strip component comprises a test strip and a platinum porphyrin marked specific antibody independently packed. The test strip comprises a bottom lining, a water absorption pad, coating analysis film and a sample pad. The coating analysis film is provided with a detection line and a quality control line, a specific antibody coated by the detection line is a C-reactive protein resistance monoclonal antibody, and a specific antibody coated by the quality control line is a rabbit intravenous gamma globulin (IgG) antibody; the detection card component comprises a test strip, a card box composed of a cover plate and a back plate and a latinum porphyrin marked specific antibody independently packed. The test strip component for detecting C-reactive protein in body fluid has the advantages of being simple in operation, rapid, sensitive, good in specificity and the like, and having good clinical application prospect.
Description
Technical field
The invention belongs to field of medical examination, relate in particular to Immunofluorescence test paper strip assembly of a kind of fast quantitative determination of C-reaction protein and preparation method thereof, and the test card assembly being supported by Immunofluorescence test paper strip assembly and preparation method thereof.
Background technology
C reactive protein (C-reactive protein, CRP) be ring-type five spheroid albumen, belong to Oligomeric calbindin, relative molecular mass approximately 120000, by 5 identical monomers, with non-covalent bond, formed, be inflammatory lymph factor interleukin-6, il-1, TNF stimulates liver epithelial cell to synthesize.Due to C reactive protein, 6～8h after infecting generation starts to raise, and 24～48h peaks, and peak value can reach normal hundreds of times, and the hurried decline of its content after infecting elimination can recover normal in one week.C reactive protein is acknowledged as the acute phase reactive protein of most worthy, and its rising can be pointed out the generation of many inflammatory episode, therefore, is widely used in for a long time the detection of clinical infectious disease.Have in recent years researcher to find, inflammation has been played the part of important role in atherosclerotic initial, formation, evolution, so this extremely sensitive inflammation index of C reactive protein has obtained research widely in all kinds angiocardiopathy.
The conventional C reactive protein method of inspection can detect C reactive protein > 10mg/L when infection or tissue damage, and still, the C reactive protein that can not detect well low-level (0.1～10mg/L) changes.The C reactive protein of this level and angiocardiopathy have a close relationship, so day by day receive researcher's concern.Appearance along with some high-sensitivity detecting methods, low-level C reactive protein also can be detected, because the detection method of applying has higher sensitivity, therefore, the c reactive protein of this level higher hs-CRP (high sensitivity C-reactive protein, hs-C-reactive protein) that is otherwise known as.Studies show that, in the healthy male of non-evident sympton, the rising degree of C reactive protein is proportionate with the first danger that coronary heart disease occurs, the following danger that apoplexy, myocardial infarction, peripheral vascular disease occur of the part crowd of hs-C-reactive protein concentration in high value is respectively 2 times of (RR=1.9 of low value, 95%CI 1.1～3.3), 3 times of (RR=2.9,95%) CI 1.8～6.0) and 4 times of (RR=4.1,95%CI 1.2～6.0), and confirmation is not subject to the impact of smoking factor and is independent of outside other risk factors of coronary heart disease.Hs-C-reactive protein is also the predictor (RR=4.4,95%CI 2.2～8.9) of a strong following cardiovascular morbidity for women.Hs-C-reactive protein also has very important effect for the diagnosis of acute coronary syndrome.The myocardial infarction patient that unstable angina or non-ST section are raised has hs-C-reactive protein to raise, show various adverse events to occur, the death that the myocardial infarction of send out again as anginal, ST section being raised or coronary heart disease cause.
Clinical labororatory's C reactive protein detects and adopts transmission immunity and scattering immunization method always, and the reference range change of these methods is very large, from 3mg/L to surpassing 200mg/L.Along with the understanding of people for low-level C reactive protein and angiocardiopathy relation, the sensitivity of these assay methods can not meet the demands far away, and even current detection method used expensive instrument and equipment and reagent, can not take into account the testing requirement of high value and low value simultaneously.Therefore, set up a kind of detection time short, and detect except can be carry out in laboratory, also requirement can be carried out the other detection of bed, simultaneously to high value and low value C reactive protein applicable detection method all, thereby for the clinical diagnosis basis accurately that provides, be very necessary.
At present, be used for measuring the label that uses in the immunochromatography technique of C reactive protein normally enzyme, collaurum and various color micro-sphere label, these labels are applied in immunochromatography technique, there is identical feature: physical adsorption way mark and judge testing result by color.Wherein the feature of physical adsorption way mark (being hydrophobicity and electrostatic absorption principle) makes it easily form nonspecific interference, need in production technology formula, add nonspecific interference and eliminate reagent, as surfactants such as polysorbas20 etc., but when using this class reagent, also easily cause false positive or false-negative result based on this class label.By color sentence read result, affected greatly by observer's subjectivity in addition, especially weak positive findings, and can only make qualitative judgement, and cannot realize accurate quantitative judgement.These shortcomings have limited the range of application of immunochromatography technique in clinical detection greatly.
And China discloses No. 102087293 patent and disclosed a kind of whole process and quantitatively detect immune chromatography test paper of C reactive protein and preparation method thereof, although it has adopted fluorescent quantitation technology, there is no clearly concrete fluorescent material; Simultaneously, it is dried fluorescence emulsion particle to be fixed on pad, must affect its dissolving and release rate on glass fibre element film, also affect its creep speed on nitrocellulose filter, also will far surpass 10 seconds minutes of its requirement whole detection time.Simultaneously, the C reactive protein antibody of the C reactive protein in detected material and fluorescent latex particles mark, and fixing C reactive protein antibody on nitrocellulose filter, within 10 seconds, be difficult to form effective antigen-antibody reaction, immunological response is inadequate, will cause detection sensitivity on the low side, detect linear narrow range, be difficult to meet the demand of clinical detection.
