CN106546744A - By the method and corresponding reagent box of fecal hemoglobin, transferrins and PKM2 joint-detection assessing colorectal cancers - Google Patents

By the method and corresponding reagent box of fecal hemoglobin, transferrins and PKM2 joint-detection assessing colorectal cancers Download PDF

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Publication number
CN106546744A
CN106546744A CN201510595002.4A CN201510595002A CN106546744A CN 106546744 A CN106546744 A CN 106546744A CN 201510595002 A CN201510595002 A CN 201510595002A CN 106546744 A CN106546744 A CN 106546744A
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China
Prior art keywords
antibody
pkm2
detection
solid phase
kit
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赵新慧
蔡勇华
郭安亮
姚见儿
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Shanghai Tou Jing Life Science Limited-Liability Co
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Shanghai Tou Jing Life Science Limited-Liability Co
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Abstract

The invention provides a kind of kit and its method by fecal hemoglobin, transferrins and PKM2 joint-detection assessing colorectal cancers.The present invention is based on streaming fluorescence platform, and the early diagnosis tumor in digestive tract such as the carcinoma of the rectum or colon cancer is assessed by hemoglobin, transferrins and PKM2 marks in quantitative determination excrement.

Description

By fecal hemoglobin, the method for transferrins and PKM2 joint-detection assessing colorectal cancers and Corresponding reagent box
Technical field
The present invention relates to field of biological detection, specifically, the invention provides it is a kind of by fecal hemoglobin, The method and corresponding reagent box of transferrins and PKM2 joint-detection assessing colorectal cancers.
Background technology
The occult blood examination of excrement, refers to hemorrhage of digestive tract amount seldom, visually loses color, and a small amount of red blood cell It is again digested to decompose so that the bleeding situation for finding of also having no way of under microscope.The a small amount of bleeding of stool with occult blood alimentary canal can Do not cause stool color change, only just can determine that by FOB Fecal Occult Blood Testing.Every disease of digestive tract causes few Blood is measured, can have a stool with occult blood, therefore occult blood examination has for diagnosing various hemorrhage of digestive tract diseases Important value, is the effective means of generaI investigation screening disease of digestive tract, and it is constantly deep that occult blood experiment has become people Enter the focus of research.
Occult blood test is that the hemorrhage of digestive tract that diagnosis a variety of causes causes and Screening Diagnosis Malignant gastrointestinal are swollen The important means of knurl.The method that predominantly detects of occult blood test has chemical method and immunological method at present:
Chemical method species is various, and conventional has ortho-aminotoluene method, phenolphthalin method, benzidine method, pyramidon Method, guaiac method.The hemosiderin part that its experimental design principles is all based in human hemoglobin has The effect that catalysis peroxide decomposes, the peroxide in energy catalytic reagent, decomposes the oxygen of release nascent state, oxygen Change above-mentioned chromogen substance and colour generation, the depth reflection hemoglobin of colour generation number.But chemical method lacks accurate True property and specificity.As contained hemoglobin, myoglobins, the work of its ferroheme in exogenous diet food With can make experiment be positive, can also be catalyzed containing activated plant superoxide in a large amount of salad vegetables , there is positive reaction in peroxide breaks down.Blood overstays in enteron aisle, hemoglobin denaturation, then occur The negative findings not being inconsistent with the state of an illness.In addition, when clothes for patients with substantial amounts of vitamin C or other have reduction Peroxide can be reduced, so as to aoxidize chromogen substance, experiment can also go out by the medicine of property in an experiment Existing false negative.
Immunological method mainly includes immune monoclonal antibody method, immune spot-ing, latex immunochemistry coacervation, puts Penetrate SRID, reverse indirect blood coagulation, colloid gold label sandwich immunoassay method of inspection etc..Such test institute It is divided into two big class, i.e. antihuman hemoglobin antibody and anti-human erythrocyte matrix antibody with antibody, at present typically all Using antihuman hemoglobin antibody.The colloidal gold immunity chromatography researched and developed in recent years, by monoclonal technigue and glue Body technology for gold is combined, can be with high degree of specificity using the monoclonal antibody and human hemoglobin of antihuman hemoglobin With reference to the characteristics of detecting fecal occult blood.This method is by the blood of animal, meat, the fresh vegetable containing peroxidase Dish, chalybeate, ascorbic impact, sensitivity are high, and reaction is quick, causes bleeding in clinical various diseases Preferable effect is served in diagnosis.
Colloidal Gold OBT has certain sensitivity, may occur in which false the moon higher or lower than this scope Property.And monoclonal antibody colloidal gold method can be sometimes failed to pinpoint a disease in diagnosis in detection hemorrhage of digestive tract, and can only be to inspection Surveying result carries out qualitative evaluation.
Up to the present, the sensitivity of single fecal occult blood (FOB) diagnostic method and specificity all need into one Step is improved.Joint-detection is had pointed out in the market and develops some related combined detection reagents.But, The sensitivity of the method and reagent of joint-detection is unsatisfactory at present, there are a large amount of missing inspections.
Therefore, this area is in the urgent need to exploitation is with high sensitivity, comprehensive recall rate and Detection accuracy height etc. The fecal occult blood detection reagent of overall merit and method.
The content of the invention
The object of the invention is just to provide a kind of comprehensive with high sensitivity, comprehensive recall rate and Detection accuracy height etc. Close the fecal occult blood detection reagent and method of advantage.
A kind of a first aspect of the present invention, there is provided kit for detecting or diagnosing lower digestive tract tumour, The kit includes：
A () solid phase binding thing, described solid phase binding thing include：Coating hemoglobin is that the captures of HB first are anti- First solid phase carrier of body, coating transferrins are the second solid phase carrier of the capture antibody of TF second, He Bao It is the 3rd solid phase carrier that PKM2 the 3rd captures antibody by pyruvate kinase M2；Wherein, described each capture Antibody is combined with solid phase carrier by covalent cross-linking mode；
B () lights conjugate, described luminous conjugate includes：For be incorporated into HB the first detection antibody, For being incorporated into second detection antibody and the 3rd detection antibody for being incorporated into PKM2 of TF, wherein, Described each detection antibody carries detectable light emitting molecule；With
Optional (c) standard items, described standard items are the standard items containing HB, TF and/or PKM2 antigen Solution.
In another preference, the first described capture antibody and the first detection antibody are incorporated into HB antigens not Same epi-position.
In another preference, the second described capture antibody and the second detection antibody are incorporated into TF antigens not Same epi-position.
In another preference, the 3rd described capture antibody and the 3rd detection antibody are incorporated into PKM2 antigens Different epitopes.
In another preference, first, second, and third described solid phase carrier is fluorescently-labeled with difference Solid phase microballoon.
In another preference, described kit also includes specification.
In another preference, herein below in described specification, is indicated：
In another preference, if the content of single albumen is higher than cutoff value, judge representated by the albumen Index is the positive；And when in the index representated by HB, TF and PKM2 three, arbitrary index is the positive, then The sample is judged for positive.
In another preference, the positive cutoff value of HB is 50-150ng/ml, is more preferably 80-120ng/ml, Most preferably about 100ng/ml.
In another preference, the positive cutoff value of TF is 20-60ng/ml, is more preferably 30-50ng/ml, Most preferably about 40ng/ml.
