CN104422773A - Immunochromatography test paper for detecting human PGI protein and preparation method thereof - Google Patents
Immunochromatography test paper for detecting human PGI protein and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a Immunochromatography test paper for detecting a human PGI protein and a preparation method thereof. The test paper employs a double antibody sandwich method for detection of the human PGI protein. The double antibody sandwich method uses a first PGI monoclonal antibody labeled with fluorescent microspheres as a capture antibody, and the first PGI monoclonal antibody is one of the sequences from the SEQ ID NO.1 and SEQ ID NO.2 in the sequence table; and the double antibody sandwich method uses a second PGI monoclonal antibody as a detection antibody, and the second PGI monoclonal antibody is another one from SEQ ID NO.1 and SEQ ID NO.2 in the sequence table. The invention of the immunochromatography test paper has the advantages of simple operation, rapidness, wide detection range, high specificity and good sensitivity.
Description
Technical field
The invention belongs to chemiluminescent polypeptide and field of immunology, be specifically related to human pepsinogen I(PGI) epitope peptide, the PGI specific antigen prepared with this epitope peptide and corresponding monoclonal antibody or polyclonal antibody, the purposes of described antibody on preparation people PGI external diagnosis reagent case, people PGI external diagnosis reagent case, and a kind of fluorescence immune chromatography test paper for quantitatively detecting people PGI albumen in determinand and preparation method thereof.
Background technology
Propepsin (PG) is pepsic inactive precursor in gastric juice.Human pepsinogen can be divided into PG1 to PG7 seven kind isozymogens by fast to slow by its electrophoretic mobility, and can be divided into PGI and PGII two subgroups according to distribution in its biochemical property, immunogenicity, cell derived and tissue.PG1 to PG5 immunogenicity in seven kinds of isozymogens is similar to, and is called pepsinogen I (PGI), its chief cell primarily of gastric gland and the secretion of mucus neck cell; PG6 to PG7 is called as pepsinogen I I(PGII), except secreting except the cell secretes of acid gland by mucous membrane at the bottom of body of stomach and stomach, the Brunner gland of the mucilage cell and duodenum epimere of secreting the pyloric gland of the mucus neck cell of acid gland, cardiac gland and stomach hole also can produce PGII.Stomach is almost the exclusive source of PG, and can change in the secretory volume in secretion stage.Serum PG I and PGII reflects the secreting function of gastric mucosa different parts.PG major part after synthesis enters gastral cavity, under acidic gastric juice effect, activate into pepsin, only has a small amount of PG(about 1%) through stomach lining hair.Therefore, serum PG concentration can reflect its secretion level.PGI in normal human serum is 6 times of PGII, and when stomach lining generation pathological change, serum PG content also changes thereupon.
Serum PG content infects to helicobacter pylori (HP), chronic gastritis, cancer of the stomach etc. are relevant.In different disease of stomach, the value generation respective change of serum PG I, PGII and PGI/PGII, it raises, reduce or constant and inconsistent, as the sensitivity of diagnosis or treatment results index and specificity also different.Therefore, for different disease of stomach, the value of serum PG I, PGII and PGI/PGII also has different criterions.
PGI is the pointer detecting oxyntic gland (fundus gland) cell function, and gastric acid secretion increases, then PGI raises; Gastric acid secretion minimizing or the atrophy of gastric mucosa body of gland, then PGI reduces.Such as, in the gastritis that superficial gastritis and the helicobacter pylori (HP) of gastroxia infect, the secretion of PGI can increase; And in chronic severe atrophy gastritis, when chief cell reduces, PGI content declines.The PGI of high concentration, and can as an index of observing HP and infect (helicobacter pylori) eradication therapy curative effect also as the subclinical indication of duodenal ulcer and complication risk.
Cancer of the stomach is one of common malignant tumour of China, and its case fatality rate occupies first of various malignant tumour, and its early diagnosis, early treatment become and improve life in patients, the unique channel that reduces case fatality rate.Chinese scholars has done quantifier elimination discovery greatly to Serum Obtained From Advance Gastric Cancer PG change: when getting a cancer of the stomach; serum PG I level reduces; PGII level substantially keeps normal or raises; and then; the mensuration level of serum PG I and the ratio of PGI/PGII contribute to the antidiastole of cancer of the stomach, and too low PGI and PGI/PGII should watch out for early carcinoma of stomach.Content due to serum PG directly reflects the function of stomach lining, and therefore Serum Obtained From Advance Gastric Cancer PGI level obviously declines, and shows that patients with gastric cancer stomach lining secretion capacity declines.The cancer of the stomach of more than 80% is with atrophic gastritis, and atrophic gastritis can cause stomach lining chief cell to be lost, thus affects its secreting function; Serum Obtained From Advance Gastric Cancer PGI level obviously declines and reduces relevant with patients with gastric cancer Mucosal atrophy, intestines thus secrete.Therefore serum PG I and PGI/PGII obviously decline to monitoring early carcinoma of stomach there is important clinical meaning, effectively can be applied to the preliminary examination of high incidence area of gastric cancer crowd.PG detects as noninvasive method, can reduce the misery of patients with gastric disease or cancer of the stomach people at highest risk inspection, and easy, economical, is significant.
The optimal method detecting the PGI level in serum is immunoassays.Therefore, find and suitable there is immunogenic PGI epitope peptide, prepare specific PGI antigen and namely antibody become emphasis.
The determinand that fluorescence immune chromatography method is surveyed in blood is easy and simple to handle, quick, and only need just can complete sample detection in 10 minutes, and sensing range is wide, sensitivity is good, can the assisted diagnosis state of an illness in time rapidly, monitors prognosis.Chinese patent application No.200910117820.8 discloses a kind of preparation method and quantitative detecting method of fluorescent micro-ball immune chromatography test paper strip; Chinese patent application No.200910047352.1 discloses a kind of fluorescence immune chromatography test paper and its preparation method and application.But, at present also not for the fluorescence immune chromatography test paper of people PGI albumen.
Summary of the invention
For solving problem existing in above-mentioned prior art, the invention provides a kind of fluorescence immune chromatography test paper for quantitatively detecting people PGI albumen in determinand and preparation method thereof.
Specifically, the invention provides:
For quantitatively detecting a fluorescence immune chromatography test paper for people PGI albumen in determinand, this test paper detects described people PGI albumen by double antibody sandwich method, wherein:
Described double antibody sandwich method adopts the PGI monoclonal antibody being marked with fluorescent microsphere as capture antibody, and a described PGI monoclonal antibody derives from the one in people PGI epitope peptide (1) and (2); And
Described double antibody sandwich method adopts the 2nd PGI monoclonal antibody as detection antibody, and described 2nd PGI monoclonal antibody derives from the another one in people PGI epitope peptide (1) and (2);
Described people PGI epitope peptide (1) and (2) are respectively:
(1)Tyr-Lys-Val-Pro-Leu-Ile-Arg-Lys-Lys-Ser-Leu-Arg-Arg；
(2)Tyr-Lys-Asn-Phe-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Gln。
Preferably, a described PGI monoclonal antibody is prepared from by a PGI antigen, and a PGI antigen is by making the one in described people PGI epitope peptide (1) and (2) and carrier protein couplet be prepared from; And
Described 2nd PGI monoclonal antibody is prepared from by the 2nd PGI antigen, and the 2nd PGI antigen is by making the another one in described people PGI epitope peptide (1) and (2) and carrier protein couplet be prepared from.
Preferably, the particle diameter of described fluorescent microsphere is 320nm to 400nm.
Preferably, the fluorescent material on described fluorescent microsphere is fluorescein isothiocynate, RB 200, TRITC or X-rhodamine, is wherein preferably X-rhodamine; The micro-sphere material of described fluorescent microsphere is the multipolymer of polystyrene, polymethylmethacrylate or methyl methacrylate.
