CN102023211A - 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 immuno-chromatographic test paper strip of a kind of omnidistance detection by quantitative 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, it is human important acute phase reactive protein, acute stage,, concentration can raise thousands of times, and the CRP half life period in the circulation is 19 hours.
C reactive protein can with the pod membrane C polysaccharide combination of streptococcus pneumonia, be the acute phase protein of body when liver cell is synthetic when being subjected 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 is determined at severity of disease such as diagnosis bacterial infection, cancer, processes, more back and result of treatment aspect have clinical meaning widely.
CRP is as the strongest dangerous index of angiocardiopathy, and its level can be predicted the danger of miocardial infarction and apoplexy in the future.Healthy people CRP 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 smaller or equal to 1mg/L person, the danger that miocardial infarction takes place in the future is 2.9 times of the latter, and the danger that ishemic stroke takes place is 1.9 times of the latter, and the danger that the peripheral arterial vascular conditions takes place is 4.1 times of the latter.The simultaneous determination of CRP and blood fat more can indicate the danger that the heart, cranial vascular disease take place than other risk factors, is the best model that carries out the coronary heart disease assessment of risks at present.Studies show that recently the CRP of low concentration measures, can also be as infection of newborn, diseases such as local infection, and the diagnosis marker of relevant disease.
C reactive protein is a diagnosis index of bacterial 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 are as rheumatic arthritis, systemic vasculitis, polymyalgia rheumatism etc.
2. the index of postoperative infection and complication: patients after surgery CRP raises, and the CRP level should descend in postoperative 7-10 days, does not reduce or raises once more as CRP, and 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 then majority does not raise.
4. there is research to point out to survey CRP in recent years, improves the susceptibility of measuring, can be used for the dangerous prediction of the coronary heart disease and heart stalk with super quick latex enhancing method.
The several method of traditional detection CRP: the sensitivity at the latex agglutination experimental method is 1.0mg/L, is semiquantitative method, less now use; Problems such as the sensitivity of immunoturbidimetry is 5.0mg/L, and sensing range is 5～230mg/L, but needs large-scale instruments such as automatic biochemistry analyzer and robotization immunoassays instrument, has complicated operation length consuming time, and required specimen amount is big.It is long detection time that chemoluminescence method detects life period, generally needs about 90mins consuming time, need be in laboratory specialty operation.Still there are problems such as isotopic contamination in the sensitivity of radioimmunoassay at 3 μ g/L.
Omnidistance detection by quantitative c reactive protein is meant 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 an immunoturbidimetry, measurement range is generally 3～200mg/L, but its sensitivity is relatively low, can't accurately measure 3mg/L with lower horizontal c reactive protein content, can not be as the dangerous index of the heart, cranial vascular disease, the danger that pre-thought-read, cranial vascular disease take place.The measurement range of clinical high-sensitive C-reactive protein is relative narrower again, can't accurately measure the content of CRP under the high concentration level, can not be used for the diagnosis of infectious diseases or the observation of curative effect.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, provide a kind of and both can measure low-level CRP content, and highly sensitive, stable strong, the omnidistance accurately c reactive protein test strip of mensuration.
For achieving the above object, the present invention has taked following technical scheme:
A kind of immuno-chromatographic test paper strip of omnidistance detection by quantitative c reactive protein, overlap in turn to stick on the base plate by sample pad, label pad, coated film, thieving paper and constitute, include the CRP monoclonal antibody of fluorescent latex particulate mark and the rabbit igg of fluorescent latex particulate mark on the described label pad; Described coated film comprises detection zone and Quality Control district, and described detection zone is coated with the another kind of CRP monoclonal antibody that is in different epi-positions with the CRP monoclonal antibody of described fluorescent latex particulate mark.
Preferably, the concentration of the CRP monoclonal antibody of described fluorescent latex particulate mark is 0.5～2mg/ml, and the consumption on test strips is 0.4～1.0 μ g/cm
2The concentration of described rabbit igg is 0.5～2mg/ml, and the consumption on test strips is 0.25-0.45 μ g/cm
2The concentration of the CRP monoclonal antibody of described detection zone bag quilt is 0.5～2mg/ml, and consumption is 20 μ l/27-35cm by film coating buffer amount.
