CN107300621A - Quantitatively detect the detection reagent card and system of human complement factor H GAP-associated protein GAPs - Google Patents
Quantitatively detect the detection reagent card and system of human complement factor H GAP-associated protein GAPs Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a kind of system for quantitatively detecting human complement factor H GAP-associated protein GAPs, including detection reagent card and immune quantitative analyzer；Reagent card is sequentially provided with sample pad, colloidal gold pad, nitrocellulose filter and adsorptive pads from left to right；Backing is provided with the lower section of sample pad, colloidal gold pad, nitrocellulose filter and adsorptive pads.Present system accuracy of reading, data are reliable；Detection efficiency is high, and whole detection process only needs 15min to complete；Sensitivity is high, it is only necessary to 100 μ L urine samples, while expanding detection range, detection range reaches as high as 32U/mL and strengthens the reference assessment to the different order of severity risks of tumor of bladder；Simple to operate, urine sample obtains simple and convenient, and noninvasive no pain, can be achieved to detect by the bed of patient.
Description
Technical field
The present invention relates to biomedicine technical field.
Background technology
CFH GAP-associated protein GAP (human complement factor H related protein), also known as
Bladder tumor antigen (Bladder tumor antigen), complement factor H（Complement Factor H）Be its it is specific into
Point, it, by cancer cell and macrophage generation, is not that normal epidermis cell is produced that complement factor H, which is, tumor cell secretion
Endogenous basement proteins and basement membrane surface protein receptor binding, and discharge proteolytic enzyme destruction basilar memebrane, basilar memebrane fragment
（Collagen fragment, glycoprotein and proteoglycan etc.）Polymer composite is polymerized in into bladder.Size is 16~165kd, with urine
Liquid is discharged, and can be detected as bladder tumor antigen.
Clinically diagnosis detection Superficial bladder cancer standard method mainly has urine sediment inspection, cystoscopy at present
And tissue biopsy.Urine sediment checks that specificity is high, but low to being classified low tumor of bladder sensitiveness, is especially easy to miss inspection C1
Level tumour.And cystoscope is invasive inspection, and it is costly, it is impossible to early diagnose and patient compliance is poor.Urine sediment is examined
Look into, indwelling twenty-four-hour urine, the urine for removing face deposition does microexamination, continuously does three days.Tumor of bladder biopsy is usually wing
One piece of tissue is taken to do pathological examination with pincers under Guang mirror.It is thin that sealing wax section microscopy urine sediment inspection is mainly used in upper urinary tract
Born of the same parents' cancer, the most important clinical characters of bladder transitional cell carcinoma be easy recurrence and with recurrent number increase tumour be more prone to it is pernicious.
Therefore select suitable molecular biology mark to predict the recurrence of bladder transitional cell carcinoma, effective remedy measures can be taken in early days, reduce
Tumour progression chance.
The examination of tumor of bladder is first using ultrasound, and ultrasound finds there is thing, pelvic cavity CT is, if to determine whether swell
Knurl, makees cystoscope and takes tissue biopsy（It is not do urine sediment）It is first ultrasonic（It is noninvasive and cheap）, then CT again（Costliness is again
There is ray）, finally it is only cystoscope（Invasive also costliness）.
At this stage, urgently need to develop a kind of can early diagnose as tumor of bladder and a kind of detection of fast quantification
System, and method can be commonly used to masses.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of detection examination of quantitatively detection human complement factor H GAP-associated protein GAPs
Agent card and system, present system accuracy of reading, data are reliable；Detection efficiency is high, and whole detection process only needs 15min complete
Into；Sensitivity is high, it is only necessary to which 100 μ L urine samples, while expanding detection range, detection range reaches as high as 32U/mL and strengthened to wing
The reference of Guang tumour difference order of severity risk is assessed；Method is simple to operate, and urine sample obtains simple and convenient and noninvasive painless
Hardship, can be achieved to detect by the bed of patient.