Preferably, the excitation wavelength of described fluorescent microsphere is 350 ~ 600nm, is preferably 390nm; Emission wavelength is 500 ~ 700nm, is preferably 615nm.
Preferably, described fluorescence immune chromatography test paper has base plate, and be provided with the way of contact successively along chromatography direction when using on which floor plate: sample pad, pad, reaction film, absorbent filter, described pad is coated with the described PGI monoclonal antibody being marked with fluorescent microsphere, described reaction film comprises detection zone and quality control region, described detection zone is coated with described 2nd PGI monoclonal antibody, and described quality control region is coated with the antiantibody that can be combined with the described PGI monoclonal antibody specificity being marked with fluorescent microsphere.
Preferably, described reaction film is not fluorescent nitrocellulose filter substantially under the wavelength being greater than 550nm.
Preferably, described base plate does not have photoluminescent property substantially.
Preferably, described antiantibody is sheep anti-mouse igg monoclonal antibody or rabbit anti-mouse igg monoclonal antibody, is wherein preferably sheep anti-mouse igg monoclonal antibody.
Present invention also offers a kind of method preparing the described fluorescence immune chromatography test paper for quantitatively detecting people PGI albumen in determinand, it comprises the following steps:
1) the PGI monoclonal antibody being marked with fluorescent microsphere is provided;
2) provide pad, wherein wrap by the described PGI monoclonal antibody being marked with fluorescent microsphere on described pad;
3) provide reaction film, wherein on described reaction film, fix the 2nd PGI monoclonal antibody and antiantibody, to form detection zone and quality control region respectively along the interval, chromatography direction when using; With
4) on base plate, successively sample pad, described pad, described reaction film, absorbent filter are set with the way of contact along chromatography direction when using, thus make described fluorescence immune chromatography test paper.
Preferably, described step 1) comprises:
A) use carbodiimide activation fluorescent microsphere, preferably, the aqueous dispersions of fluorescent microsphere or MES damping fluid dispersion liquid are mixed with carbodiimide through ultrasound wave process, thus activates described fluorescent microsphere;
B) fluorescent microsphere of activation that a) obtains of washing step, preferably, fluorescent microsphere N-hydroxy thiosuccinimide-citrate buffer solution washing of the activation that step a) is obtained, dispersion, and through ultrasound wave process;
C) by fluorescent microsphere mark the one PGI monoclonal antibody that step b) obtains, preferably, fluorescent microsphere step b) obtained mixes with a PGI monoclonal antibody, with BSA-monoethanolamine buffer blind, centrifugal, with the dispersion of BSA-Tween solution, through ultrasound wave process, thus obtain the PGI monoclonal antibody being marked with fluorescent microsphere.
Preferably, in described step 2) in, the described bag being marked with a PGI monoclonal antibody of fluorescent microsphere is 0.5 ~ 2mg/ml by concentration.
Preferably, in described step 3), described detection zone and described quality control region interval 3mm to 8mm, the bag of described 2nd PGI monoclonal antibody and described antiantibody is respectively 0.5 ~ 2mg/ml by concentration.
The present invention compared with prior art has the following advantages and good effect:
1. people PGI epitope peptide of the present invention has good antigenicity, and antigen (immunogene) immune animal of preparing with it can produce monoclonal antibody and the polyclonal antibody of high degree of specificity.
2. the PGI monoclonal antibody prepared with the present invention and polyclonal antibody can high special PGI in blood sample be combined.
3. people PGI external diagnosis reagent case of the present invention can monitor the level of the PGI in serum effectively, and preparation method is simple, and cost is low, is suitable for large-scale production.
4. fluorescence analysis combines with flash chromatography immunological technique by the present invention, provide a kind of fluorescence immune chromatography test paper for quantitatively detecting people PGI albumen in determinand, with the people PGI albumen in this detection paper determinand, easy and simple to handle, quick, only need just can complete sample detection in 10 minutes, and sensing range is wide, specificity is high, sensitivity is good, can the assisted diagnosis state of an illness in time rapidly, monitoring prognosis.
5. the present invention is in the process of the fluorescence immune chromatography test paper of the described people PGI albumen of preparation, groped by a large amount of tests, optimize the preparation condition of each side, when making to detect with fluorescence immune chromatography test paper of the present invention, fluorescence signal-to-background ratio improves greatly, thus improves detection sensitivity and credible result degree; In addition, the present invention also carrys out the content of PGI in response sample by the change of the detection zone of test paper and the fluorescence intensity ratio of quality control region, compared with the absolute fluorescence intensity that this and traditional chromatographic technique only examine or check detection zone, decrease the impact of external condition and background etc. to the full extent, further increase testing result confidence level.
Accompanying drawing explanation
Fig. 1 is the figure of the correlativity that the testing result of people PGI external diagnosis reagent case of the present invention and the testing result of known agent box are shown, wherein horizontal ordinate is the value of PGI concentration in the sample recorded with known agent box, unit is ng/ml, ordinate is the value of PGI concentration in the sample recorded with kit of the present invention, and unit is ng/ml.
Fig. 2 schematically shows the structural representation of the fluorescence immune chromatography test paper of one embodiment of the invention.
Embodiment
Below by way of embodiment description and the invention will be further described with reference to accompanying drawing, but this is not limitation of the present invention, those skilled in the art are according to basic thought of the present invention, various amendment or improvement can be made, but only otherwise depart from basic thought of the present invention, all within the scope of the present invention.
One, people PGI epitope peptide
People PGI albumen described is herein known in the art, and its amino acid sequence is known in the art, can find in the specialized databases such as NCBI.
The invention provides a kind of people PGI epitope peptide (1) and (2), its amino acid sequence respectively as shown in sequence table SEQ ID No.1 and SEQ ID No.2, for:
(1) Y-K-V-P-L-I-R-K-K-S-L-R-R; With
(2)Y-K-N-F-T-V-F-D-R-A-N-N-Q。
The present inventor gropes through a large amount of theoretical researches and experiment, and screening obtains two and has good antigenic epitope peptide.
PGI epitope peptide (1) holds one section of the 19 to 30 containing the peptide section of 12 amino acid residues as antigenic determinant using people PGI albumen (PG3) N, and is added with Y at its N end.Adding Y at N end is to make this epitope peptide by BDB(Bis-diazotizedbenzidine dichloride) be linked on carrier protein (such as hemocyanin (KLH)), thus carry out Dispersal risk as antigen.PGI epitope peptide (1) has the advantages that water wettability is high, antigenicity strong and be easy to synthesis.
PGI epitope peptide (2) holds one section of the 372 to 381 containing the peptide section of 10 amino acid residues as antigenic determinant using people PGI albumen (PG3) C, and is added with 3 amino acid residues at its N end: Y, K, N.The PGI epitope peptide (2) of such formation also has the feature that water wettability is high, antigenicity strong and be easy to synthesis.
At present, the present invention studies discovery, and PGI epitope peptide of the present invention has following function:
1. there is antigenicity; 2. after being connected with carrier protein, produce specific antibody as immunogene stimulating animal; 3. can be combined with people PGI specifically with antibody prepared by epitope peptide.
Preparation method's useful chemical synthetic method of PGI epitope peptide of the present invention: utilize American AB I431A type polypeptide automatic synthesizer, by Solid phase synthesis epitope peptide.The molecular weight of epitope peptide of the present invention (1) and (2) is respectively 1657.24 and 1616.93, and available mass spectrum is determined, and measures the epitope peptide sequence synthesized by qualification by peptide sequence.The purity thin-layer chromatography of peptide section and high performance liquid chromatography are evaluated, and measure the concentration of epitope peptide.