And metalloporphyrin is the class macrocyclic compound that occurring in nature extensively exists, Porphyrin Molecule is linked by methine by 4 pyrrole rings, forms a four-coordination porphyrin core.Porphyrin ring is highly stable, can be the metal generation coordination of 3.7 dusts with diameter.The electronic of porphyrin mainly contains Soret band (having another name called B band) and Q band.Soret band is between 380～450nm, and molar absorptivity is high by (2～5 * 10
5mol
-1.L.cm
-1).And a little less than the absorption of the Soret of metalloporphyrin band, when ring side has electrophilic group, Soret band will move to long wave direction.The Q band of porphyrin generally, between 450～650nm, has 4 relevant peaks; Hydrogen on pyrrole ring nitrogen is replaced and forms after metalloporphyrin by metallic ion, and 4 relevant peaks weaken or disappear.Porphyrin and metalloporphyrin be owing to having the large π delocalization of 18 electronics structure, so, using its B band or Q band as excitation wavelength, all between 500～800nm, (or longer wavelength coverage) has fluorescent emission in various degree.Under room temperature, porphyrin itself is not phosphorescent, when forming complex compound with some metals and coexisting with ordered media biomacromolecules such as (as:) surfactant, protein and nucleic acid just at near-infrared region transmitting phosphorescence; But only have the metalloporphyrin of only a few phosphorescent, modal is palladium porphyrin and platinum porphyrins.Palladium/platinum porphyrins has extremely strong phosphorescence, is characterized in the long-life (ms), long wavelength (600～1000nm).Modal have water-soluble meso-tetra-(4-sulphenyl) porphyrin (H
2tSPP
4-) and meso-tetra-(4-N-front three aminophenyl) porphyrin (H
2tMAP
4+) palladium/platinum complex, and non-water-soluble octaethylporphyrin (OEP) and tetraphenyl-Tetrabenzoporphyrin (Ph
4tBP) palladium/platinum complex etc.The near-infrared region of 600～1000nm is an extremely useful region of the biological material luminescence probe of research and Optochemical sensor.So the palladium/platinum porphyrins with special phosphorescent characteristics just becomes the very effective probe molecule in bioanalysis aspect, just can provide very high sensitivity and selectivity in conjunction with some simple detecting instruments.
Thisly take platinum as basic porphyrin is under the external world excites, at 650nm, send strong phosphorescence, duration 100-microsecond (absorbing ripple scope 390-410nm), sends strong phosphorescence duration 500-microsecond (absorbing ripple scope 400-420nm) at 670nm.These porphyrin particles also have very large stokes displacement (being 280nm).Compare with other luminescent material, the advantage of platinum porphyrins is atomic photobleaching, uses cheap intense light source, as light emitting diode just can effectively excite.In addition, the background fluorescence of biological specimen and nitrocellulose filter is all low when take the excitation wavelength 365nm of the time-resolved fluorescence that europium ion is representative at 390-420nm rate of induced polarization.Although 390-420nm light transmission nitrocellulose filter is also desirable not to the utmost, be better than 365nm light, be more suitable for measuring in transmission-type.Platinum porphyrins also can covalent labeling antibody, for detecting various samples, provides a sensitive Fast Detection Technique.
Luminescent material platinum porphyrins and bioactive molecule are prepared to label by covalent bond coupling, and this label is fixed on the surface of solid phase carrier in the process of chromatography by immune response, and launches infrared light under the exciting of green laser.Realizing thus platinum porphyrins luminescent material combines with immunochromatography technique the target detected material in biological sample is detected.
Therefore, how platinum porphyrins luminescent material is combined with immunochromatography technique, shorten detection time as far as possible, and detect except can be carry out in laboratory, also requirement can be carried out the other detection of bed, the C reactive protein of energy quantitative measurement simultaneously method, thus for the clinical diagnosis basis accurately that provides, be very necessary.
Summary of the invention
For solving above-mentioned the deficiencies in the prior art, the present invention combines platinum porphyrins luminescent material with immunochromatography technique, provide a kind of test strips assembly with C reactive protein level in human body, the present invention only needs the special-purpose fluorescence detector of supporting routine to use, can detect high value and low value C reactive protein simultaneously, especially low value C reactive protein, its cost of manufacture is cheap, simple to operate, quick, sensitive, and specificity is good, the prevention occurring for cardiocerebrovasculaevents events has very important meaning.
Concrete, for achieving the above object, the present invention has taked following technical scheme:
An Immunofluorescence test paper strip assembly for fast quantitative determination of C-reaction protein, comprises the platinum porphyrins mark specific antibody of test strips and independent packaging.Wherein, test strips is comprised of end liner and the adsorptive pads engaging successively on end liner, coated analyzing film, sample pad; Described coated analyzing film is provided with detection line and nature controlling line; The coated specific antibody of described detection line is anti-C reactive protein monoclonal antibody, and the coated specific antibody of described nature controlling line is rabbit igg antibody.Described platinum porphyrins mark specific antibody is anti-c reactive protein monoclonal antibody and anti-rabbit igg antibody, and in addition, the sealing of the platinum porphyrins mark specific antibody of independent packaging is divided in plastics reagent bottle.
Preferably, the surface of described end liner is coated with viscose or specific double faced adhesive tape, in order to fixing described adsorptive pads, coated analyzing film and sample pad, coated analyzing film sticks to the centre of described end liner, and coated analyzing film both sides are connected with described sample pad with described adsorptive pads respectively simultaneously.
Preferably, described adsorptive pads is a kind of filter paper, and concrete is thieving paper or filter paper for oil; ; Described adsorptive pads sticks to a described side at end liner, simultaneously the overlapping 1-2mm in junction of adsorptive pads and coated analyzing film.
Preferably, described sample pad is the slow glass fibre membrane soaking of phosphate buffer, and described sample pad sticks to the overlapping 1-2mm in junction of opposite side, sample pad and the coated analyzing film of described end liner.
Preferably, described coated analyzing film is comprised of nitrocellulose filter, sprays anti-C reactive protein monoclonal antibody and rabbit igg antibody on coated analyzing film simultaneously.
Preferably, described platinum porphyrins mark specific antibody is anti-C reactive protein monoclonal antibody and anti-rabbit igg antibody, by the 0.01-0.1M phosphate buffer dilution containing following component for described platinum porphyrins mark specific antibody, then pack and obtain with plastics reagent bottle: 5%-20% glycerine, 1%-5% bovine serum albumin(BSA), 0.1-2% glycocoll and 0.01%-0.05% surfactant.
Preferably, the excitation light source range of described platinum porphyrins mark is 390-420nm, and wavelength of transmitted light scope is 600-700nm.
The present invention also provides a kind of method of preparing the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein, comprises following six steps.
1) preparation of antibody
Select the anti-C reactive protein monoclonal antibody A of gene engineering expression and the rabbit igg of B, anti-rabbit igg antibody and purifying of purifying.Anti-C reactive protein monoclonal antibody A and anti-C reactive protein monoclonal antibody B can be incorporated into the different loci of C reactive protein.The rabbit igg of anti-rabbit igg antibody and purifying is reaction bonded directly.
2) preparation of coated analyzing film
Adopt Membrane jetter on a nitrocellulose membrane, to draw respectively detection line and nature controlling line, rule careful even, detection line and nature controlling line interval 5mm;
With the coated damping fluid of detection line dilute anti-C reactive protein monoclonal antibody A to concentration be 5--20ug/ml, adopt Membrane jetter by anti-myoglobins monoclonal antibody spray printing on the detection line of nitrocellulose membrane;
By rabbit igg antibody to the concentration of the coated damping fluid dilution of nature controlling line purifying, be 5--20ug/ml, adopt Membrane jetter by the rabbit igg antibody spray printing of purifying on the nature controlling line of nitrocellulose membrane;
Nitrocellulose membrane after spray film is put into the vacuum drying chamber of 30-50 ℃, be dried taking-up after 20-30 minute and seal standby.
