CN107918015A

CN107918015A - Immune chromatographic semiquantitative test paper bar, kit and detection method

Info

Publication number: CN107918015A
Application number: CN201710576803.5A
Authority: CN
Inventors: 刘密; 王镕
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-07-14
Filing date: 2017-07-14
Publication date: 2018-04-17

Abstract

The present invention provides immune chromatographic semiquantitative test paper bar, corresponding kit and the relevant detection method that energy is quick, content that is accurate and easily treating testing goal thing carries out half-quantitative detection, immune chromatographic semiquantitative test paper bar therein is based on antigen-antibody reaction principle, for being chromatographed to detected sample and in chromatography side upwardly through different antigen-antibody reactions, and to label cure the detection for the concentration progress sxemiquantitative that colour developing comes to the purpose thing to be detected in detected sample, it is characterised in that：Quality control region includes a plurality of nature controlling line, a plurality of nature controlling line is coated with the Quality Control thing of the standard purpose thing corresponding with purpose thing to be detected of corresponding different content respectively, the standard purpose thing of different content corresponds to the concentration of different standard purpose things, first scheduled volume be more than or equal to total amount and the second scheduled volume of all standard purpose things and, and the second scheduled volume is more than the content of the standard purpose thing of content maximum.

Description

Immune chromatographic semiquantitative test paper bar, kit and detection method

Technical field

The invention belongs to field of immunodetection, and in particular to one kind can be quick, accurate and easily treats testing goal The content of thing carries out the immune chromatographic semiquantitative test paper bar including its kit and detection method of half-quantitative detection.

Background technology

With the continuous improvement of various diseases incidence, the popular need for easy, quick, high specificity detection technique Ask increasing.

Immunochromatography technique is to be based on antigen-antibody reaction principle, is applied to using label as tracer label thing anti-in vivo A kind of former or antibody test new detection technique.Due to the technology have cost it is relatively low it is easy to use, quick, easy to basic unit Using with the advantage such as onsite application, applied to human chorionic gonadotrophin (HCG), alpha-fetoprotein (AFP), prostate-specific The detection of antigen (PSA), bird flu, the plague, Human Hydatidosis, mycoplasma pneumoniae etc..

But due to there was only a nature controlling line in existing product, and current immunochromatography technique is mainly used for Carry out qualitative detection, and when for needing to judge disease property by concentration range, especially for C peptides, follicular stimulating hormone, Lutropin etc. has the clinical detection index of different term of reference, even if its testing result is all positive but concentration level Different then meaning differs greatly, and represents the different phase for whether suffering from different disease or illnesses.Thus detecting As a result to further determine that scope residing for its testing result during positive it is particularly important that.

Since the immunochromatography technique of current application and development comprises only a nature controlling line, thus for necessarily referring to model The detection of the body index enclosed, needs additional colorimetric card to be compared carry out half-quantitative detection at present, and it is current due to Colorimetric card all prepares in advance, due to colorimetric card preparation condition and detection when condition it is inconsistent, in this way, can cause compared with Big error so that testing result is often inaccurate, easily judges by accident；And if using more accurate quantitative detection side Method, such as the methods of chemoluminescence method, time-resolved fluorescence method, process is complicated, cumbersome, causes to spend the time longer, and Need to carry out last result reading by expensive instrument, cause to spend cost larger.

The content of the invention

Offer of the invention is a kind of can content progress half-quantitative detection that is quick, accurate and easily treating testing goal thing Immune chromatographic semiquantitative test paper bar and include its kit and detection method.

To achieve these goals, present invention employs following technical solution：

The present invention provides a kind of immune chromatographic semiquantitative test paper bar, based on antigen-antibody reaction principle, for to be checked Sample is chromatographed and in chromatography side upwardly through different antigen-antibody reactions, and label is carried out to cure next pair of colour developing The concentration of purpose thing to be detected in detected sample carries out end to end in the detection of sxemiquantitative, including edge chromatography direction add Sample area, label land, colour developing area and suction zones, are disposed with detection zone and quality control region in the area that develops the color along chromatography direction, Label land include label and can with purpose thing to be detected with reference to the first scheduled volume the first homologue with reference to and The mark conjugate of formation, detection zone are coated with the second homologue of the second scheduled volume that can be combined with purpose thing to be detected, its It is characterized in that including：Quality control region includes a plurality of nature controlling line, a plurality of nature controlling line be coated with corresponding different content respectively with it is to be detected The Quality Control thing of the corresponding standard purpose thing of purpose thing, the standard purpose thing of different content correspond to the dense of different standard purpose things Degree, the first scheduled volume be more than or equal to total amount and the second scheduled volume of all standard purpose things and, and the second scheduled volume is more than containing Measure the content of maximum standard purpose thing.

Immune chromatographic semiquantitative test paper bar provided by the invention, also has the feature that：Purpose thing to be detected is to be checked Antigen is surveyed, the first homologue is antibody a corresponding with determined antigen, and the second homologue is antibody b corresponding with antigen to be detected, Antibody a and antibody b identify the different antigenic determinants on the antigen to be detected respectively and respectively with the antigen binding to be detected.

Immune chromatographic semiquantitative test paper bar provided by the invention, also has the feature that：Quality Control thing for standard purpose thing, Conjugate, the block polymer of standard purpose thing and macromolecular or the antibody a's that its corresponding antibody binding of standard purpose thing is formed One or more in secondary antibody.

Immune chromatographic semiquantitative test paper bar provided by the invention, also has the feature that：Purpose thing to be detected is to be checked Antibody is surveyed, the first homologue is secondary antibody corresponding with test antibodies, and the second homologue is antigen corresponding with detection antibody.

Immune chromatographic semiquantitative test paper bar provided by the invention, also has the feature that：Purpose thing to be detected is to be checked Antibody is surveyed, the first homologue is antigen corresponding with test antibodies, and the second homologue is secondary antibody corresponding with detection antibody.

Immune chromatographic semiquantitative test paper bar provided by the invention, also has the feature that：Purpose thing to be detected is to be checked Antibody is surveyed, the first homologue is antigen a corresponding with detection antibody, and the second homologue is antigen corresponding with detection antibody B, antigen a and antigen b can be combined with the different parts of detection antibody respectively.

Immune chromatographic semiquantitative test paper bar provided by the invention, also has the feature that：Quality Control thing is standard purpose thing.

Immune chromatographic semiquantitative test paper bar provided by the invention, also has the feature that：Multiple nature controlling lines are coated with respectively Quality Control thing in the corresponding concentration of standard purpose thing raised successively along chromatography direction.

Immune chromatographic semiquantitative test paper bar provided by the invention, also has the feature that：Multiple nature controlling lines are coated with respectively Quality Control thing in the corresponding concentration of standard purpose thing reduced successively along chromatography direction.

Immune chromatographic semiquantitative test paper bar provided by the invention, also has the feature that：Label is colloidal gold, chemistry Luminescence reagent, chemochromic reagent, metallic, carbon nano-particle, latex particle, magnetic particle, quantum dot, fluorescent material or One or more in rare earth ion.

Immune chromatographic semiquantitative test paper bar provided by the invention, also has the feature that：Multiple nature controlling lines are coated with respectively Quality Control thing in the corresponding concentration of standard purpose thing in Cmax be purpose thing to be detected reference upper level concentration.

Immune chromatographic semiquantitative test paper bar provided by the invention, also has the feature that：Multiple nature controlling lines are coated with respectively Quality Control thing in the corresponding concentration of standard purpose thing in Cmin be purpose thing to be detected reference lower limit concentration.

Present invention also offers a kind of immunochromatography half-quantitative detection kit, it is characterised in that including：On at least one The immune chromatographic semiquantitative test paper bar of any one in stating.

