CN110531077A - Mesothelin immunologic combined detection reagent kit - Google Patents
Mesothelin immunologic combined detection reagent kit Download PDFInfo
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Abstract
This application involves a kind of mesothelin (Mesothelin, MSLN) immunologic combined detection reagent kits, contain MSLN monoclonal antibody 3-2G6.
Description
Technical field
The present invention relates to a kind of immunohistochemical kits of detection mesothelin (Mesothelin, MSLN).
Background technique
Mesothelin (Mesothelin, MSLN) is a kind of cell surface glycoprotein.Mesothelin gene coding is one
The preceding albumen of 71KD, through modification cutting become two sections, be respectively 41KD glycolsyl-phosphatidylinositol cell membrane anchorin and
The free segment for being referred to as megakaryocyte-potentiating factor of 30KD.There is research to confirm that the film anchorin of 41KD is comprising one section and another
The combined area of one tumor markers CA125 albumen, may be related to cell adherence, thus the anchorin potentially contributes to
The transfer of tumour, and cause outcome undesirable.Recent studies have shown that: MSLN in most celiothelioma and cancer of pancreas,
It is highly expressed in most oophoromas and lung cancer, in the normal tissue seldom expression, it is general only in pleural mesothelial cell lining, peritonaeum
There is a certain amount of expression with pericardium.Although the function of Mesothelin is still inaccurate at present, either cell membrane combined area is also
It is that free area is all proved to can be used as the marker and therapy target of the kinds of tumors such as carcinoma mesothelial, cancer of pancreas, oophoroma.
The cardinal principle of ImmunohistochemistryMethods Methods is: applied immunology basic principle --- antigen-antibody reaction, i.e. antigen with
The principle that antibody specificity combines makes color developing agent (fluorescein, enzyme, metal ion, the same position of labelled antibody by chemically reacting
Element) it develops the color to determine histocyte endoantigen (peptide and protein), it is positioned, qualitative and relative quantification research side
Formula.
Immunohistochemistry technique is mainly characterized by: 1. high specificities: immunologic basic principle determines antigen and antibody
Between combination have high degree of specificity；2. sensibility is high: antibody, which dilutes thousands of times, up to ten thousand times even more than one hundred million times, can still organize
With antigen binding in cell, the antibody antigen of such hypersensitivity reacts, and is conveniently used in ImmunohistochemistryMethods Methods increasingly
Routine pathology diagnostic work；3. accurate positioning: the technology, can be in tissue and cell by antigen-antibody reaction and color reaction
The accurate positionin of antigen is carried out, thus position observation can be carried out in same tissue or cell to not synantigen simultaneously, thus
The research that form and function combines can be carried out, to deeply having great importance for pathological research.In targeted drug
During research and clinical use, positioning and relative quantification are carried out to the target antigen in pathological tissue using ImmunohistochemistryMethods Methods
Detection, can predict patient whether be possible to from the treatment of the targeted drug be benefited, in targeted drug clinical test process
In success of the test rate can be improved, medication precision can be improved during targeted drug clinical use.Currently, having had more
Money turns to the adjoint diagnostic kit listing of principle with immune group, such as: VENTANA ALK IHC detection kit is to obtain
FDA approval for identification be suitble to targeted drug Trastuzumab treatment patient in-vitro diagnosis IHC detection kit, 2014
The granted listing of China, obtains extensive concern.But still lacks can satisfy the quick to mesothelin protein of market needs at present
Express the immunohistochemistry antibody reagent and related detecting method effectively detected.
Summary of the invention
In order to overcome the drawbacks of the prior art, the present invention provides a kind of for the people through the fixed paraffin embedding of formalin
The positioning of MSLN and the immunohistochemistry antibody reagent of half-quantitative detection in the tissue such as cancer of pancreas, oophoroma, celiothelioma, can be to swollen
Tumor treats mesothelin targeting medication and provides guidance.
Due to changing through MSLN antigenic structure in the fixed paraffin-embedded tissue of formalin, the antibody of commercially available anti-MSLN
Be difficult to effectively combine and realize detection, for this phenomenon, the present invention provides a kind of hybridoma cell strain, the cell can production capacity it is enough
The monoclonal antibody of specific recognition denaturation MSLN antigen.And during the hybridoma cell strain is preserved on December 20th, 2017
State's Type Tissue Collection is (referred to as: CCTCC；Address: Wuhan, China city Wuchang Luo Jia Shan), classification naming is that hybridoma is thin
Born of the same parents 3-2G6, deposit number are CCTCC C201811.