Preferably, the preparation method of the coated damping fluid of detection line: use the phosphate buffer dilution of the pH value 7.6 of 50mM, this phosphate buffer is containing 1% methyl alcohol, 0.5% trehalose, 0.5% bovine serum albumin(BSA) and the anti-C reactive protein antibody A of 10ug/ml;
Preferably, the preparation method of the coated damping fluid of nature controlling line: with the phosphate buffer dilution of 50mM, pH value 7.6, this phosphate buffer is containing 1% methyl alcohol, 0.8% bovine serum albumin(BSA) and 0.5mg/ml rabbit igg; Be coated with the preparation of analyzing film: debugging Membrane jetter, making its film liquid measure is 25ul/35cm, machine line makes detection line 4 and nature controlling line 5 intervals keep 5mm, rules careful even.
3) preparation of sample pad
With containing the 0.01-0.1M of 0.01%-0.5% polyglycol, 1%-5% bovine serum albumin(BSA) and 0.01%-0.05% surfactant, the phosphate buffer immersion glass fibre membrane that pH value is 7.2-7.6, after immersion treatment, put it in the vacuum drying chamber of 60-80 ℃, dry after 40-60 minute, to take out sealing standby;
4) preparation of Immunofluorescence test paper strip:
First will be coated with analyzing film and stick on end liner centre position, in coated analyzing film one side, adhere to adsorptive pads, the overlapping 1-2mm in junction of coated analyzing film and adsorptive pads; At coated analyzing film opposite side, adhere to sample pad, the overlapping 1-2mm in junction of coated analyzing film and sample pad; Again the end liner of pasting coated analyzing film, adsorptive pads and sample pad is cut into the wide slice of 4mm.
5) preparation of platinum porphyrins labelled antibody
With the sodium bicarbonate solution of 0.05-0.2M, pH value 9.5-11.0 respectively by the concentration dilution of anti-C reactive protein monoclonal antibody B and anti-rabbit igg antibody to 0.8-2mg/ml, then in anti-C reactive protein monoclonal antibody B and anti-rabbit igg antibody, add respectively the platinum porphyrins lysate of 30-50mg to stir evenly, at room temperature hatch 1-2 hour, wherein in the process of hatching, every 15-20 minute, mix respectively once simultaneously; After having hatched, the gel column that is G25 by specifications and models carried out respectively column separating purification, collect respectively again anti-C reactive protein monoclonal antibody B and anti-rabbit igg antibody that platinum porphyrins mark is good, after the phosphate buffer dilution of each personal 0.01-0.1M, pH value 7.2-7.6 mixes, in-20 ℃, preserve.
6) preparation of the platinum porphyrins labelled antibody working fluid of independent packaging:
Determine anti-C reactive protein monoclonal antibody B and the anti-rabbit igg antibody of the platinum porphyrins mark of appropriate concentration, adopt the 0.01-0.1M phosphate buffer containing 5%-20% glycerine, 1%-5% bovine serum albumin(BSA), 0.1-2% glycocoll and 0.01%-0.05% surfactant to dilute in the lump, be divided in plastics reagent bottle and seal bottle cap, wherein the dispensed loading amount of each plastics reagent bottle is 0.3-3ml, is unified at 2-8 ℃ and preserves.
In addition, the present invention also provides a kind of test card assembly of being made by the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein, and described test card assembly comprises the cartridge that backboard that test strips assembly and the cover plate of being made by polystyrene or Polyvinylchloride and polystyrene or Polyvinylchloride are made forms.
Wherein said backboard comprises draw-in groove and the latch for being combined with described cover plate of placing described test strips assembly, and described cover plate comprises the detection window of Observable testing result, the well that can drip sample and the fixed orifice for being combined with the latch of described backboard; Described test strips assembly is combined with described fixed orifice and is entrenched between described backboard and described cover plate by described latch; Especially, described coated analyzing film is over against described detection window, and described sample pad is over against described well.The platinum porphyrins mark specific antibody of independent packaging is divided in plastics reagent bottle and seal bottle cap.
Further, prepare the method for the test card assembly that the Immunofluorescence test paper strip assembly of described fast quantitative determination of C-reaction protein makes, comprise following three steps:
1) make backboard and cover plate
By polystyrene or Polyvinylchloride, make backboard and cover plate, described backboard comprises draw-in groove and the latch for being combined with described cover plate of placing described test strips assembly, and described cover plate comprises can survey the detection window of result, the well that can drip sample and the fixed orifice for being combined with the latch of described backboard.
2) assembling
Test strips assembly is placed in the described draw-in groove of described backboard, by the latch of described backboard and the fixed orifice of described cover plate, be combined, by test strips component chimeric between backboard and cover plate, wherein, coated analyzing film is over against described detection window, and sample pad is over against described well.
3) packing:
The test strips of 1 person-portion, a cartridge and drying prescription of being responsible for a task until it is completed is encapsulated in aluminium foil polybag, and pack the platinum porphyrins mark specific antibody of organizing many groups of described independent packagings of described aluminium foil polybag and correspondence into a packing box, not frozen the preserving of lucifuge under 2 ℃ of-8 ℃ of environment more.
The present invention has following usefulness:
C reactive protein level in the present invention combines platinum porphyrins luminescent material ELISA test strip human body with immunochromatography technique, for traditional immunochromatography technique has brought breakthrough change:
1), the feature of platinum porphyrins luminous marker, make can be combined with instrument with the immuno-chromatographic test paper strip of its thing that serves as a mark, target detected material is carried out to the high accurate quantification of sensitivity and detects;
2), the characteristic spectrum that has of platinum porphyrins, make the immuno-chromatographic test paper strip based on platinum porphyrins luminescence technology can carry out the high multiple analysis of sensitivity, disposable a plurality of target detected materials in biological sample are detected;
3), the luminescence phenomenon of the large Stock displacement of platinum porphyrins uniqueness, make the testing process of its thing that serves as a mark got rid of due to target detected material autofluorescence cause interference may, improved signal to noise ratio (S/N ratio), thereby improved the sensitivity and the specificity that detect;
4) reliability and stability of detection system, by covalent manner, be cross-linked bioactive molecule, under the prerequisite that guarantees detection sensitivity, have been improved.
Further, the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein provided by the invention, utilize the highly sensitive feature of fluorescence, and sample adds test card after reacting with platinum porphyrins labelled antibody again, make sample and the comprehensive engagement in liquid phase of platinum porphyrins labelled antibody, sufficient reacting, therefore can increase substantially reaction sensitivity, increased the extension rate of sample simultaneously, removed the matrix effect of sample, make quantitative result have good repeatability, and improved the preci-sion and accuracy of quantitative result, can meet the clinical diagnosis requirement that simultaneously detects high value and low value.