Two, PGI antigen
Present invention also offers a kind of PGI antigen, it is prepared from by making the one in people PGI epitope peptide (1) of the present invention and (2) and carrier protein couplet.Specifically, the invention provides PGI antigen (1) and (2), described PGI antigen (1) is prepared from by making people PGI epitope peptide (1) of the present invention and carrier protein couplet; Described PGI antigen (2) is prepared from by making people PGI epitope peptide (2) of the present invention and carrier protein couplet.PGI antigen of the present invention has immunogenicity and specificity, is a kind of immunogene, can be used to immune animal thus prepares specific PGI antibody.In the present invention, the example of available carrier protein comprises KLH(keyhole limpet hemocyanin), bovine serum albumin(BSA) (BSA), ovalbumin OVA etc.Due to KLH(keyhole limpet hemocyanin) immunogenicity is strong, and binding site is many, and immune effect is better, and comparatively far away with immune animal sibship, and not easily causing cross reaction with it as carrier protein, is therefore preferred.
Three, PGI monoclonal antibody, PGI polyclonal antibody and people PGI external diagnosis reagent case
Present invention also offers people PGI monoclonal antibody and people PGI polyclonal antibody, described antibody can utilize PGI antigen (1) of the present invention and (2) (immunogene) immune animal prepare and obtain respectively.Preparation method can adopt the ordinary skill in the art, specifically can see embodiment 2.
PGI monoclonal antibody of the present invention and polyclonal antibody may be used for preparation people PGI external diagnosis reagent case, and this kit can detect the PGI in blood preparation based on immunization method.
Therefore, the invention provides a kind of people PGI external diagnosis reagent case, it comprises people PGI monoclonal antibody of the present invention or polyclonal antibody.
The known immunization experiment method that can be used for clinical examination mainly comprises following several at present: ELISA method, chemoluminescence method, fluorescent chromatographic method, colloid gold immune determination method etc.
And ELISA method comprises following several types: double antibody sandwich method detectable antigens, dual-antigen sandwich method detect antibody, indirect method surveys antibody, competition law surveys antibody, competition law surveys antigen, catch bag is surveyed antibody etc. by method.
People PGI external diagnosis reagent case of the present invention preferably adopts ELISA double antibody sandwich method to detect PGI albumen.This kit can comprise coated antibody, binding antibody, the second antibody of enzyme labeling and/or the instrument of necessity and reagent etc.
Preferably, described people PGI external diagnosis reagent case adopts people PGI monoclonal antibody of the present invention as coated antibody.At this, term " coated antibody " refers to the antibody be coated in the ELISA Plate of solid phase.In addition, described people PGI external diagnosis reagent case also preferably comprises people PGI polyclonal antibody using as binding antibody, wherein, when described binding antibody derives from the one in people PGI epitope peptide (1) of the present invention and (2), described coated antibody derives from the another one in described epitope peptide (1) and (2).At this, term " binding antibody " refers to the specific antibody that can be combined with determined antigen and enzyme-labeled secondary antibody in kit.Described kit can also comprise the second antibody of enzyme labeling, and this second antibody can be goat anti-rabbit igg antibody, and described enzyme labeling can be horseradish peroxidase, alkaline phosphatase etc.
In kit of the present invention, any reagent needed for detection or instrument can also be comprised, such as pre-coated plate, cleansing solution, developer, stop buffer etc.
Four, for quantitatively detecting the fluorescence immune chromatography test paper of people PGI albumen
Present invention also offers a kind of fluorescence immune chromatography test paper for quantitatively detecting people PGI albumen in determinand, this test paper detects described people PGI albumen by double antibody sandwich method, wherein:
Described double antibody sandwich method adopts the PGI monoclonal antibody being marked with fluorescent microsphere as capture antibody, and a described PGI monoclonal antibody derives from the one in people PGI epitope peptide (1) and (2); And
Described double antibody sandwich method also adopts the 2nd PGI monoclonal antibody as detection antibody, and described 2nd PGI monoclonal antibody derives from the another one in people PGI epitope peptide (1) and (2);
Described people PGI epitope peptide (1) and (2) are respectively:
(1)Tyr-Lys-Val-Pro-Leu-Ile-Arg-Lys-Lys-Ser-Leu-Arg-Arg；
(2)Tyr-Lys-Asn-Phe-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Gln。
In the present invention, in double antibody sandwich method fluorescence immune chromatography test paper, " capture antibody " refers to can the antibody of first specific recognition determined antigen, and it is coated on pad usually; " detection antibody " refers to that another kind can the antibody of specific recognition determined antigen, and itself and capture antibody identify the different epitope on determined antigen molecule respectively, and it is fixed on the detection zone of reaction film usually.
In the present invention, a described PGI monoclonal antibody can be prepared from by a PGI antigen, and a PGI antigen can be prepared from by making the one in described people PGI epitope peptide (1) and (2) and carrier protein couplet; And described 2nd PGI monoclonal antibody can be prepared from by the 2nd PGI antigen, the 2nd PGI antigen can be prepared from by making the another one in described people PGI epitope peptide (1) and (2) and carrier protein couplet.
In the present invention, the example of available carrier protein comprises KLH(keyhole limpet hemocyanin), bovine serum albumin(BSA) (BSA), ovalbumin OVA etc.Due to KLH(keyhole limpet hemocyanin) immunogenicity is strong, and binding site is many, and immune effect is better, and comparatively far away with immune animal sibship, and not easily causing cross reaction with it as carrier protein, is therefore preferred.
Preferably, the particle diameter of fluorescent microsphere used in fluorescence immune chromatography test paper of the present invention is 320nm to 400nm, be preferably 360nm, fluorescent material on fluorescent microsphere can be fluorescein isothiocynate, RB 200, TRITC or X-rhodamine etc., is wherein preferably X-rhodamine (can purchased from Shanghai Jing Chun company).The micro-sphere material of fluorescent microsphere can be the multipolymer formed by polystyrene, polymethylmethacrylate or methyl methacrylate and other monomer copolymerization, and the example of other monomer is styrene etc.The excitation wavelength of fluorescent microsphere can be 350 ~ 600nm, is preferably 390nm; Emission wavelength can be 500 ~ 700nm, is preferably 615nm.
In the present invention, the maximum excitation wavelength of fluorescent microsphere and emission wavelength difference are comparatively large, illustrate that fluorescent microsphere has larger Stokes (Stokes) displacement, like this, the background interference of fluorescent test paper is lower, and doing immunochromatography label with this microballoon has stronger advantage.
In a specific embodiment, fluorescence immune chromatography test paper of the present invention has base plate, and be provided with the way of contact successively along chromatography direction when using on which floor plate: sample pad, pad, reaction film, absorbent filter, described sample pad is used for loading testing sample in use, described pad is coated with the described PGI monoclonal antibody being marked with fluorescent microsphere, described reaction film comprises detection zone and quality control region, described detection zone is coated with described 2nd PGI monoclonal antibody, described quality control region is coated with the antiantibody that can be combined with the described PGI monoclonal antibody specificity being marked with fluorescent microsphere.
Preferably, the sample pad of fluorescence immune chromatography test paper of the present invention, pad, reaction film, absorbent filter can overlap successively along chromatography direction when using and be arranged on (see Fig. 2) on base plate.On reaction film, spaced detection zone and quality control region can be, but are not limited to, the forms such as line, band, block, detection zone and quality control region preferred interval 3mm to 8mm.
In the present invention, reaction film is preferably not fluorescent nitrocellulose filter substantially under the wavelength being greater than 550nm.In addition, base plate does not preferably have photoluminescent property substantially.
Usually, conventional chromatographic test paper assembly (reaction film, base plate etc.) has obvious fluorescence background under 550nm wavelength, and this detection to fluorescence signal produces very large interference.The present invention by adopting the base plate of not fluorescent nitrocellulose filter and low Poison character substantially under the wavelength being greater than 550nm, thus overcomes the defect of conventional fluorescent test paper.In addition, the present invention's fluorescent material X-rhodamine used can produce stronger fluorescence signal, thus substantially increases fluorescence signal-to-background ratio further, makes it possible to distinguish signal and background well, and then improves detection sensitivity.