Present invention also offers a kind of immunochromatography semi-quantitative detection method, based on antigen-antibody reaction principle, pass through by Detected sample by chromatograph successively by sample application zone, label land, the area that develops the color detection zone and quality control region and successively The detection of different antigen-antibody reactions and the concentration progress sxemiquantitative to the purpose thing to be detected in detected sample is carried out, its It is characterized in that, comprises the following steps：Step 1, it is loaded, detected sample is added to sample application zone, needs when containing in detected sample Testing goal thing, then enter step 2, when not containing purpose thing to be detected in detected sample, then enters step 4；Step 2, mark It is to be checked after remembering that thing land antigen-antibody reaction, detected sample reach label land under chromatography effect from sample application zone Survey on the mark conjugate that purpose thing passes through the part in the first scheduled volume of antigen-antibody reaction and label land First homologue combines the conjugate for obtaining the purpose thing to be detected and marking conjugate；Step 3, detection zone antigen-antibody reaction The mark conjugate that the conjugate and another part of colour developing, purpose thing to be detected and mark conjugate are not combined is acted in chromatography Under migrated towards detection zone, the purpose thing to be detected combined with the first homologue states detection zone coated second in detection zone and this Formation is corresponding with detection zone after the second homologue combination curing label of scheduled volume detects developed band；Step 4, quality control region resists Antigen-antibody reaction develops the color, and the mark conjugate that another part is not combined continues to move to quality control region under chromatography effect, successively By and be solidificated in different content corresponding with various concentrations respectively in the Quality Control thing on each bar nature controlling line of quality control region Various criterion purpose thing with reference to and form be of different shades corresponding with each nature controlling line after curing the label of different content Quality Control developed band；Step 5, compare and judge, by the way that detection developed band is compared with the color of each Quality Control developed band To comparison result, according to the corresponding concentration range of the corresponding standard purpose thing of comparison result, detected sample is semi-quantitatively determined In purpose thing to be detected concentration range, wherein, the first scheduled volume is more than or equal to the total amount of all standard purpose things and second pre- Quantitative sum, and the second scheduled volume is more than the content of the standard purpose thing of content maximum.

Immunochromatography semi-quantitative detection method provided by the invention, also has the feature that：Using above-mentioned any one Immune chromatographic semiquantitative test paper bar carry out.

Immunochromatography semi-quantitative detection method provided by the invention, also has the feature that：Purpose thing to be detected is to treat When detecting antigen, immunochromatography semi-quantitative detection method can use double antibody sandwich method.

Immunochromatography semi-quantitative detection method provided by the invention, also has the feature that：Purpose thing to be detected is to treat When detecting antibody, immunochromatography semi-quantitative detection method can use one kind in indirect method, prize law or dual-antigen sandwich method.

Invention effect

Immune chromatographic semiquantitative test paper bar including its immunochromatography half-quantitative detection kit provided by the invention, Multiple nature controlling lines are provided with the colour developing area included due to immune chromatographic semiquantitative test paper bar therein, these nature controlling lines are distinguished again It is coated with the Quality Control thing of the standard purpose thing corresponding with purpose thing to be measured of the different content containing corresponding various concentrations, Er Qie One scheduled volume be more than or equal to total amount and the second scheduled volume of all standard purpose things and, and the second scheduled volume is more than content maximum Standard purpose thing content, so can cure in chromatography side upwardly through different antigen-antibody reactions to label The content for developing the color to treat testing goal thing carries out the detection of sxemiquantitative, so reduces the mistake come due to color condition different band Difference, improves testing result, and can farthest avoid the unnecessary inconvenient and use cost for accurately detecting and bringing, And it is easy to operate, it is easy to use.

And immunochromatography semi-quantitative detection method provided by the invention, due to by by detected sample by chromatograph successively Different antigen-antibody reactions is carried out by sample application zone, label land, the detection zone in the area that develops the color and quality control region and successively, And the detection developed band for the antigen-antibody reaction formation for passing through detection zone, developing the color with quality control region antigen-antibody reaction, it is each to be formed Quality Control developed band corresponding with various concentrations carries out color comparison, just can be to the concentration of the purpose thing to be detected in detected sample The detection of sxemiquantitative is carried out, so half-quantitative detection can be carried out by colorimetric at the same time in the detection, similarly brings above-mentioned effect Fruit.

Brief description of the drawings

Fig. 1 is the overall structure diagram of the immunochromatography half-quantitative detection kit involved by embodiment 1；

Fig. 2 is immunochromatography half-quantitative detection kit involved by embodiment 1 when detecting Diagnostic Value of Fasting Serum C peptide levels pair An immune chromatographic semiquantitative test paper bar color status schematic diagram of normal level should be less than；

Fig. 3 is immunochromatography half-quantitative detection kit involved by embodiment 1 when detecting Diagnostic Value of Fasting Serum C peptide levels pair Another immune chromatographic semiquantitative test paper bar color status schematic diagram of normal level should be less than；

Fig. 4 is immunochromatography half-quantitative detection kit involved by embodiment 1 when Diagnostic Value of Fasting Serum C peptide levels detect pair Answer the color status schematic diagram of another immune chromatographic semiquantitative test paper bar of normal level；

Immunochromatography sxemiquantitative kits of the Fig. 5 involved by embodiment 2 is in the release of C peptides during Serum C-peptide detection An immune chromatographic semiquantitative test paper bar color status schematic diagram；

Fig. 6 is that the immunochromatography sxemiquantitative kit one when follicular stimulating hormone detect involved by embodiment 3 is immunized Chromatograph the color status schematic diagram of semiquantitative test paper bar；

Immunochromatography sxemiquantitative kits of the Fig. 7 involved by embodiment 4 is when detecting cyclic citrullinated peptid content An immune chromatographic semiquantitative test paper bar color status schematic diagram.

Embodiment

Below by label be colloidal gold exemplified by, and with reference to attached drawing come illustrate the present invention embodiment.For reality Apply in example used specific method or material, those skilled in the art can on the basis of the technology of the present invention thinking, according to Existing technology carries out conventional replacement selection, is not limited solely to the specific record of the embodiment of the present invention.

Method used in embodiment is conventional method unless otherwise specified；Used material, reagent etc., such as Without specified otherwise, it is commercially available.

Embodiment 1

The purpose of the immunochromatography sxemiquantitative kit of the present embodiment is to detect people in sky using double antibody sandwich method C peptide levels in serum during abdomen, namely the present embodiment purpose thing to be detected to be detected are the C peptides in serum detected sample Antigen, namely antigen to be detected are C peptides.

Fig. 2 is immunochromatography half-quantitative detection kit involved by embodiment 1 when detecting Diagnostic Value of Fasting Serum C peptide levels pair An immune chromatographic semiquantitative test paper bar color status schematic diagram of normal level should be less than.

As depicted in figs. 1 and 2, immunochromatography half-quantitative detection kit 1 includes immune chromatographic semiquantitative test paper bar 100.

Immune chromatographic semiquantitative test paper bar 100 is end to end successively including antiadhesive stent 10 and along chromatography direction and is adhered to Sample application zone 20, label land 30, colour developing area 40 and suction zones 50 on antiadhesive stent 10.The immunochromatography half-quantitative detection Kit 100 promotes detected sample successively by sample application zone 20, label through capillary action under the absorption of suction zones 50 Land 30, colour developing area 40 simultaneously eventually arrive at suction zones 50, in detected sample moving process by antigen-antibody reaction and Colloidal gold develops the color to complete to detect.Here head and the tail, what is said is from sample application zone 20 into suction zones 50, and each area is in chromatography direction On, former and later two areas are end to end.Relation between each area in each embodiment below, all as the present embodiment.For Reach preferable chromatography, above-mentioned each area poststack of attaching most importance in junction connects again, for the purposes of reaching preferable chromatography, above-mentioned Each area is parallel distribution preferably in chromatography direction.

PVC board can be selected in the matrix material of antiadhesive stent 10, and polymer PET or glass fibers can be selected in 20 matrix material of sample application zone Dimension.50 matrix of suction zones can be the absorbent materials such as commercially available absorbent filter.

Polymer PET, glass fibre element film or filter paper fibre, matrix material can be selected in the matrix material of label land 30 On include label and can with purpose thing to be detected with reference to the first homologue with reference to and formed mark conjugate.This implementation In example, label is colloidal gold, and the first homologue is specially the anti-human C in mouse source for antigen to be detected corresponding antibody a, antibody a Peptide monoclonal antibody a.Antibody a by identify the specific antigen determinant on the antigen to be detected and with the antigen to be detected Antibody response and combine.