The detection reagent card of human complement factor H GAP-associated protein GAPs is quantitatively detected, sample pad, colloid are sequentially provided with from left to right
Gold pad, nitrocellulose filter and adsorptive pads；
Backing is provided with the lower section of sample pad, colloidal gold pad, nitrocellulose filter and adsorptive pads.
It is preferred that reagent card on the outside of be additionally provided with and get stuck.Get stuck is used to fix sample pad, colloidal gold pad, nitric acid fibre with backing
The plain film of dimension and adsorptive pads.
Sample pad is the polyester fibers of mat through the 0.1M PBSs processing containing 0.2% Tween-20；
The antibody of the anti-human complement factor H of colloid gold label is fixed with colloidal gold pad；The particle diameter of the colloid gold particle is
30nm；The amount of the anti-human complement factor H antibody of the colloid gold label is 30 μ g/mL；
There are the coated detection line of antibody and the coated nature controlling line of IgG antibody of anti-human complement factor H on nitrocellulose filter；Institute
It is 0.5mg/mL to state detection line line concentration.
The antibody of anti-human complement factor H is the anti-human complement factor H antibody of mouse.
IgG antibody is rabbit anti-mouse igg antibody.
The antibody of anti-human complement factor H is monoclonal antibody, and IgG antibody is polyclonal antibody.
Colloid gold particle is prepared by the chlorauric acid solution of 1% mass concentration and the citric acid three sodium solution of 1% mass concentration, chlorine
The volume ratio of auric acid solution and citric acid three sodium solution is 1:2.8.
30 μ g anti-human complement factor H antibody is added in per mL colloidal gold solutions, with 0.1mol/L K2CO3Adjust pH.
Quantitatively the system of detection human complement factor H GAP-associated protein GAPs includes detection reagent card and immune quantitative analyzer；Examination
Agent card is sequentially provided with sample pad, colloidal gold pad, nitrocellulose filter and adsorptive pads from left to right；
Backing is provided with the lower section of sample pad, colloidal gold pad, nitrocellulose filter and adsorptive pads.The outside of reagent card can also be set
Have and get stuck.Get stuck is used to fix sample pad, colloidal gold pad, nitrocellulose filter and adsorptive pads with backing.
Also include the test chip for storing the detection reagent card standard curve inside immune quantitative analyzer；Standard is bent
The acquisition methods of line are：The human complement factor H protein titer of various concentrations is detected using immune quantitative analyzer, mark is drawn
Directrix curve, is stored in test chip.
The operation principle of present system is：Detection sample is added drop-wise in sample pad, sample and the glue in colloidal gold pad
The conjugate of body gold and antibody fully reacts, and forms sample-collaurum-antibody complex；Due to the capillary of nitrocellulose filter
Pipe is acted on, and its sample-collaurum-antibody complex will be moved forward along the film, and fixed when being moved to detection line T, nature controlling line C
When having on the region of antibody, sample-collaurum-antibody complex and the antibody in its region are specifically bound, so as to realize
Specific immunodiagnosis.
The application method of present system is：
Step one：It will detect that sample is added on the sample application region of detection reagent card sample pad；
Step 2：The optical density of the detection reagent card detection zone is read using immune quantitative analyzer, when complement factor H albumen
The U/mL of ＞ 0.5, represent that the risk with tumor of bladder is larger；As the U/mL of complement factor H albumen ＜ 0.5, represent to suffer from bladder
The risk of tumour is smaller.
Sample described in the detection method of the present invention is human urine, and the sample-adding amount of the sample is 100~300 μ L.
Immune quantitative analyzer is used for the optical density of detection reagent card detection zone.
The effect of sample pad is to be used to absorb sample.