In the present invention, the material of sample pad and pad can adopt the normally used material in this area, and such as, sample pad and pad can be glass fibre.
The antiantibody that can be combined with the PGI monoclonal antibody specificity being marked with fluorescent microsphere of the present invention can be sheep anti-mouse igg monoclonal antibody or rabbit anti-mouse igg monoclonal antibody, wherein be preferably sheep anti-mouse igg monoclonal antibody, compared with polyclonal antibody, monoclonal antibody specificity is higher.
In a specific embodiment, fluorescence immune chromatography test paper of the present invention in use, sample pad drips sample liquid (blood sample as containing PGI), under capillarity, sample liquid moves to absorbent filter one end, immune complex is formed at pad place and the described PGI monoclonal antibody being marked with fluorescent microsphere, the movement further of this immune complex, detection line is combined with described 2nd PGI monoclonal antibody the immune complex forming double-antibody sandwich, the antiantibody of a PGI monoclonal antibody then on nature controlling line being marked with fluorescent microsphere not forming immune complex is combined.This process need 10 minutes to 15 minutes, afterwards, detects with fluorescence detector, if band does not appear in nature controlling line place, then illustrates that test paper lost efficacy; If band appears in nature controlling line place, and band does not appear in detection line place, then do not contain people PGI albumen in interpret sample; If all there is band on nature controlling line and detection line, then contain people PGI albumen in interpret sample.
In yet another aspect, the invention provides a kind of for the preparation of the quantitative method detecting the fluorescence immune chromatography test paper of people PGI albumen in determinand, it comprises the following steps:
1) the PGI monoclonal antibody being marked with fluorescent microsphere is provided;
2) provide pad, wherein wrap by the described PGI monoclonal antibody being marked with fluorescent microsphere on described pad;
3) provide reaction film, wherein on described reaction film, fix the 2nd PGI monoclonal antibody and antiantibody, to form detection zone and quality control region respectively along the interval, chromatography direction when using; With
4) on base plate, successively sample pad, described pad, described reaction film, absorbent filter are set with the way of contact along chromatography direction when using, thus make described fluorescence immune chromatography test paper.
It will be appreciated by persons skilled in the art that and can adjust the order of above-mentioned steps according to actual needs, such as step 3) can before step 1), or in step 1) and 2) between.
Method of the present invention can also comprise the step 5) fluorescence immune chromatography test paper made being cut into proper width.
The present inventor is groped by a large amount of tests, optimize the condition of each step of the method preparing the fluorescence immune chromatography test paper for detecting people PGI albumen of the present invention, thus make fluorescence immune chromatography test paper of the present invention can obtain for people PGI albumen the result meeting clinical detection and require, that is, sensing range is wide, specificity is high, sensitivity is good.
Therefore, preferably, in the method for the invention, described step 1) comprises:
A) use carbodiimide activation fluorescent microsphere, preferably, the aqueous dispersions of fluorescent microsphere or MES damping fluid dispersion liquid are mixed with carbodiimide through ultrasound wave process, thus activates described fluorescent microsphere;
B) fluorescent microsphere of activation that a) obtains of washing step, preferably, fluorescent microsphere N-hydroxy thiosuccinimide-citrate buffer solution washing of the activation that step a) is obtained, dispersion, and through ultrasound wave process;
C) by fluorescent microsphere mark the one PGI monoclonal antibody that step b) obtains, preferably, fluorescent microsphere step b) obtained mixes with a PGI monoclonal antibody, with BSA-monoethanolamine buffer blind, centrifugal, with the dispersion of BSA-Tween solution, through ultrasound wave process, thus obtain the PGI monoclonal antibody being marked with fluorescent microsphere.
In a specific embodiment of the present invention, described step 1) comprises:
A) carbodiimide activation fluorescent microsphere is used, wherein, get the fluorescent microsphere aqueous dispersions of 1 (w/v) %, centrifugal 5 to 10 minutes of 10000rpm to 15000rpm low temperature (such as 10 DEG C), removes supernatant, sediment is distributed to 500 μ l distilled water or just in wash buffer (the MES aqueous solution of 0.1M), ultrasound wave (240W) processes 1 to 2 minute, repeats above process three times, adds carbodiimide 10mg to 50mg, stir 10 ~ 15 minutes, thus activate described fluorescent microsphere;
B) fluorescent microsphere of activation that a) obtains of washing step, wherein, the fluorescent microsphere of the activation that step a) is obtained under 1000rpm to 15000rpm centrifugal 5 to 10 minutes, sediment is distributed in 1ml coupling buffer (the N-hydroxy thiosuccinimide-citrate buffer solution of 20 ~ 100mM), ultrasound wave (240W) processes 1 to 2 minute, repeats above process three times;
C) by fluorescent microsphere mark the one PGI monoclonal antibody that step b) obtains, wherein, according to the ratio of the fluorescent microsphere activated described in 1 μ l to 3 μ l antibody (10mg/ml)/100 μ l, fluorescent microsphere step b) obtained mixes with a PGI monoclonal antibody, 1.5 ~ 3 hours (preferably 2 hours) are stirred under room temperature (25 DEG C), add 1ml Block buffer (1 (w/v) %BSA-0.05M monoethanolamine), continue stirring 1 hour, under 10000rpm to 15000rpm centrifugal 5 to 10 minutes, repeated centrifugation 3 times, sediment is distributed in the whole wash buffer (0.5 (w/v) %BSA-0.11 (v/v) % Tween solution) of 500 μ l, ultrasound wave (240W) processes 1 to 2 minute, 500 μ l are settled to described whole wash buffer.
Preferably, in the method for the invention, described step 2) comprising: will a PGI monoclonal antibody antibody diluent (1% (w/v) BSA-0.01M PBS(pH7.2) damping fluid of fluorescent microsphere be marked with) dilute, to be diluted to 0.5 ~ 2mg/ml, be preferably 1mg/ml, then use micropipettor even application on pad, post-drying or vacuum freeze drying.Step 2 in method of the present invention) in, the described bag being marked with a PGI monoclonal antibody of fluorescent microsphere is 0.5 ~ 2mg/ml by concentration, is preferably 1mg/ml.
Preferably, described step 3) comprises: described 2nd PGI monoclonal antibody and antiantibody to be drawn on nitrocellulose filter (solid phase carrier) with metal spraying machine using as detection zone and quality control region, what make detection zone and quality control region is spaced apart 3mm to 8mm, the concentration of described 2nd PGI monoclonal antibody and described antiantibody is respectively 0.5 ~ 2mg/ml, is preferably 1mg/ml.
Preferably, result detects and utilizes special fluorescence detector (can purchased from Anqun Bioengineering Co., Ltd., Shenzhen, model AQ-3000) quality control region and detection zone are detected, the content of the PGI in the ratio of detection zone and quality control region fluorescence intensity and testing sample is directly proportional.Adopt the ratio of detection zone and quality control region fluorescence intensity instead of directly adopt the absolute fluorescence value of detection zone can reduce the impact of reaction conditions, matrix etc. as much as possible, and avoiding background interference as far as possible.
Mode below by way of example further explains and describes content of the present invention, but these examples should not be understood to the restriction to protection scope of the present invention.
Embodiment
Except as otherwise noted, the following stated solution is aqueous solution, and the percentage in solution is percent by volume.
The preparation of embodiment 1:PGI epitope peptide (1) and (2).