Accompanying drawing explanation
Fig. 1 is the structural representation of test strips of the present invention;
Fig. 2 is cartridge decomposition texture schematic diagram of the present invention;
Fig. 3 is the spectral radiation curves of platinum porphyrins mark specific antibody luminescent material of the present invention;
Fig. 4 is reactive mode schematic diagram of the present invention;
Fig. 5 is testing result schematic diagram of the present invention;
Wherein, a: the positive; B: feminine gender; C: invalid;
Fig. 6 is C reactive protein examination criteria working curve of the present invention;
Wherein, Y=1.0177X+0.8727, R
2=0.9976;
Fig. 7 is C reactive protein testing result correlation analysis curve of the present invention;
Wherein, Y=0.9993X-2.6157, r=0.9988.
Embodiment
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1:
With reference to accompanying drawing 1, a kind of Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein, comprises test strips and is used in conjunction with test strips and the platinum porphyrins mark specific antibody of independent packaging; Wherein test strips is by end liner 1 be located at the adsorptive pads 2 engaging successively on end liner 1, coated analyzing film 3, sample pad 6 and form; Described coated analyzing film 3 is provided with detection line 4 and nature controlling line 5; The coated specific antibody of described detection line 4 is anti-C reactive protein monoclonal antibody, and the coated specific antibody of nature controlling line 5 is anti-rabbit igg antibody;
Wherein, end liner 1 surface is coated with viscose or double faced adhesive tape, for fixing described adsorptive pads 2, coated analyzing film 3 and sample pad 6, concrete, coated analyzing film 3 sticks to the centre of described end liner 1, and coated analyzing film 3 both sides are connected with described sample pad 6 with described adsorptive pads 2 respectively simultaneously.
Coated analyzing film 3 is comprised of nitrocellulose filter, on coated analyzing film 3, sprays anti-c reactive protein monoclonal antibody and anti-rabbit igg antibody.
Sample pad 6 is the slow glass fibre membrane soaking of phosphate buffer, and sample pad 6 sticks on end liner 1, wherein, and the overlapping 1-2mm in junction of sample pad 6 and coated analyzing film 3.
Adsorptive pads 2 is filter paper, and concrete is thieving paper or filter paper for oil; Adsorptive pads 2 sticks on end liner 1, simultaneously the overlapping 1-2mm in junction of adsorptive pads 2 and coated analyzing film 3.
Described platinum porphyrins mark specific antibody is anti-C reactive protein monoclonal antibody B and anti-rabbit igg antibody, by the 0.01-0.1M phosphate buffer dilution containing following component for described platinum porphyrins mark specific antibody, then pack and obtain with plastics reagent bottle: 5%-20% glycerine, 1%-5% bovine serum albumin(BSA), 0.1-2% glycocoll and 0.01%-0.05% surfactant.
The preparation method of the Immunofluorescence test paper strip assembly of above-mentioned Quantitative detection c reactive protein, step is:
1, the preparation of antibody
Select the rabbit igg antibody of the anti-C reactive protein monoclonal antibody of the gene engineering expression of purifying, anti-rabbit igg antibody and purifying.
2, the preparation of coated analyzing film 3
Adopt Membrane jetter on a nitrocellulose membrane, to draw respectively detection line 4 and nature controlling line 5, rule careful even, detection line 4 and nature controlling line 5 interval 5mm;
With the coated damping fluid of detection line, diluting anti-C reactive protein monoclonal antibody to concentration is 5--20ug/ml, adopt Membrane jetter by anti-myoglobins monoclonal antibody spray printing on the detection line of nitrocellulose membrane;
By rabbit igg antibody to the concentration of the coated damping fluid dilution of nature controlling line purifying, be 5--20ug/ml, adopt Membrane jetter by the rabbit igg antibody spray printing of purifying on the nature controlling line of nitrocellulose membrane;
Nitrocellulose membrane after spray film is put into the vacuum drying chamber of 30-50 ℃, be dried taking-up after 20-30 minute and seal standby.
Wherein, the phosphate buffer of the pH value 7.6 that the coated damping fluid of detection line is 50mM, this phosphate buffer is containing 1% methyl alcohol, 0.5% trehalose, 0.5% bovine serum albumin(BSA) and 12D8 antibody 1mg/ml.
The coated damping fluid of nature controlling line is the phosphate buffer of 50mM, pH value 7.6, and this phosphate buffer is containing 1% methyl alcohol, 0.8% bovine serum albumin(BSA) and 0.5mg/ml rabbit igg antibody.
3, the preparation of sample pad 6:
With the 0.01M phosphate buffer that contains 0.2% polyglycol, 2.5% bovine serum albumin(BSA), 0.03% surfactant, soak glass fibre membrane, the glass fibre membrane after processing is put into the vacuum drying chamber of 30-50 ℃, be dried taking-up after 3 hours and seal standby.
4, preparation detects the Immunofluorescence test paper strip of C reactive protein:
According to reaction sequence, first will be coated with analyzing film 3 and stick on end liner 1 centre position, in coated analyzing film 3 one sides, adhere to adsorptive pads 2, the overlapping 1-2mm in junction of coated analyzing film 3 and adsorptive pads 2; At coated analyzing film 3 opposite sides, adhere to sample pad 6, the overlapping 1-2mm in junction of coated analyzing film 3 and sample pad 6; Again the end liner 1 of pasting coated analyzing film 3, adsorptive pads 2 and sample pad 6 is cut into the wide slice of 4mm.
5, the preparation of platinum porphyrins labelled antibody
To resist C reactive protein monoclonal antibody 7D9,10C11 and anti-rabbit igg antibody, with 0.1M sodium bicarbonate solution, be diluted to 1mg/ml respectively, respectively get 5ml antibody-solutions, add respectively 30mg fluorescein platinum porphyrins lysate, stir evenly, incubated at room 1 hour, mixed once every 15 minutes.Finally with G25 gel column, cross column separating purification, collect the good platinum porphyrins labelled antibody of mark, with the 0.01M phosphate buffer dilution containing 0.01%-0.5% polyglycol, 1%-5% bovine serum albumin(BSA), 5%-20% glycerine, 0.01%-0.05% surfactant, with plastic bottle, pack, in 4 ℃ of preservations.
6, the preparation of platinum porphyrins labelled antibody working fluid
According to test and use, need to determine anti-C reactive protein monoclonal antibody and the anti-rabbit igg antibody of the platinum porphyrins mark of appropriate concentration, adopt the 0.01-0.1M phosphate buffer containing 5%-20% glycerine, 1%-5% bovine serum albumin(BSA), 0.1-2% glycocoll and 0.01%-0.05% surfactant to dilute in the lump, be divided in plastics reagent bottle and seal bottle cap, wherein the dispensed loading amount of each plastics reagent bottle is 0.3-3ml, is unified at 2-8 ℃ and preserves.
