CN101377506A - Monophosphoinositide proteoglycans-3 chemiluminescence immune analysis determination reagent kit and preparing method thereof - Google Patents
Monophosphoinositide proteoglycans-3 chemiluminescence immune analysis determination reagent kit and preparing method thereof Download PDFInfo
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Abstract
The invention relates to the medical field of immunoassay, more specially, the invention provides a chemiluminescent immunoassay detection kit for phosphatidylinositol proteoglycan-3(GPC-3) and a preparation method thereof, and realizes the simultaneous serological detection of GPC-3 N terminal and C-terminal protein with the chemiluminescent immunoassay method. The kit has the advantages of simple sampling, convenient detection and accurate and specific technical method. The invention adopts a biotin-strapavidin system to coat antibodies and improve the efficiency of antibody coating and the linear range of detection as well as sensitivity, and can be conveniently used for the tracing observation of early diagnosis or treatment effect for primary carcinoma of liver.
Description
Technical field
The present invention relates to the immunoassay medical domain, concrete, the invention provides an a kind of step immune analysis method fast, accurately, relative broad range measures chemical luminescence immune analysis reagent box of Monophosphoinositideproteoglycans proteoglycans-3 (GPC3) and preparation method thereof.This kit is measured the protein content of GPC3 in the serum sample accurately, really, comprises N end protein and C end protein, can truly reflect generation, the development of primary hepatocarcinoma patient in-vivo tumour.
Background technology
Liver cancer is divided into primary carcinoma of liver (Hepatocellular carcinoma HCC) and secondary carcinoma of liver, and (Cholangiocarcinoma, Ccc) and three kinds of mixed carcinoma of liver, wherein HCC accounts for more than 90%.HCC is a fifth-largest common cancer in the world wide, and lethality occupy the 3rd; Liver cancer mainly is distributed in Africa, East Asia Region.In China, the onset of liver cancer rate is that other regional three-to-four-fold .HCC of the world is that hepatitis B, third liver and cirrhosis further develop, and symptom shows as the expansion of stomachache (upper right portion), belly, easily produce stasis of blood green grass or young crops bleeds and jaundice.Mistaken diagnosis mostly is late period when finding HCC because HCC often is accompanied by liver diseases such as hepatitis, perhaps because.So need special TM as the auxiliary diagnosis standard.But because the AFP false positive is than higher; Other primary carcinoma of liver (HCC) patient AFP more than 30% does not raise.So the researchist constantly seeks more special, sensitiveer TM all the time.If can check in early days, can reduce probability that HCC takes place or the generation of thoroughly eliminating HCC.
GPC-3 has prospect liver cancer tumor markers very much.GPC-3 belongs to the glypican that is connected on the heparan phosphate proteoglycan, and GPC can promote cell growth, cell differentiation and cell migration.Report GPC has six kinds of different types, and the probability that occurs in various disease is inequality, therefore can characterize different diseases.GPC-3mRNA molecular weight 2.4kb wherein, the molecular weight 35～66kDa of albumen can occur in the HCC patient body He in the melanoma patient body, reports that maximum is the diagnosis that is applied to HCC.In reporting at present, GPC-3 is the most special mark in the HCC diagnosis, can reach 80%, both can be used for early diagnosis, also can be used for the treatment of the observation of process curative effect.GPC-3 can distinguishing benign and pernicious liver diseases in addition, and the variation of GPC-3 content in blood can reflect HCC growth of tumor situation.
The initial mRNA that utilizes it that detects of GPC-3, mainly be to carry out GPC-3 at GPC-3mRNA content increase in the body to detect, the main Northern of employing blot, RT-PCR, SABC and original position are divided technology such as hybridization, these technology have determined that the mensuration to GPC-3 can only be qualitative determination, and concrete quantitative data can't draw.The mRNA content that detects the GPC-3 correspondence in the tissue samples of primary hepatocarcinoma patient and postoperative patient has increased by 75% than the normal person.The corresponding mRNA of the GPC-3 that raises also is present among the blood, and the sensitivity of its detection has surpassed AFP; Tumour caking is less than 3cm, and sensitivity is higher.But tissue detection, sampling is complicated, and testing process is consuming time, is unfavorable for clinical practice and popularization.