Preferably, the diameter of described fluorescent latex particulate is 0.1 μ m～1 μ m.The wavelength of emission was 180nm～800nm after described fluorescent latex particulate was stimulated.
Preferably, the Quality Control district on the described coated film comprise bag by the C1 line of anti-human IgG and bag by the C2 line of 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 film coating buffer amount.
The present invention also provides a kind of method for preparing the immuno-chromatographic test paper strip of omnidistance detection by quantitative c reactive protein, has taked following technical scheme:
May further comprise the steps:
A. fluorescent latex is covalent activated
Ultrasonic Treatment fluorescent latex microsphere is 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 solutions, and ultrasound wave 200W handled 30 seconds; 20～100mg/ml the carbodiimide that adds earlier 10～100 μ l, mixing adds 20～100mg/mlN-hydroxy thiosuccinimide of 10～100 μ l, mixing again; 10000～15000xg after incubated at room 20-40 minute, centrifugal 5～15 minutes, precipitation is dissolved with the citrate buffer solution of 20～100mM, pH5.0～6.0, is positioned under 2～8 ℃ of conditions standby;
B. the preparation of fluorescent latex particulate labelled protein
After fluorescent latex ultrasound wave 200W after the above-mentioned activation handled 30 seconds, ratio according to 1 μ g～125 μ g albumen/100 μ l fluorescent latex adds CRP monoclonal antibody and rabbit igg respectively, the stirring at room reaction is 1.5-3 hour behind the mixing, centrifuge washing 2-4 time, each 10000～15000xg, centrifugal 5～15 minutes, precipitation was handled 30 seconds with PBS-TBN dissolving and ultrasound wave 100W, and recovering centrifugal front volume with PBS-TBN, to be adjusted to concentration be 0.5～2mg/ml, by 0.4～1.0 μ g/cm
2Consumption be coated on the label pad;
C. the preparation of coated film
Respectively another kind of CRP monoclonal antibody and anti-rabbit igg being cushioned liquid with bag, to be adjusted to concentration be 0.5～2mg/ml, to resist human IgG to be cushioned liquid with bag, to be adjusted to concentration be 0.5～1mg/ml, the CRP monoclonal antibody is sprayed onto detection zone on the coated film (3), to resist rabbit igg and anti-human IgG to be sprayed onto Quality Control district on the coated film (3), the consumption of described CRP monoclonal antibody, anti-rabbit igg and anti-human IgG is 20 μ l/27-35cm by film coating buffer amount, detection zone and Quality Control interval are every 3-8mm, under the room temperature of humidity＜30% airing 12-24 hour, envelope, standby;
D. on end liner, paste sample pad, label pad, coated film and thieving paper mutually in turn obtain test paper plate to overlap joint, cut into the test strips of proper width as requested.
The detection principle of the immuno-chromatographic test paper strip of c reactive protein of the present invention is a double antibody sandwich method, latex microsphere and a kind of CRP antibody and all kinds of different fluorescein covalent bond with diameter range 0.01 μ m～1 μ m, the carboxyl that utilizes the latex microsphere macromolecular substances to have, amino, groups such as hydroxyl are in conjunction with a kind of antibody of CRP antigen, utilize the fluorescein can emitting fluorescence under the exciting light effect, when the CRP of this fluorescent latex mark antibody with detect sample in enough CRP antigen combine and form compound, this compound moves to the detection zone of coated film under the chromatography effect, the detection zone of coated film be coated with can with the another kind of antibody of CRP antigen, this antibodies forms the compound of double-antibody sandwich on another epi-position of CRP antigen.Compound accumulates in the T line place of coated film, is subjected to the emission light that light source activation discharges respective wavelength, by fluorescence detecting system the light signal of catching is converted into digital signal, thereby can be used in the accurately quantitative tachysynthesis detection.