On the one hand, the present invention provides a kind of immunohistochemistry antibody compositions for detecting MSLN, contains anti-MSLN monoclonal
Antibody 3-2G6, the antibody is by being preserved in Chinese Typical Representative culture on December 20th, 2017 with deposit number CCTCC C201811
The hybridoma of object collection generates.
In some embodiments, antibody compositions of the invention are by mutually mixing antibody of the invention with antibody diluent
Conjunction obtains, wherein the antibody diluent includes phosphoric acid (PB) buffer of PH 7.2, it is preferable that the phosphate buffer density
For 0.005-0.5M, more preferable 0.01-0.1M.
In some embodiments, the antibody diluent also includes: protein protectant, the 0.05%- of 0.5%-5%
0.5% surfactant and the preservative of 0.01%-0.2%；Preferably, the protein protectant of 1%-3%, 0.05%-
0.2% surfactant and the preservative of 0.02%-0.1%；It is highly preferred that 1% protein protectant, 0.05% table
Face activating agent and 0.05% preservative.
In some embodiments, the protein protectant includes bovine serum albumin(BSA), and the surfactant includes
Polysorbas20, the preservative include 2-methyl-4-isothiazolin-3-one and 5-Chloro-2-methyl-4-isothiazolin-3-one.
In some embodiments, the antibody diluent also includes 0.05-0.5M NaCl, preferably 0.1-0.3M
NaCl, more preferable 0.15M NaCl.
In some embodiments, the antibody diluent includes: 0.05M phosphate buffer, 0.15MNaCl, 1% ox blood
Pure albumen, 0.05% polysorbas20 and 0.05% ProClin300.
In some embodiments, the anti-MSLN monoclonal antibody exists in the form of antibody-solutions, and wherein antibody is dense
Degree is 0.05-20 μ g/ml, more preferable 0.2-10 μ g/ml, most preferably 0.3-5 μ g/ml.
In some embodiments, the volume ratio of the antibody-solutions and the antibody diluent is 1: 200-1: 18000,
It is preferred that 1: 1600-1: 6400, more preferable 1: 3200.
On the other hand, this application involves immunohistochemistry antibody compositions of the invention to prepare mesothelin detection immunohistochemistry
Application in kit.
On the other hand, this application involves the immunohistochemical kits for detecting MSLN, exempt from it includes described herein
Epidemic disease group antibody compositions.
In some embodiments, immunohistochemical kit of the invention also includes: antigen retrieval buffers, immunohistochemistry pen with
And cytoplasm control wafer.
In some embodiments, antigen retrieval buffers described in immunohistochemical kit of the invention include the lemon of 0.01M
Lemon acid or its salt, pH 6.0.
In some embodiments, immunohistochemical kit of the invention includes cytoplasm control wafer, wherein the cytoplasm
Control wafer includes 4 kinds of cells as control, is respectively as follows: 293 cells of mesothelin-negative expression, mesothelin weakly positive is expressed
The OVCAR-3-MSLN cell that CFPAC-1 cell, the OVCAR-3 cell of mesothelin positive expression, mesothelin strong positive are expressed,
Described in OVCAR-3-MSLN cell be genetically modified to express the OVCAR-3 cell of external source MSLN.
In some embodiments, the protein protectant is preferably BSA (as bovine serum albumin(BSA)).
In some embodiments, the surfactant is preferred Tween-20.
In some embodiments, the preservative is preferred ProClin300.The ProClin300 includes: 2- first
Base -4- isothiazoline -3- ketone and 5-Chloro-2-methyl-4-isothiazolin-3-one.