In another preference, the positive cutoff value of PKM2 is 2-8ng/ml, is more preferably 3-7ng/ml, Most preferably about 5ng/ml.
In another preference, described solid phase carrier is microspheres with solid, preferably magnetic microsphere.
In another preference, described solid phase carrier resists to be crosslinked HB capture antibody, TF captures respectively Body and PKM2 capture the magnetic microsphere of antibody.
In another preference, in the kit, also containing cutting corresponding to HB, TF and PKM2 Break and be worth the standard solution of (ng/ml), the ratio of the cutoff value V2 of the cutoff value V1 and TF of wherein HB is 1.5-3.5, preferably 2-3.
In another preference, in described standard solution, the concentration of HB is 50-150ng/ml, more preferably Ground is 80-120ng/ml, most preferably about 100ng/ml.
In another preference, in described standard solution, the concentration of TF is 20-60ng/ml, more preferably For 30-50ng/ml, most preferably about 40ng/ml.
In another preference, in described standard solution, the concentration of PKM2 is 2-8ng/ml, more preferably For 3-7ng/ml, most preferably about 5ng/ml.
In another preference, described standard items are included respectively containing a kind of antigen in HB, TF or PKM2 Multiple independent standard solution.
In another preference, described standard items are while containing the same of tri- kinds of antigens of HB, TF and PKM2 One standard solution for merging.
In another preference, described standard items include that concentration is different and in gradient a series of Standard solution.
In another preference, described standard items include：Known n1 containing 0～3000ng/ml is not With the HB antigen standard solution of concentration, the TF of the known n2 variable concentrations containing 0～2000ng/ml Antigen standard solution, and/or the PKM2 antigens of the known n3 variable concentrations containing 0～1000ng/ml Standard solution, wherein n1, n2 and n3 are each independently 2-10, preferably the positive integer of 3-8.
In another preference, described solid phase binding thing is placed in the first container.
In another preference, in described kit, the luminous conjugate is solid or liquid form, And the solid or liquid are located in second container.
In another preference, the first described container and second container are independent or same containers.
In another preference, the kit has one or more features being selected from the group：
The first capture antibody of the HB, the first detection antibody are anti-human HB murine monoclonals detection antibody；
The second capture antibody of the TF, the second detection antibody are anti-human TF murine monoclonals detection antibody；Or
The capture antibody of the PKM2 the 3rd, the 3rd detection antibody are anti-human PKM2 murine monoclonals detection antibody.
In another preference, described light emitting molecule is selected from the group：Phycoerythrin (PE), isothiocyanic acid are glimmering Light element (FITC), TRITC (TRITC), cyanine type dye (Cy2, Cy3 or Cy5), Amino methylcoumarin acetic acid esters (Aminomethylcoumarin Acetate, AMCA), or its combination.
In another preference, described solid phase binding thing solution captures antibody, TF to be crosslinked HB first respectively Second capture antibody and PKM2 the 3rd capture the magnetic microsphere mixed solution of antibody；And/or
Described light-emitting junction polymer solution is the first detection antibodies of HB, the TF that marked phycoerythrin (PE) respectively The mixed solution of the 3rd detection antibody of the second detection antibody and PKM2.
In another preference, in described kit, described solid phase binding thing is containing solid phase binding thing Solution；And/or
Described light-emitting junction polymer solution includes：The anti-human HB murine monoclonals detection antibody of phycoerythrin mark, The anti-human TF murine monoclonals detection antibody of phycoerythrin mark, the anti-human PKM2 mouse Dan Ke of phycoerythrin mark Grand detection antibody；And/or
Described standard items include：The standard solution of the HB antigens of n1 gradient concentration, n2 gradient are dense The TF antigen standard solution of degree, the standard solution of the PKM2 antigens of n3 gradient concentration, wherein, n1, N2 and n3 are each independently the positive integer of 3-8.
In another preference, the described solution containing solid phase binding thing, light-emitting junction polymer solution and standard items Solution includes PBS buffer systems and/or preservative (such as NaN independently of one another3)。
In another preference, described solid phase binding thing solution includes：Coating HB first captures the magnetic of antibody Property microballoon, coating TF second capture antibody magnetic microsphere, coating PKM2 the 3rd capture antibody magnetic it is micro- Ball, PBS and NaN3。
In another preference, pH=7.2～7.4 of described PBS buffer systems.
In another preference, in described kit, described solid phase binding thing solution includes：
8×103～4 × 104Individual/mL coating HB capture the magnetic microsphere of antibody,
8×103～4 × 104Individual/mL coating TF capture the magnetic microsphere of antibody,
8×103～4 × 104Individual/mL coating PKM2 capture the magnetic microsphere of antibody,
And 0.005-0.02M PBS, 0.2～1.0g/L NaN3；
Described light-emitting junction polymer solution includes：
The anti-human HB murine monoclonals detection antibody of the phycoerythrin mark of 2ug/mL～10ug/mL concentration,
The anti-human TF murine monoclonals detection antibody of the phycoerythrin mark of 2ug/mL～10ug/mL concentration,
The anti-human PKM2 murine monoclonals detection antibody of the phycoerythrin mark of 2ug/mL～10ug/mL concentration,
And 0.005-0.02M PBS, 0.2～1.0g/L NaN3；
And/or described standard items include：The standard solution of the HB antigens of n1 gradient concentration, n2 terraced The TF antigen standard solution of degree concentration, the standard solution of the PKM2 antigens of n3 gradient concentration；And 0.005-0.02M PBS, 0.2～1.0g/L NaN3, n1, n2 and n3 are each independently the whole of 3-6 Number.
In another preference, described solid phase binding thing solution includes：8×103～4 × 104Individual/mL coatings HB captures the magnetic microsphere (AC of antibody：100～400ug/mL), 8 × 103～4 × 104Individual/mL bags Magnetic microsphere (the AC of antibody is captured by TF：100～400ug/mL), 8 × 103～4 × 104Individual/mL Coating PKM2 captures the magnetic microsphere (AC of antibody：100～400ug/mL), 0.005-0.02M PBS, 0.2～1.0g/L NaN3。
In another preference, the reagent component of described kit includes following one or more compositions：
1) solid phase binding thing solution：4×104Individual/mL coating HB capture the microballoon (AC of antibody： 200ug/mL), 4 × 104Individual/mL coating TF capture the microballoon (AC of antibody：200ug/mL), 4 × 104 Individual/mL coating PKM2 capture the microballoon (AC of antibody：200ug/mL), 0.01M PBS (pH7.2～ 7.4), 0.2～1.0g/L NaN3；
2) light-emitting junction polymer solution：The anti-human HB murine monoclonals detection of the phycoerythrin mark of 5ug/mL concentration Antibody, the anti-human TF murine monoclonals detection antibody of the phycoerythrin mark of 5ug/mL concentration, 5ug/mL concentration Phycoerythrin mark anti-human PKM2 murine monoclonals detection antibody, 0.01M PBS (pH7.2), 0.5g/L NaN3。
3) standard items include following standard solution：
Std1 standard solutions：0.01M PBS (pH7.2) buffer solution, 0.5g/LNaN3；
Std2 standard solutions：Containing concentration for 40ng/mL HB antigens, the TF antigens of 20ng/mL, The PKM2 antigens of 5ng/mL, 0.01M PBS (pH7.2) buffer solution, 0.5g/L NaN3；
Std3 standard solutions：Containing concentration for 200ng/mL HB antigens, the TF antigens of 80ng/mL, The PKM2 antigens of 20ng/mL, 0.01M PBS (pH7.2) buffer solution, 0.5g/L NaN3；
Std4 standard solutions：Containing concentration for 625ng/mL HB antigens, the TF antigens of 160ng/mL, The PKM2 antigens of 50ng/mL, 0.01M PBS (pH7.2) buffer solution, 0.5g/L NaN3；
Std5 standard solutions：Containing concentration for 1250ng/mL HB antigens, the TF antigens of 400ng/mL, The PKM2 antigens of 100ng/mL, 0.01M PBS (pH7.2) buffer solution, 0.5g/L NaN3；
Std6 standard solutions：Containing the HB antigens that concentration is 2000ng/mL, the TF of 1000ng/mL resists Original, the PKM2 antigens of 200ng/mL, 0.01M PBS (pH7.2) buffer solution, 0.5g/L NaN3。
In another preference, described lower digestive tract tumour is selected from the group：Colon tumor, rectal neoplasm, Or its combination.