Consult shown in Fig. 2, a test card assembly of being made by the Immunofluorescence test paper strip assembly of the anti-c reactive protein of Quantitative detection, it comprises test strips, the cartridge being comprised of the cover plate 11 of making by polystyrene or Polyvinylchloride and the backboard 13 made by polystyrene or Polyvinylchloride and the platinum porphyrins mark specific antibody 15 of independent packaging.Described backboard 13 comprises draw-in groove 12 and the latch 14 for being combined with cover plate of placing test strips; Described cover plate 11 comprise Observable testing result detection window 8, can drip the well 7 of sample and for the fixed orifice 10 of latch 14 combinations with backboard 13.Described test strips is entrenched between described backboard 13 and described cover plate 11 by described latch 14 and 10 combinations of described fixed orifice, has formed test card.Wherein, described coated analyzing film 3 is over against described detection window 8, and described sample pad 6 is over against described well 7; In addition, the platinum porphyrins mark specific antibody 15 of independent packaging is divided in plastics reagent bottle and seal bottle cap.
The method that the invention provides the test card assembly that a kind of Immunofluorescence test paper strip assembly of preparing the anti-c reactive protein of Quantitative detection makes, it comprises the following steps:
1) make backboard 13 and cover plate 11
By polystyrene or Polyvinylchloride, make backboard 13 and cover plate 11, described backboard 13 comprises places the draw-in groove 12 of described test strips assembly and for the latch 14 with described cover plate 11 combinations, described cover plate 11 comprise can survey result detection window 8, can drip the well 7 of sample and for the fixed orifice 10 of latch 14 combinations with described backboard.
2) assembling
Test strips assembly is placed in the described draw-in groove of described backboard 13 to 12, by the latch 14 of described backboard 13 and fixed orifice 10 combinations of described cover plate 11, by test strips component chimeric between backboard 13 and cover plate 11, wherein, coated analyzing film 3 is over against described detection window 8, and sample pad 6 is over against described well 7.
3) assembling, packing
The test strips of 1 person-portion, a cartridge and drying prescription of being responsible for a task until it is completed is encapsulated in aluminium foil polybag, and pack the platinum porphyrins mark specific antibody of 25 bottles of described independent packagings of the described aluminium foil polybag of 25 person-portions and correspondence into a packing box, not frozen the preserving of lucifuge under 2 ℃ of-8 ℃ of environment.
Detection principle of the present invention
Spectral radiation curves to platinum porphyrins luminescent material is as shown in Figure 3 analyzed, and finds that the excitation light source range of the characteristic spectrum that platinum porphyrins has is 390-420nm, and wavelength of transmitted light scope is 600-700nm.Feature due to platinum porphyrins luminous marker, make can be combined with instrument with the Immunofluorescence test paper strip of its thing that serves as a mark, make the immuno-chromatographic test paper strip based on platinum porphyrins luminescence technology can carry out the high multiple analysis of sensitivity, and target detected material is carried out to the high accurate quantification of sensitivity and detect.
In addition, the Immunofluorescence test paper strip of fast quantitative determination of C-reaction protein of the present invention and test card, utilize the highly sensitive feature of fluorescence, testing sample and platinum porphyrins mark are first reacted in reagent bottle, because testing sample and platinum porphyrins are marked at comprehensive engagement in liquid phase, sufficient reacting, therefore can increase substantially reaction sensitivity, increased the extension rate of testing sample simultaneously, removed the matrix effect of testing sample, made quantitative result have good repeatability; And the specific antibody on platinum porphyrins mark and the abundant combination formation of testing sample one compound, then compound is turned in the sample pad 6 of the well 7 that is added to test card, thereby realize the platinum porphyrins luminescent material C reactive protein level in human body that combines with immunochromatography technique.As shown in Figure 4, after compound is added in the sample pad 6 of well 7 of test card, on the coated analyzing film 3 that platinum porphyrins labelled antibody 15 is flowed through in test card under the suction of adsorptive pads 2, if this compound has C reactive protein 16, anti-C reactive protein monoclonal antibody 17 on the tested survey line 4 of its energy is caught, form with infrared light light signal under green light is irradiated shows, the fluorescence available dedicated instrument quantitative measurement of sending, fluorescence intensity is directly proportional to the concentration of C reactive protein in sample.If C reactive protein is lower than lowest detection standard in compound, detection line 4 can not send fluorescence.Outside two, consult shown in a, b, the c of Fig. 5, in the effective situation of test strips, the anti-C reactive protein monoclonal antibody 17 on detection line 4 all can be reacted and send fluorescence with compound with the rabbit igg antibody 18 on nature controlling line 5, be that detection line 4 is all luminous with nature controlling line 5, ELISA test strip result is positive; Only have nature controlling line 5 luminous, ELISA test strip result is negative; Article two, line is all not luminous, and ELISA test strip result is invalid.
The drafting of standard working curve of the present invention
First, using the c reactive protein standard items of purifying, with the normal human serum (adopting pH7.20.02M PB damping fluid to dilute) of dilution in 1: 10, as diluent preparing series concentration standard items, concentration is: 6 duplicate samples of 0ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 100ng/ml, 200ng/ml.Secondly, each sample is used respectively 10 C reactive protein ELISA test strips 10 times, and the sample detection T value of No. 10 detecting instrument interpretations and contrast C value are averaged respectively, finally according to the ratio of the two, draw the T/C result that each concentration is corresponding, list in table 1.
Using T/C value as X coordinate, using C reactive protein concentration as Y coordinate drawing standard working curve, the expression formula of matching standard working curve is by statistics: Y=1.0177X+0.8727, fitting coefficient square be R
2=0.9976.The results are shown in accompanying drawing 6:C-reactive protein examination criteria working curve.
Embodiment 2:
The preparation method of the present embodiment and embodiment mono-are basic identical, and difference is in step 2:
The phosphate buffer of the pH value 7.2 that the coated damping fluid of detection line is 20mM, this phosphate buffer is containing 0.8% methyl alcohol, 1.0% trehalose, 0.6% bovine serum albumin(BSA) and the anti-C reactive protein antibody of 15ug/ml.
The coated damping fluid of nature controlling line 5 is the phosphate buffer of 50mM, pH value 7.4, and this phosphate buffer is containing 0.7% methyl alcohol, 0.5% bovine serum albumin(BSA) and 0.5mg/ml rabbit igg antibody.
The preparation of coated analyzing film 3: debugging BIO-DOT Membrane jetter, making its film liquid measure is 20ul/40cm, the nitrocellulose membrane after spray film is placed on to 50 ℃ of vacuum drying chambers and processes 20 minutes, pack sealing is standby.
Embodiment 3:
The preparation method of the present embodiment and embodiment mono-are basic identical, and difference is:
In step 3, with the sodium bicarbonate solution of 0.05M, pH value 10 respectively by the concentration dilution of anti-C reactive protein monoclonal antibody and anti-rabbit igg antibody to 2mg/ml, respectively get 5ml antibody-solutions, then in anti-C reactive protein monoclonal antibody and anti-rabbit igg antibody, add respectively 40mg fluorescent material platinum porphyrins lysate to stir evenly,, incubated at room 1.5 hours, wherein in the process of hatching, every 15 minutes, mix once simultaneously.