On the other hand, the present invention provides produce anti-MSLN monoclonal antibody for detecting warp by hybridoma 3-2G6
The detection method on the non-diagnostic mesh ground of MSLN in the fixed paraffin-embedded tissue of formalin, it is characterised in that:
1. tumor sample is fixed in formalin solution, graded ethanol dehydration is then carried out, dewatered tissue is set
It is transparent in dimethylbenzene, it immerses in the low melt point paraffin of thawing, carries out paraffin embedding later, after wax stone solidification, be cut into thickness
3-5 μm of thin slice uses drift to dry instrument drying after picking up tissue using the slide for being coated with poly-D-lysine；
2. histotomy after roasting piece 1 hour, carries out dewaxing treatment in 65 DEG C in dimethylbenzene, graded ethanol water is then carried out
Change；
3. antigen retrieval buffers are added in pressure cooker, the pressure cooker high fire on electromagnetic oven of uncapping is heated to antigen retrieval buffers boiling
It rises, ceases fire, slice is submerged into and repairs pot cover on liquid middle cover, pressure valve is buckled, is heated to jet, since jet, be adjusted to
Fiery timing 2.5 minutes ceases fire, pulls up power supply；Pressure cooker is transferred on experimental bench, pot cover is opened, to antigen retrieval buffers nature
It is cooled to room temperature taking-up slide, PBST impregnates；The PBST are as follows: phosphate Tween buffer；
4. getting rid of the liquid being sliced around tissue, drawn a circle around tissue with immunohistochemistry oil pike, in delineation region
3% peroxidase of Fresh is added to block liquid, 37 DEG C are incubated for 10 minutes, and PBST cleaning slice 2 times；Add 3-2G6 antibody molten
Liquid, while negative control, blank control and positive control are set, 37 DEG C are incubated for 60 minutes, and PBST cleaning slice 2 times；
5. 1-2 drop detection reagent I covering, which is added dropwise, is sliced upper tissue, 37 DEG C incubation 10 minutes, embathe 2 times with PBST, carefully
The liquid being sliced around tissue is got rid of, 1-2 drop detection reagent II covering is added dropwise and is sliced upper tissue, 37 DEG C are incubated for 10 minutes, use
PBST embathes 2 times；Above-mentioned detection reagent I, II is respectively polymer iodine secondary antibody system DAB Detection Kit (good fortune
State steps new, article No.: KIT-0016) in reaction amplification agent and Gao Min type enzyme mark anti-mouse/rabbit igg polymer；Then using new
The DAB chromogenic reagent of fresh preparation 1 minute, PBST impregnate color development stopping；Mayer ' s bush uniformly dyeing core 1 minute, is rinsed with water more
Transparent after remaining haematoxylin, then through being dehydrated, mounting carries out the reading of coloration result with optical microscopy and takes pictures.
In some embodiments, the antigen retrieval condition of sample to be detected may is that pH6.0 can be used in sample to be measured
Citric acid solution carry out Pressure method, repair time is 2.5 minutes, and the buffer also can be used, and to carry out hot repair multiple,
Repairing condition is 95-97 DEG C, 20 minutes.
In some embodiments, the sample should be fixed in formalin solution in vitro 30 minutes.
In some embodiments, the antigen retrieval buffers are as follows: the citrate buffer of 0.01M pH 6.0 repairs item
Part is: being repaired under high pressure using above-mentioned reparation liquid.
In some embodiments, the high fire condition are as follows: 210 DEG C of 1600W；The moderate heat condition are as follows: 800W 130
℃。
In some embodiments, herein described 3-2G6 antibody-solutions are instant 3-2G6 antibody-solutions, and antibody is dense
Degree is preferably 0.05-2 μ g/mL, more preferable 0.2-1 μ g/mL, most preferably 0.31 μ g/mL；Negative control antibody is mouse monoclonal
1 Isotype control of IgG antibody (CST, CatalogNo.5415), clone G3A1；Positive control and negative control sample can
1 cytoplasm control wafers are closed with 4, and Quality Control piece is that the cell of 4 different mesothelin expressions is prepared to paraffin mass, and according to tissue core
The requirement of piece is prepared in again in a new paraffin mass, is sliced；Wherein the cell line of MSLN feminine gender expression is 293
(0)；MSLN weakly positive expression cell is CFPAC-1 (1)；MSLN positive expression cell OVCAR-3 (2)；The expression of MSLN strong positive
Cell OVCAR-3-MSLN cell (3).