In a second aspect of the present invention, there is provided the preparation method of the kit described in first aspect present invention, Characterized in that, including step：
A () provides solid phase binding thing and luminous conjugate,
Wherein, described solid phase binding thing includes：Coating hemoglobin is that the first of the capture antibody of HB first is consolidated Phase carrier, coating transferrins are that the second solid phase carrier and coating pyruvic acid of the capture antibody of TF second swash Enzyme M2 is the 3rd solid phase carrier that PKM2 the 3rd captures antibody；Wherein, described each capture antibody is by altogether Valency crosslinking method is combined with solid phase carrier；
Described luminous conjugate includes：For being incorporated into first detection antibody of HB, for being incorporated into TF The second detection antibody and the 3rd detection antibody for being incorporated into PKM2, wherein, described each detection Antibody carries detectable light emitting molecule；
B the solid phase binding thing is placed in the first container by (), described luminous conjugate is placed in second container, And it is assembled into the kit described in first aspect present invention.
In another preference, described method includes：In step (a), (c) standard items, institute are also provided The standard items stated are the standard solution containing HB, TF and/or PKM2 antigen；And in step (c), will The standard items are placed in the 3rd container, and together with the first container and second container are assembled into kit.
In another preference, described solid phase binding thing is prepared with following solid phase carrier coating：Pass through Carbodiimide (EDC) condensation method, carries out cross-linking reaction with described solid phase carrier with described capture antibody, Obtain being coated with the solid phase carrier of capture antibody；Wherein, described solid phase carrier is the solid phase of surface carboxyl groups Carrier；
In another preference, the detection antibody is carried out as follows mark：It is sub- by N- hydroxysuccinimidyls acyl Amine activation method is marked to described detection antibody.
In a fourth aspect of the present invention, there is provided a kind of method of non-diagnostic and non-therapeutic ground detection sample, Including step：
(1) kit described in a first aspect present invention is provided；
(2) sample for being derived from excrement is provided, and optionally, described sample is processed, separate Protein component in the sample；
(3) antibody-antigene reaction is carried out with described solid phase binding thing and described sample, obtain containing solid phase First reactant liquor of carrier-capture antibody-protein complexes；
(4) resisted with described solid phase carrier-capture antibody-protein complexes with described luminous conjugate Body-antigen-reactive, obtains containing solid phase carrier-capture Antibody-protein-detection antibody-light emitting molecule compound The second reactant liquor；
(5) optical detection is carried out to the second described reactant liquor, detected value is obtained；
Wherein, described albumen is the combination of following albumen：HB, TF and PKM2.
In another preference, described optical detection is detected for fluorescent value (MFI).
In another preference, described sample behaviour fecal sample.
In another preference, also including step：Will be the detected value bent with the detected value or standard of standard items Line is compared.
In another preference, the detected value or calibration curve of described standard items are obtained as below：
I () carries out antibody-antigene reaction with described solid phase binding thing and calibration object solution, obtain containing solid phase First calibration solution of carrier-capture antibody-protein complexes；
(ii) carried out with described solid phase carrier-capture antibody-protein complexes with described luminous conjugate Antibody-antigene reacts, and obtains compound containing solid phase carrier-capture Antibody-protein-detection antibody-light emitting molecule The calibration solution of thing；
(iii) optical detection is carried out to the second described calibration solution, obtains standard items detected value or further Obtain calibration curve.
In another preference, if the content of single albumen is higher than cutoff value, judge representated by the albumen Index is the positive.
In another preference, the positive cutoff value of HB is 50-150ng/ml, is more preferably 80-120ng/ml, Most preferably about 100ng/ml.
In another preference, the positive cutoff value of TF is 20-60ng/ml, is more preferably 30-50ng/ml, Most preferably about 40ng/ml.
In another preference, the positive cutoff value of PKM2 is 2-8ng/ml, is more preferably 3-7ng/ml, Most preferably about 5ng/ml.
In another preference, when in the index representated by HB, TF and PKM2 three, arbitrary index is the positive, The sample is then judged for positive.
In another preference, described lower digestive tract tumour is selected from the group：Colon tumor, rectal neoplasm, Or its combination.
In a fourth aspect of the present invention, there is provided a kind of purposes of combination, it is described be combined as Hb H B, Transferrins is the combination constituted by TF and pyruvate kinase M2 (i.e. PKM2), or described is combined as HB The combination constituted by antibody, TF antibody and PKM2 antibody, under the combination is used for preparing detection or diagnosing The kit of tumor in digestive tract.
In another preference, described antibody includes capturing antibody and detection antibody.
In another preference, described antibody includes the first capture antibody and the first detection for being incorporated into HB Antibody, the second capture antibody and the second detection antibody for being incorporated into TF, the 3rd capture for being incorporated into PKM2 are anti- Body and the 3rd detection antibody.
In another preference, described lower digestive tract tumour is selected from the group：Colon tumor, rectal neoplasm, Or its combination.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and below (such as enforcement Example) in can be combined with each other between each technical characteristic for specifically describing, so as to constitute new or preferred skill Art scheme.As space is limited, here is no longer tired out one by one and is stated.
Description of the drawings
Fig. 1 shows the HB calibration curves in an example of the invention.
Fig. 2 shows the TF calibration curves in an example of the invention.
Fig. 3 shows the PKM2 calibration curves in an example of the invention.
Fig. 4 shows the PKM2/HB/TF detection sample ROC curves in an example of the invention.
Specific embodiment
The present inventor exceeds meaning through extensively in-depth study by the screening to a large amount of unlike signal things Material ground is found, carries out joint inspection and combine specific cutoff value when being combined using tri- mark of HB, TF and PKM2 When, the high specific of detection is on the one hand maintained, the sensitivity of detection is also dramatically improved, so as to have There are the overall merits such as comprehensive recall rate height and Detection accuracy height.On this basis, inventor completes this It is bright.
Specifically, double-antibody sandwich immunization method of the present inventor based on streaming fluorescence platform, joint HB, TF and PKM2 carries out quantitative determination to fecal occult blood, by the optimization to detection and cutoff value such that it is able to The content of corresponding mark in sample is preferably pointed out, is contributed to the objectivity of result judgement, is improved early stage straight The accuracy rate of intestinal cancer or diagnosis of colon cancer, also provides quick, easy means and timely to early diagnose this disease Treatment.