Preparation method's chemical synthesis: utilize American AB I431A type polypeptide automatic synthesizer, synthesize PGI epitope peptide (1) and (2) respectively by solid phase method.The purity high performance liquid chromatography of epitope peptide is evaluated, and measures the concentration of peptide section.The molecular weight of epitope peptide of the present invention (1) and (2) is respectively 1657.24,1616.93, utilizes mass spectrum to determine, measures the peptide sequence synthesized by qualification by peptide sequence.
One, the synthesis of PGI epitope peptide (1) and (2)
Above-mentioned peptide section adopts Solid phase synthesis.The main thought of Solid phase peptide synthesis is: be first connected with the same insoluble macromolecular compound (resin) of covalent bond form by the carboxyl that will synthesize the carboxyl-terminus amino acid of peptide chain; then amino acid on solid phase carrier is combined in as moiety using this; through deaminate protecting group and with the reaction of excessive activated carboxyl component, spreading peptide chain.Such step can repeatedly go on repeatedly, finally reaches the length of the peptide chain of required synthesis.This building-up process is as follows.
The respective concrete preparation process of PGI epitope peptide (1) of the present invention and (2) is as follows:
1. raw materials used:
HMP resin (P-hydroxymethyl phenoxy methyl poly vinyl, can purchased from sigma company)
Fmoc-AA (amino acid of 9-fluorenylmethoxycarbonyl carbonyl acyl group protection, can purchased from Merck company)
NMP(nitrogen methyl pyrrolidone, can purchased from sigma company)
DCM(methylene chloride, can purchased from Central Plains chemical company)
MeoH(methyl alcohol, can purchased from Central Plains chemical company)
Piperidine(piperidines, can purchased from sigma company)
DMAP(dimethyl aminopyridine, can purchased from sigma company)
HOBT(hydroxybenzotriazole, can purchased from sigma company)
DCC(dicyclohexylcarbodiimide, can purchased from sigma company)
TFA(trifluoroacetic acid, can purchased from sigma company)
EDT(1,2-dithioglycol, can purchased from sigma company)
Thioanisole, can purchased from Guangzhou Wei Bai Chemical Co., Ltd.
Crystalline phenol, can purchased from Chemical Reagent Co., Ltd., Sinopharm Group
Acetonitrile, can purchased from Chemical Reagent Co., Ltd., Sinopharm Group
2. use instrument:
Polypeptide automatic synthesizer, model 431A, can purchased from ABI company
Rotary Evaporators, model R-201, can purchased from Shanghai Shen Shun company
High performance liquid chromatograph, Waters600, can purchased from American Waters company
Freeze drier, model VFD-2000, can purchased from Beijing rich doctor Kanggong department
3. synthetic method and process:
Take HMP resin 100mg, replacing equivalent is 1.0meq, and be placed in by 0.1mmol in the reaction chamber of American AB I431A type polypeptide automatic synthesizer, be automatically linked in sequence by different by specific amino acid by synthesizer, Conjugate ratio reaches 99%.React as follows:
(1) amino acid whose activation (HOBt/DCC method)
The amino acid of Fmoc protection
(2) amino acid is connected on resin
(3) the Fmoc protecting group of deaminate acid
(4) another amino acid whose activation (HOBt/DCC method)
(5) coupling
Peptide-the resin of new coupling
(6) step (3) to (5) is repeated until end of synthesis.
(7) peptide resin:
By resin transfer in beaker, use TFA(trifluoroacetic acid) cut peptide chain, with EDT(2.5 volume %), thioanisole (2.5 volume %) makes scavenger, at room temperature react 3.0 hours, removing cutting reagent, use extracted with diethyl ether again, obtain the crude product 124.3mg of PGI peptide section (1) and the crude product 119.8mg of PGI peptide section (2) respectively.
Two, the purifying of PGI epitope peptide (1) and (2) crude product:
Adopt high performance liquid chromatography separation and purification:
Condition: chromatographic column: C810 × 100mm, can purchased from American Waters company
Chromatograph: Waters600, Waters, US
Mobile phase: A:0.1%TFA(trifluoroacetic acid) aqueous solution
B:0.1%TFA(trifluoroacetic acid) in 60% acetonitrile
Determined wavelength: 214nm
Flow velocity: 4ml/ minute
Gradient: 20-60%B, 30 minutes
HPLC(high performance liquid chromatography) analyze
Chromatographic column: C184.6 × 150mm, can purchased from American Waters company
Mobile phase: A:0.1%TFA(trifluoroacetic acid) aqueous solution
B:0.1%TFA(trifluoroacetic acid) in acetonitrile
Determined wavelength: 214nm
Flow velocity: 1ml/ minute
Gradient: 0-60%B, 30 minutes
The purity that peptide piecewise analysis result shows PGI epitope peptide (1) of the present invention and (2) is more than 95%.
Three, the qualification of PGI epitope peptide (1) and (2)
1. utilize mass spectrum to measure the PGI epitope peptide (1) of purifying gained and the molecular weight of (2) respectively.
(1) reagent raw material
TFA(trifluoroacetic acid, can purchased from sigma company)
HCCA(alpha-cyano-4-hydroxycinnamic acid, can purchased from sigma company)
Acetonitrile (can purchased from Chemical Reagent Co., Ltd., Sinopharm Group)
(2) instrument
Matrix Assisted Laser Desorption ionization time-of-flight mass spectrometer MALDI-TOF-MS(model: REFLEX III, German Bruker company);
(3) matrix liquid: α-CCA is dissolved in the 50%ACN solution containing 0.1%TFA, makes saturated solution, centrifugal, get supernatant;
(4) instrument testing conditions: reflection detection mode; Flight pipe range 3m; Nitrogen laser: wavelength 337nm, accelerating potential 20KV; Reflected voltage 23KV.
(5) operation steps: the sample getting the above-mentioned purified polypeptide of 1 μ L (1) and (2) respectively, mixes with the saturated stromal supernatant mixing equal-volume of 1 μ L separately, gets 1 μ L point respectively on sample target, sends in ion gun and detects.
As a result, the molecular weight recording gained PGI epitope peptide (1) is the molecular weight of 1658.5, PGI epitope peptide (2) is 1618.4, with theoretical molecular 1657.24,1616.93 consistent, proves product for the purpose of improvement on synthesis namely.
2. the sequence identifying gained PGI epitope peptide (1) and (2) is respectively measured by peptide sequence.
(1) principle: the ultimate principle of polypeptid acid sequence analysis is Edman degraded is a circulating chemical reaction process.Comprise three main chemical steps: (1) coupling: the N-of isothiocyanic acid benzene fat and proteins and peptides holds residue to react, form phenylamino formyl sulfide (PTC) derivant, i.e. PTC-peptide.(2) cyclisation cracking: PTC-peptide cyclisation cracking.(3) transform: thiazole purine ketone phenylamino (ATZ) is converted into the different sulphur urine amino acid of benzene (PTH-amino acid).Stay the peptide decreasing an amino acid residue in the solution to repeat above-mentioned course of reaction again, whole sequencing procedure is all automatically carried out by sequenator now.
(2) instrument: American AB I company 491 type protein/polypeptide-terminal amino acid sequenator
(3) reagent raw material
Phenyl isothiocyanate PITC, can purchased from sigma company
Normal heptane, can purchased from Chemical Reagent Co., Ltd., Sinopharm Group
Trimethylamine TMA aqueous solution, can purchased from Chemical Reagent Co., Ltd., Sinopharm Group
TFA(trifluoroacetic acid, can purchased from sigma company)
Ethyl acetate, can purchased from Chemical Reagent Co., Ltd., Sinopharm Group
Chlorobutane, can purchased from sigma company
Acetonitrile, can purchased from Chemical Reagent Co., Ltd., Sinopharm Group
(4) measure
Undertaken by instrument instructions.
Result: through qualification, the sequence of gained PGI epitope peptide (1) and (2) is respectively:
(1) Y-K-V-P-L-I-R-K-K-S-L-R-R; With
(2)Y-K-N-F-T-V-F-D-R-A-N-N-Q。
This result is consistent with target section of synthesized peptide.