GPC3 antibody research beginning is ripe and the immuno analytical method of GPC-3 occurs after 2003, comprises the ELISA detection technique, and detection by quantitative just begins to occur, quantitative technique have people the begin one's study variation of GPC-3 content and a relation of HCC developing stage.Have in the research and report, although in the blood in GPC-3 protein content and the tissue mRNA expression not to be dominance relevant, the rising of protein content and situation of change still reflect generation, the development of HCC in the blood, so can belong to the serology tumor markers.Have and report it mainly is that the N end form of GPC-3 albumen enters blood circulation, relevant report for work and patent is set up the ELSIA method and detected N end protein in the blood being arranged.But follow-up report afterwards shows its C end protein and also enters blood circulation, so the GPC3 serology detection accuracy that previous patent adopts N end protein antibody to carry out is challenged.
The GPC3 protein content has in primary hepatocarcinoma patient serum significantly and raises in addition, can reach thousands of ng/ml, compares broad so it is detected the range of linearity that requires to detect.
Summary of the invention
(1) technical issues that need to address
The present invention for the clinical diagnosis basis accurately that provides, has solved the problems referred to above in order to satisfy simple, fast detecting requirement, promptly replaces loaded down with trivial details histology, has set up GPC3 serology detection method accurately, detects when having realized N end and C end protein.And solved the requirement that conventional coated in microporous plate antibody can't realize that wide region detects, be that this kit is on the chemiluminescence immunoassay technology platform base of sensitivity, microwell plate adopts streptavidin to modify special being combined on the solid phase carrier of biotin labeled then antibody.
The purpose of this invention is to provide a kind of GPC3 serology detection kit, adopt chemiluminescence immunoassay technology, quantitatively, fast detecting GPC3.
Another object of the present invention is to realize the detection of broad linear scope, improves the range of application and the using value of kit.
(2) technical scheme
Comprise in detection GPC3 chemiluminescence immune analysis method of the present invention and the assembling kit: the GPC3 standard items; The microwell plate of GPC3 polyclonal antibody bag quilt; The GPC3 monoclonal antibody of horseradish peroxidase-labeled; Luminous substrate night.
GPC3 chemical luminescence immune analysis reagent box of the present invention, preparation process is as follows:
1. select appropriate calibration product dilution, and with the pure product preparation of GPC-3 calibration object, concrete GPC3 standard items concentration is respectively: 0ng/ml, 10ng/ml, 50ng/ml, 150ng/ml, 500ng/ml, 1500ng/ml.
2.GPC-3 the polyclonal antibody bag is by microwell plate, concrete step comprises:
1) microwell plate of Streptavidin modification;
2) biotin labeling GPC3 polyclonal antibody;
3) with biotin labeled GPC3 polyclonal antibody bag by on the microwell plate of modifying in Streptavidin;
4) sealing
3. prepare the GPC-3 polyclonal antibody of horseradish peroxidase-labeled, concrete step comprises:
1) 12mgHRP is dissolved among the 1ml PBS (0.1Mol/L pH6.8) that contains the 5mgGPC3 monoclonal antibody, slowly stirs adding 1% glutaraldehyde solution 4mL down, put room temperature 3 hours.
2) add 0.1mL 0.2mol/L lysine (0.29g is dissolved in 10mL distilled water), place 2h for 4 ℃, to seal residual aldehyde radical, cessation reaction.
3) bag filter of packing into, with 0.01mol/L, pH7.2 PBS, 4 ℃ of dialysed overnight.
4) add equivalent 60% glycerine, packing in a small amount ,-20 ℃ of preservations.
4. prepare the chemical luminous substrate liquid that horseradish peroxidase acted on, concrete comprises:
1) based on the described chemical luminous substrate A of 1000mL liquid, comprise 1.7716g luminol, 0.051g 4-xenol, 0.012g 4-iodobenzene boric acid, 11.4g boric acid, 4.9g borax, its pH value is 8.0～10.0;
2) based on the described chemical luminous substrate B of 1000mL liquid, comprise 0.329g urea peroxide, 1ml Tween20,51.58gNa2HPO412H2O, 8.74g NaH2PO42H2O, its pH value is 7.0～7.6.