Hemoglobin (Hb) in excrement
Hemoglobin (Hb, Hemoglobin) in excrement be called fecal occult blood (Feces occult Blood, FOB mark), is counted as one of tumor markers of the carcinoma of the rectum, colon cancer at present in clinic.Inspection Fecal occult blood is broadly divided into chemical measure (Guaiac test) and immunodetection.Chemical method is cumbersome And whole process patient endures hardships very much, immunodetection is generally adopted at present, i.e., by anti-human ferroheme globulin Specific antibody detects human red blood's element globulin using immuno-sandwich, and this method is because being for human red blood's element Globulin reaction, therefore specificity is high.Thus relevant fecal occult blood detection is almost based on this mark of excrement HB Carry out.
Research shows that excrement HB is more sensitive to hemorrhage of lower digestive tract.And hemorrhage of digestive tract is often colorectal cancer Early symptom, thus fecal occult blood is tested frequently as early screening colorectal cancer common method.But determine excrement Just HB has 20%-30% false negatives to the carcinoma of the rectum or colon cancer, causes this result main cause:By alimentary canal A small amount of bleeding, the hemoglobin for disengaging (Hb) when through longer enteron aisle with excrement in Hp be combined into it is compound Thing causes occult blood false negative, it is clear that whether the hemoglobin in inspection excrement suffers from the carcinoma of the rectum or colon cancer to diagnose Tumor markers has serious missing inspection.
The research of the present inventor shows, using specific detection method of the invention, not only detect excrement it is this into During point extremely complex sample, the detectable lower limit of HB is low, and by the cutoff value of HB be arranged at for 50-150ng/ml, is more preferably 80-120ng/ml, during most preferably about 100ng/ml, can be more preferably regional The fecal sample of point tumor patient (or susceptible person) and the fecal sample of normal population, with high sensitivity, low False positive rate and have comprehensive recall rate high when carrying out three joint inspections with TF and PKM2 and Detection accuracy height etc. Overall merit.
Transferrins (TF)
Transferrins (TF, Transferrin) is primarily present in blood plasma, is that one kind is released by neutrophil cell Exoergic combines ferric glycoprotein, belongs to 1 microglobulins of β, and its molecular weight is 77kD, accounts for blood plasma egg The 0.3%-0.5% of white total amount, mainly synthesizes in liver.Research shows, in hemorrhage of digestive tract disease, with Hemoglobin is similar, and transferrins also can be entered in enteron aisle in hemorrhage of digestive tract, and is discharged with excrement.
Research finds that transferrins (TF) is there's almost no in healthy human faecal mass, and in hemorrhage of gastrointestinal tract Excrement is present in a large number, therefore the content of transferrins is higher than normal person in intestinal canal tumour patient's excrement.And, excrement Just the stability in excrement of the transferrins in is high compared with hemoglobin, therefore in methodology has one with immunization Fixed complementarity.
The research of the present inventor shows, using specific detection method of the invention, not only detect excrement it is this into During point extremely complex sample, the detectable lower limit of TF is low, and by the cutoff value of TF be arranged at for 20-60ng/ml, is more preferably 30-50ng/ml, during most preferably about 40ng/ml, can be better discriminated between The fecal sample of tumor patient (or susceptible person) and the fecal sample of normal population, with high sensitivity, low vacation Positive rate and have comprehensive recall rate high when carrying out three joint inspections with HB and PKM2 and Detection accuracy height etc. Overall merit.
Pyruvate kinase (PK)
Pyruvate kinase (pyruvate kinase, PK) is the key enzyme of cell glycolysis path, and swollen The abnormal key enzyme of cell glucose metabolism in knurl generation.Pyruvate kinase is known to have L, R, M1, M24 kind Isodynamic enzyme (PKL, PKR, PKM1, PKM2), its differential expression depend on the metabolic condition of nucleus tissue. Wherein M2 types pyruvic acid expression synthesizes vigorous tissue in nucleic acid, and such as embryonic cell, stem cell and tumour is thin Born of the same parents.Tumour is usually associated with the other conversion of tumor locus pyruvate kinase isozymes expression, because comparing In the synthesis of ATP, the synthesis of tumour cell large biological molecule is more vigorous, is to maintain the fast breeding of its own. PKM2 early diagnosis clinically to tumour and judge that tumor progression has great importance, be a kind of new The tumor markers that has wide application prospects of possibility, its sensitivity is when tumor in digestive tract occurs than classical CA19-9, CA72-4 and CEA are higher.In tumour generating process, due to the necrosis and transfer of tumour cell, PKM2 in tumour cell can be discharged in blood, and the PKM2 of tumor in digestive tract also can suffer from tumour The excrement of person is discharged.
The research of the present inventor shows, using specific detection method of the invention, not only detect excrement it is this into During point extremely complex sample, the detectable lower limit of PKM2 is low, and by the cutoff value of PKM2 be arranged at for The positive cutoff value of PKM2 is 2-8ng/ml, is more preferably 3-7ng/ml, during most preferably about 5ng/ml, The fecal sample of tumor patient (or susceptible person) and the fecal sample of normal population can be better discriminated between, is had High sensitivity, low false positive rate and have comprehensive recall rate high and detection when carrying out three joint inspections with HB and TF The overall merits such as accuracy rate height.
Three furnace process fecal occult blood diagnostic kit and detection
The invention provides the three furnace process of a kind of easy, accurate, quick, high sensitivity and high specific is just dived Blood diagnostic kit, the kit are suitable for the purposes such as clinical diagnosis or examination, by quantitative determination excrement Diagnosis of the particular combination (HB, TF and PKM2) of mark for colorectal cancer.
The quantitative determination reagent kit of colorectal cancer of the present invention, is reacted based on antibody-antigene, for Some specific marker proteins of colorectal cancer carry out half-quantitative detection, according to the positive/the moon of three kinds of albumen Implementations, judge that the sample whether there is colorectal cancer.
In a preference, kit of the present invention is specifically included：
(a) solid phase binding thing；Described solid phase binding thing includes：The capture of the albumen that coating is selected from the group resists The solid phase carrier of body：HB, TF and PKM2；Wherein, described capture antibody by covalent cross-linking mode with Solid phase carrier is combined；
(b) light-emitting junction polymer solution；Described light-emitting junction polymer solution includes：Marked the choosing of light emitting molecule From the solution of the detection antibody of the albumen of the following group：HB, TF and PKM2；With
Optional (c) standard items, described standard items are HB, the TF and PKM2 antigen containing concentration known Solution or solution combination.
Wherein, described solid phase carrier has no particular limits, and can be the detection side of any suitable present invention The solid phase carrier of method, is preferably crosslinked the magnetic microsphere mixed solution that HB, TF and PKM2 capture antibody respectively, The solid phase binding thing solution of HB, TF and PKM2 antigen can be captured.It is highly preferred that the solid phase binding thing Microspheres with solid in solution is magnetic microsphere.
Described light-emitting junction polymer solution can be any Protein Detection antibody for being marked with light emitting molecule, wherein Light emitting molecule can select the light emitting molecule commonly used in the art of arbitrarily suitable detection method, preferably algae red Albumen (PE).