Embodiment 2: respectively the PGI epitope peptide (1) of embodiment 1 gained is connected to prepare PGI antigen (1) and (2) with (2) with carrier protein, utilize gained antigen (1) and (2) immune animal respectively, thus utilize antigen (1) to prepare specific monoclonal antibody and polyclonal antibody, and antigen (2) is utilized to prepare specific monoclonal antibody and polyclonal antibody.
1. the preparation of antigen: with BDB(Bis-diazotizedbenzidine dichloride) method by PGI peptide section (1) and (2) respectively with carrier protein KLH(keyhole limpet hemocyanin) be connected and be prepared into PGI antigen (1) and (2).
Get PGI peptide section (1) or (2) 10.0mg, dissolve with 1ml0.1M PBS damping fluid (pH7.4); KLH10mg, dissolves with 0.2M borate buffer solution (pH9.0) 20ml; Then both are mixed, be cooled to 0 DEG C, get BDBCl
2110 μ L, react 1.5h under room temperature, packing after dialysed overnight ,-20 DEG C of preservations.
In the present embodiment, the formula of PBS damping fluid is: the Na of 0.2mol/L
2hPO
481ml adds the NaH of 0.2mol/L
2pO
419ml mixes.
The formula of borate buffer solution is: 0.05mol/L borax 80ml, adds 0.2mol/L boric acid 20ml and mixes.
2. immune animal prepares monoclonal antibody:
2.1. get after the PGI antigen (1) of above-mentioned preparation and (2) (immunogene) fully mix with isopyknic Freund's complete adjuvant (purchased from Shanghai Yuan Ju biotech firm) respectively, individually immune Balb/c mouse, 50 μ g antigens/only, subcutaneous multi-point injection.Serum titer is surveyed after 4 weeks, select the good mouse booster immunization again of immunoreactivity: get after antigen fully mixes with isopyknic incomplete Freund's adjuvant, antigen dose 25 μ g/ only, subcutaneous multi-point injection, the number of times of booster immunization is 6 times, continuous booster immunization twice before merging, extracting spleen cell and Sp2/0 myeloma cell use 50%PEG(MW4000 according to a conventional method afterwards) (purchased from Central Plains chemical company) mediate and merges, and with HAT conditioned medium (purchased from sigma company) selection cultivation.CO is put into after fusion
2cultivate after 9 ~ 11 days for 37 DEG C in incubator, in hole, occur larger cell clone.Within 11 days, start to screen with indirect ELISA.Utilize limiting dilution assay to carry out 4 time cloningizations to the hole of the primary dcreening operation positive to cultivate (even if a large amount of schizogamy of cell after screening), afterwards amplifying cells, frozen, preparation ascites.
2.2. Balb/c mouse norphytane (purchased from sigma company) 0.5ml/ is only processed, one week pneumoretroperitoneum inoculation hybridoma 2 × 10
6individual/only, collect ascites after 10 days.
2.3. measure antibody titer: measure tiring of monoclonal antibody (1) utilizing PGI antigen (1) to prepare with indirect ELISA method, tiring of monoclonal antibody of result display reaches more than 1:32000.
Tiring of the monoclonal antibody (2) utilizing PGI antigen (2) to prepare also utilizes identical method to measure, and it is tired and also reaches more than 1:32000.
3. immune animal prepares polyclonal antibody:
3.1. select three monthly ages, body weight is about the New Zealand white rabbit of about 2kg as immune animal.In fundamental immunity, the PGI antigen (1) of above-mentioned for 1-2mg preparation and (2) (immunogene) are mixed with isopyknic Freund's complete adjuvant respectively-fully emulsified after individually carry out multiple spot hypodermic injection at rabbit back.Every 4 weeks booster immunizations once, antigen and incomplete Freund's adjuvant fully emulsified after, with 100 μ g/ only in back multiple spot hypodermic injection.Arteria carotis bloodletting in 10th day after final boost, separation of serum.
3.2. measure antibody titer: measure tiring of polyclonal antibody (1) utilizing PGI antigen (1) to prepare with indirect elisa method, result display antibody titer reaches more than 1:16000.
Tiring of the polyclonal antibody (2) utilizing PGI antigen (2) to prepare also utilizes identical method to measure, and it is tired and also reaches more than 1:16000.
3.3. blood and separation of serum is got: arteria carotis intubate gets blood, separation of serum.
4. separation and purification antibody: after ammonium sulfate precipitation, then through Protein G(purchased from sigma company) affinity purification.
5. freeze-drying after antibody packing, Cord blood.
Embodiment 3: the specificity identification of people PGI monoclonal antibody (1) and (2)
Detect with ELISA.Respectively with people PGI albumen and PGII albumen (all purchased from osmanthus, Shanghai Kanggong department) for detectable antigens bag is by elisa plate, the specific reaction of prepared PGI monoclonal antibody (1) and (2) and this people PGI albumen is detected respectively by ELISA, make negative control with normal BALB/c mouse serum, PBS liquid makes blank.
Result: PGI monoclonal antibody (1) and (2) are only reacted with PGI respectively for positive (P/N>2.1), and react for negative with PGII, illustrate that PGI monoclonal antibody (1) of the present invention and (2) have specificity respectively.
Embodiment 4: the specificity identification of people PGI polyclonal antibody (1) and (2)
The method identical with above-mentioned qualification monoclonal antibody specificity is utilized to identify.
Result shows: PGI polyclonal antibody (1) and (2) are reacted for positive (P/N>2.1) respectively with PGI, and react for negative with PGII, illustrate that PGI polyclonal antibody (1) of the present invention and (2) have specificity respectively.
Embodiment 5: utilize PGI monoclonal antibody and PGI polyclonal antibody preparation PGI external diagnosis reagent case.
In the present embodiment, monoclonal antibody (1) coated antibody in this kit will PGI epitope peptide (1) being utilized in embodiment 2 to prepare; Using utilize PGI epitope peptide (2) to prepare in embodiment 2 polyclonal antibody (2) as binding antibody.
Preparation and the operation of PGI external diagnosis reagent case are as follows:
1. the preparation of various damping fluid and reagent:
A, bag are buffered liquid: the CB(carbonate buffer solution of 0.050M, pH9.6)
Na
2cO
3: 16.0 grams
NaHCO
3: 29.0 grams
Distill water-soluble to 1000ml
10 × the PBS-Tween20 of B, sample/lavation buffer solution: pH7.2
Na
2hPO
412H
2o:58 gram
KH
2pO
4: 4 grams
NaCl:100 gram
KCl:4 gram
Distill water-soluble to 1000ml
Add Tween20:20ml
C, enzyme marker dilution:
10×PBS-Tween20：10ml
FCS(calf serum): 20ml
Distill water-soluble to 1000ml
Enzyme stabilizers (can purchased from Shanghai Xi Bao company): 1 gram
Biological preservative (can purchased from Shanghai Xi Bao company): 1ml
D, developer A:
Citric acid: 35.5 grams
Urea peroxide: 10 grams
Distill water-soluble to 1000ml
Tween20：10ml
E, developer B:
Citric acid: 120 grams
EDTA-2Na:1 gram
TMB2HCl:2 gram
Distill water-soluble to 1000ml
F, stop buffer: 2M H
2sO
4
The concentrated sulphuric acid (95-98%): 22.2ml
Distilled water: 177.3ml
The concentrated sulphuric acid slowly instills in distilled water by timing, and limit edged shakes up.
2. the preparation of pre-coated plate:
PGI monoclonal antibody (1) is dissolved in the carbonate buffer solution of the 0.05M of pH=9.6, make pre-coated liquid, 100 μ l are added by 0.1 μ g/ hole in the upper every hole of ELISA Plate (can purchased from Shenzhen Jin Canhua company), put 4 DEG C and place 18-24 hour, take out, get rid of coating buffer, washing, load in aluminide-coating bag after closed 16 hours of BSA, dried overnight and vacuumize sealing, and be placed in 4 DEG C of preservations.