It is using the beneficial effect produced by above-mentioned technical proposal：
Present system includes detection reagent card and immune quantitative analyzer, and immune quantitative analyzer detects the reagent card inspection
The optical density in area is surveyed, the quantitative detection to human complement factor H protein can be achieved, and then realize the reference to tumor of bladder risk
Assess, as the U/mL of complement factor H Protein G T.GT.GT 0.5, represent that the risk with tumor of bladder is larger；As complement factor H albumen ＜
0.5 U/mL, represents that the risk with tumor of bladder is smaller.
Immune quantitative analyzer also includes the test chip for storing the standard curve of the reagent card.Pass through immune quantitative
Analyzer reads the standard curve in chip, can save the time of the standard curve of clinical acquisition so that whole detection process is only
Need 15min to complete, improve detection efficiency.
Reagent card contains the monoclonal antibody of two plants of anti-human complement factor Hs.The anti-human complement factor H albumen of first mouse
Monoclonal antibody is incorporated into gold pad with colloidal gold conjugate, and it can be combined with urine.The anti-human complement factor H albumen of second mouse
Monoclonal antibody is placed on nitrocellulose filter, is reacted with the immune conjugate of first monoclonal antibody, so as to realize detection
The purpose of specific complement factor H albumen in urine, which raises sensitivity, it is only necessary to 100 μ L urine samples, while expanding detection model
Enclose, detection range reaches as high as 32U/mL and strengthens the reference assessment to the different order of severity risks of tumor of bladder.
The particle diameter of the colloid gold particle of the detection reagent card of the present invention is 30nm, and the potency of labelled antibody is 1：2-4×
106, the antibody line concentration of the anti-human complement factor H albumen of detection line mouse is 0.5mg/mL, can ensure the present invention under this technique
Immune quantitative analyzer can realize accurate reading, its data is reliable.
The system operatio of the present invention is simple, and detection sample is urine, and sample obtains simple and convenient, and noninvasive no pain, can
Realize and detected by the bed of patient.
Brief description of the drawings
Fig. 1 is the structural representation of detection reagent card of the present invention；
Fig. 2 is collaurum form schematic diagram；
Fig. 3 is that collaurum UV scanning identifies schematic diagram；
Fig. 4 is collaurum binding antibody principle schematic；
Fig. 5 is the standard curve of detection chip；
Wherein, 1 is sample pad, and 2 be colloidal gold pad, and 3 be nitrocellulose filter, and 4 be adsorptive pads, and 5 be backing, and 6 be detection line T, 7
For nature controlling line C.
Embodiment
Embodiment 1
A kind of system for quantitatively detecting human complement factor H GAP-associated protein GAPs, including detection reagent card and immune quantitative analyzer；
Reagent card is sequentially provided with sample pad 1, colloidal gold pad 2, nitrocellulose filter 3 and adsorptive pads 4 from left to right；
Backing 5 is provided with the lower section of sample pad 1, colloidal gold pad 2, nitrocellulose filter 3 and adsorptive pads 4；
Also include the test chip for storing the detection reagent card standard curve inside immune quantitative analyzer.
The preparation process of detection reagent card of the present invention is：
First, the preparation of anti-human complement factor H antibody and the determination of pairing antibody
The preparation and purification of mouse anti-human's complement factor H protein monoclonal antibody：Human complement factor H protein is used as immunizing antigen
Immune 6 week old health BALB/c female mices, prepared by hybridoma technology and limiting dilution method routinely and screening monoclonal is anti-
Body cell strain, and then by anti-human complement factor H protein specific antibody cell line Multiplying culture, it is expelled to preparation in Mice Body
Ascites, gained ascites carries out antibody purification through octanoic acid-ammonium sulfate precipitation method.