In a preferred embodiment of the present invention, the reagent of described kit is respectively by following component group Into：
1) solid phase binding thing solution：Coating HB captures the microspheres with solid of antibody, is coated with consolidating for TF capture antibody Body microballoon, is coated with the mixture solution of the microspheres with solid of PKM2 capture antibody, described microspheres with solid with catch Antibody is obtained by being covalently crosslinked；
2) light-emitting junction polymer solution：The anti-human HB murine monoclonals detection antibody of phycoerythrin mark, phycoerythrin The anti-human TF murine monoclonals detection antibody of mark, the anti-human PKM2 murine monoclonals detection of phycoerythrin mark are anti- Body；
3) standard items：The HB of the known n variable concentrations containing 0～3000ng/ml, containing 0～ The TF of the known n variable concentrations of the 2000ng/ml and known n containing 0～1000ng/ml Bu Tong dense The PKM2 antigenic solutions of degree, positive integers of the wherein n for 3-8.
Further, the reagent of kit includes following component：
1) solid phase binding thing solution：8×103～4 × 104The magnetic microsphere of individual/mL coating HB capture antibody is (anti- Bulk concentration：100～400ug/mL), 8 × 103～4 × 104The magnetic of individual/mL coating TF capture antibody is micro- Ball (AC：100～400ug/mL), 8 × 103～4 × 104Individual/mL coating PKM2 capture antibody Magnetic microsphere (AC：100～400ug/mL), 0.01M PBS (pH7.2～7.4), 0.5g/L NaN3。
2) light-emitting junction polymer solution：The anti-human HB mouse list of the phycoerythrin mark of 2ug/mL～10ug/mL concentration Clone's detection antibody, the anti-human TF murine monoclonals detection of the phycoerythrin mark of 2ug/mL～10ug/mL concentration Antibody, the anti-human PKM2 murine monoclonals detection antibody of the phycoerythrin mark of 2ug/mL～10ug/mL concentration, Such as 0.01M PBS (pH7.2～7.4), 0.2～1.0g/L NaN3。
3) standard items：Containing concentration range the n level of 0～2000ng/mL HB antigens, containing dense TF antigen of the degree scope in the n level of 0～1000ng/mL, containing concentration range in 0～500ng/mL N level PKM2 antigens, 0.01M PBS (pH7.2～7.4), 0.2～1.0g/L NaN3, n is The integer of 3-6.
It is highly preferred that the reagent component of the kit includes following component：
1) solid phase binding thing solution：4×104Individual/mL coating HB capture the microballoon (AC of antibody： 200ug/mL), 4 × 104Individual/mL coating TF capture the microballoon (AC of antibody：200ug/mL), 4 × 104 Individual/mL coating PKM2 capture the microballoon (AC of antibody：200ug/mL), 0.01M PBS (pH7.2～ 7.4), 0.2～1.0g/L NaN3。
2) light-emitting junction polymer solution：The anti-human HB murine monoclonals detection of the phycoerythrin mark of 5ug/mL concentration Antibody, the anti-human TF murine monoclonals detection antibody of the phycoerythrin mark of 5ug/mL concentration, 5ug/mL concentration Phycoerythrin mark anti-human PKM2 murine monoclonals detection antibody, 0.01M PBS (pH7.2), 0.5g/L NaN3。
3) standard items：
Std1：0.01M PBS (pH7.2) buffer solution, 0.5g/L NaN3；
Std2：Containing the HB antigens that concentration is 40ng/mL, the TF antigens of 20ng/mL, the PKM2 of 5ng/mL Antigen, 0.01M PBS (pH7.2) buffer solution, 0.5g/L NaN3；
Std3：Containing concentration for 200ng/mL HB antigens, the TF antigens of 80ng/mL, 20ng/mL's PKM2 antigens, 0.01M PBS (pH7.2) buffer solution, 0.5g/L NaN3；
Std4：Containing concentration for 625ng/mL HB antigens, the TF antigens of 160ng/mL, 50ng/mL's PKM2 antigens, 0.01M PBS (pH7.2) buffer solution, 0.5g/L NaN3；
Std5：Containing the HB antigens that concentration is 1250ng/mL, the TF antigens of 400ng/mL, 100ng/mL PKM2 antigens, 0.01M PBS (pH7.2) buffer solution, 0.5g/L NaN3；
Std6：Containing the HB antigens that concentration is 2000ng/mL, the TF antigens of 1000ng/mL, 200ng/mL PKM2 antigens, 0.01M PBS (pH7.2) buffer solution, 0.5g/L NaN3；
In the present invention, preferred solid phase carrier is polystyrene material, and sphere diameter about 4-10 microns are (such as from about 6.5 μm), microsphere surface possesses carboxylic group, can cross-linking antibody.In prepared by solid phase carrier-antibody complex, Carbodiimide condensation method can be adopted.
Currently preferred capture antibody and detection antibody are mouse monoclonal antibody, with for the difference of antigen Epi-position.Wherein capture antibody and detection antibody can specificity with mark (HB, TF and PKM2) antigen binding.
Include step using the concrete grammar of the kit detection of the present invention：
The first step, by the coding microball suspension mixing of sample and crosslinking specificity capture antibody, the analysis in sample Thing is combined with capture antibody.
Second step, adds the detection antibody solution of phycoerythrin mark, ultimately forms for (HB, TF and PKM2) " detection antibody of the antibody linked coding microball-antigen of capture-phycoerythrin mark " compound.
When being detected to which using multi-functional streaming dot matrix instrument, coding microball can be led to by transmission system defiled The region of two beam laser detections is crossed, to determine detection project, another beam determines algae red egg to the coding of a branch of judgement microballoon White fluorescent value (MFI).By the quantity for determining the reporter fluorescence molecule combined on microballoon, determine for brief introduction micro- The content of the fecal occult blood mark combined on ball.
In another preference, the kit and three joint inspection methods of the present invention also compare the cutoff value V1 of HB with The ratio R 1 of the cutoff value V2 of TF, and the R1 cutoff values are set as 1.5-3.5, preferably 2-3 is (low In this).The R1 cutoff values contribute to reduction false positive rate, and (it is sun to be difficult the pattern detection of normal population Property).
It is preferred that the kit of the present invention is also containing the cutoff value (ng/ml) corresponding to HB, TF and PKM2 Standard solution, the ratio of the cutoff value V2 of the cutoff value V1 and TF of wherein HB is 1.5-3.5, preferably Ground is 2-3.
Main advantages of the present invention include：
A () the inventive method and kit have high detection sensitivity, delicately can detect in FOB Multiple markers.
When () carries out joint inspection using the combination of tri- mark of HB, TF and PKM2 and combines specific cutoff value b, The inventive method had both maintained the high specific of detection, also dramatically improved the sensitivity of detection, so as to With the overall merit such as comprehensive recall rate height and Detection accuracy height.
C double-antibody sandwich immunization method of the () present invention based on streaming fluorescence platform, combines HB, TF and PKM2 Quantitative determination is carried out to fecal occult blood, three kinds of marks can be detected simultaneously in same system, It is not only time-consuming, and the process due to three kinds of marks and association reaction carried out in same system, because This is easy to the ratio relation for accurately measuring and comparing in the middle of the measured value and measured value of three kinds of marks (outstanding Which is the cutoff value of the ratio R 1 of the cutoff value V2 of the cutoff value V1 and TF based on HB), so as to sensitiveer And detected exactly.
