CN102023211B - Immunochromatographic test strip for full quantitative detection of C-reactive protein and preparation method thereof - Google Patents
Immunochromatographic test strip for full quantitative detection of C-reactive protein and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an immunochromatographic test strip for full quantitative detection of C-reactive protein and a preparation method thereof. The test strip is composed of a sample pad, a mark pad, a coating film and absorbent paper which are successively overlapped and adhered on a baseplate, wherein the mark pad is coated with C-reactive protein (CRP) monoclonal antibody labelled by fluorescent latex particles and rabbit IgG labelled by fluorescent latex particles; and the coating film is composed of a detection region and a quality control region, and the detection region is coated with another CRP monoclonal antibody which is at different epitope with the CRP monoclonal antibody labelled by fluorescent latex particles. The C-reactive protein immunochromatographic test strip can perform sensitive quantitative detection on the full CRP in 10 seconds, can diagnose diseases and identify infection more rapidly and accurately, and can detect infectious illness state and determine the curative effect of antibiotics; and the full CRP has two detection results, namely hs-CRP and conventional CRP, comprehensive linear range and good detection sensitivity and less required sample amount, and is very convenient to operate.
Description
Technical field
The invention belongs to field of medical examination, relate in particular to a kind of whole process and quantitatively detect immuno-chromatographic test paper strip of c reactive protein and preparation method thereof.
Background technology
C reactive protein (C-reactive protein, CRP), nineteen thirty Tillet and Francis in acute lobar pneumonia patients serum, find can be when calcium ion exists and CPS play precipitation reaction and gain the name, the important acute phase reactive proteins of the mankind, acute stage,, concentration can raise thousands of times, and the CRP half life period in circulation is 19 hours.
C reactive protein can with the pod membrane C polysaccharide combination of streptococcus pneumonia, the acute phase protein of body when liver cell is synthetic when being subject to microorganism invasion or tissue damage etc. and stimulating, when be inflamed disease or tissue fester or are downright bad, can in blood, raise rapidly, but along with the recovery of institutional framework and function, it is normal that its content also recovers.Therefore, the CRP of high concentration be determined at the seriousness, process of the diseases such as diagnosis bacterium infections, cancer, more afterwards and result for the treatment of aspect there is clinical meaning widely.
CRP is as the strongest Danger Indexes of angiocardiopathy, and its level can be predicted the danger of miocardial infarction and apoplexy in the future.Healthy Human C-reactiveprotein term of reference is 0.58-1.13mg/L substantially.CRP content is greater than the people of 2.1mg/L, relatively CRP content is less than or equal to 1mg/L person, the danger that miocardial infarction occurs is in the future the latter 2.9 times, the danger that ishemic stroke occurs be the latter 1.9 times, the danger of generation peripheral arterial vascular conditions is the latter 4.1 times.The simultaneous determination of CRP and blood fat, more can indicate the danger that the heart, cranial vascular disease occur compared with other risk factors, be to carry out at present the best model of coronary heart disease assessment of risks.Study and show recently, the CRP of low concentration measures, can also be as infection of newborn, and the diseases such as local infection, and the diagnosis marker of relevant disease.
C reactive protein is a diagnosis index of bacterium infection and serious tissue damage, and its rising is found in:
1. tissue damage, infection, tumour, miocardial infarction and a series of active chronic inflammation disease, as rheumatic arthritis, systemic vasculitis, polymyalgia rheumatism etc.
2. the index of postoperative infection and complication: patients after surgery CRP raises, and CRP level should decline in postoperative 7-10 days, as CRP does not reduce or again raises, prompting may accompanying infection or thromboembolism.
3. can be used as the antidiastole of bacterial infection and viral infection: most of bacterial infections can cause that patients serum CRP raises, and viral infection majority does not raise.
4. there is in recent years research to point out to survey CRP by super quick latex enhancing method, improve the susceptibility of measuring, can be used for coronary heart disease and the heart and obstruct dangerous prediction.