The screening of monoclonal antibody：Screening obtains 80 plants of monoclonal cell strain antibodies, and being added testing inspection through ELISA draws
Monoclonal antibody 1H8 and 6C9 is matches antibody, and additivity index highest, and its additivity index AI=97.6%, two strain antibodies are combined
The different comformational epitopes of antigen and nitrocellulose filter and mark collaurum are coated with respectively apart from far, and by its two strain antibody,
Human complement factor H protein is measured, as a result shows that 1H8 mark collaurums are shown with 6C9 coating nitrocellulose filter detections
Preferably, wherein 1H8 monoclonal antibodies potency is 1 to effect:2.8 × 106,6C9 monoclonal antibody potency are 1：4 × 106, hypotype is IgG1, albumen
Concentration is respectively 5mg/mL, 6.2mg/mL, is suitable for the preparation of next step gold-immunochromatographyreagent reagent for assay card device.
2nd, the preparation of collaurum
Collaurum（Colloidal gold）It is gold chloride（HAuCl4）The hydrosol, gold chloride in the presence of reducing agent, gather
The gold grain of particular size is synthesized, and because electrostatic interaction turns into a kind of colloidal state of stabilization.The preparation of collaurum is typically adopted
With reducing process, conventional reducing agent has sodium citrate, tannic acid, ascorbic acid, white phosphorus, sodium borohydride etc..According to different preparations
Method, can prepare diameter 1-500nm colloidal gold particle, but as immune labeled probe, its diameter should be in 3-30nm models
In enclosing.The good collaurum of the quality judging standard quality of collaurum：Solution takes on a red color, and colloid gold particle is spherical, and size is homogeneous,
Without corner angle and particle diameter distribution is uniform.（Such as Fig. 2, shown in Fig. 3）Ropy collaurum：Solution is in purple, and not of uniform size, shape is each
It is different.
In chlorauride（HAuCl4）Reducing agent is added in the aqueous solution to be allowed to reduce and build up to form colloidal gold particle.Collaurum
It is negatively charged under mild alkaline conditions, can be with protein molecule（Immunoglobulin）Positive charge group formed and firmly combine to form
Colloid gold label thing, because this combination is electrostatical binding, so not influenceing the biological nature of protein.（Such as Fig. 4）
The present invention prepares 30 nm collaurums using sodium citrate, takes 250 ml triangular flasks one, plus 100 ml distilled waters and
The chloraurides of 1 ml 1%, ebuillition of heated；Different amounts of 1% sodium citrate is taken to add in above-mentioned solution.Mix, then keep boiling 30
Min, solution colour blackening first, then gradually redden, when particle volume is smaller, solution is in salmon pink, and particle volume it is larger when,
Then color is inclined to purple.The present invention adds 2.8mL trisodium citrate, and obtaining 30nm colloid gold particle is used for next step reality
Test. SHAPE \* MERGEFORMAT
Collaurum suitable label concentration, PH determination
Antibody is determined with collaurum combination Optimal pH：Because albumen optimum PH range is narrower, the gradient of setting can not be too big.Take one
96 well culture plates, take 100 μ L to add in hole above-mentioned collaurum, with 0.1mol/L K respectively respectively from low to high by pH2CO3Adjust
Section pH value is 7.2,7.6,7.9,8.2 (multiple holes), 8.5（Multiple holes）, 8.8,9.2 and blank well, with 0.22 μm of miillpore filter mistake
Filter or high speed centrifugation remove residual thing or polymer in antibody, and its each hole is quantitatively adding, and mixed room temperature places 15min, add respectively
Enter and 10 min are placed under 10% NaCl solution, mixed room temperature；Observing colloid gold color change, recording the minimum pH for keeping red is
8.2, it does not occur color change and coagulation phenomenon, and 8.2 be optimum pH.