D () is by the optimization to detection and cutoff value such that it is able to preferably point out corresponding mark in sample Content, contribute to the objectivity of result judgement, improve the accuracy rate of Early rectal tumor or diagnosis of colon cancer, Also for early diagnosis, this disease provides quick, easy means and treatment in time.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only used for The bright present invention rather than restriction the scope of the present invention.The experiment side of unreceipted actual conditions in the following example Method, generally according to normal condition, such as Sambrook et al., molecular cloning：Laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989) described in condition, or according to manufactory Condition proposed by business.Unless otherwise indicated, otherwise percentage and number are percentage by weight and parts by weight.
Embodiment 1：The preparation of tri- joint inspection kit of HB, TF and PKM2
1. critical component information needed for kit
Capture antibody that hemoglobin, transferrins and PKM2 are used, detection antibody are commercial antibody, HB standard items are purchased from Meridian companies, and TF standard items are purchased from Sigma companies, and PKM2 standard items are by JaRa Company (Shanghai) synthesizes.
2. kit of the present invention includes following components：
1) solid phase binding thing solution：
It has been coated with the 26# microballoons (4 × 10 that HB captures antibody4Individual/mL), coating TF capture antibody 28# it is micro- Ball (4 × 104Individual/mL) and it is coated with the 30# microballoons (4 × 10 that PKM2 captures antibody4Individual/mL), other components, 0.01M PBS (pH7.2), 0.5g/L NaN3。
2) light-emitting junction polymer solution：
Respectively with phycoerythrin mark HB detection antibodies (5ug/mL), TF detection antibodies (5ug/mL) and PKM2 detection antibodies (5ug/mL), 0.01M PBS (pH7.2), 0.5g/L NaN3。
3) standard items, are the mixture of HB, TF, PKM2 antigen standard of one group of 6 concentration known, Wherein：
Std1：0.01M PBS (pH7.2, similarly hereinafter) buffer solution, 0.5g/L NaN3；
Std2：Containing the HB antigens that concentration is 40ng/mL, the TF antigens of 20ng/mL, the PKM2 of 5ng/mL Antigen, 0.01M PBSs, 0.5g/L NaN3；
Std3：Containing the HB antigens that concentration is 200ng/mL, the TF antigens of 80ng/mL, the PKM2 of 20ng/mL Antigen, 0.01M PBS (pH7.2) buffer solution, 0.5g/L NaN3；
Std4：Containing the HB antigens that concentration is 625ng/mL, the TF antigens of 160ng/mL, the PKM2 of 50ng/mL Antigen, 0.01M PBSs, 0.5g/L NaN3；
Std5：Containing concentration for 1250ng/mL HB antigens, the TF antigens of 400ng/mL, 100ng/mL's PKM2 antigens, 0.01M PBSs, 0.5g/L NaN3；
Std6：Containing the HB antigens that concentration is 2000ng/mL, the TF antigens of 1000ng/mL, 200ng/mL PKM2 antigens, 0.01M PBSs, 0.5g/L NaN3；
3. mentioned reagent box is prepared, is comprised the steps：
1) it is to realize the crosslinking of coding microball and capture antibody to be coated with technique main target.The technique is based on carbon two Imines (EDC) condensation method, by EDC as bridge crosslinking structure at N- hydroxy thiosuccinimides (Sulfo-NHS) Catalysis under condensation microballoon carboxyl (- COOH) and the amino (- NH of antibody2) realize coating.
Through coated operation link, main raw material(s) antibody and coding microball are prepared as middle product Beads-Ab, is then used to be configured to microsphere suspensions.
2) main target of marking process is to realize the mark with phycoerythrin (PE) to detection antibody.The technique Based on N-hydroxy-succinamide activation method, PE is activated by Sulfo-SMCC, there is provided maleic di-imidogen Group；Detection antibody is reduced by DTT after SPDP activating antibodies so as to sulfhydrylation, sulfydryl and maleic diimine The mark of PE antagonists is realized in group condensation.
Through the operation link for marking, main raw material(s) antibody and phycoerythrin are prepared as middle product Ab-PE, It is subsequently used for preparing PE labelled antibody solution.
4) calibration object：Titer 1 (Std1), wherein without any mark；Titer 2-6 (Std2, Std3, Std4, Std5, Std6), the mark antigen (HB, TF, PKM2) containing three kinds of concentration knowns.
4. sample process
1) excrement is taken with adopting just rod multiple spot, sampling amount is that 15～20mg (adopts closet distal end spiral shell with all coverings Rotation shape groove is advisable)；
2) just rod will be adopted to put back in feces collection container, lid is tightened, is fully mixed；
3) should be detected using fresh excreta；The difference sample that Jing dilutions or distilled water are mixed should be in 1h Inside detect as early as possible.
5. detection process：
Using front, by reagent bottle fully shaking, 10s or so, it is well mixed reagent；Cleaning fluid pure water 10 times of uses of dilution.
1) this product and Fit Models Luminex200 or Luminex MAGPIX are ready to.Will be to be tested Sample is processed on request.
2) sequentially add on 96 hole reaction plates respectively：20 μ L/ holes of sample or calibration object, microsphere suspensions 50 μ L/ holes.1～6 hole of left side first row of reaction plate is set gradually the fixed bit for Std1～Std6 by suggestion Put.Shrouding paper is added a cover, is fully mixed in micropore plate oscillator, put 37 DEG C of insulating boxs, lucifuge reaction 15min.
3) 96 hole reaction plates are taken out, is first placed in micropore plate oscillator fully mixing 1min, on magnetic sheet After standing 1min, then knockout plate adds 200 μ L/ holes of washing lotion successively, and concussion mixes 1min, quiet on magnetic sheet After putting 1min, knockout plate is washed 3 times altogether.Secondly 100 μ L/ holes of PE labelled antibodies are added, shrouding is added a cover Paper, fully mixes in micropore plate oscillator, puts 37 DEG C of insulating boxs, lucifuge reaction 15min.
4) 96 hole reaction plates are taken out, is washed 3 times, added 100 μ L/ holes of washing lotion, add a cover shrouding paper, micropore Fully mix in plate oscillator.
5) 96 hole reaction plates are put into into reading on multi-functional streaming dot matrix instrument.
6. the calculating of testing result
Using Std1～6 calibration object concentration as X-axis, using calibration object fluorescent value as Y-axis, using five parameters Logistic equation model calibration curves, calculate analyte in sample according to the fluorescent value of calibration curve and sample Concentration.
The each index calibration point of 1 detection kit of table is arranged and signal Distribution value
Can release from accompanying drawing 1-3 and above-mentioned table 1, each criterion curve detection of HB, TF and PKM2 is dense R >=0.99 in the range of degree, and each calibration point rate of recovery is between 85%～115% (data are not arranged).
The calibration curve of the present invention is generated by the method that multiple spot is calibrated, bent with five parameter logi stic Line is fitted, and according to the curve of fitting, substitutes into pattern detection fluorescent value, calculates the fitting token thing of sample Concentration value, with reference to cutoff value (cutoff value by great amount of samples detection determine), can HB in truer reflected sample, The actual concentrations of TF and PKM2, are conducive to sample more directly perceived, objective assessment.