A liquid B liquid 1:1 mixes use during use.
5. preparation concentrated cleaning solution;
6. a packing component, and be assembled into finished product.
The present invention's " Monophosphoinositideproteoglycans proteoglycans-3 (GPC3) chemical luminescence immune assay determination reagent kit " can detect Monophosphoinositideproteoglycans proteoglycans-3 (GPC3) the form of ownership protein content in the primary hepatocarcinoma patient body on the one hand exactly, provides reliable clinical reference value for small liver cancer patient early diagnosis, early treatment; On the other hand owing to use specific bond bag between streptavidin-biotin by solid phase, therefore effective quantity of the coated antibody that improves both can be saved antibody, quantity that again can Zheng family's coated antibody, thereby the saving cost, the range of linearity of realization relative broad range.
Be somebody's turn to do " Monophosphoinositideproteoglycans proteoglycans-3 (GPC3) chemical luminescence immune assay determination reagent kit ", the Monophosphoinositideproteoglycans proteoglycans-3 of the antibody of bag quilt and sample forms the sandwich complex structure of " coated antibody-Ag-Ab-enzyme " on the antibody of enzyme labeling and the carrier, so " double-antibody sandwich single stage method " reaction pattern of the present invention's employing.The present invention has effectively utilized the chemiluminescence principle, has guaranteed the sensitivity that detects.And applicability easy and simple to handle is wide, both can be applicable to the open luminous measuring instrument of semi-automatic chemistry, also can be used for full automatic measuring system, can realize fast detecting in enormous quantities, use cost is low, easier applying is particularly suitable for vast hospital and promotes the use of, for clinical diagnosis and research work provide a kind of very valuable detection means.
Description of drawings
Fig. 1 is the calibration object linear graph in the prepared kit of embodiment 1
Fig. 2 is the together external clinical blood sample measured value of the kit comparison chart of the present invention
Embodiment
Embodiment 1 preparation Monophosphoinositideproteoglycans proteoglycans-3 chemiluminescence immune analysis determination kit of the present invention
One. the configuration of standard items
With horse serum the pure product of GPC-3 are diluted to calibration object, packing concentration is respectively 0ng/ml, 15ng/ml, 50ng/ml, 150ng/ml, 500ng/ml, 1500ng/ml, totally 6 bottles.
Two. the preparation of antibody sandwich plate
1. the microwell plate modified of Streptavidin: Streptavidin with 10mg/L (pH 9.6 0.05mol/L carbonic acid buffers) concentration bag by micropore (100 μ g/ hole), 4 ℃ are spent the night, take out next day, wash 3 times (cleansing solution 0.05%tween20, pH7.4,0.01mol/L PBS), with 37 ℃ of sealings of 10% calf serum 30min, wash 3 times.
2. biotin labeling GPC3 polyclonal antibody: with the biotin succinimide ester under the alkalescence condition with monoclonal antibody generation coupling reaction, fully dialyse with PBS, biotinylated antibody dilutes with calf serum, adds proclin300, preserves below-20 ℃.
3. the 0.046M pH value of bag quilt: 1000mL is that 4.6 citrate buffer solution comprises the citric acid of 4.44g and the trisodium citrate deionized water solution of 7.3g is made damping fluid, with finite concentration, approximately the biotin labeling GPC3 polyclonal antibody of 2-4 μ g/mL is mixed and made into coating buffer.The every hole of microwell plate adds 150 μ L coating buffers, and the bag quilt spends the night;
4. sealing: the preparation confining liquid, as follows,
NaH
2PO
4·2H
2O 0.2g
Na
2HPO
4·12H
2O 2.9g
BSA 10g
Proclin300 1ml
Distilled water is settled to 1000ml
The mentioned reagent weighing is put into clean container well, add the distilled water constant volume, the dissolving mixing, measuring the pH value is 7.0.Every hole adds confining liquid 300 μ L respectively, and room temperature was placed 3 hours.Get rid of confining liquid, on thieving paper, pat dry.Room temperature removal moisture drying 24 hours.Carry out vacuum sealing bag, behind the envelope, labeling is put 4 ℃ of preservations.