40 host material of area that develops the color is nitrocellulose filter, and chromatography direction is provided with detection zone and quality control region thereon.

A detection line 41 is provided with detection zone, which is coated with can be combined with purpose thing to be detected second Homologue, the second homologue is antibody b corresponding with antigen to be detected in the present embodiment, i.e. the anti-C-P monoclonal in mouse source resists Body b.Antibody b is known by identifying the specific antigen determinant on the antigen to be detected with the antigen binding to be detected, antibody b Other antigenic determinant is different from the antigenic determinant that antibody a is identified.

Quality control region includes two nature controlling lines, is respectively nature controlling line 42 and nature controlling line 43, is coated with corresponding different content respectively Standard purpose thing corresponding with purpose thing to be detected Quality Control thing, due to antigen-antibody reaction when will ensure that content can expire Foot requires, and it is by concentration, so the standard purpose thing of different content corresponds to different standard mesh again to carry out deciphering after detecting Thing concentration, can direct corresponding concentration during in order to use.Quality Control thing can be standard purpose thing, standard purpose thing and its phase Conjugate, the block polymer of standard purpose thing and macromolecular or the secondary antibody of antibody a that the antibody binding answered is formed, when Quality Control thing is mark The block polymer of conjugate, standard purpose thing and macromolecular that its corresponding antibody binding of quasi- purpose thing, standard purpose thing is formed When, the content and concentration of setting are exactly the standard purpose thing wherein contained, and when Quality Control thing is the secondary antibody of antibody a, setting Content and concentration be all the secondary antibody, but the content of secondary antibody at this time and concentration are corresponding with standard purpose thing.This implementation The coated Quality Control thing of two nature controlling lines in example is standard purpose thing, namely the standard items of the C peptide antigens to be detected prepared, is used To compare the concentration range of purpose thing to be detected.

In the present embodiment, by chromatography direction, namely from close detection line 41 to the direction away from detection line 41, nature controlling line In the coated coated content of standard purpose thing, namely corresponding concentration reduces successively, so coated matter in nature controlling line 42 It is corresponding with the reference upper level concentration of purpose thing to be detected to control the Cmax in the corresponding concentration of standard purpose thing in thing Concentration, the Cmin in the concentration of the standard purpose thing in nature controlling line 43 in coated Quality Control thing is and purpose thing to be detected The corresponding concentration of reference lower limit concentration.In reference of the reference upper level concentration for C peptides in normal human serum in the present embodiment Concentration 0.6nmol/L is limited, reference lower limit concentration is the reference lower limit concentration 0.25nmol/L of C peptides in normal human serum.

In above-mentioned, the setting of the total amount of the first scheduled volume, the second scheduled volume and all standard purpose things follows following rule Then：First scheduled volume be more than or equal to total amount and the second scheduled volume of all standard purpose things and, and the second scheduled volume is more than containing The content of maximum standard purpose thing is measured, " total amount and second of first scheduled volume more than or equal to all standard purpose things here The content of the first homologue combined in the mark conjugate that the sum of scheduled volume ", i.e. label land 30 include, is greater than The summation of content and containing for coated second homologue of detection line in the coated Quality Control thing Plays purpose thing of all nature controlling lines The sum of amount；Here " content of the standard purpose thing of content maximum ", as in multiple nature controlling lines, is tied in coated Quality Control thing The nature controlling line of most standard purpose things, namely the nature controlling line of the highest standard purpose thing of corresponding concentration are closed.

It by above-mentioned setting, can guarantee that in detection is applied there are enough mark conjugates pass through detection line, respectively successively A nature controlling line, and can mark what is combined on conjugate to treat when the immobilised band formed after these lines coating The content of testing goal thing, it is sufficient to carry out antigen-antibody reaction with the second homologue in detection line, and mark and tied on conjugate The content of the first homologue closed, it is sufficient to carry out antigen-antibody reaction, such ability with each standard purpose thing on nature controlling line The matter of immune chromatographic semiquantitative test paper bar 100 is judged by redissolution situation to chromatography situation, mark conjugate etc. in the detection Whether amount meets requirement, while nature controlling line is shown normally, could semi-quantitatively judge the concentration range of purpose thing to be detected.

Such as in the present embodiment, it is respective in the preparation with label land 30, detection line 41 and each nature controlling line Comprising or coated solution be 10 μ L, according to reference upper level concentration and reference lower limit concentration, then 42 coated standard of nature controlling line The content of purpose thing is 0.6 × 10^-5Nmol, the content of the coated standard purpose thing of nature controlling line 43 is 0.25 × 10^-5Nmol, institute The total amount for having standard purpose thing is 0.85 × 10^-5Nmol, then correspondingly, by the content of the second homologue, namely the second scheduled volume It is set greater than 0.6 × 10^-5Nmol, such as 300 × 10^-5Nmol, in this way, the second scheduled volume and all standard purpose things is total Amount and for 300.85 × 10^-5nmol；Then the content of the first homologue, namely the first scheduled volume are set greater than 300.85 ×10^-5Nmol, such as 800 × 10^-4nmol。

In this way, in the present embodiment, the first scheduled volume 800 × 10^-4Nmol is more than the total amount and second of all standard purpose things Scheduled volume and (300.85 × 10^-5), and the second scheduled volume 300 × 10 nmol^-5Nmol is more than the standard purpose thing of content maximum Content 0.6 × 10^-5Nmol, can meet testing requirements.

The preparation process of immunochromatography sxemiquantitative kit 1

How detailed description below prepares the immunochromatography sxemiquantitative kit 1 of the present embodiment, specifically comprises the following steps：

Step 1, the mark conjugate using colloidal gold as label is prepared

The heating of 100mL chlorauric acid solutions is boiled and is rapidly added the citric acid trisodium that 0.75mL mass percentages are 1% Or trisodium citrate aqueous solution, treat that colour stable continues to boil about 5-30 minutes, cooling can obtain colloidal gold solution.

Colloidal gold solution pH value is adjusted to can be electric with the grade of the first homologue of the antigen binding to be detected in the present embodiment Near point or meta-alkalescence (optimal pH, the first homologue is slowly added by most suitable protein labeling amount into colloidal gold solution). The first homologue in the present embodiment is the corresponding antibody a of antigen to be detected, and the antibody a in the present embodiment is specially the anti-of mouse source C-P monoclonal antibody a.

Then, magnetic stirring apparatus after mixing, adds BSA or polyglycol solution, centrifuges 20-50 minutes, sediment Redissolved with the PB liquid containing 1%BSA or the buffer solution containing polyethylene glycol, repeated centrifugation 2-3 times, final precipitation redissolves volume for original The 1/10 of volume, you can conjugate must be marked, colloidal gold labeled monoclonal antibody is obtained in the present embodiment.

Most suitable protein labeling amount determines that method is：With 0.1mol/L K₂CO₃Colloidal gold solution pH value is adjusted to 8.0- In the range of 9.2, the colloidal gold solution of different content is sequentially added graded is allowed into some centrifuge tubes, mixed and stand 2- 4 hours, add 100 μ L of 10%NaCl solution in every pipe, using the protein content of the constant pipe of color as minimum protein stabilized amount, It is most suitable protein labeling amount that 10%-20% is added on the basis of this.

Optimal pH is the isoelectric point or more slightly larger than isoelectric pH value of the first homologue, it determines that method is：By colloidal gold If solution is added in main pipe, often colloidal gold solution addition is 1mL. 0.1mol/L K in pipe₂CO₃Adjust respectively in each pipe Colloidal gold solution pH value, be allowed into graded.The most suitable protein labeling amount of equivalent is added in each Guan Zhongjun, mixes and stands 2 Hour, 100 μ L of 10%NaCl solution are added, using the pH value of the constant pipe of color as optimal pH.