Detected by great amount of samples, finally determine that by ROC curve (accompanying drawing 4) the positive cutoff value of HB is The positive cutoff value of 100ng/ml, TF is 40ng/ml, and the positive cutoff value of PKM2 is 5ng/ml, three In arbitrary result it is positive, that is, judge that clinical samples are the positive.Three index joint inspections can be completed in 1 hour Detection.
Embodiment 2 HB, TF and PKM2 tri- joint inspection detect positive sample result
From the tri- joint inspection detection reagent of HB, TF and PKM2 described in embodiment 1, to 124 excrement altogether Sample carries out immunoassay, wherein 55 from outpatient service and inpatient excrement, and Jing endoscopies are true Think colorectal cancer and progressive stage adenoma (a kind of pathological state before colorectal cancer pathology) sample, 69 is normal Control negative sample.
Joint-detection is carried out to 55 positive samples wherein, as a result as shown in table 2.
The each index of 2 kit of table is to positive sample screening results
3 55 positive samples of table are in PKM2, HB and TF testing result distribution situation
Sample NAlways=55 PKM2 HB TF
26 + + +
3 + + -
2 + - -
3 + - +
5 - + +
3 - + -
8 - - +
10 - - -
The single detection of PKM2, HB and TF is to the straight cancer of total 55 colons and progressive stage adenoma sample as seen from Table 2 This recall rate is respectively 61.8% and 67.3% and 76.4%, and triple combination is detected to total 55 colon cancers 81.8% is up to the recall rate of progressive stage adenoma patients' sample, positive rate is compared singly to examine to have and significantly carried It is high.And false negative rate reduces 20%, 14.5% and 5.3% respectively than alone PKM2, HB or TF.
In table 3, the yin and yang attribute distribution of 55 samples in PKM2, HB and TF Indexs measure shows, PKM2 Positive 34, HB is positive 37, TF 42 cases.Single inspection and joint inspection of the single index to other indexs There is certain effect of reexamining, the positive detections of wherein clinical samples TF compare the positive detections of HB and PKM2 to be had Notable supplementary function.Detection of the joint inspection of as shown by data HB, PKM2 and TF to colorectal cancer has notable clinic The missing inspection for supplementing detection, reducing that the mono- inspection of HB, PKM2 or joint inspection are produced of meaning, especially TF indexs, Significantly improve patient's positive rate.
Embodiment 3 analyzes the susceptibility of detection sample (n=124) with kit cutoff value
It is that colorectal cancer or progressive stage adenoma positive sample and 69 normal samples carry out sensitivity to 55 confirmations Degree, specificity analysis.
During by for the analysis and optimization of detection data, finding using cutoff value shown in table 4 below, can be with Detection sensitivity is effectively improved, especially the sensitivity of three joint inspections and accuracy.
Sensitivity analysis result of 4 kit of table to cancer pattern detection
PKM2 HB TF HB+TF+PKM2
Cutoff value 5ng/ml 100ng/ml 40ng/ml —
Susceptibility 61.8% 67.3% 76.4% 81.8%
Especially when the cutoff value of TF is set to 30-50ng/ml samples (such as 40ng/ml), can be well Indicative function of the TF marks to colon cancer is played, not only with very high sensitivity, also will not or be difficult to lead Normal sample is caused to produce false positive.
Additionally, when the cutoff value of PKM2 is set to 4-8ng/ml samples (such as 54-8ng/ml), can be fine Ground plays indicative function of the PKM2 marks to colon cancer, not only with certain sensitivity, will not also lead Normal sample is caused to produce false positive.
Also show from 4 result of table, during using above-mentioned cutoff value (cut-off value), reagent of the present invention is to suffering from The single detection and analysis susceptibilitys of person sample HB, TF and PKM2 are up to 67.3%, 76.4% and 61.8% respectively, And tested and analyzed by triple combination, the susceptibility of joint-detection is 81.8%, and more single detection and analysis is sensitive Degree is improved, and has been respectively increased 14.5%, 5.4% and 20.0%.
4 kit of the present invention of embodiment reagent similar with market is tested and analyzed to clinical samples
To clinical samples and check sample, (55 confirmations are comparison kit of the present invention reagent similar with market Colorectal cancer or progressive stage adenoma positive sample and 69 normal samples) testing result is analyzed, wherein PKM2 marks are using commercially availableTumor M2PK kits are used as reference method of testing, HB marks Will thing uses fecal occult blood detection reagent (latex immunoturbidimetry) and colloidal gold strip as reference test side Method, TF marks use transferrins colloidal gold diagnosis test paper as reference method of testing.
When three joint inspection of the present invention is carried out, using the cutoff value shown in table 4.As a result it is as shown in table 5.
Testing result of the different qualitative and quantitative product of table 5 to 124 samples
As seen from Table 5,
1) three joint inspection kits of the invention are all kinds of for the recall rate (susceptibility) of positive sample is significantly higher than Qualitative or quantitative single inspection product, false positive ＜ 10% to 69 normal samples, with single index when (PKM2, HB or TF) false positive rate no difference of science of statistics.
2) difference on susceptibility is little with commercially available basis weight products for single PKM2 and HB of the invention.3) present invention With the qualitative products of existing HB, the qualitative products of TF compare, no matter single inspection or joint inspection, susceptibility exists larger Difference.
Found by above-mentioned difference sample, wherein there are 7 samples, present invention detection HB is positive, and HB is fixed Property be detected as feminine gender, by checking fluorescent measurement of the present invention to HB, derive the HB of this 7 samples Detected value major part is in the range of 135～183ng/mL, it is clear that by sample fluorescence detected value MFI, leads to Quantitation curves are crossed, fitting concentration can be more objectively derived, with reference to cutoff value, in true reflected sample The concentration of HB, reduces false negative, be easy to give sample intuitively, objective assessment.
The comparison of 6 16 HB difference samples of table
Equally, by 9 TF difference samples, i.e. present invention detection is positive, and the qualitative reagent detections of TF are negative, The TF derived by fluorescent value is fitted concentration, find the concentration of present invention detection positive 9 40～ 50ng/mL or so (data are not arranged), the content in true reflected sample, contributes to the clinic to sample again Assessment.
Embodiment 5：The screening of detection mark
40 colorectal cancer positive samples are gathered again, with the detection reagent described in the embodiment of the present invention 1 The detection reagent of box and various other marks, detects to each sample.Based on HB, PKM2, TF And the selection result is shown in Table 7 in the several carcinoma markers of CEA (carcinomebryonic antigen).
Table 7：The selection result of HB, PKM2, TF, CEA mark
Sample N PKM2 HB TF CEA
8 + + + +
3 + + + -
3 + + - +
3 + + - -
1 + - - +
3 + - - -
2 + - + +
1 + - + -
1 - + + +
3 - + + -
1 - + - +
3 - + - -
2 - - + +
3 - - + -
1 - - - +
2 - - - -
Visible by table 7, wherein single inspection PKM2 is positive 24, HB is positive 25, and TF is positive 23, CEA is positive 19.It is most to combine PKM2 (reexamining 7) and joint TF (reexamining 8) based on HB The effectively best of breed of comprehensive detection lower digestive tract tumour.