Three. the preparation of enzymic-labelled antibody:
1, Gai Liang glutaraldehyde cross-linking method mark horseradish peroxidase (HRP)
1) 12mgHRP is dissolved among the 1ml PBS (0.1Mol/L pH6.8) that contains the 5mgDCP monoclonal antibody, slowly stirs adding 1% glutaraldehyde solution 4mL down, put room temperature 3 hours.
2) add 0.1mL0.2mol/L lysine (0.29g is dissolved in 10mL distilled water), place 2h for 4 ℃, to seal residual aldehyde radical, cessation reaction.
3) bag filter of packing into, with 0.01mol/L, pH7.2 PBS, 4 ℃ of dialysed overnight.
4) product of will dialysing moves into reagent bottle and adds equivalent 60% glycerine, and mixing adds 1% BSA again, packing in a small amount ,-20 ℃ of preservations.
2, enzyme labelled antibody concentration is selected
Adopting the square formation method to select the working concentration of enzyme labelled antibody is 1:4000.
3, the selection of enzymic-labelled antibody dilution
With the dilution of BSA albumen damping fluid as the enzyme labeling thing, concrete protein concentration adopts the dilution of the definite enzymic-labelled antibody of chessboard test to be specially through evidence:
| Reagent | Molecular formula | Final concentration | Molecular weight | The 1000mL consumption |
| Sodium dihydrogen phosphate | NaH 2PO 4·2H 2O | 3.8mM | 156.01 | 0.6g |
| Dibastic sodium phosphate | Na 2HPO 4·12H 2O | 16.2mM | 358.14 | 5.8g |
| Potassium chloride | KCl | 0.076％ | 58.5 | 0.76g |
| Bovine serum albumin(BSA) | BSA | 1.0％ | 10g | |
| Biological preservative | Proclin300 | 0.1％ | 1.0ml | |
| Food Red | 0.05‰ | 1.0ml | ||
| Purified water | H 2O | Add to 1000mL |
Four, chemical luminous substrate A liquid
Luminol 10mM 1.7716g
4-xenol 0.3mM 0.051g
4-iodobenzene boric acid 0.05mM 0.012g
Boric acid 11.4g
Borax 4.9g
The fixed molten 1000mL of distilled water
pH 8.0～10.0
Five, chemical luminous substrate B liquid
Urea peroxide 3.5mM 0.329g
Tween20 0.1％ 1ml
Na
2HPO
4·12H
2O 51.58g
NaH
2PO
4·2H
2O 8.74g
Distilled water constant volume 1000mL
pH 7.0～7.6
A liquid mixes with B liquid equal proportion during use.
Six, cleansing solution damping fluid
Na
2HPO
4·12H
2O 116g
NaH
2PO
4·2H
2O 4g
NaCl 320g
Tween-20 2ml
Deionized water 1000ml
Adjust pH to 7.2 ︿ 7.4,30 times of uses of dilution during use.
Seven, semi-manufacture and finished product are formed
Through the above-mentioned steps products obtained therefrom, be all compositions of kit of the present invention.But will meet requirements for clinical application as qualified kit, promptly satisfy specificity, accuracy, certain sensitivity and the requirement of stability, through being up to the standards, labelling is assembled into kit.
Embodiment 2 Monophosphoinositideproteoglycans proteoglycans-3 chemiluminescence immune analysis determination kit using method of the present invention
One. sample pre-treatments
Get people's empty stomach serum, the centrifugal 10min of 2000-4000rpm gets supernatant, need not other special processing, analyzes.
Two. detect step
Before using this kit to detect, need to take out earlier and divide each medicine that installs in the kit: bag is left standstill by plate, enzyme labeling thing, standard items room temperature, uses after equilibrating to room temperature; Afterwards, constant temperature incubator or water-bath are transferred to 37 ℃; Again, be ready to suitable micro sample adding appliance and corresponding suction nozzle and check whether operate as normal of Chemiluminescence Apparatus.