Embodiment 4:
The preparation method of the present embodiment and embodiment mono-are basic identical, and difference is:
In step 3, with the sodium bicarbonate solution of 0.2M, pH value 11 respectively by the concentration dilution of anti-C reactive protein monoclonal antibody and anti-rabbit igg antibody to 1mg/ml, respectively get 5ml antibody-solutions, then in anti-C reactive protein monoclonal antibody and anti-rabbit igg antibody, add respectively 50mg fluorescent material platinum porphyrins lysate to stir evenly,, incubated at room 2 hours, wherein in the process of hatching, every 15 minutes, mix once simultaneously.After having hatched, the gel column that is G25 by specifications and models carried out respectively column separating purification and collected respectively more anti-C reactive protein monoclonal antibody and the anti-rabbit igg antibody that platinum porphyrins mark is good, each personal 0.01M, the 7.4 phosphate buffer dilutions of pH value mix, preserve in-20 ℃, wherein said phosphate buffer is containing 0.01% polyglycol, 5% bovine serum albumin(BSA), 10% glycerine and 0.02% surfactant.
Embodiment 5:
The preparation method of the present embodiment and embodiment mono-are basic identical, and difference is:
In step 4, with the 0.02M containing 0.4% PEG 8000,1.5% bovine serum albumin(BSA), 0.02% surfactant, pH value 7.2 phosphate buffers, soak, after immersion treatment, sample pad 6 is put into the vacuum drying chamber of 60 ℃, be dried taking-up after 40 minutes and seal standby.
The mensuration of the test strips of embodiment 1-5 being carried out to aspect of performance, lowest detection is limited to 0.01ng/ml.Clinical sample is detected simultaneously.The C reactive protein clinical sample (wherein positive 37 parts, negative 21 parts) that 58 examples are collected from hospital carries out double-blind study detection with colloidal gold immune chromatography test and native system simultaneously:
Colloidal gold immune chromatography test method---31 parts of positives, 27 parts of feminine genders (6 parts of positives are undetected);
Platinum porphyrins test paper and instrumental method---37 parts of positives, 21 parts of feminine genders, fit like a glove with actual result., compare with the qualitative detection of colloidal gold immune chromatography test, platinum porphyrins test paper and instrumental method have provided the final accurately concentration of every duplicate samples meanwhile.
This 58 example is collected to the C reactive protein clinical sample from hospital, simultaneously detect and carry out correlation analysis with U.S. Roche (Roche) company C reactive protein chemoluminescence method reagent, using chemiluminescence detection result as X coordinate, platinum porphyrins test paper and instrumental method result are drawn correlation analysis curve as Y coordinate, expression formula is Y=0.9993X-2.6157, and relative coefficient is r=0.9988.Analysis result is shown in accompanying drawing 7.According to statistical analysis, r > 95%, P < 0.01, has positive correlation.
Aspect withinrun precision, utilize the test strips of embodiment 1-5, content is respectively to the sample of high value, intermediate value and low value, carry out continuously at least 10 times and detect, calculate the coefficient of variation (CV).For the high value (200ng/ml) of c reactive protein content, intermediate value (90ng/ml), each portion of low value (15ng/ml) sample, measure respectively 10 times, according to the data of its mensuration, adopt the analysis of SPSS statistical method, with measurement result means standard deviation, represent, high value 201.8 ± 2.7ng/ml, CV2.3%; Intermediate value 89.6 ± 1.3ng/ml, CV5.2%; Low value 15.1 ± 1.2ng/ml, CV7.9%; Testing result CV value is all less than 15%.
Aspect betweenrun precision, utilize the test strips of embodiment 1-5, to 10 times of pH7.20.02M PB damping fluid dilutions for the clinical positive of a C reactive protein, carry out continuously at least 10 times and detect, the results are shown in table 2.The coefficient of variation (CV) of calculating this duplicate samples duplicate detection is 1.83%.
From above-mentioned detection, detection method of the present invention has higher sensitivity, and in realizing batch, batch between accurate quantification have when detecting repeated well.
Above embodiment is only for setting forth the present invention, and protection scope of the present invention is not only confined to above embodiment.Those skilled in the art make suitable modification and change according to above embodiment to content disclosed by the invention and the parameters scope of getting, and all belong to protection scope of the present invention.
Claims (15)
1. an Immunofluorescence test paper strip assembly for fast quantitative determination of C-reaction protein, is characterized in that, it comprises test strips and is used in conjunction with test strips and the platinum porphyrins mark specific antibody of independent packaging; Wherein test strips is comprised of end liner (1) and the adsorptive pads (2) engaging successively on end liner (1), coated analyzing film (3), sample pad (6); Described coated analyzing film (3) is provided with detection line (4) and nature controlling line (5); The coated specific antibody of described detection line (4) is anti-C reactive protein monoclonal antibody, and the coated specific antibody of described nature controlling line (5) is rabbit igg antibody, and described platinum porphyrins mark specific antibody is anti-c reactive protein monoclonal antibody and anti-rabbit igg antibody;
The preparation method of the Immunofluorescence test paper strip assembly of wherein said fast quantitative determination of C-reaction protein comprises the following steps:
1) preparation of antibody:
Select the rabbit igg of the anti-C reactive protein monoclonal antibody A of the gene engineering expression of purifying, anti-C reactive protein monoclonal antibody B, anti-rabbit igg antibody and purifying, wherein anti-C reactive protein monoclonal antibody A and anti-C reactive protein monoclonal antibody B are incorporated into the different loci of C reactive protein, and the rabbit igg of anti-rabbit igg antibody and purifying is reaction bonded directly;
2) preparation of coated analyzing film (3):
Adopt Membrane jetter on a nitrocellulose membrane, to draw respectively detection line (4) and nature controlling line (5), rule careful even, detection line (4) and nature controlling line (5) interval 5mm;
With the coated damping fluid of detection line dilute anti-C reactive protein monoclonal antibody A to concentration be 5--20 μ g/ml, adopt Membrane jetter by anti-C reactive protein monoclonal antibody spray printing on the detection line of nitrocellulose membrane;
By rabbit igg antibody to the concentration of the coated damping fluid dilution of nature controlling line purifying, be 5--20 μ g/ml, adopt Membrane jetter by the rabbit igg antibody spray printing of purifying on the nature controlling line of nitrocellulose membrane;
Nitrocellulose membrane after spray film is put into the vacuum drying chamber of 30-50 ℃, be dried taking-up after 20-30 minute and seal standby;
3) preparation of sample pad (6):
With containing the 0.01-0.1M of 0.01%-0.5% polyglycol, 1%-5% bovine serum albumin(BSA) and 0.01%-0.05% surfactant, the phosphate buffer immersion glass fibre membrane that pH value is 7.2-7.6, after immersion treatment, put it in the vacuum drying chamber of 60-80 ℃, dry after 40-60 minute, to take out sealing standby;
4) preparation of Immunofluorescence test paper strip:
First will be coated with analyzing film (3) and stick on end liner (1) centre position, in coated analyzing film (3) one sides, adhere to adsorptive pads (2), the overlapping 1-2mm in junction of coated analyzing film (3) and adsorptive pads (2); At coated analyzing film (3) opposite side, adhere to sample pad (6), the overlapping 1-2mm in junction of coated analyzing film (3) and sample pad (6); Again the end liner (1) of pasting coated analyzing film (3), adsorptive pads (2) and sample pad (6) is cut into the wide slice of 4mm;