Antibody is determined with collaurum combination optium concentration：By collaurum 0.1mol/L K2CO3PH is adjusted to 8.2, takes 1
Well culture plate, every 100 μ L add each hole, and residual thing or poly in antibody are removed with 0.22 μm of filtering with microporous membrane or high speed centrifugation
Body, it is 0.5 μ g, 1.0 μ g, 1.5 μ g, 2.0 μ g, 2.5 μ g to add each hole amount of antibody（Multiple holes）, 3.0 μ g（Multiple holes）, 3.5 μ g（It is multiple
Hole）, 5min is placed under mixed room temperature, adds after 10% NaCl and observes, color still keeps red minimum albumen consumption i.e. minimum
Protein concentration, in actual probes preparation work, protein concentration is often the 120% of Cmin.Five, the six holes of the invention are
Minimum protein concentration is the μ L collaurums of 2.5 μ g antibody labelings 100, and it is 3 μ g that reality Jia 20% again on this basis, that is, stablizes 1mL
Collaurum optimum mark protein content is 30 μ g.
Colloid gold label anti-human complement factor H monoclonal antibody（Fig. 3）：PH8.2 30nm colloidal gold solutions are taken out
1mL, the 1H8 antibody purifications for adding 30 μ g are mixed, and room temperature places 10min, add 10 μ L 2% PEG20000, and room temperature places 5
min；10000 rpm centrifuge 20 min, gently absorb supernatant；The loose collaurum precipitation of 0.1M PBST solution resuspensions, union
In into new pipe, fully mix, -20 DEG C save backup.
3rd, on nitrocellulose filter detection line concentration determination
Nature controlling line in the present embodiment（C lines）It is coated with rabbit anti-mouse igg antibody, detection line（T lines）It is coated with for 6C9 antibody.Fixed C lines
Concentration, changes T line concentration, is reference with the T lines depth, sensitivity etc., selects the optium concentration of T lines line.T lines line concentration point
Not Wei 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, by detection experiment draw when T lines line concentration be 0.5mg/mL when, colour developing very
Deep, the easy interpretation of instrument will not waste antibody, therefore T lines most preferably rule concentration for 0.5mg/mL.
4th, prepared by test chip
Human complement factor H protein is configured to 0.5 U/mL, 1 U/mL, 2 U/mL, 4 U/mL, 8 U/mL, 16 U/mL, 32
The series standard solution such as U/mL.With the detection reagent card of the present invention, each concentration is repeated 3 times, and carries out immune quantitative analyzer reading
Number, so as to obtain the standard curve of human complement factor H protein optical density and concentration.It is final to determine standard curve（Such as Fig. 5 institutes
Show）.The detection sensitivity of detection reagent card prepared by the present invention is linearly in the range of 0.5-32 U/mL up to 0.5 U/mL
R2=0.9987。
, need to be in the interior repeatability of same concentration batch because the detection reagent card of every batch has the difference manufactured with operation, will
Human complement factor H protein is configured to 1 U/mL standard liquids, does 15 repetitions, is detected, and observes its repeatability, such as table
Shown in 1, batch interior CV=10.3%
The reperformance test data of the different detection reagent cards of table 1
5th, the detection of actual sample is carried out using present system
The monitoring of Hebei hospital journals, 2016.11-2017.5 totally 80 suspected patients because of blood urine（53）Or other main suits arrive
Certain hospital outpatient is gone to a doctor.
This group 80, man 61, female 19, age 20-88 Sui.
All patients carry out the imageological examinations such as conventional preoperative planning, and B ultrasound, CT or CTU after being admitted to hospital.
The urine specimen of all patients is left and taken before cystoscopy, carries out the detection of detection reagent card of the present invention.
It is positive 64：Including urothelial tumor 51（Carcinoma of urinary bladder 34, tumor of renal pelvis 10, tumor of ureter 7)；
Urothelium papillary hyperplasia is with different increasing 4, ureteral calculi 5, urinary infection 2, chyluria 1, cystectomy art
Uronephrosis 1 afterwards；Detection reagent card ＞ 0.5U/mL of the present invention.
It is negative 16：Including carcinoma of urinary bladder 1, clear cell carcinoma of kidney 1, prostate cancer example, renal cyst 3, caruncle of urethra 1
Example, hyperplasia of prostate 4, ureteral calculi 1, the postoperative check no abnormality seen 5 of carcinoma of urinary bladder；Detection reagent card detection of the present invention
＜ 0.5U/mL.