Use this kit as follows according to the concrete operations step that the method for embodiment 1 experimentizes:
1) takes out kit and equilibrate to room temperature;
2) will test the lath that needs is placed on the hollow plate frame;
3) add 25 μ L blood serum samples or series standard product solution (standard items 0ng/ml in the reacting hole, 15ng/ml, 50ng/ml, 150ng/ml, 500ng/ml, 1500ng/ml), add label 50 μ L again, blank 1 hole is established in each test, and except that blank well, every hole adds horseradish peroxidase-labeled antibody-solutions 50 μ L then;
4) 30 seconds mixings of vibration on the micro oscillator;
5) 37 ℃ of incubations are 30 minutes;
6) discard solution in the hole, with the washing lotion washing, automatic washer or craft are washed plate 5 times, pat dry on thieving paper;
7) every hole adds luminous substrate 100 μ L, the vibration mixing, and room temperature (22～28 ℃) left standstill 5 minutes;
8) survey relative light unit (RLU) with Chemiluminescence Apparatus, 1 second/hole of Measuring Time;
9) respectively calibration object concentration and corresponding RLU are taken the logarithm, on the double logarithmic curve of setting up, set up typical curve, on typical curve, find the concentration of the PSA of this serum, calculating testing result with each test serum RLU value;
10) print test results report.
The calibration curve of this kit is seen accompanying drawing 1, and wherein, I is a luminous intensity, and C is AFP concentration (unit is μ g/L), related coefficient Y=1.6640+1.3263X, r=0.9983.
The methodology of embodiment 3 kits of the present invention is identified
According to manufacturing conventional in this area and vertification regulation the kit of preparation in the embodiment example 1 is examined and determine, the result is as follows:
1. kit precision
(1) standard items precision experiment
With the kit of preparation among the embodiment 1, get three batches respectively and carry out the precision experiment, every batch is extracted 10 kits.With the GPC3 standard items of the kit measurement 150ng/mL that extracted among the embodiment 15 times.Calculate the coefficient of variation of measuring concentration.The measurement result of three batches of kits among the embodiment 1 is as shown in table 1, the result show the coefficient of variation 2.0%～5.5% between.
The repeatable experiment of table 1 GPC3 standard
(2) sample precision experiment
Precision is the reappearance of analysis result, prepares each 8 parts in three parts of Gao Zhong low value samples, with detecting simultaneously in once analyzing every increment this, every increment this coefficient of variation and the ratio of standard deviation promptly analyze within variance coefficient; If high, normal, basic three increments are originally, the coefficient of variation of measured value and the ratio of standard deviation are the coefficient of variation between analysis in three times are analyzed. the result shows that variation makes a variation between 5.6～8.3% between analysis in analyzing between 8.1～9.6%.
2, kit accuracy determination
Get two clinical definite value serum of GPC3, concentration is respectively 172.3ng/ml, 289.9ng/ml and 30.6ng/ml, respectively to wherein adding isopyknic certain density GPC3 standard solution, to each concentration do 3 parallel, calculate recovery rate.The result is as shown in table 4, and the interpolation recovery that shows GPC3 is between 90～109%.
The table 2 serum sample recovery
| Human serum background values ng/ml | Normal concentration value ng/ml | Actual detected value ng/ml | Recovery % |
| 172.3 | 650 | 886.6 | 109 |
| 289.9 | 280 | 537.8 | 87 |
| 30.6 | 112 | 131.58 | 90 |
3, kit stability experiment
After the kit of embodiment 1 carried out 37 ℃ of 7 days accelerated tests, the results such as recovery of standard addition that measure the maximum of kit, minimum luminous intensity, determinand showed that the kit index of embodiment 1 is all within normal range.The kit key component of embodiment 1 is carried out 2～8 ℃ of 8 months tracking tests, and the result shows that every index meets the requirements fully.Consider the generation of the freezing situation of kit, with the kit of embodiment 1 put into-20 ℃ freezing 7 days, measurement result shows that also every index of kit is normal fully.Kit can be preserved more than 6 months at least at 2～8 ℃ as can be seen from the above results.