Step 2, the preparation of label land 30

Polymer PET, glass fibre element film or filter paper fibre, the present embodiment can be selected in the matrix material of label land 30 Middle selection glass fibre element film.Before by colloidal gold labeled monoclonal antibody point sample to label land 30, with the treatment fluid containing BSA Handle glass fibre element film.To then be combined with the mark conjugate solution even application of the first homologue of the first scheduled volume in On glass fibre element film, to complete to include process to mark conjugate.In the present embodiment, such as, add the mark included The total amount of note conjugate solution be 10 μ L, the first scheduled volume for comprising concentration multiply in comprising material total amount, and the first scheduled volume For 800 × 10^-4Nmol, so after calculating, the first homologue should be 8 × 10 comprising concentration³nmol/L。

Step 3, sample application zone 20 is prepared

Sample application zone 20 is selected from polymer PET or glass fibre.Stayed overnight by treatment fluid processing sample application zone 20.

Step 4, the coating in colour developing area 40

Develop the color the selection of area 40 nitrocellulose filter, by the chromatography direction in use, rules in colour developing area 40 and is divided into detection Area (T lines area) and quality control region (C lines area).

The anti-human C-peptide monoclonal antibody b in certain density mouse source is coated with T lines area, is coated in colour developing area 40 into wire, Obtain detection line 41.In the present embodiment, the second scheduled volume multiplies in coated material total amount for coating concentration, and the second scheduled volume For example it is 300 × 10^-5Nmol, coated solution total amount are 10 μ L, so after calculating, the coating concentration of the second homologue should be 300nmol/L。

It is coated with the Quality Control thing of the standard purpose thing corresponding with purpose thing to be measured containing different content successively in C lines area, In the present embodiment, according to the reference upper level concentration 0.6nmol/L of C peptides in normal human serum and reference lower limit concentration 0.25nmol/L And coated solution total amount is coated with for 10 μ L, Quality Control thing is coated with respectively in colour developing area 40 into wire along chromatography direction, Obtain the nature controlling line 42 and nature controlling line 43 of standard purpose thing with respective amount.

Step 5, suction zones 50

The absorbent materials such as filter paper are selected in suction zones 50.Suction zones 50 can promote detected sample to pass through successively through capillary action Cross sample application zone 20, label land 30, colour developing area 40 and eventually arrive at suction zones 50 and chromatographed, to complete to detect.

Step 6, the assembling of detection kit

The sample application zone 20 being prepared, label land 30, colour developing area 40 and suction zones 50 are pasted successively viscous Immune chromatographic semiquantitative test paper bar 100 is obtained on attached stent, further assembles and just obtains immunochromatography half-quantitative detection reagent Box 1.

Detection application

The immunochromatography half-quantitative detection kit 1 being prepared using the present embodiment is to people in serum on an empty stomach C peptide levels are detected.Laboratory is when carrying out clients routine C peptides measure, normal human serum C peptide radiommunoassays Method is generally 0.2~0.6nmol/L, and average is 0.56 ± 0.29nmol/L.As testing result shows detected person's serum C peptide water It is flat be less than lower limit, then show that detected person's insulin secreting ability is insufficient, prompt for type 1 diabetes or；If C peptide levels are located at Between bound, then show that detected person's insulin secretion is normal；Detected person's insulin is prompted if C peptide levels are higher than the upper limit Secretion level is higher than ordinary person, such as diabetes B patient.When the detection kit is used for the detection of people Diagnostic Value of Fasting Serum C peptide levels, It is type 1 diabetes patient, non-patient or diabetes B patient that clients, which can be distinguished, according to this so as to diabetes into Row diagnosis typing.

Specific detection reaction process comprises the following steps：

Step 1, it is loaded

Detected sample is added to sample application zone 20：The detected sample of certain volume, namely blood sample are added dropwise to and added In sample area 20.

When containing purpose thing to be detected in detected sample, namely during antigens c peptide to be detected in the present embodiment, complete In entering step 2 after step 1, when not containing purpose thing to be detected in detected sample, then step 4 is directly entered；

Step 2, label land antigen-antibody reaction

After detected sample reaches label land 30 under chromatography effect from sample application zone 20, then the antigens c to be detected First on mark conjugate that peptide passes through the part in the first scheduled volume of antigen-antibody reaction and label land 30 Homologue, namely antibody a are combined, and are obtained the antigens c peptide to be detected and are marked the conjugate of conjugate, that is,：Test sample to be checked Antigens c peptide to be detected in product marks the antibody a in conjugate to be treated by the specific bound fraction of antigen-antibody reaction Antigens c peptide is detected with marking the conjugate of conjugate, subsequently into step 3；

Step 3, detection zone antigen-antibody reaction develops the color

The mark that antigens c peptide to be detected is not combined with the conjugate and another part for marking conjugate in step 2 is combined Thing migrates after being redissolved under chromatography effect towards detection zone 41, and the antigens c peptide to be detected combined with the first homologue is detecting The second homologue that area states coated second scheduled volume of detection line 41 with this is combined, and thus combining label is solidificated in inspection Survey area, namely detection line 41, so formed it is corresponding with detection zone namely detection line 41 detect developed band, detected in the present embodiment Claret is shown in developed band, subsequently into step 4；

Step 4, quality control region antigen-antibody reaction develops the color

After step 3, the mark conjugate that another part is not combined continues to move to quality control region under chromatography effect, Pass through successively and difference corresponding with various concentrations respectively contains with the Quality Control thing on each bar nature controlling line for being solidificated in quality control region The various criterion purpose thing of amount with reference to and form shade corresponding with each nature controlling line after curing the label of different content Different Quality Control developed band, claret is shown in Quality Control developed band in the present embodiment；

After step 1, when not containing purpose thing to be detected in detected sample, then mark whole in the step combines Thing all chromatographs quality control region,

5 are entered step after step 4；

Step 5, compare and judge

By by detect developed band and the color of each Quality Control developed band be compared obtain comparison result, according to comparison As a result the corresponding concentration range of corresponding standard purpose thing, semi-quantitatively determine detected sample in purpose thing to be detected it is dense Spend scope：When the nature controlling line most more shallow than color of detection line 41 is shallow, show that antigenic content to be detected is less than in this in detected sample The corresponding concentration of nature controlling line；When detection line 41 does not have color, show not containing antigen to be detected in detected sample；Work as detection line The color nature controlling line most deeper than color is deep, shows that antigenic content to be detected is corresponding dense higher than the nature controlling line in detected sample Degree.It thus can semi-quantitatively determine the concentration range of antigen to be detected in sample.It is specific as follows for the present embodiment：

Fig. 4 is immunochromatography half-quantitative detection kit involved by embodiment 1 when Diagnostic Value of Fasting Serum C peptide levels detect pair Answer the color status schematic diagram of another immune chromatographic semiquantitative test paper bar of normal level.

If in testing result, nature controlling line 42 and nature controlling line 43 all occur without corresponding Quality Control developed band, or only There is Quality Control developed band in a wherein nature controlling line, or the color of Quality Control developed band corresponding with nature controlling line 42 is shallower than and nature controlling line The color of 43 corresponding Quality Control developed band, then it is invalid to detect.It is why invalid, it may be possible to for the immunochromatography sxemiquantitative detected Chromatography problem or mark conjugate redissolution problem etc., which occurs, in immune chromatographic semiquantitative test paper bar in kit to be caused to fail, Be probably purpose thing to be detected concentration beyond estimating, cause no enough mark conjugate chromatographies to be carried out to each bar nature controlling line With reference to colour developing, immunochromatography sxemiquantitative kit can more be renewed according to actual conditions or concentration range bigger is resurveyed.

As shown in Figures 2 and 3, testing result shows that the color of detection developed band corresponding with nature controlling line 42 is better than and Quality Control The color of the corresponding Quality Control developed band of line 43, and it is corresponding with detection line 41 detection developed band not occur or with detection line 41 The color of corresponding detection developed band is weaker than the color of Quality Control developed band corresponding with nature controlling line 43, then it represents that detected person's serum C peptide levels are less than 0.25nmol/L less than the level of insulin secretion of β cells in normal person and pancreas, and detected person may be 1 Diabetes mellitus type or pancreas excision person.

As shown in figure 4, testing result shows that the color of Quality Control developed band corresponding with nature controlling line 42 is better than and nature controlling line 43 The color of corresponding Quality Control developed band, and the color of detection developed band corresponding with detection line 41 is between nature controlling line 42 and nature controlling line Among the color of 43 respective Quality Control developed band, then it represents that detected person's Serum C-peptide is in 0.25nmol/L- Between 0.6nmol/L, belong to normal level.