3. the dilution ratio of binding antibody (PGI polyclonal antibody (2)) and enzyme connection thing (goat anti-rabbit igg antibody (purchased from Beijing company of Zhong Shan Golden Bridge) of horseradish peroxidase-labeled) is determined by square formation titration experiments.
4. the composition of kit:
Pre-coated plate: 48/96 hole
PGI calibration object (raw material is purchased from osmanthus, Shanghai Kanggong department): 6: 6 × 1.0ml(concentration is respectively 0ng/ml, 20ng/ml, 40ng/ml, 80ng/ml, 160ng/ml, 320ng/ml)
PGI binding antibody: 1 × 10ml(dilutes through 1:5000)
Enzyme connection thing: 1 × 10ml(dilutes through 1:5000)
Concentrated cleaning solution (25 × PBS-Tween20): 1 × 20ml
Developer A:1 × 6.0ml
Developer B:1 × 6.0ml
Stop buffer: 1 × 6.0ml
5. the operation steps of kit:
In each hole of pre-coated plate, add blood sample to be checked and standard items 100 μ l/ hole respectively, be diplopore, hatch 60 minutes for 37 DEG C, wash 5 times with 1 × lavation buffer solution, pat dry.In each hole, add PGI binding antibody 100 μ l/ hole, hatch 30 minutes for 37 DEG C, wash 5 times with 1 × lavation buffer solution, pat dry.In each hole, add enzyme connection thing 100 μ l/ hole again, hatch 30 minutes for 37 DEG C, wash 5 times with 1 × lavation buffer solution, pat dry.Add developer A, B liquid, each 50 μ l in every hole, mixing, hatches 15 minutes for 37 DEG C.Add stop buffer 50 μ l/ hole cessation reaction, join detector (model RT-6000, can purchased from Lei Du company) with enzyme and detect absorbance with dual wavelength (450nm, 620nm).
6. result judges:
1. utilize kit of the present invention to carry out serum PG I detection
Table 1: standard concentration and corresponding mean light absorbency (OD) value
| Concentration ng/ml | 0 | 20 | 40 | 80 | 160 | 320 |
| Mean OD value | 0.068 | 0.143 | 0.320 | 0.585 | 1.023 | 1.867 |
With standard concentration and corresponding absorbance drawing standard curve, the R of typical curve
2=0.997.
The PGI concentration results in the sample detected is calculated according to typical curve.
Carry out serum PG I detection in a manner described to 28 routine superficial gastritis patients, 30 routine atrophic gastritis patients, 34 routine Patients with Gastric Cancer and 70 routine healthy persons, testing result is in table 2.
Table 2: four groups of sample PGI concentration compare
Table 2 result shows, the difference between each group has statistical significance (P<0.01), can be corresponding with clinical detection result.
2. the correlation analysis of the testing result of the testing result of kit of the present invention and the ELISA kit of known detection PGI
Utilize DRG kit (
human Pepsinogen I ELISA, purchased from ALPCO Diagnostics company) and kit of the present invention detect same 50 parts of blood samples (comprising superficial gastritis blood sample of patient, patients with atrophic gastritis blood sample and patients with gastric cancer blood sample) respectively.With the measured value of DRG kit be horizontal ordinate, with the measured value of kit of the present invention for ordinate, set up straight-line equation calculate related coefficient.
Carry out paired t-test to the result of two kinds of kits, whether there were significant differences for both judgements quantitative result.
As shown in Figure 1, the coefficient R of the measured value of kit of the present invention and DRG kit
2=0.997, dependent equation is y=0.9968x+0.0144, and wherein y represents the measured value of kit of the present invention, and x represents the measured value of DRG kit.As shown in Figure 1, the correlativity of the measured value of kit of the present invention and the measured value of DRG kit is good.
Statistical procedures is carried out to the measured value paired t-test of two kinds of kits, obtains | t|=0.996, v=n-1=49, look into t dividing value table and obtain P>0.05.This result shows that the testing result of two kinds of kits is without significant difference, illustrate that the correlativity of the measured value of kit of the present invention and the measured value of DRG kit is good, thus kit of the present invention is with a high credibility, and can be further used for PGI/PGII joint-detection.
Embodiment six: for detecting the preparation of the fluorescence immune chromatography test paper of people PGI albumen in determinand.
One, be marked with the preparation of the monoclonal antibody of fluorescent microsphere and wrap combined pad
1, the preparation of the monoclonal antibody of fluorescent microsphere is marked with
1.1, the activation of fluorescent microsphere:
Get fluorescent microsphere (purchased from the Guangzhou Growth hormone secretagogue company) aqueous dispersions of 500 μ l, content 1 (w/v) %, at 10 DEG C, with 12000rpm centrifugal 10 minutes, remove supernatant, sediment is distributed to the distilled water of 500 μ l or just in wash buffer (the MES aqueous solution of 0.1M), ultrasound wave (240W) processes 2 minutes, repeat above process three times, add carbodiimide (purchased from Shanghai Jing Chun company) 50mg, stir 15 minutes, thus activate described fluorescent microsphere.
1.2, with the fluorescent microsphere labelled antibody activated:
By the fluorescent microsphere that activated under 12000rpm centrifugal 10 minutes, remove supernatant, sediment is distributed in 1ml coupling buffer (citrate buffer solution of the N-hydroxy thiosuccinimide of 50mM), ultrasound wave (240W) processes 2 minutes, repeat above process three times, obtain the damping fluid 1ml being dispersed with fluorescent microsphere.The ratio of the fluorescent microsphere activated according to 3 μ l antibody (10mg/ml)/100 μ l, add the PGI monoclonal antibody (1) prepared by embodiment 2 wherein, stir 2 hours at normal temperatures, add 1ml Block buffer (1 (w/v) %BSA-0.05M monoethanolamine), continue stirring 1 hour, afterwards, under 12000rpm centrifugal 10 minutes, repeated centrifugation 3 times, sediment is distributed in the whole wash buffer (0.5 (w/v) %BSA-0.11 (v/v) % Tween solution) of 500 μ l, ultrasound wave (240W) processes 2 minutes, 500 μ l are settled to above-mentioned whole wash buffer.
2, combined pad is wrapped
PGI monoclonal antibody (1) antibody diluent (1% (w/v) BSA-0.01M PBS(pH7.2) damping fluid being marked with fluorescent microsphere by above-mentioned preparation) be diluted to 1mg/ml, obtain working fluid, then micropipettor (purchased from labsystems company) is used by the amount even application of 4 μ l/cm on pad, use 37 DEG C of oven for drying afterwards, save backup under 45% humidity.
Two, the preparation of reaction film
The PGI monoclonal antibody (2) prepared according to embodiment 2 and sheep anti-mouse igg monoclonal antibody (purchased from Beijing company of Zhong Shan Golden Bridge) are diluted to 1mg/ml respectively with the PBS damping fluid of 50mM pH7.2, the detection line of metal spraying machine (purchased from Hangzhou Feng Hang company) and nature controlling line spacing parameter are set to 6mm, package amount is set to respectively 1.0 μ l/cm, on nitrocellulose filter, draw PGI monoclonal antibody (2) and sheep anti-mouse igg monoclonal antibody with metal spraying machine, normal temperature dries for subsequent use.
Three, the assembling of test paper and cutting
On base plate, overlap joint pastes sample pad, pad, reaction film and absorbent filter mutually successively, obtains test paper plate, is cut to the test strips that width is 5mm.