Kit method index of the present invention is as follows through a large amount of experiment showed:
Sensing range: 0～1500ng/ml;
Sensitivity: minimum detects and is limited to 3.2ng/mL;
Precision: less than 9% (n=5);
Accuracy: average recovery rate is between 93.0%～102.0%;
Specificity: with the cross reacting rate of common tumor markers such as AFP, CA724, CA15-3 less than 0.1%;
Stability: each reagent set splits 37 ℃, investigates after 7 days, and each component is still stable.
Kit of the present invention is mainly used in Monophosphoinositideproteoglycans proteoglycans-3 (GPC3) serology and detects, and replaces traditional tissue detection, avoids complicated, the more defective consuming time of tissue detection process sampling.Kit of the present invention in addition can detect N end and the C end protein form in the serum sample simultaneously, accurately detects the expression of GPC3 albumen in the serum sample, the detection sensitivity raising, thus provide reliable reference data for clinical diagnosis.The present invention adopts chemiluminescence immunoassay technology, combines with biotin-avidin solid phase carrier coating technique, and sensitivity and sensing range improve greatly, and the detecting instrument cost is relatively low.Therefore this kit is convenient to clinical expansion, has good marketable value.
Claims (10)
1, a kind of Monophosphoinositideproteoglycans proteoglycans-3 (GPC-3) chemical luminescence immune assay determination reagent kit is characterized in that described kit comprises:
1) GPC-3 calibration object;
2) microwell plate of GPC-3 polyclonal antibody bag quilt;
3) the anti-GPC-3 monoclonal antibody of horseradish peroxidase-labeled;
4) luminous substrate liquid;
5) concentrated cleaning solution;
2, kit as claimed in claim 1 is characterized in that, described GPC-3 standard items are the people GPC3 albumen of reorganization, have high affinity with the solid phase polyclonal antibody.
3. kit as claimed in claim 1 is characterized in that, the microwell plate of described GPC3 polyclonal antibody bag quilt by the covalent bonds bag by on solid phase carrier.
4. bag as claimed in claim 3 be is characterized in that by mode described covalent bond is meant, the coated in microporous plate Streptavidin, and GPC3 polyclonal antibody mark biotin carries out the bag quilt of antibody then by the specific bond of biotin-Streptavidin.
5, a kind of method for preparing the described kit of claim 1 is characterized in that preparation process may further comprise the steps:
1) with the pure product preparation of GPC-3 calibration object;
2) GPC-3 polyclonal antibody bag is by microwell plate;
3) the GPC-3 monoclonal antibody of preparation horseradish peroxidase-labeled;
4) the preparation chemical luminous substrate liquid that horseradish peroxidase acted on;
5) preparation concentrated cleaning solution;
6) packing said components, and be assembled into the finished product kit.
6. method as claimed in claim 5 is characterized in that described bag is by the step 2 of solid phase carrier) may further comprise the steps:
1) the biotinylated GPC3 monoclonal antibody of preparation;
2) microwell plate of preparation streptavidin modification;
3) bag quilt;
4) washing rear enclosed and drying.
7. as described in the claim 6, it is characterized in that described confining liquid is a compound closure liquid: the 1000mL phosphate buffer contains 0.2g NaH
2PO
42H
2O, 2.9g NaH
2PO
412H
2O adds 10g BSA, 1g gelatin hydrolysate and 1mL proclin300, and the pH value of described confining liquid is 7.0～7.6, then the gained confining liquid is carried on the solid phase carrier after the above-mentioned washing.
8. kit according to claim 1 is characterized in that, needs with 30 times of dilutions of deionized water before described concentrated cleaning solution uses.
9. kit according to claim 1 is characterized in that, can detect the N end protein and the C end protein form of the GPC3 albumen in the serum simultaneously.
10, kit as claimed in claim 1 is characterized in that, described chemical luminous substrate liquid comprises A liquid and B liquid, wherein,
Described chemical luminous substrate A liquid comprises 10mM luminol, 0.3mM 4-xenol, 0.05mM 4-iodobenzene boric acid, 0.2M pH8.7 boric acid-borate buffer solution, and its pH value is 8.0～10.0;
Described chemical luminous substrate B liquid comprises 3.5mM urea peroxide, 0.1% Tween20,0.2M pH7.2 phosphate buffer, and its pH value is 7.0～7.6.
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