It is corresponding with nature controlling line 43 that if testing result shows that the color of Quality Control developed band corresponding with nature controlling line 42 is better than The color of Quality Control developed band, and the color of detection developed band corresponding with detection line 41 is better than and is shown with 42 corresponding Quality Control of nature controlling line The color of colour band, then it represents that detected person's Serum C-peptide is higher than 0.6nmol/L, may suffer from diabetes B.

Immunochromatography sxemiquantitative kit 1 is kept flat standing 10 to 20 minutes by above-mentioned whole process when detecting, just can be complete Into fast and convenient and accurate.

Embodiment 2

It is the explanation to embodiment 2 below.

In example 2, for the structure identical with embodiment 1, identical symbol is given, and omits identical say It is bright.

The purpose of the immunochromatography sxemiquantitative kit of the present embodiment is to detect people in C peptides using double antibody sandwich method C peptide levels in serum in release, namely the present embodiment purpose thing to be detected to be detected are the C in serum detected sample Peptide antigen, also antigen as to be detected is C peptides.

Immunochromatography sxemiquantitative kits of the Fig. 5 involved by embodiment 2 is in the release of C peptides during Serum C-peptide detection An immune chromatographic semiquantitative test paper bar color status schematic diagram.

As shown in figure 5, immunochromatography sxemiquantitative kit includes immune chromatographic semiquantitative test paper bar 200.

Immune chromatographic semiquantitative test paper bar 200 is end to end successively including antiadhesive stent and along chromatography direction and is adhered to viscous Sample application zone 20, label land 230, colour developing area 240 and suction zones 50 on attached stent.

Develop the color area 240 also be nitrocellulose filter with embodiment 1 as, thereon also along chromatograph direction be provided with detection zone with Quality control region.

Quality control region includes four nature controlling lines, is respectively nature controlling line 242, nature controlling line 243, nature controlling line 244 and nature controlling line 245. In the present embodiment, nature controlling line 242, nature controlling line 243, nature controlling line 244 and nature controlling line 245 are distinguished coated corresponding with purpose thing to be measured Standard purpose thing Quality Control thing it is identical with embodiment 1, unlike, by chromatography direction, namely from close to detection line 241 Onto the direction away from detection line 241, the content of the coated standard purpose thing in the nature controlling line in the present embodiment raises successively.

The Quality Control thing and different reference concentrations that nature controlling line 242, nature controlling line 243, nature controlling line 244 and nature controlling line 245 are distinguished Corresponding, the corresponding concentration of content of the coated standard purpose thing of nature controlling line 242 is in normal human serum under the reference of C peptide concentrations 0.25nmol/L is limited, the corresponding concentration of content of the coated standard purpose thing of nature controlling line 243 is C peptide concentrations in normal human serum Reference upper level 0.6nmol/L；When the corresponding concentration of content of the coated standard purpose thing of nature controlling line 244 detects for the release of c peptides just The reference lower limit 1.25nmol/L of C peptide concentrations in ordinary person's serum；The content of the coated standard purpose thing of nature controlling line 245 is corresponding Concentration discharges the reference upper level 3.6nmol/L of C peptide concentrations in normal human serum when detecting for c peptides.

Likewise, the total amount of the first scheduled volume, the second scheduled volume and all standard purpose things in the present embodiment is set Surely rule in the same manner as in Example 1 is followed.

So in the present embodiment, the solution that label land 230 includes in the preparation is 100 μ L, detection line 41 with And each coated solution is 10 μ L to each nature controlling line in the preparation, according to reference upper level concentration and reference lower limit concentration, then The content of the coated standard purpose thing of nature controlling line 242 is 0.25 × 10^-5Nmol, the coated standard purpose thing of nature controlling line 243 contain Measure as 0.6 × 10^-5Nmol, the content of the coated standard purpose thing of nature controlling line 244 is 1.25 × 10^-5Nmol, nature controlling line 245 wrap The content of the standard purpose thing of quilt is 3.6 × 10^-5nmol；The total amount of all standard purpose things is 5.7 × 10^-5Nmol, then accordingly Ground, 3.6 × 10 are set greater than by the content of the second homologue, namely the second scheduled volume^-5Nmol, such as 400.0 × 10^- ⁵Nmol, such second scheduled volume and all standard purpose things total amount and be 405.7 × 10^-5nmol；Then first is corresponded to The content of thing, namely the first scheduled volume are set greater than 405.7 × 10^-5Nmol, such as 900.0 × 10^-4nmol。

In this way, in the present embodiment, the first scheduled volume 900.0 × 10^-4Nmol is more than the total amount and the of all standard purpose things Two scheduled volumes and (405.7 × 10^-5), and the second scheduled volume 400.0 × 10 nmol^-5Nmol is more than the standard mesh of content maximum Thing content 3.6 × 10^-5nmol.It can meet testing requirements.

The preparation process of immunochromatography sxemiquantitative kit

In the present embodiment, the preparation process of immunochromatography sxemiquantitative kit is similar to the preparation process in embodiment 1, inspection Coated solution is 10 μ L when surveying area and quality control region preparation, unlike, in the present embodiment prepared by label land When the solution that includes be 100 μ L, the coating concentration of the first homologue is 900nmol/L, the coating concentration of the second homologue should For 400nmol/L；

Nature controlling line also in the present embodiment is respectively nature controlling line 242, Quality Control 243, nature controlling line 244 and nature controlling line 245, it Be coated with the Quality Control thing of the standard purpose thing corresponding with purpose thing to be measured containing different content successively, in the present embodiment, according to The Quality Control thing of each concentration of 10 μ L, is coated on colour developing area by above-mentioned several reference upper level values and reference lower limit value respectively respectively Into wire on 240, that is, obtain nature controlling line 242, Quality Control 243, nature controlling line 244 and the Quality Control of standard purpose thing with corresponding content Line 245.

Detection application

The immunochromatography half-quantitative detection kit that the present embodiment obtains can be not only used for the detection of people empty stomach C peptide levels, also may be used Detected for C peptide levels in C peptide release experiments, be used in the present embodiment to the C peptide levels in serum of the people when C peptides discharge into Row detection, when carrying out routine C peptides measure, normal human serum C peptides are generally 0.2~0.6nmoL/L with radioimmunoassay, Average is 0.56 ± 0.29nmoL/L；After glucosieloading test (C peptides release experiment) is carried out, change of serum C peptide content peak goes out The existing time is consistent with insulin, than 5~6 times high on an empty stomach.

Identical in detection reaction process and embodiment 1 in the present embodiment, the nature controlling line of different simply quality control regions has Four, so in the detection, quality control region can show four Quality Control being of different shades developed band, the red detection of the present embodiment is shown The color of colour band and each Quality Control developed band is all claret.

Colour developing to the present embodiment judges specific as follows：

If in testing result, nature controlling line 242, nature controlling line 243, nature controlling line 244 and nature controlling line 245 do not occur accordingly Quality Control developed band, either not every nature controlling line all occur corresponding Quality Control developed band or along chromatography direction, four Quality Controls The color of developed band is not gradually to deepen, then it is invalid to detect.

As shown in figure 5, color of the testing result display along chromatography direction Quality Control developed band corresponding with each nature controlling line It is effective gradually to deepen then detection.The color of detection developed band corresponding with detection line 41 is shallower than corresponding with nature controlling line 44 in Fig. 5 The color of Quality Control developed band, then it represents that the C peptide levels in detected person's serum are less than 1.25nmol/L, which is 1 type Diabetic or pancreas excision person.

Embodiment 3

It is the explanation to embodiment 3 below.

In embodiment 3, for the structure identical with embodiment 1, identical symbol is given, and omits identical say It is bright.

The purpose of the immunochromatography sxemiquantitative kit of the present embodiment is in order to using double antibody sandwich method detection ovarian follicle thorn Hormone (Follicle-stimulating hormone, FSH) content, namely the present embodiment purpose thing to be detected to be detected is Follicular stimulating hormone antigen in serum detected sample, namely antigen to be detected is follicular stimulating hormone.