5) preparation of platinum porphyrins labelled antibody:
With the sodium bicarbonate solution of 0.05-0.2M, pH value 9.5-11.0 respectively by the concentration dilution of anti-C reactive protein monoclonal antibody B and anti-rabbit igg antibody to 0.8-2mg/ml, then in anti-C reactive protein monoclonal antibody B and anti-rabbit igg antibody, add respectively the platinum porphyrins lysate of 30-50mg to stir evenly, at room temperature hatch 1-2 hour, wherein in the process of hatching, every 15-20 minute, mix respectively once simultaneously; After having hatched, the gel column that is G25 by specifications and models carried out respectively column separating purification, collect respectively again anti-C reactive protein monoclonal antibody B and anti-rabbit igg antibody that platinum porphyrins mark is good, after the phosphate buffer dilution of each personal 0.01-0.1M, pH value 7.2-7.6 mixes, in-20 ℃, preserve;
6) preparation of the platinum porphyrins mark specific antibody working fluid of independent packaging:
Determine anti-C reactive protein monoclonal antibody B and the anti-rabbit igg antibody of the platinum porphyrins mark of appropriate concentration, adopt the 0.01-0.1M phosphate buffer containing 5%-20% glycerine, 1%-5% bovine serum albumin(BSA), 0.1-2% glycocoll and 0.01%-0.05% surfactant to dilute in the lump, be divided in plastics reagent bottle and seal bottle cap, wherein the dispensed loading amount of each plastics reagent bottle is 0.3-3ml, is unified at 2-8 ℃ and preserves.
2. the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein according to claim 1, the end liner of described test strips (1) surface is coated with viscose or double faced adhesive tape, for fixing described adsorptive pads (2), described coated analyzing film (3) and described sample pad (6), described coated analyzing film (3) sticks to the centre of described end liner (1), and described coated analyzing film (3) both sides are connected with described sample pad (6) with described adsorptive pads (2) respectively simultaneously.
3. the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein according to claim 1 and 2, it is characterized in that described coated analyzing film (3) is comprised of nitrocellulose filter, at described coated analyzing film (3) the anti-C reactive protein monoclonal antibody of upper spraying and rabbit igg antibody.
4. the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein according to claim 1 and 2, is characterized in that described adsorptive pads (2) is filter paper, and concrete is thieving paper or filter paper for oil; It is upper that described adsorptive pads (2) sticks to described end liner (1), simultaneously the overlapping 1-2mm in junction of described adsorptive pads (2) and described coated analyzing film (3).
5. according to the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein described in claim 1 or 2, it is characterized in that the glass fibre membrane that described sample pad (6) was soaked for phosphate buffer, it is upper that described sample pad (6) sticks to described end liner (1), the overlapping 1-2mm in junction of described sample pad (6) and described coated analyzing film (3).
6. the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein according to claim 1, it is characterized in that described platinum porphyrins mark specific antibody is anti-C reactive protein monoclonal antibody and anti-rabbit igg antibody, by the 0.01-0.1M phosphate buffer dilution containing following component for described platinum porphyrins mark specific antibody, then pack and obtain with plastics reagent bottle: 5%-20% glycerine, 1%-5% bovine serum albumin(BSA), 0.1-2% glycocoll and 0.01%-0.05% surfactant.
7. the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein according to claim 1, the exciting light sources wavelength coverage that it is characterized in that described platinum porphyrins mark is 390-420nm, wavelength of transmitted light scope is 600-700nm.
8. a method of preparing the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein claimed in claim 1, is characterized in that comprising the following steps:
1) preparation of antibody:
Select the rabbit igg of the anti-C reactive protein monoclonal antibody A of the gene engineering expression of purifying, anti-C reactive protein monoclonal antibody B, anti-rabbit igg antibody and purifying, wherein anti-C reactive protein monoclonal antibody A and anti-C reactive protein monoclonal antibody B are incorporated into the different loci of C reactive protein, and the rabbit igg of anti-rabbit igg antibody and purifying is reaction bonded directly;
2) preparation of coated analyzing film (3):
Adopt Membrane jetter on a nitrocellulose membrane, to draw respectively detection line (4) and nature controlling line (5), rule careful even, detection line (4) and nature controlling line (5) interval 5mm;
With the coated damping fluid of detection line dilute anti-C reactive protein monoclonal antibody A to concentration be 5--20 μ g/ml, adopt Membrane jetter by anti-C reactive protein monoclonal antibody spray printing on the detection line of nitrocellulose membrane;
By rabbit igg antibody to the concentration of the coated damping fluid dilution of nature controlling line purifying, be 5--20 μ g/ml, adopt Membrane jetter by the rabbit igg antibody spray printing of purifying on the nature controlling line of nitrocellulose membrane;
Nitrocellulose membrane after spray film is put into the vacuum drying chamber of 30-50 ℃, be dried taking-up after 20-30 minute and seal standby;
3) preparation of sample pad (6):
With containing the 0.01-0.1M of 0.01%-0.5% polyglycol, 1%-5% bovine serum albumin(BSA) and 0.01%-0.05% surfactant, the phosphate buffer immersion glass fibre membrane that pH value is 7.2-7.6, after immersion treatment, put it in the vacuum drying chamber of 60-80 ℃, dry after 40-60 minute, to take out sealing standby;
4) preparation of Immunofluorescence test paper strip:
First will be coated with analyzing film (3) and stick on end liner (1) centre position, in coated analyzing film (3) one sides, adhere to adsorptive pads (2), the overlapping 1-2mm in junction of coated analyzing film (3) and adsorptive pads (2); At coated analyzing film (3) opposite side, adhere to sample pad (6), the overlapping 1-2mm in junction of coated analyzing film (3) and sample pad (6); Again the end liner (1) of pasting coated analyzing film (3), adsorptive pads (2) and sample pad (6) is cut into the wide slice of 4mm;
5) preparation of platinum porphyrins labelled antibody:
With the sodium bicarbonate solution of 0.05-0.2M, pH value 9.5-11.0 respectively by the concentration dilution of anti-C reactive protein monoclonal antibody B and anti-rabbit igg antibody to 0.8-2mg/ml, then in anti-C reactive protein monoclonal antibody B and anti-rabbit igg antibody, add respectively the platinum porphyrins lysate of 30-50mg to stir evenly, at room temperature hatch 1-2 hour, wherein in the process of hatching, every 15-20 minute, mix respectively once simultaneously; After having hatched, the gel column that is G25 by specifications and models carried out respectively column separating purification, collect respectively again anti-C reactive protein monoclonal antibody B and anti-rabbit igg antibody that platinum porphyrins mark is good, after the phosphate buffer dilution of each personal 0.01-0.1M, pH value 7.2-7.6 mixes, in-20 ℃, preserve;
6) preparation of the platinum porphyrins mark specific antibody working fluid of independent packaging:
Determine anti-C reactive protein monoclonal antibody B and the anti-rabbit igg antibody of the platinum porphyrins mark of appropriate concentration, adopt the 0.01-0.1M phosphate buffer containing 5%-20% glycerine, 1%-5% bovine serum albumin(BSA), 0.1-2% glycocoll and 0.01%-0.05% surfactant to dilute in the lump, be divided in plastics reagent bottle and seal bottle cap, wherein the dispensed loading amount of each plastics reagent bottle is 0.3-3ml, is unified at 2-8 ℃ and preserves.