Four, the preparation of PGI fluorescence immunoassay test card:
Be fixed on by the test paper of above-mentioned well cutting on plastic bottom card, test paper surface face card compresses, and face is stuck on the sample pad of test strips and the position of reaction film and has well and view window.Test card assembles in rear loading aluminium foil bag, adds drying agent sealing and preserves, can preserve more than 1 year under drying at room temperature condition.
Five, the detection of sample
PGI standard items (purchased from osmanthus, Shanghai Kanggong department) sample diluting liquid (1% (w/v) BSA-0.01M PBS(pH7.2) damping fluid) be mixed with the calibration object of following series concentration: 400ng/ml, 200ng/ml, 100ng/ml, 50ng/ml, 20ng/ml, 10ng/ml, 0ng/ml, the above calibration object of 50 μ l is added drop-wise on well respectively, use fluorescence detector (purchased from Anqun Bioengineering Co., Ltd., Shenzhen after 10 minutes, model AQ-3000) detect, fluorescence can be collected on detection line and nature controlling line position.Take sample concentration as horizontal ordinate, the ratio of the fluorescence intensity at detection line and nature controlling line place is that ordinate draws calibration curve, R
2be 0.996.Nature controlling line is used for test paper Effective judgement and does corresponding correction to detection line signal, as band does not appear in nature controlling line, then illustrates that test paper lost efficacy.
Get the blood sample to be checked of 50 μ l, be added drop-wise on well, detect after 10 minutes with fluorescence detector, if band appears in detection line, containing PGI in interpret sample, its concentration can obtain according to calibration curve.
Six, PGI fluorescence immunoassay test paper performance evaluation
1. evaluate the index of test paper performance
1) range of linearity: each concentration calibration product duplicate detection 3 times, draw calibration curve, through data fitting and statistical study, test paper linear detection range of the present invention is 5ng/ml-400ng/ml.
2) minimum detectability: PGI null value blood sample (without PGI composition) (purchased from Central Plains, Shenzhen company) is divided into 20 parts and detects, calculating concentration mean value and 2 times of standard deviation sums, obtain test paper lowest detection of the present invention and be limited to 3.1ng/ml.
3) precision: detect with PGI fluorescence immunoassay test paper of the present invention the blood sample that PGI concentration is respectively 300ng/ml, 100ng/ml, 30ng/ml respectively, duplicate detection 10 times, carries out withinrun precision mensuration.Every day, the sample to above-mentioned 3 concentration measured, 1 day 1 time, and survey 20 days continuously, carry out betweenrun precision mensuration, result is as shown in table 3 below:
Table 3
Batch in the CV(coefficient of variation) and batch between CV be all less than 8%, illustrate that this reagent accurate is good.
In addition, as seen from the above table, the range of linearity of this detection paper PGI is wide, sensitivity good.
Claims (13)
1., for quantitatively detecting a fluorescence immune chromatography test paper for people PGI albumen in determinand, this test paper detects described people PGI albumen by double antibody sandwich method, wherein:
Described double antibody sandwich method adopts the PGI monoclonal antibody being marked with fluorescent microsphere as capture antibody, and a described PGI monoclonal antibody derives from the one in people PGI epitope peptide (1) and (2); And
Described double antibody sandwich method adopts the 2nd PGI monoclonal antibody as detection antibody, and described 2nd PGI monoclonal antibody derives from the another one in people PGI epitope peptide (1) and (2);
Described people PGI epitope peptide (1) and (2) are respectively:
(1)Tyr-Lys-Val-Pro-Leu-Ile-Arg-Lys-Lys-Ser-Leu-Arg-Arg；
(2)Tyr-Lys-Asn-Phe-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Gln。
2. fluorescence immune chromatography test paper according to claim 1, a wherein said PGI monoclonal antibody is prepared from by a PGI antigen, and a PGI antigen is by making the one in described people PGI epitope peptide (1) and (2) and carrier protein couplet be prepared from; And
Described 2nd PGI monoclonal antibody is prepared from by the 2nd PGI antigen, and the 2nd PGI antigen is by making the another one in described people PGI epitope peptide (1) and (2) and carrier protein couplet be prepared from.
3. fluorescence immune chromatography test paper according to claim 1, the particle diameter of wherein said fluorescent microsphere is 320nm to 400nm.
4. fluorescence immune chromatography test paper according to claim 1, the fluorescent material on wherein said fluorescent microsphere is fluorescein isothiocynate, RB 200, TRITC or X-rhodamine, is wherein preferably X-rhodamine; The micro-sphere material of described fluorescent microsphere is the multipolymer of polystyrene, polymethylmethacrylate or methyl methacrylate.
5. fluorescence immune chromatography test paper according to claim 1, the excitation wavelength of wherein said fluorescent microsphere is 350 ~ 600nm, is preferably 390nm; Emission wavelength is 500 ~ 700nm, is preferably 615nm.
6. fluorescence immune chromatography test paper according to claim 1, wherein said test paper has base plate, and be provided with the way of contact successively along chromatography direction when using on which floor plate: sample pad, pad, reaction film, absorbent filter, described pad is coated with the described PGI monoclonal antibody being marked with fluorescent microsphere, described reaction film comprises detection zone and quality control region, described detection zone is coated with described 2nd PGI monoclonal antibody, described quality control region is coated with the antiantibody that can be combined with the described PGI monoclonal antibody specificity being marked with fluorescent microsphere.
7. fluorescence immune chromatography test paper according to claim 6, wherein said reaction film is not fluorescent nitrocellulose filter substantially under the wavelength being greater than 550nm.
8. fluorescence immune chromatography test paper according to claim 6, wherein said base plate does not have photoluminescent property substantially.
9. fluorescence immune chromatography test paper according to claim 6, wherein said antiantibody is sheep anti-mouse igg monoclonal antibody or rabbit anti-mouse igg monoclonal antibody, is wherein preferably sheep anti-mouse igg monoclonal antibody.
10. prepare a method for fluorescence immune chromatography test paper as claimed in any of claims 1 to 9, it comprises the following steps:
1) the PGI monoclonal antibody being marked with fluorescent microsphere is provided;
2) provide pad, wherein wrap by the described PGI monoclonal antibody being marked with fluorescent microsphere on described pad;
3) provide reaction film, wherein on described reaction film, fix the 2nd PGI monoclonal antibody and antiantibody, to form detection zone and quality control region respectively along the interval, chromatography direction when using; With
4) on base plate, successively sample pad, described pad, described reaction film, absorbent filter are set with the way of contact along chromatography direction when using, thus make described fluorescence immune chromatography test paper.
11. methods according to claim 10, wherein said step 1) comprises:
A) use carbodiimide activation fluorescent microsphere, preferably, the aqueous dispersions of fluorescent microsphere or MES damping fluid dispersion liquid are mixed with carbodiimide through ultrasound wave process, thus activates described fluorescent microsphere;
B) fluorescent microsphere of activation that a) obtains of washing step, preferably, fluorescent microsphere N-hydroxy thiosuccinimide-citrate buffer solution washing of the activation that step a) is obtained, dispersion, and through ultrasound wave process;
C) by fluorescent microsphere mark the one PGI monoclonal antibody that step b) obtains, preferably, fluorescent microsphere step b) obtained mixes with a PGI monoclonal antibody, with BSA-monoethanolamine buffer blind, centrifugal, with the dispersion of BSA-Tween solution, through ultrasound wave process, thus obtain the PGI monoclonal antibody being marked with fluorescent microsphere.
12. methods according to claim 10, wherein in described step 2) in, the described bag being marked with a PGI monoclonal antibody of fluorescent microsphere is 0.5 ~ 2mg/ml by concentration.
13. methods according to claim 10, wherein in described step 3), described detection zone and described quality control region interval 3mm to 8mm, the bag of described 2nd PGI monoclonal antibody and described antiantibody is respectively 0.5 ~ 2mg/ml by concentration.
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