Claims (10)
1. a kind of detection reagent card for quantitatively detecting human complement factor H GAP-associated protein GAPs, it is characterised in that：
The reagent card is sequentially provided with sample pad from left to right（1）, colloidal gold pad（2）, nitrocellulose filter（3）And adsorptive pads
（4）；
In sample pad（1）, colloidal gold pad（2）, nitrocellulose filter（3）And adsorptive pads（4）Lower section be provided with backing（5）.
2. the detection reagent card according to claim 1 for quantitatively detecting human complement factor H GAP-associated protein GAPs, its feature exists
In：It is additionally provided with and gets stuck on the outside of the reagent card.
3. the reagent card according to claim 1 for quantitatively detecting human complement factor H GAP-associated protein GAPs, it is characterised in that：Institute
The sample pad stated（1）It is the polyester fibers of mat through the 0.1M PBSs processing containing 0.2% Tween-20；
Described colloidal gold pad（2）On be fixed with colloid gold label anti-human complement factor H antibody；The colloid gold particle
Particle diameter be 30nm；The amount of the anti-human complement factor H antibody of the colloid gold label is 30 μ g/mL；
Described nitrocellulose filter（3）On have the coated detection line of antibody and IgG antibody of anti-human complement factor H coated
Nature controlling line；The detection line line concentration is 0.5mg/mL.
4. the reagent card according to claim 3 for quantitatively detecting human complement factor H GAP-associated protein GAPs, it is characterised in that：Institute
The antibody for the anti-human complement factor H stated is the anti-human complement factor H antibody of mouse.
5. the reagent card according to claim 3 for quantitatively detecting human complement factor H GAP-associated protein GAPs, it is characterised in that：Institute
The IgG antibody stated is rabbit anti-mouse igg antibody.
6. the reagent card according to claim 3 for quantitatively detecting human complement factor H GAP-associated protein GAPs, it is characterised in that：Institute
The antibody for the anti-human complement factor H stated is monoclonal antibody, and IgG antibody is polyclonal antibody.
7. the reagent card according to claim 3 for quantitatively detecting human complement factor H GAP-associated protein GAPs, it is characterised in that：Institute
The colloid gold particle stated is prepared by the chlorauric acid solution of 1% mass concentration and the citric acid three sodium solution of 1% mass concentration, gold chloride
The volume ratio of solution and citric acid three sodium solution is 1:2.8.
8. the experimental rig according to claim 7 for quantitatively detecting human complement factor H GAP-associated protein GAPs, it is characterised in that：
30 μ g anti-human complement factor H antibody is added in per mL colloidal gold solutions, with 0.1mol/L K2CO3Adjust pH.
9. a kind of system for quantitatively detecting human complement factor H GAP-associated protein GAPs, it is characterised in that：Including detection reagent card and immune
Quantitative analysis instrument；
The reagent card is sequentially provided with sample pad from left to right（1）, colloidal gold pad（2）, nitrocellulose filter（3）And adsorptive pads
（4）；
In sample pad（1）, colloidal gold pad（2）, nitrocellulose filter（3）And adsorptive pads（4）Lower section be provided with backing（5）.
10. the system of human complement factor H GAP-associated protein GAPs is quantitatively detected according to claim 9, it is characterised in that：It is immune fixed
Also include the test chip for storing the detection reagent card standard curve inside amount analyzer.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111579786A (en) * | 2020-05-27 | 2020-08-25 | 郑州大学第一附属医院 | Test strip for screening early esophageal squamous carcinoma of high risk group |
| CN111650371A (en) * | 2019-03-04 | 2020-09-11 | 深圳市第二人民医院 | Bladder cancer BTA test strip based on nanoenzyme |
| CN114605519A (en) * | 2020-12-03 | 2022-06-10 | 四川远大蜀阳药业有限责任公司 | Method for extracting H factor |
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