9. the method for preparing the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein according to claim 8, it is characterized in that described step 2) in, the preparation method of the coated damping fluid of detection line: use the phosphate buffer dilution of the pH value 7.6 of 50mM, this phosphate buffer is containing 1% methyl alcohol, 0.5% trehalose, 0.5% bovine serum albumin(BSA) and the anti-C reactive protein antibody A of 10 μ g/ml;
The preparation method of the coated damping fluid of nature controlling line: with the phosphate buffer dilution of 50mM, pH value 7.6, this phosphate buffer is containing 1% methyl alcohol, 0.8% bovine serum albumin(BSA) and 0.5mg/ml rabbit igg antibody; Be coated with the preparation of analyzing film (3): debugging Membrane jetter, making its film liquid measure is 25 μ l/35cm, machine line makes detection line 4 and nature controlling line 5 intervals keep 5mm, rules careful even.
10. the method for preparing the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein according to claim 8, it is characterized in that in step 2) in, the preparation method of the coated damping fluid of detection line (4): use the phosphate buffer of the pH value 7.2 of 20mM, this phosphate buffer is containing 0.8% methyl alcohol, 1.0% trehalose, 0.6% bovine serum albumin(BSA) and the anti-C reactive protein antibody A of 15 μ g/ml; The preparation method of the coated damping fluid of nature controlling line (5): with the phosphate buffer of 50mM, pH value 7.4, this phosphate buffer is containing 0.7% methyl alcohol, 0.5% bovine serum albumin(BSA) and 0.5mg/ml rabbit igg antibody.
11. methods of preparing the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein according to claim 8, it is characterized in that in step 5) in, collect respectively after the anti-C reactive protein monoclonal antibody B and anti-rabbit igg antibody that platinum porphyrins mark is good, the phosphate buffer dilution of each personal 0.01M, pH value 7.4 mixes, preserve in-20 ℃, wherein said phosphate buffer is containing 0.5% polyglycol, 5% bovine serum albumin(BSA), 15% glycerine and 0.01% surfactant.
12. methods of preparing the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein according to claim 8, it is characterized in that in step 5) in, collect respectively after the anti-C reactive protein monoclonal antibody B and anti-rabbit igg antibody that platinum porphyrins mark is good, the phosphate buffer dilution of each personal 0.01M, pH value 7.4 mixes the rear while and preserves in-20 ℃, and wherein said phosphate buffer is containing 0.01% polyglycol, 5% bovine serum albumin(BSA), 10% glycerine and 0.02% surfactant.
13. methods of preparing the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein according to claim 8, it is characterized in that in step 4) in, with the 0.02M containing 0.4% PEG 8000,1.5% bovine serum albumin(BSA), 0.02% surfactant, pH value 7.2 phosphate buffers, soak, after immersion treatment, sample pad (6) is put into the vacuum drying chamber of 60 ℃, be dried taking-up after 40 minutes and seal standby.
14. 1 kinds of test card assemblies of being made by the Immunofluorescence test paper strip assembly of fast quantitative determination of C-reaction protein claimed in claim 1, is characterized in that: the cartridge that the backboard (13) that described test card assembly comprises test strips assembly and the cover plate (11) made by polystyrene or Polyvinylchloride and polystyrene or Polyvinylchloride are made forms; Wherein, described backboard (13) comprises places the draw-in groove (12) of described test strips assembly and for the latch (14) with described cover plate (11) combination, described cover plate (11) comprise Observable testing result detection window (8), can drip the well (7) of sample and for the fixed orifice (10) of the latch with described backboard (14) combination; Described test strips assembly is entrenched between described backboard (13) and described cover plate (11) by described latch (14) and described fixed orifice (10) combination; Especially, described coated analyzing film (3) is over against described detection window (8), and described sample pad (6) is over against described well (7); The platinum porphyrins mark specific antibody of independent packaging is divided in plastics reagent bottle and seal bottle cap.
The method of the test card assembly that 15. 1 kinds of Immunofluorescence test paper strip assemblies of preparing the fast quantitative determination of C-reaction protein described in claim 14 are made, is characterized in that comprising the following steps:
1) make backboard (13) and cover plate (11):
By polystyrene or Polyvinylchloride, make backboard (13) and cover plate (11), described backboard (13) comprises places the draw-in groove (12) of described test strips assembly and for the latch (14) with described cover plate (11) combination, described cover plate (11) comprise can survey result detection window (8), can drip the well (7) of sample and for the fixed orifice (10) of the latch with described backboard (14) combination;
2) assembling:
Test strips assembly is placed on to the described draw-in groove interior (12) of described backboard (13), fixed orifice (10) combination of latch (14) Yu described cover plate (11) by described backboard (13), by test strips component chimeric between backboard (13) and cover plate (11), wherein, coated analyzing film (3) is over against described detection window (8), and sample pad (6) is over against described well (7);
3) packing:
The test strips of 1 person-portion, a cartridge and drying prescription of being responsible for a task until it is completed is encapsulated in aluminium foil polybag, and pack the platinum porphyrins mark specific antibody of organizing many groups of described independent packagings of described aluminium foil polybag and correspondence into a packing box, not frozen the preserving of lucifuge under 2 ℃ of-8 ℃ of environment more.
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Denomination of invention: Immune-fluorescence test strip component for rapidly detecting C-reactive protein quantitatively, detection card component produced by same and method for preparing same Effective date of registration: 20190304 Granted publication date: 20141217 Pledgee: Guangzhou Branch of Guangzhou Bank Co.,Ltd. Pledgor: GUANGZHOU HONGQI OPTICAL INSTRUMENT TECHNOLOGY Co.,Ltd. Registration number: 2019440000084 |
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