Fig. 6 is that the immunochromatography sxemiquantitative kit one when follicular stimulating hormone detect involved by embodiment 3 is immunized Chromatograph the color status schematic diagram of semiquantitative test paper bar.

As shown in fig. 6, immunochromatography sxemiquantitative kit 3 includes immune chromatographic semiquantitative test paper bar 300.

Immune chromatographic semiquantitative test paper bar 300 is end to end successively including antiadhesive stent and along chromatography direction and is adhered to viscous Sample application zone 20, label land 330, colour developing area 340 and suction zones 50 on attached stent.

Label land 330 include label and can with purpose thing to be detected with reference to the first homologue with reference to and formed Mark conjugate.In the present embodiment, label is colloidal gold, and the first homologue is the corresponding antibody a of antigen to be detected, antibody A is specially the anti-human follicular stimulating hormone monoclonal antibody a in mouse source.Antibody a is by identifying the specific antigen on the antigen to be detected Determinant and combined with the antigen antibody response to be detected.

Develop the color area 340 also be nitrocellulose filter with embodiment 1 as, thereon also along chromatograph direction be provided with detection zone with Quality control region.

Detection zone also includes a detection line 41, and the detection line 41 is coated with can be combined with purpose thing to be detected second Homologue, the second homologue is the anti-human follicular stimulating hormone list of antibody b corresponding with antigen to be detected, i.e. mouse source in the present embodiment Clonal antibody b.Antibody b by identify the specific antigen determinant on the antigen to be detected and with the antigen antibody to be detected React and combine, the antigenic determinant of antibody b identifications is different from the antigenic determinant that antibody a is identified.

Quality control region includes three nature controlling lines, is respectively nature controlling line 342, nature controlling line 343 and nature controlling line 344, they are wrapped respectively There are the Quality Control thing of the standard purpose thing corresponding with purpose thing to be detected of corresponding different content, similarly, the mark of different content Quasi- purpose thing corresponds to the concentration of different standard purpose things again, can direct corresponding concentration during in order to use.Quality Control thing can be The Qian He of conjugate, standard purpose thing and macromolecular that its corresponding antibody binding of standard purpose thing, standard purpose thing is formed The secondary antibody of thing or antibody a, the content of each Quality Control thing and the correspondence of concentration are the same as embodiment 1.Three nature controlling lines in the present embodiment Coated Quality Control thing is standard purpose thing, namely the standard items of the follicular stimulating hormone to be detected prepared, to be detected for comparing The concentration range of purpose thing.

In the present embodiment, nature controlling line 342, the content of nature controlling line 343 and the respective Quality Control thing of nature controlling line 344 and different ginsengs It is corresponding to examine concentration, corresponding concentration is 20.0U/L, 10.0U/L and 1.3U/L, correspondingly, by chromatography direction, namely On direction from close to detection line 341 to away from detection line 341, the content of the coated standard purpose thing in each nature controlling line according to Secondary reduction.

The setting of the total amount of the first scheduled volume, the second scheduled volume and all standard purpose things in the present embodiment follow with Rule as embodiment 1.

So in the present embodiment, it is each in the preparation with label land 330, detection line 341 and each nature controlling line Self-contained or coated solution is 10 μ L, according to the corresponding concentration of each nature controlling line, then the coated standard purpose of nature controlling line 242 The content of thing is 20.0 × 10^-5U, the content of the coated standard purpose thing of nature controlling line 243 is 10.0 × 10^-5U, nature controlling line 244 wrap The content of the standard purpose thing of quilt is 1.3 × 10^-5U；The total amount of all standard purpose things is 30.3 × 10^-5U, then correspondingly, will The content of second homologue, namely the second scheduled volume are set greater than 20 × 10^-5U, such as 300.0 × 10^-5U, such second is pre- It is quantitative with the total amount of all standard purpose things and be 330.3 × 10^-5U；Then by the content of the first homologue, namely first pre- Quantitatively it is set greater than 330.3 × 10^-5U, such as 200.0 × 10^-4U。

In this way, in the present embodiment, the first scheduled volume 200.0 × 10^-4U is pre- more than the total amount of all standard purpose things and second It is quantitative and (330.3 × 10^-5), and the second scheduled volume 300.0 × 10 U^-5U is more than the content of the standard purpose thing of content maximum 20.0×10^-5U.It can meet testing requirements.

The preparation process of immunochromatography sxemiquantitative kit 3

In the present embodiment, the preparation process of immunochromatography sxemiquantitative kit 3 is similar to the preparation process in embodiment 1, Each position includes or coated solution is also 10 μ L, unlike, in the present embodiment, each coated material in position with The difference of embodiment 1, and the first homologue is 2.0 × 10 comprising concentration³U/L, the coating concentration of the second homologue are 300.0U/L；Nature controlling line also in the present embodiment is respectively nature controlling line 342, Quality Control 343 and nature controlling line 344, they are wrapped successively By the Quality Control thing of the standard purpose thing corresponding with purpose thing to be measured containing different content, in the present embodiment, according to above-mentioned several The Quality Control thing of 10 μ L, is coated in colour developing area 340 into wire, that is, obtains the standard with respective amount by reference concentration respectively respectively Nature controlling line 342, Quality Control 343 and the nature controlling line 344 of purpose thing.

Detection application

The immunochromatography half-quantitative detection kit that the present embodiment obtains is used for follicular stimulating hormone content, with to checking women Endocrine status.

Identical in detection reaction process and embodiment 1 in the present embodiment, the nature controlling line of different simply quality control regions has Three, so in the detection, quality control region can show three Quality Control being of different shades developed band, and the detection in the present embodiment is shown The color of colour band and each Quality Control developed band is all claret.

Colour developing to the present embodiment judges specific as follows：

As shown in fig. 6, if testing result shows that each nature controlling line shows band, and color gradually becomes along chromatography direction Shallow, then detection is effective.41 color of detection line is shallower than the band color of nature controlling line 344 in figure, then it represents that detected person's ovarian follicle stimulates Element secretion is less.

If the band color of detection line 41 is among the band color of nature controlling line 343 and nature controlling line 344, then prompting is tested Survey person is located at follicular phase or the onset of ovulation.

If the band color of detection line 41 is among the band color of nature controlling line 342 and nature controlling line 343, then prompting is tested Survey person is located at the onset of ovulation.

Band color such as detection line 41 is deeper than the band color of nature controlling line 342, then prompts detected person to be located at menopause Or diacrisis.

Embodiment 4

It is the explanation to embodiment 4 below.

In example 4, for the structure identical with embodiment 1, identical symbol is given, and omits identical say It is bright.

The purpose of the immunochromatography sxemiquantitative kit of the present embodiment is to detect anti-cyclic citrullinated peptide using indirect method Antibody content, namely the present embodiment purpose thing to be detected to be detected resist for the anti-cyclic citrullinated peptide in serum detected sample Body, namely detection antibody are cyclic citrullinated peptids.

Fig. 7 is that the immunochromatography sxemiquantitative kit involved by embodiment 4 is examined in detection cyclic citrullinated peptid content The color status schematic diagram of immune chromatographic semiquantitative test paper bar during survey.

As shown in fig. 7, immunochromatography sxemiquantitative kit 4 includes immune chromatographic semiquantitative test paper bar 400.

Immune chromatographic semiquantitative test paper bar 400 is end to end successively including antiadhesive stent and along chromatography direction and is adhered to viscous Sample application zone 20, label land 430, colour developing area 440 and suction zones 50 on attached stent.

Label land 430 include label and can with purpose thing to be detected with reference to the first homologue with reference to and formed Mark conjugate.In the present embodiment, label is colloidal gold, and the first homologue is the corresponding secondary antibody of detection antibody, this two Anti- is specially the anti-human cyclic citrullinated peptid secondary antibody in rabbit source.Above-mentioned secondary antibody is to be detected with this by specific recognition body to be detected Antibody-antigen-antibody is reacted and combined.

Colour developing area 440 is also nitrocellulose filter such as embodiment 1, and chromatography direction is also provided with detection zone thereon And quality control region.