The several method of traditional detection CRP: the sensitivity at latex agglutination experimental method is 1.0mg/L, is semiquantitative method, now less use; The problems such as the sensitivity of immunoturbidimetry is 5.0mg/L, and sensing range is 5～230mg/L, but needs the large-scale instruments such as automatic biochemistry analyzer and robotization immunoassays instrument, has complicated operation length consuming time, and required specimen amount is large.It is long detection time that chemoluminescence method detects life period, generally needs 90mins consuming time left and right, need to be in laboratory specialty operation.At 3 μ g/L, still there is the problems such as isotopic contamination in the sensitivity of radioimmunoassay.
The omnidistance c reactive protein that quantitatively detects refers to that its assay method is more responsive than classic method, and measurement range is broader.The method of routine clinical mensuration c reactive protein is immunoturbidimetry, measurement range is generally 3～200mg/L, but its sensitivity is relatively low, cannot Accurate Determining 3mg/L with lower horizontal c reactive protein content, can not be as the Danger Indexes of the heart, cranial vascular disease, the danger that pre-thought-read, cranial vascular disease occur.The measurement range of clinical high-sensitive C-reactive protein is relative narrower again, cannot Accurate Determining high concentration level under the content of CRP, can not be for the diagnosis of infectious diseases or the observation of curative effect.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, provide a kind of and both can measure low-level CRP content, and highly sensitive, stability strong, measure omnidistance c reactive protein test strip accurately.
For achieving the above object, the present invention has taked following technical scheme:
A kind of whole process quantitatively detects the immuno-chromatographic test paper strip of c reactive protein, by sample pad, label pad, coated film, thieving paper, overlapped in turn to stick on base plate and form, in described label pad, include the CRP monoclonal antibody of fluorescent latex particulate mark and the rabbit igg of fluorescent latex particulate mark; Described coated film comprises detection zone and Quality Control district, and described detection zone is coated with CRP monoclonal antibody from the described fluorescent latex particulate mark another kind of CRP monoclonal antibody in different epi-positions.
Preferably, the concentration of the CRP monoclonal antibody of described fluorescent latex particulate mark is 0.5～2mg/ml, and the consumption in test strips is 0.4～1.0 μ g/cm
2.The concentration of described rabbit igg is 0.5～2mg/ml, and the consumption in test strips is 0.25-0.45 μ g/cm
2.The concentration of the CRP monoclonal antibody that described detection zone is coated is 0.5～2mg/ml, and consumption is 20 μ l/27-35cm by the coated liquid measure of film.
Preferably, the diameter of described fluorescent latex particulate is 0.1 μ m～1 μ m.After described fluorescent latex particulate is stimulated, the wavelength of transmitting is 180nm～800nm.
Preferably, the Quality Control district on described coated film comprises the C1 line of coated anti-human IgG and the C2 line of coated anti-rabbit igg.The concentration of described anti-human IgG is 0.5～1mg/ml, and the concentration of anti-rabbit igg is 0.5～2mg/ml, and the consumption of anti-human IgG and anti-rabbit igg is 20 μ l/27-35cm by the coated liquid measure of film.