Fluorescent latex mark CRP antibody is with latex microsphere and the CRP antibody of diameter range at 0.01 μ m～1 μ m, form the CRP antigen-antibody complex when the CRP of this fluorescent latex mark antibody and corresponding CRP antigen in detecting sample combine the back, this fluorescent latex compound acts on coated film by chromatography detection zone T line place combines with the another kind of CRP antibody specificity of wrapping quilt in advance and accumulation in a large number.
Utilize fluorescent quantitation spectral detection system, fluorescein by detection zone T line place accumulation on the light source activation coated film, the fluorescence that fluorescein is launched is received by corresponding detecting system, and photosignal is changed into electric signal by processes such as light-to-current inversion, photoelectricity conversions, and by the automatic control system that is provided with in the system signal is exported, demonstrate final quantitative result.Because 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 is compared with radioimmunoassay, euzymelinked immunosorbent assay (ELISA) detection CRP antibody, has handling safety (no radiation pollution), easy (one step of simple operations finishes), suitable single part of detection and quick advantages such as (about 3 minutes the result can be arranged); Compare with the immuno-gold labeling test strips, the present invention has that sensitivity is higher, accurately quantitative, many indexs detect advantages such as (the fluorescent latex tag application of different-waveband is in the synchronous detection by quantitative of many indexs), 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, and faster more precise diagnosis disease and discriminating are infected, and 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 the comprehensive range of linearity (0.5-200mg/L) and good detection sensitivity (Pg/ML level) are arranged, and the sample size that needs is few, operates very easy.
Description of drawings
Fig. 1 is the structural representation of omnidistance detection by quantitative CRP immuno-chromatographic test paper strip of the present invention;
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
Describe the present invention in detail below in conjunction with the drawings and specific embodiments.
In embodiments of the present invention, the CRP antibody that is adopted is the monoclonal antibody of conventional monoclonal antibody technique preparation, and the principle of utilizing double antibody sandwich method to detect CRP antigen detects sample.
As shown in Figure 1, in this embodiment, omnidistance detection by quantitative CRP immuno-chromatographic test paper strip, 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 a sample application zone, is used to draw CRP to be detected and detects sample.Between sample pad 1 and the far-end, be overlapped with the label pad 2 of the plain membrane material of glass fibre, the coated film 3 and the thieving paper 4 of cellulose nitrate membrane material successively.Sample pad 1, label pad 2, coated film 3 and thieving paper 4 all are arranged on the base plate 7.
In this embodiment, be to use fluorescent latex (the about 300nm of diameter) the mark 1 strain CRP monoclonal antibody and the rabbit igg of specific exciting light (470nm)/emission light (525nm) wavelength on the label pad 2; (0.5～2mg/ml) wraps quilt to another strain monoclonal antibody of the detection zone T line place employing CRP of coated film 3.At the Quality Control district C1 of coated film 3 line place working concentration is that the anti-human IgG of 0.5～1mg/ml wraps quilt, be used for and serum in non-specific human IgG, play the effect of filtration, prevent the influence that the non-specific human IgG in the serum reacts Quality Control C1, C2 line, T line place, the C2 line is coated with anti-rabbit igg, concentration is 0.5～2mg/ml, the rabbit igg that is used for combined with fluorescent latex mark, the validity that is used for test strip, the consumption of CRP monoclonal antibody, anti-rabbit igg and anti-human IgG is 20 μ l/27-35cm by film coating buffer amount.