Detection zone includes a detection line 441, which is coated with the second couple that can be combined with purpose thing to be detected Answer thing, the second homologue is antigen corresponding with detection antibody, i.e. cyclic citrullinated peptide antigen in the present embodiment.The antigen passes through The specific recognition detection antibody is combined with the detection antibody antigen-antibody reaction.

Quality control region includes two nature controlling lines, is respectively nature controlling line 442 and nature controlling line 443, they are coated with respectively corresponds to not With the Quality Control thing of the standard purpose thing corresponding with purpose thing to be detected of content, similarly, the standard purpose thing of different content is again The concentration of corresponding different standard purpose thing, during in order to use can direct corresponding concentration, the Quality Control thing in the present embodiment is Standard purpose thing, namely the standard items of the cyclic citrullinated peptid to be detected prepared, for comparing purpose thing to be detected Concentration range.

In the present embodiment, the content for the standard purpose thing that nature controlling line 442 and the respective Quality Control thing of nature controlling line 443 contain with not Same reference concentration is corresponding, and corresponding concentration is 20U/mL and 60U/mL.Correspondingly, by chromatography direction, Ye Jicong In detection line 441 to the direction of remote detection line 441, the content of the coated standard purpose thing in nature controlling line rises successively It is high.

So in the present embodiment, the solution that label land 430 includes in the preparation is 100 μ L, detection line 441 And each coated solution is 10 μ L to each nature controlling line in the preparation, according to the corresponding concentration of each nature controlling line, then Quality Control The content of the coated standard purpose thing of line 442 is 0.2U, and the content of the coated standard purpose thing of nature controlling line 443 is 0.6U；It is all The total amount of standard purpose thing is 0.8U, then correspondingly, the content of the second homologue, namely the second scheduled volume are set greater than 0.8U, such as 50.0U, such second scheduled volume and all standard purpose things total amount and be 50.8U；Then first is corresponded to The content of thing, namely the first scheduled volume are set greater than 50.8U, such as 1000.0U,

In this way, in the present embodiment, the first scheduled volume 1000.0U is more than the total amount and the second scheduled volume of all standard purpose things And (50.8U), and the second scheduled volume 50.0U be more than content maximum standard purpose thing content 0.6U.It can meet that detection will Ask.

The preparation process of immunochromatography sxemiquantitative kit 4

In the present embodiment, the preparation process of immunochromatography sxemiquantitative kit 4 is similar to the preparation process in embodiment 1, Detection line and nature controlling line solution coated in the preparation are also 10 μ L, unlike, label land includes in the preparation Solution be 100 μ L, in the present embodiment, each position includes or the difference of coated material and embodiment 1, and first corresponds to Thing is 1.0 × 10 comprising concentration⁴U/mL, the coating concentration of the second homologue are 5000.0U/mL；Also in the present embodiment Nature controlling line is respectively nature controlling line 442 and Quality Control 443, they are coated with containing different content mark corresponding with purpose thing to be measured successively The Quality Control thing of quasi- purpose thing, in the present embodiment, according to above-mentioned several reference concentrations, is coated on colour developing area 440 by Quality Control thing respectively On into wire, that is, the nature controlling line 442 for the standard purpose thing for obtaining there is corresponding content and Quality Control 443.

Detection application

The immunochromatography half-quantitative detection kit that the present embodiment obtains is used for the detection of cyclic citrullinated peptid content. Cyclic citrullinated peptid is present in the serum of rheumatoid arthritis people, is a kind of using cyclic citrullinated peptide as target antigen Autoantibody.Cyclic citrullinated peptid has rheumatoid arthritis (RA) good Sensitivity and Specificity, and anti- The more anti-cyclic citrullinated peptid negative patient of rheumatoid arthritis people's osteoclasia of cyclic citrullinated peptide antibodies positive is serious. In clinical practice, the cyclic citrullinated peptid tests positive of most of patient with rheumatoid arthritis, this causes anti-ring Citrulling peptide antibody becomes a good rheumatoid arthritis biomarker, it, which is detected, can be used for rheumatoid arthritis to suffer from Examination of person etc..

It is identical in detection process and embodiment 1 in the present embodiment, detect developed band and each Quality Control developed band is all same Sample is claret, and the different antigen-antibody reactions simply occurred is different, and various pieces occur anti-in specific the present embodiment Antigen-antibody reaction is：

It is to be detected when the detected sample containing detection antibody reaches label land 430 by sample application zone 420 Antibody can be with the secondary antibody in specific binding marker conjugate by antigen-antibody reaction；

When the conjugate of detection antibody and secondary antibody reaches detection zone, the conjugate of detection antibody and secondary antibody is detecting Line antigen binding corresponding with being coated with immobilised detection antibody；

When the mark conjugate arrival quality control region not combined with detection antibody, then successively with being fixed on each bar nature controlling line Standard purpose thing combine.

Similarly, detection antibody content of the present embodiment in detected sample, the color that detection line 441 is presented are deep Shallow difference：Detection antibody content is fewer in the bright sample of color more superficial；Color more deeply feels detection antibody content in bright sample More, the colour developing to the present embodiment judges specific as follows：

As shown in fig. 7, if the band of testing result nature controlling line 442 and nature controlling line 443 all develops the color and the bar of nature controlling line 443 The band color of nature controlling line 442 is deeper than with color, then detection is effective.In Fig. 7, the band color of detection line 441 is shallower than nature controlling line 442 band color, then it represents that the concentration of the cyclic citrullinated peptid of detected person is less than 20U/mL, and testing result is the moon Property, possibility of the detected person with rheumatoid arthritis is relatively low.

If the band color of detection line 441 is detected among the band color of nature controlling line 442 and nature controlling line 443 The concentration of the cyclic citrullinated peptid of survey person prompts the detected person to be suffered from for the positive between 20U/mL-60U/mL Rheumatoid arthritis.

If the band color of detection line 441 is deeper than the band color of nature controlling line 443, the anti-cyclic citrulline of detected person The concentration of peptide antibody is more than 60U/mL, and it is strong positive to prompt the detected person, and the possibility with rheumatoid arthritis is very big.

Embodiment effect

The immune chromatographic semiquantitative test paper bar that is there is provided according to embodiment 1 to 4 and include its immunochromatography sxemiquantitative Kit, is provided with multiple nature controlling lines in the colour developing area included due to immune chromatographic semiquantitative test paper bar therein, these Quality Controls Line is coated with the Quality Control of the standard purpose thing corresponding with purpose thing to be measured of the different content containing corresponding various concentrations respectively again Thing, and the first scheduled volume be more than or equal to total amount and the second scheduled volume of all standard purpose things and, and the second scheduled volume is big In the content of the standard purpose thing of content maximum, so can be in chromatography side upwardly through different antigen-antibody reactions and to mark Thing carries out curing the detection that the content to develop the color to treat testing goal thing carries out sxemiquantitative, so reduces due to color condition not With the error brought, testing result is improved, and it is inconvenient farthest to avoid unnecessary accurate detection from bringing And use cost, and it is easy to operate, it is easy to use；

Embodiment 1 to 4 in the immunochromatography semi-quantitative detection method that carries out, due to by the way that detected sample is passed through layer Analysis carries out by sample application zone, label land, the detection zone in the area that develops the color and quality control region and successively different antigen and resists successively Precursor reactant, and pass through the detection developed band of the antigen-antibody reaction formation of detection zone, with quality control region antigen-antibody reaction colour developing shape Into each corresponding with various concentrations Quality Control developed band carry out color comparison, just can be to the purpose to be detected in detected sample The concentration of thing carries out the detection of sxemiquantitative, so half-quantitative detection can be carried out by colorimetric at the same time in the detection, similarly brings The effect above.

In addition, in embodiment 1 to 4, selected label is colloidal gold, can be with as the label of the present invention Select similar chemical illuminating reagent, chemochromic reagent, metallic, carbon nano-particle, latex particle, magnetic particle, quantum Any one or a few in point and fluorescent material etc. can redissolve and with chromatography movement and cured display material.

In addition, in embodiment 1 to 4, the corresponding concentration of the coated standard purpose thing of nature controlling line according to chromatography direction successively Reduce or raise, as the present invention, the distributing order in the edge chromatography direction of corresponding concentration can arbitrarily be set according to using needs Put.