The present invention also provides a kind of omnidistance method that quantitatively detects the immuno-chromatographic test paper strip of c reactive protein of preparing, and has taked following technical scheme:
Comprise the following steps:
A. fluorescent latex is covalent activated
Ultrasound wave was processed fluorescent latex microsphere after 30 seconds, and regulating fluorescent latex microsphere concentration is 1.0 * 10
12～1.0 * 10
13/ ml, centrifugal 5～15 minutes of 10000～15000xg, sediment dissolves with distilled water or 50～200mM pH6.0～7.0 sodium radio-phosphate,P-32 solution, and ultrasound wave 200W processes 30 seconds; 20～100mg/ml the carbodiimide that first adds 10～100 μ l, mixes, then adds 20～100mg/mlN-hydroxy thiosuccinimide of 10～100 μ l, mixes; 10000～15000xg after incubated at room 20-40 minute, centrifugal 5～15 minutes, the citrate buffer solution dissolving of 20～100mM, pH5.0～6.0 for precipitation, is positioned under 2～8 ℃ of conditions standby;
B. the preparation of fluorescent latex particulate labelled protein
Fluorescent latex ultrasound wave 200W after above-mentioned activation was processed after 30 seconds, ratio according to 1 μ g albumen/100, μ g～125 μ l fluorescent latex adds respectively CRP monoclonal antibody and rabbit igg, mix rear stirring at room reaction 1.5-3 hour, centrifuge washing 2-4 time, each 10000～15000xg, centrifugal 5～15 minutes, precipitation is dissolved with PBS-TBN and ultrasound wave 100W processes 30 seconds, and with PBS-TBN, recovering centrifugal front volume, to be adjusted to concentration be 0.5～2mg/ml, by 0.4～1.0 μ g/cm
2consumption be coated in label pad;
C. the preparation of coated film
Respectively another kind of CRP monoclonal antibody and anti-rabbit igg being adjusted to concentration with coated damping fluid is 0.5～2mg/ml, it is 0.5～1mg/ml that anti-human IgG is adjusted to concentration with coated damping fluid, CRP monoclonal antibody is sprayed onto to the detection zone on coated film (3), to resist rabbit igg and anti-human IgG to be sprayed onto the Quality Control district on coated film (3), described CRP monoclonal antibody, the consumption of anti-rabbit igg and anti-human IgG is 20 μ l/27-35cm by the coated liquid measure of film, detection zone and Quality Control interval are every 3-8mm, airing 12-24 hour under the room temperature of humidity < 30%, envelope, standby,
D. on end liner, pasting sample pad, label pad, coated film and thieving paper obtains test paper plate overlap joint mutually in turn, cuts into as requested the test strips of proper width.
The detection principle of the immuno-chromatographic test paper strip of c reactive protein of the present invention is double antibody sandwich method, by the latex microsphere of diameter range 0.01 μ m～1 μ m and a kind of CRP antibody and all kinds of different fluorescein covalent bond, the carboxyl that utilizes latex microsphere macromolecular substances to have, amino, the groups such as hydroxyl are in conjunction with a kind of antibody of CRP antigen, utilize the fluorescein can emitting fluorescence under exciting light effect, when the CRP of this fluorescent latex mark antibody enough CRP antigen in detecting sample, be combined and form compound, this compound moves to the detection zone of coated film under chromatography effect, in the detection zone of coated film, be coated with can with the another kind of antibody of CRP antigen, this antibody is combined in another epi-position of CRP antigen, form the compound of double-antibody sandwich.Compound accumulates in the T line place of coated film, is subject to the utilizing emitted light that light source activation discharges respective wavelength, by fluorescence detecting system, the light signal of seizure is converted into digital signal, thereby can be used in the tachysynthesis detection of accurate quantitative analysis.
Fluorescent latex mark CRP antibody is latex microsphere and the CRP antibody at 0.01 μ m～1 μ m by diameter range, after the corresponding CRP antigen of the CRP of this fluorescent latex mark antibody in detecting sample is combined, form CRP antigen-antibody complex, this fluorescent latex compound acts on the T line place, detection zone of coated film by chromatography and builds up in a large number with coated in advance another kind of CRP antibody specific binding.
Utilize fluorescent quantitation spectral detection system, the fluorescein of being built up by T line place, detection zone on light source activation coated film, the fluorescence that fluorescein is launched is received by corresponding detection system, and by processes such as light-to-current inversion, photoelectric conversions, photosignal is changed into electric signal, and the automatic control system arranging exports signal, demonstrate final quantitative result in system.Due to the difference of the kind of the fluorescein of selecting, its excitation/emission light wavelength λ and automatic control system also can be different.