In this embodiment, the preparation of the immuno-chromatographic test paper strip of omnidistance detection by quantitative c reactive protein may further comprise the steps:
1. fluorescent latex is covalent activated
Ultrasonic Treatment latex microsphere body is after 30 seconds, and regulating the latex microsphere bulk concentration is 1.0 * 10
12～1.0 * 10
13/ ml, centrifugal 10 minutes of 10000～15000xg, centrifugal back collecting precipitation thing dissolves with distilled water or 100mM pH6.0 sodium radio-phosphate,P-32 solution, and ultrasound wave 200W handled 30 seconds; Add the 100mg/mlEDC of 50 μ l earlier, shake mixing, add the 50mg/ml N-hydroxy thiosuccinimide (S μ lfo-NHS) of 50 μ l again, the concussion mixing; Incubated at room 10000～15000xg after 30 minutes, centrifugal 5～15 minutes, precipitation is placed under 2～8 ℃ of conditions standby with the citrate buffer solution dissolving of 100mM, pH5.0～6.0;
2. the preparation of fluorescent latex particulate labelled protein
After fluorescent latex ultrasound wave 200W after the above-mentioned activation handled 30 seconds, ratio according to 50 μ g albumen/100 μ l fluorescent latex adds CRP antibody and rabbit igg, the stirring at room reaction is 2 hours behind the Votex mixing, centrifuge washing 3 times, each 10000～15000xg, centrifugal 10 minutes, precipitation was handled 30 seconds with PBS-TBN dissolving and ultrasound wave 100W, and recovering centrifugal front volume with PBS-TBN, to be adjusted to concentration be 1.0mg/ml, by 0.8 μ g/cm
2Consumption be coated on the label pad;
3.CRP antibody sandwich is to coated film
Another kind of CRP monoclonal antibody and anti-rabbit igg are cushioned liquid with bag, and to be adjusted to concentration be 1.0mg/ml, to resist human IgG to be cushioned liquid with bag, to be adjusted to concentration be 0.8mg/ml, the consumption that by film coating buffer amount is 20 μ l/30cm is sprayed onto detection zone on the coated film 3 with the CRP monoclonal antibody, be that the consumption of 20 μ l/27cm and consumption that film coating buffer amount is 20 μ l/34cm will resist rabbit igg and anti-human IgG to be sprayed onto Quality Control district 6 on the coated film 3 by film coating buffer amount respectively, detection zone 5 and Quality Control district 6 be 5mm at interval, airing is 24 hours under the room temperature of humidity＜30%, envelope, standby;
4. on base plate, paste sample pad 1, label pad 2, coated film 3 and thieving paper 4 mutually successively obtain test paper plate to overlap joint, cut into the test strips of proper width as requested.
In one embodiment of the invention, omnidistance detection by quantitative CRP immuno-chromatographic test paper strip, in use, be assembled in by plastics upper casing and plastics lower casing and fasten in the plastic casing that forms, the plastics upper casing is provided with two perforates, application of sample window and display aperture, the application of sample window is corresponding to described omnidistance detection by quantitative CRP immuno-chromatographic test paper strip sample pad 1, display window is corresponding to the detection zone 5 and the Quality Control district 6 of described omnidistance detection by quantitative CRP immuno-chromatographic test paper strip as a result, and this whole process detection by quantitative CRP immuno-chromatographic test paper strip can take out from this plastic casing.
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.
The fluorescence light source system sends monochromatic excitation light, be radiated at the detection zone of test strips, detection zone passes through the fluorescent latex of reaction aggegation under the effect of exciting light, launch fluorescence signal, the detected system acquisition of this fluorescence signal, detection system is made up of photomultiplier and solid-state detector, detection system receives the incident fluorescence signal, realize the amplification of light signal by photomultiplier, by solid-state detector light signal is converted to electric signal then, by the electric signal sensing circuit electric signal is exported, passed through automatic software analysis and Control system handles again, display result on display screen.Realization is to the accurate quantification of sample.
The present invention and moral spirit DN-100 special proteins analyzer comparison shows that to the measurement result of 417 routine clinical CRP samples (whole blood/blood plasma): measure CRP (0.5～200mg/L) accuracy height, the correlativity R of two kinds of product testing results in global extent
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 (9)
1. the immuno-chromatographic test paper strip of an omnidistance detection by quantitative c reactive protein, described test strips is overlapped to stick on the base plate in turn by sample pad (1), label pad (2), coated film (3), thieving paper (4) and constitutes, it is characterized in that, be coated with the CRP monoclonal antibody of fluorescent latex particulate mark and the rabbit igg of fluorescent latex particulate mark on the described label pad (2); Described coated film (3) comprises detection zone (5) and Quality Control district (6), and described detection zone (5) is coated with the another kind of CRP monoclonal antibody that is in different epi-positions with the CRP monoclonal antibody of described fluorescent latex particulate mark.