In order to further verify above-mentioned optimal joint inspection combination, and the combination side that consideration various combination may bring 4 marks are re-started combination, can obtain following testing result by the omission of case：
The positive detection result of 8 unlike signal thing of table combination
No Combination Positive sample detects number of cases
Combination one HB+PKM2+TF 37
Combination two HB+PKM2+CEA 35
Combination three HB+TF+CEA 35
Combination four HB+TF 33
Combination five PKM2+TF+CEA 35
Combination six TF+CEA 29
Combination seven TF+PKM2 33
Combination eight PKM2+CEA 29
Obviously, it is optimal scheme with the combination one of HB, TF and PKM2.
The all documents referred in the present invention are all incorporated as reference in this application, just as each document It is individually recited such as reference.In addition, it is to be understood that after the above-mentioned instruction content for having read the present invention, Those skilled in the art can be made various changes or modifications to the present invention, and these equivalent form of values equally fall within this Shen Please appended claims limited range.

Claims (10)

1. a kind of kit for detecting or diagnosing lower digestive tract tumour, it is characterised in that the kit Including：
A () solid phase binding thing, described solid phase binding thing include：Coating hemoglobin is that the captures of HB first are anti- First solid phase carrier of body, coating transferrins are the second solid phase carrier of the capture antibody of TF second, He Bao It is the 3rd solid phase carrier that PKM2 the 3rd captures antibody by pyruvate kinase M2；Wherein, described each capture Antibody is combined with solid phase carrier by covalent cross-linking mode；
B () lights conjugate, described luminous conjugate includes：For be incorporated into HB the first detection antibody, For being incorporated into second detection antibody and the 3rd detection antibody for being incorporated into PKM2 of TF, wherein, Described each detection antibody carries detectable light emitting molecule；With
Optional (c) standard items, described standard items are the standard items containing HB, TF and/or PKM2 antigen Solution.
2. kit as claimed in claim 1, it is characterised in that described solid phase carrier is to be crosslinked respectively HB capture antibody, TF capture antibody and PKM2 capture the magnetic microsphere of antibody.
3. kit as claimed in claim 1, it is characterised in that in the kit, also containing right Should in the standard solution of the cutoff value (ng/ml) of HB, TF and PKM2, the cutoff value V1 of wherein HB with The ratio of the cutoff value V2 of TF is 1.5-3.5, preferably 2-3.
4. kit as claimed in claim 1, it is characterised in that described light emitting molecule is selected from the group： Phycoerythrin (PE), fluorescein isothiocynate (FITC), TRITC (TRITC), cyanines Class dyestuff (Cy2, Cy3 or Cy5), amino methylcoumarin acetic acid esters (Aminomethylcoumarin Acetate, AMCA), or its combination.
5. kit as claimed in claim 1, it is characterised in that described solid phase binding thing solution for point Not Jiao Lian HB first capture antibody, TF second capture antibody and PKM2 the 3rd capture antibody magnetic microsphere mix Close solution；And/or
Described light-emitting junction polymer solution is the first detection antibodies of HB, the TF that marked phycoerythrin (PE) respectively The mixed solution of the 3rd detection antibody of the second detection antibody and PKM2.
6. kit as claimed in claim 1, it is characterised in that in described kit, described consolidates The thing that combines is the solution containing solid phase binding thing；And/or
Described light-emitting junction polymer solution includes：The anti-human HB murine monoclonals detection antibody of phycoerythrin mark, The anti-human TF murine monoclonals detection antibody of phycoerythrin mark, the anti-human PKM2 mouse Dan Ke of phycoerythrin mark Grand detection antibody；And/or
Described standard items include：The standard solution of the HB antigens of n1 gradient concentration, n2 gradient are dense The TF antigen standard solution of degree, the standard solution of the PKM2 antigens of n3 gradient concentration, wherein, n1, N2 and n3 are each independently the positive integer of 3-8.
7. kit as claimed in claim 6, it is characterised in that in described kit：
Described solid phase binding thing solution includes：
8×103～4 × 104Individual/mL coating HB capture the magnetic microsphere of antibody,
8×103～4 × 104Individual/mL coating TF capture the magnetic microsphere of antibody,
8×103～4 × 104Individual/mL coating PKM2 capture the magnetic microsphere of antibody,
And 0.005-0.02M PBS, 0.2～1.0g/L NaN3；
Described light-emitting junction polymer solution includes：
The anti-human HB murine monoclonals detection antibody of the phycoerythrin mark of 2ug/mL～10ug/mL concentration,
The anti-human TF murine monoclonals detection antibody of the phycoerythrin mark of 2ug/mL～10ug/mL concentration,
The anti-human PKM2 murine monoclonals detection antibody of the phycoerythrin mark of 2ug/mL～10ug/mL concentration,
And 0.005-0.02M PBS, 0.2～1.0g/L NaN3；
And/or described standard items include：The standard solution of the HB antigens of n1 gradient concentration, n2 terraced The TF antigen standard solution of degree concentration, the standard solution of the PKM2 antigens of n3 gradient concentration；And 0.005-0.02M PBS, 0.2～1.0g/L NaN3, n1, n2 and n3 are each independently the whole of 3-6 Number.
8. the preparation method of kit as claimed in claim 1, it is characterised in that including step：
A () provides solid phase binding thing and luminous conjugate,
Wherein, described solid phase binding thing includes：Coating hemoglobin is that the first of the capture antibody of HB first is consolidated Phase carrier, coating transferrins are that the second solid phase carrier and coating pyruvic acid of the capture antibody of TF second swash Enzyme M2 is the 3rd solid phase carrier that PKM2 the 3rd captures antibody；Wherein, described each capture antibody is by altogether Valency crosslinking method is combined with solid phase carrier；
Described luminous conjugate includes：For being incorporated into first detection antibody of HB, for being incorporated into TF The second detection antibody and the 3rd detection antibody for being incorporated into PKM2, wherein, described each detection Antibody carries detectable light emitting molecule；
B the solid phase binding thing is placed in the first container by (), described luminous conjugate is placed in second container, And it is assembled into the kit described in claim 1.
9. a kind of method that non-diagnostic and non-therapeutic ground detect sample, it is characterised in that including step：
(1) described kit arbitrary just like claim 1-7 is provided；
(2) sample for being derived from excrement is provided, and optionally, described sample is processed, separate institute State the protein component in sample；
(3) antibody-antigene reaction is carried out with described solid phase binding thing and described sample, obtain containing solid phase First reactant liquor of carrier-capture antibody-protein complexes；
(4) resisted with described solid phase carrier-capture antibody-protein complexes with described luminous conjugate Body-antigen-reactive, obtains containing solid phase carrier-capture Antibody-protein-detection antibody-light emitting molecule compound The second reactant liquor；
(5) optical detection is carried out to the second described reactant liquor, detected value is obtained；
Wherein, described albumen is the combination of following albumen：HB, TF and PKM2.
10. a kind of purposes of combination, described is combined as Hb H B, transferrins i.e. TF and acetone The combination constituted by acid kinase M2 (i.e. PKM2), or described it is combined as HB antibody, TF antibody and PKM2 The combination constituted by antibody, it is characterised in that for preparing detection or diagnosing the kit of lower digestive tract tumour.
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Application publication date: 20170329