In addition, in embodiment 1 to 4, detected sample is blood sample, such as serum, blood plasma or whole blood, is used as this hair Bright, detected sample can also be urine, saliva, excrement etc..

In addition, in embodiment 1 to 3, double antibody sandwich method is employed, indirect method is employed in embodiment 4, is used as this hair It is bright, prize law or dual-antigen sandwich method can also be used, when using prize law, purpose thing to be detected is detection antibody, the One homologue is antigen corresponding with test antibodies, and the second homologue is secondary antibody corresponding with detection antibody；When using dual anti- During former sandwich method, purpose thing to be detected is detection antibody, and the first homologue is antigen a corresponding with detection antibody, second Homologue is that antigen b corresponding with detection antibody, antigen a and antigen b can be combined with the different parts of detection antibody respectively.

Claims

A kind of 1. immune chromatographic semiquantitative test paper bar, based on antigen-antibody reaction principle, for being chromatographed to detected sample And in chromatography side upwardly through different antigen-antibody reactions, and label cure developing the color and is come to the detected sample In the concentration of the purpose thing to be detected carry out the detection of sxemiquantitative, including the end to end sample-adding along the chromatography direction Area, label land, colour developing area and suction zones, are disposed with detection zone and Quality Control in the area that develops the color along the chromatography direction Area, the label land include the first couple of label and the first scheduled volume that can be combined with the purpose thing to be detected Answer thing with reference to and formed mark conjugate, the detection zone be coated with can be combined with the purpose thing to be detected second make a reservation for Second homologue of amount, it is characterised in that including：

The quality control region includes a plurality of nature controlling line, a plurality of nature controlling line be coated with corresponding different content respectively with it is described to be checked The Quality Control thing of the corresponding standard purpose thing of purpose thing is surveyed, the standard purpose thing of different content corresponds to the different standard mesh Thing concentration,

First scheduled volume be more than or equal to total amount and second scheduled volume of all standard purpose things and, it is and described Second scheduled volume is more than the content of the standard purpose thing of content maximum.
2. immune chromatographic semiquantitative test paper bar according to claim 1, it is characterised in that：

The purpose thing to be detected is antigen to be detected, and first homologue is antibody a corresponding with the determined antigen, institute It is that antibody b, the antibody a and the antibody b corresponding with the antigen to be detected identify that this is to be checked respectively to state the second homologue Survey antigen on different antigenic determinants and respectively with the antigen binding to be detected.
3. immune chromatographic semiquantitative test paper bar according to claim 2, it is characterised in that：

Conjugate that the Quality Control thing is formed for its corresponding antibody binding of the standard purpose thing, the standard purpose thing, The block polymer of the standard purpose thing and macromolecular or the secondary antibody of the antibody a.
4. the immune chromatographic semiquantitative test paper bar according to right wants 1, it is characterised in that：

The purpose thing to be detected is detection antibody, and first homologue is secondary antibody corresponding with the test antibodies, institute It is antigen corresponding with the detection antibody to state the second homologue.
5. immune chromatographic semiquantitative test paper bar according to claim 1, it is characterised in that：

The purpose thing to be detected is detection antibody, and first homologue is antigen corresponding with the test antibodies, institute It is secondary antibody corresponding with the detection antibody to state the second homologue.
6. immune chromatographic semiquantitative test paper bar according to claim 1, it is characterised in that：

The purpose thing to be detected is detection antibody, and first homologue is antigen a corresponding with the detection antibody, Second homologue be antigen b corresponding with the detection antibody, the antigen a and the antigen b respectively can with it is described The different parts of detection antibody combine.
7. the immune chromatographic semiquantitative test paper bar according to claim 4 to 6 any one, it is characterised in that：

The Quality Control thing is the standard purpose thing.
8. immune chromatographic semiquantitative test paper bar according to claim 1, it is characterised in that：

Multiple nature controlling lines distinguish the corresponding concentration of the standard purpose thing in coated Quality Control thing along the chromatography direction Raise successively.
9. immune chromatographic semiquantitative test paper bar according to claim 1, it is characterised in that：

Multiple nature controlling lines distinguish the corresponding concentration of the standard purpose thing in the coated Quality Control thing along the chromatography Direction reduces successively.
10. the immune chromatographic semiquantitative test paper bar according to any one in claim 1 to 6,8 or 9, it is characterised in that：

The label is colloidal gold, chemical illuminating reagent, chemochromic reagent, metallic, carbon nano-particle, latex One or more in grain, magnetic particle, quantum dot, fluorescent material or rare earth ion.
11. the immune chromatographic semiquantitative test paper bar according to any one in claim 1 to 6,8 or 9, it is characterised in that：

Multiple nature controlling lines are distinguished maximum dense in the corresponding concentration of the standard purpose thing in the coated Quality Control thing Spend for the reference upper level concentration of the purpose thing to be detected.
12. the immune chromatographic semiquantitative test paper bar according to any one in claim 1 to 6,8 or 9, it is characterised in that：

Multiple nature controlling lines are distinguished minimum dense in the corresponding concentration of the standard purpose thing in the coated Quality Control thing Spend for the reference lower limit concentration of the purpose thing to be detected.
A kind of 13. immunochromatography half-quantitative detection kit, it is characterised in that including：

At least one immune chromatographic semiquantitative test paper bar as described in any one in claim 1 to 12.
A kind of 14. immunochromatography semi-quantitative detection method, based on antigen-antibody reaction principle, by the way that detected sample is passed through layer Analysis carries out by sample application zone, label land, the detection zone in the area that develops the color and quality control region and successively different antigen and resists successively Precursor reactant and the detection that sxemiquantitative is carried out to the concentration of the purpose thing to be detected in the detected sample, it is characterised in that bag Include following steps：

Step 1, it is loaded, detected sample is added to the sample application zone, when containing purpose thing to be detected in the detected sample, 2 are then entered step, when not containing the purpose thing to be detected in the detected sample, then enters step 4；

Step 2, label land antigen-antibody reaction, the detected sample reach under chromatography effect from the sample application zone Behind the label land, the purpose thing to be detected is first pre- by antigen-antibody reaction and the label land The first homologue on the mark conjugate of a part in quantitative is combined with reference to the purpose thing to be detected is obtained with the mark The conjugate of thing；

Step 3, detection zone antigen-antibody reaction develop the color, the purpose thing to be detected with it is described mark conjugate conjugate and separately The mark conjugate that a part is not combined migrates under chromatography effect towards the detection zone, with first homologue With reference to the purpose thing to be detected the second homologue knot of coated second scheduled volume of detection zone is stated in the detection zone with this Formation is corresponding with the detection zone after the conjunction curing label detects developed band；

Step 4, quality control region antigen-antibody reaction develops the color, and the mark conjugate that another part is not combined is under chromatography effect Continue to move to the quality control region, successively by and the Quality Control thing with being solidificated on each bar nature controlling line of quality control region in respectively with The various criterion purpose thing of the corresponding different content of various concentrations with reference to and cure formed after the label of different content with The Quality Control developed band that each nature controlling line is of different shades accordingly；

Step 5, compare and judge, by the way that the detection developed band to be compared to obtain with the color of each Quality Control developed band Comparison result, according to the corresponding concentration range of the corresponding standard purpose thing of the comparison result, semi-quantitatively determines described The concentration range of purpose thing to be detected described in detected sample,

Wherein, first scheduled volume be more than or equal to total amount and second scheduled volume of all standard purpose things and, And second scheduled volume is more than the content of the standard purpose thing of content maximum.
15. immunochromatography semi-quantitative detection method according to claim 14, it is characterised in that：

Carried out using the immune chromatographic semiquantitative test paper bar described in claim 1 to 12 any one.
16. the immunochromatography semi-quantitative detection method according to claims 14 or 15, it is characterised in that：

When the purpose thing to be detected is antigen to be detected, the immunochromatography semi-quantitative detection method uses double-antibody sandwich Method.
17. the immunochromatography semi-quantitative detection method according to claims 14 or 15, it is characterised in that：

When the purpose thing to be detected is detection antibody, the immunochromatography semi-quantitative detection method is using indirect method, capture One kind in method or dual-antigen sandwich method.