C reactive protein immuno-chromatographic test paper strip of the present invention and radioimmunoassay, euzymelinked immunosorbent assay (ELISA) detect CRP antibody and compare, and have handling safety (polluting without radiation), easy (simple operations one step completes), be applicable to single part and detect and advantage such as (can have result about 3 minutes) fast; Compare with immuno-gold labeling test strips, the present invention has that sensitivity is higher, accurate quantitative analysis, many indexs detect the advantages such as (the fluorescent latex tag application of different-waveband quantitatively detects in many indexs are synchronous), mark stability is better.
C reactive protein immuno-chromatographic test paper strip of the present invention only can reach just can carry out sensitive quantitative measurement to omnidistance CRP in 10 seconds, diagnoses the illness more accurately sooner and differentiates infection, can detect and infect the state of an illness and determine antibiotic curative effect; Omnidistance CRP can detect simultaneously and provide hs-CRP and two results of conventional CRP, and has the comprehensive range of linearity (0.5-200mg/L) and good detection sensitivity (Pg/ML level), and the sample size needing is few, operates very easy.
Accompanying drawing explanation
Fig. 1 is the structural representation that whole process of the present invention quantitatively detects CRP immuno-chromatographic test paper strip;
Reference numeral: 1, sample pad; 2, label pad; 3, coated film; 4, thieving paper; 5, detection zone; 6, Quality Control district; 7, base plate.
Embodiment
Below in conjunction with the drawings and specific embodiments, describe the present invention in detail.
Embodiment 1
In embodiments of the present invention, the CRP antibody adopting is monoclonal antibody prepared by conventional monoclonal antibody technique, and the principle of utilizing double antibody sandwich method to detect CRP antigen detects sample.
As shown in Figure 1, in this embodiment, the omnidistance CRP immuno-chromatographic test paper strip that quantitatively detects, comprise far-end and near-end, sample pad 1 is positioned at the near-end of test strips, and it contains a hydrophilic poroid barrier film, sample pad 1 is sample application zone, for drawing CRP to be detected, detects sample.Between sample pad 1 and far-end, be overlapped with successively the glass fibre element label pad 2 of membrane material, the coated film 3 of cellulose nitrate membrane material and thieving paper 4.Sample pad 1, label pad 2, coated film 3 and thieving paper 4 are all arranged on base plate 7.
In this embodiment, in label pad 2, be fluorescent latex (diameter 300nm) mark 1 strain CRP monoclonal antibody and the rabbit igg that uses specific exciting light (470nm)/utilizing emitted light (525nm) wavelength; The T line place, detection zone of coated film 3 adopts another strain monoclonal antibody (0.5～2mg/ml) of CRP to be coated with.The anti-human IgG that is 0.5～1mg/ml at the Quality Control district C1 line place of coated film 3 working concentration is coated with, be used for and serum in non-specific human IgG, play the effect of filtration, prevent the impact that the non-specific human IgG in serum reacts Quality Control C1, C2 line, T line place, C2 line is coated with anti-rabbit igg, concentration is 0.5～2mg/ml, rabbit igg for combined with fluorescent latex mark, for detection of the validity of test strips, the consumption of CRP monoclonal antibody, anti-rabbit igg and anti-human IgG is 20 μ l/27-35cm by the coated liquid measure of film.