2. the immuno-chromatographic test paper strip of omnidistance detection by quantitative c reactive protein according to claim 1 is characterized in that, the concentration of the CRP monoclonal antibody of described fluorescent latex particulate mark is 0.5～2mg/ml, and the consumption on test strips is 0.4～1.0 μ g/cm
2
3. the immuno-chromatographic test paper strip of omnidistance detection by quantitative c reactive protein according to claim 1 is characterized in that, the concentration of described rabbit igg is 0.5～2mg/ml, and the consumption on test strips is 0.25-0.45 μ g/cm
2
4. the immuno-chromatographic test paper strip of omnidistance detection by quantitative c reactive protein according to claim 1 is characterized in that, the concentration of the CRP monoclonal antibody of described detection zone bag quilt is 0.5～2mg/ml, and consumption is 20 μ l/27-35cm by film coating buffer amount.
5. the immuno-chromatographic test paper strip of omnidistance detection by quantitative c reactive protein according to claim 1 is characterized in that, the diameter of described fluorescent latex particulate is 0.1 μ m～1 μ m.
6. the immuno-chromatographic test paper strip of omnidistance detection by quantitative c reactive protein according to claim 1 is characterized in that, the wavelength of emission was 180nm～800nm after described fluorescent latex particulate was stimulated.
7. the immuno-chromatographic test paper strip of omnidistance detection by quantitative c reactive protein according to claim 1 is characterized in that, the Quality Control district on the described coated film comprise bag by the C1 line of anti-human IgG and bag by the C2 line of anti-rabbit igg.
8. the immuno-chromatographic test paper strip of omnidistance detection by quantitative c reactive protein according to claim 7, it is characterized in that, the concentration of described anti-human IgG is 0.5～1mg/ml, 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 film coating buffer amount.
9. the preparation method of the immuno-chromatographic test paper strip of the described omnidistance detection by quantitative c reactive protein of claim 1 is characterized in that, may further comprise the steps:
A. fluorescent latex is covalent activated
Ultrasonic Treatment fluorescent latex microsphere is 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 solutions, and ultrasound wave 200W handled 30 seconds; 20～100mg/ml the carbodiimide that adds earlier 10～100 μ l, mixing adds 20～100mg/mlN-hydroxy thiosuccinimide of 10～100 μ l, mixing again; 10000～15000xg after incubated at room 20-40 minute, centrifugal 5～15 minutes, precipitation is dissolved with the citrate buffer solution of 20～100mM, pH5.0～6.0, is positioned under 2～8 ℃ of conditions standby;
B. the preparation of fluorescent latex particulate labelled protein
After fluorescent latex ultrasound wave 200W after the above-mentioned activation handled 30 seconds, ratio according to 1 μ g～125 μ g albumen/100 μ l fluorescent latex adds CRP monoclonal antibody and rabbit igg respectively, the stirring at room reaction is 1.5-3 hour behind the mixing, centrifuge washing 2-4 time, each 10000～15000xg, centrifugal 5～15 minutes, precipitation was handled 30 seconds with PBS-TBN dissolving and ultrasound wave 100W, and recovering centrifugal front volume with PBS-TBN, to be adjusted to concentration be 0.5～2mg/ml, by 0.4～1.0 μ g/cm
2Consumption be coated on the label pad;
C. the preparation of coated film
Respectively another kind of CRP monoclonal antibody and anti-rabbit igg being cushioned liquid with bag, to be adjusted to concentration be 0.5～2mg/ml, to resist human IgG to be cushioned liquid with bag, to be adjusted to concentration be 0.5～1mg/ml, the CRP monoclonal antibody is sprayed onto detection zone on the coated film (3), to resist rabbit igg and anti-human IgG to be sprayed onto Quality Control district on the coated film (3), the consumption of described CRP monoclonal antibody, anti-rabbit igg and anti-human IgG is 20 μ l/27-35cm by film coating buffer amount, detection zone and Quality Control interval are every 3-8mm, under the room temperature of humidity＜30% airing 12-24 hour, envelope, standby;
D. on end liner, paste sample pad (1), label pad (2), coated film (3) and thieving paper (4) mutually in turn obtain test paper plate to overlap joint, cut into the test strips of proper width as requested.
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