In this embodiment, the omnidistance preparation that quantitatively detects the immuno-chromatographic test paper strip of c reactive protein comprises the following steps:
1. fluorescent latex is covalent activated
Ultrasound wave was processed latex microsphere body after 30 seconds, and regulating latex microsphere bulk concentration is 1.0 * 10
12～1.0 * 10
13/ ml, centrifugal 10 minutes of 10000～15000xg, centrifugal rear collecting precipitation thing dissolves with distilled water or 100mM pH6.0 sodium radio-phosphate,P-32 solution, and ultrasound wave 200W processes 30 seconds; The 100mg/mlEDC that first adds 50 μ l, concussion mixes, then adds the 50mg/ml N-hydroxy thiosuccinimide (S μ lfo-NHS) of 50 μ l, and concussion mixes; Incubated at room 10000～15000xg after 30 minutes, centrifugal 5～15 minutes, for precipitation, the citrate buffer solution of 100mM, pH5.0～6.0 dissolves, and is placed under 2～8 ℃ of conditions standby;
2. the preparation of fluorescent latex particulate labelled protein
Fluorescent latex ultrasound wave 200W after above-mentioned activation was processed after 30 seconds, ratio according to 50 μ g albumen/100 μ l fluorescent latex adds CRP antibody and rabbit igg, Votex mixes rear stirring at room reaction 2 hours, centrifuge washing 3 times, each 10000～15000xg, centrifugal 10 minutes, precipitation is dissolved with PBS-TBN and ultrasound wave 100W processes 30 seconds, and with PBS-TBN, recovering centrifugal front volume, to be adjusted to concentration be 1.0mg/ml, by 0.8 μ g/cm
2consumption be coated in label pad;
3.CRP antibody is coated with coated film
It is 1.0mg/ml that another kind of CRP monoclonal antibody and anti-rabbit igg are adjusted to concentration with coated damping fluid, it is 0.8mg/ml that anti-human IgG is adjusted to concentration with coated damping fluid, the consumption that is 20 μ l/30cm by the coated liquid measure of film is sprayed onto the detection zone on coated film 3 by CRP monoclonal antibody, the consumption that the consumption that is 20 μ l/27cm by the coated liquid measure of film respectively and the coated liquid measure of film are 20 μ l/34cm is sprayed onto the Quality Control district 6 on coated film 3 by anti-rabbit igg and anti-human IgG, detection zone 5 and Quality Control district 6 interval 5mm, under the room temperature of humidity < 30%, airing is 24 hours, envelope, standby,
4. on base plate, pasting sample pad 1, label pad 2, coated film 3 and thieving paper 4 obtains test paper plate overlap joint mutually successively, cuts into as requested the test strips of proper width.
In one embodiment of the invention, the omnidistance CRP immuno-chromatographic test paper strip that quantitatively detects, in use, be assembled in by plastics upper casing and plastics lower casing and fasten in the plastic casing forming, plastics upper casing is provided with two perforates, application of sample window and demonstration hole, application of sample window quantitatively detects CRP immuno-chromatographic test paper strip sample pad 1 corresponding to described whole process, result display window quantitatively detects detection zone 5 and the Quality Control district 6 of CRP immuno-chromatographic test paper strip corresponding to described whole process, this whole process quantitatively detects CRP immuno-chromatographic test paper strip and can from this plastic casing, take out.
In one embodiment of the present of invention, be used for testing the fluorescent quantitation spectral detection system of immuno-chromatographic test paper strip, mainly comprise fluorescence light source system, detection system and automatic software analysis and Control system.
Fluorescence light source system is sent monochromatic excitation light, be radiated at the detection zone of test strips, the fluorescent latex of reaction aggegation is passed through under the effect of exciting light in detection zone, launch fluorescence signal, this fluorescence signal is detected system acquisition, detection system is comprised of photomultiplier and solid-state detector, detection system receives incident fluorescence signal, by photomultiplier, realized the amplification of light signal, then by solid-state detector, light signal is converted to electric signal, by electric signal sensing circuit, electric signal is exported, through automatic software analysis and Control system, process again, showing screen display result.The accurate quantification of realization to sample.
The present invention and moral spirit DN-100 special proteins analyzer relatively show the measurement result of 417 routine clinical CRP samples (whole blood/blood plasma): the accuracy of measuring CRP (0.5～200mg/L) in global extent is high, the correlativity R of two kinds of product testing results
2> 0.98 (y=1.0035x+0.7457).Calibrate the linear dependence R of serial quality-control product with Britain's Landau (RANDOX) CRP
2> 0.9.
Claims (2)
1. a whole process quantitatively detects the immuno-chromatographic test paper strip of c reactive protein, described test strips is overlapped to stick on base plate in turn by sample pad (1), label pad (2), coated film (3), thieving paper (4) and forms, it is characterized in that, in described label pad (2), be coated with the CRP monoclonal antibody of fluorescent latex particulate mark and the rabbit igg of fluorescent latex particulate mark; Described coated film (3) comprises detection zone (5) and Quality Control district (6), and described detection zone (5) are coated with CRP monoclonal antibody from the described fluorescent latex particulate mark another kind of CRP monoclonal antibody in different epi-positions; Quality Control district on described coated film comprises the C1 line of coated anti-human IgG and the C2 line of coated anti-rabbit igg; The concentration of described anti-human IgG is 0.8mg/ml, and the concentration of anti-rabbit igg is 1.0mg/ml, and the consumption of anti-human IgG and anti-rabbit igg is respectively 20 μ l/34cm and 20 μ l/27cm by the coated liquid measure of film; The concentration of the CRP monoclonal antibody of described fluorescent latex particulate mark is 1.0mg/ml, and the consumption in test strips is 0.8 μ g/cm
2; The concentration of the rabbit igg of described fluorescent latex particulate mark is 1.0mg/ml, and the consumption in test strips is 0.8 μ g/cm
2; The concentration of the CRP monoclonal antibody that described detection zone is coated is 1.0mg/ml, and consumption is 20 μ l/30cm by the coated liquid measure of film; The diameter of described fluorescent latex particulate is 300nm; The excitation wavelength of described fluorescent latex particulate is 470nm, and after described fluorescent latex particulate is stimulated, the wavelength of transmitting is 525nm.
2. whole process claimed in claim 1 quantitatively detects a preparation method for the immuno-chromatographic test paper strip of c reactive protein, it is characterized in that, comprises the following steps:
A. fluorescent latex is covalent activated
Ultrasound wave was processed fluorescent latex microsphere after 30 seconds, and regulating fluorescent latex microsphere concentration is 1.0 * 10
12～1.0 * 10
13/ ml, centrifugal 10 minutes of 10000～15000xg, sediment dissolves with distilled water or 100mMpH6.0 sodium radio-phosphate,P-32 solution, and ultrasound wave 200W processes 30 seconds; The 100mg/ml carbodiimide that first adds 50 μ l, mixes, then adds the 50mg/ml N-hydroxy thiosuccinimide of 50 μ l, mixes; Incubated at room 10000～15000xg after 30 minutes, centrifugal 5～15 minutes, for precipitation, the citrate buffer solution of 100mM, pH5.0～6.0 dissolves, and is positioned under 2～8 ℃ of conditions standby;
B. the preparation of fluorescent latex particulate labelled protein
Fluorescent latex ultrasound wave 200W after above-mentioned activation was processed after 30 seconds, ratio according to 50 μ g albumen/100 μ l fluorescent latex adds respectively CRP monoclonal antibody and rabbit igg, mix rear stirring at room reaction 2 hours, centrifuge washing 3 times, each 10000～15000xg, centrifugal 10 minutes, precipitation is dissolved with PBS-TBN and ultrasound wave 100W processes 30 seconds, and with PBS-TBN, recovering centrifugal front volume, to be adjusted to concentration be 1mg/ml, by 0.8 μ g/cm
2consumption be coated in label pad;
C. the preparation of coated film
Respectively another kind of CRP monoclonal antibody and anti-rabbit igg being adjusted to concentration with coated damping fluid is 1.0mg/ml, it is 0.8mg/ml that anti-human IgG is adjusted to concentration with coated damping fluid, CRP monoclonal antibody is sprayed onto to the detection zone on coated film (3), to resist rabbit igg and anti-human IgG to be sprayed onto the Quality Control district on coated film (3), described CRP monoclonal antibody, the consumption of anti-rabbit igg and anti-human IgG is respectively 20 μ l/30cm by the coated liquid measure of film, 20 μ l/27cm, 20 μ l/34cm, detection zone and Quality Control interval are every 5mm, under the room temperature of humidity < 30%, airing is 24 hours, envelope, standby,
D. on end liner, pasting sample pad (1), label pad (2), coated film (3) and thieving paper (4) obtains test paper plate overlap joint mutually in turn, cuts into as requested the test strips of proper width.
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