Tissue specimen to be detected is detected (for the tumor group performed the operation or punctured using the immunohistochemistry antibody reagent of detection MSLN
Knit and be cut to the thin slice with a thickness of 3-5 μm after the fixed paraffin embedding of formalin) in MSLN expression, testing result can be right
Oncotherapy MSLN targets medication and provides guidance.
On the other hand, further testing result is that the expression value of MSLN is, virologist passes through immunohistochemical staining feelings
Condition combination cell Quality Control piece carries out being obtained by the integer marking of 0-3 for sxemiquantitative to the tumour cell in examined tissue specimen.In
Before the common clinic of drug, first using staining power as standards of grading.Wherein 0 point of expression: tumour cell does not occur any shape
The dyeing of formula；1 point of expression: there is cell membrane dyeing in tumour cell, but the staining power of staining power < CFPAC-1 cell；2
Dividing indicates: there is cell membrane dyeing in tumour cell, and staining power is greater than CFPAC-1, is less than OVCAR-3-MSLN；3 points of expressions: swollen
Oncocyte cell membrane dyes, staining power >=OVCAR-3-MSLN.It should be clear that participating in scoring is tumour living
Cell, and give a mark be carried out based on the MSLN that is located on cell membrane (that is: the cell for only having cytoplasm individually to dye is not included in
Count range).
It mentions cytoplasm control wafer in above-mentioned standards of grading to be prepared by the cell of known MSLN expression, including 4
Cell mass: wherein 293 cells are MSLN negative cells, and CFPAC-1 cell is the cell of MSLN weakly positive expression, and OVCAR-3 is
MSLN positive expression cell, three of the above cell can be bought by commercialization approach；OVCAR-3-MSLN is MSLN strong positive
Expression cell, preparation method are as follows:
1. the amplification of target gene: design primer obtains amplifying target genes MSLN overall length from target gene carrier；
2. target gene and expression plasmid digestion: the target gene MSLN after amplification carries out cutting glue using plastic recovery kit
Recycling, and digestion is carried out with restriction enzyme, while using endonuclease digestion expression vector pHBLV-CMV- of the same race
ZsGreen-puro；
3. the building of destination gene expression plasmid: target gene fragment and expression vector are attached；By connection product
It is transformed into competent cell, spread plate after amplification；Picking single bacterium colony carries out bacterium colony PCR；It is correct to choose amplified production
Bacterium colony is sequenced；It is the expression plasmid built that correct recombinant plasmid, which is sequenced,；
4. the packaging of target gene slow virus: inoculation 1x107A 293T cell is in T75 culture bottle, after being incubated overnight more
Change serum free medium, by expression plasmid, packaging plasmid and envelope plasmid mix after again with Lipofectamine2000
(LipofectamineTM2000 Transfection Reagent article No.s: 11668027) being added in 293T cell after mixing,
Culture 48 was as a child collected supernatant and was dispensed after being concentrated；
5. aim cell infects: logarithmic growth phase aim cell 5*104The hole a/mL, 0.5mL/ is added in 24 orifice plates,
It is incubated overnight；The original culture medium of cell is sucked, the polybrene solution that viral dilution is added and has diluted, infection is totally
Product is 250 μ L, is mixed gently；After infection 24 hours, the culture solution containing virus is removed, fresh complete medium is changed and continues to train
It supports；Virus for carrying puromycin resistance gene changes the fresh complete medium of purine-containing mycin, obtains stable transfection
Cell strain；
6. the acquisition of aim cell: the overexpression MSLN target gene stable cell strain of above-mentioned acquisition is used limiting dilution
Method obtains single clone, according to MSLN expression intensity in pathological tissue, selects MSLN high in MSLN expression intensity and pathological tissue
The consistent cell strain of expression intensity, so that obtaining OVCAR-MSLN is MSLN strong positive expression cell.
Finally, the application further relate to related immune group antibody reagent or immunohistochemistry antibody kit be used to prepare it is swollen
Purposes in tumor detection reagent.
The present invention achieves following technical effect: instant antibody used in this application, i.e., by antibodies buffer
The ingredients such as protected protein and preservative are added, can be realized following effect: reducing the non-specific binding of antibody and tissue, thus
It can be omitted BSA serum closing step and its similar step；Antibody is set to guarantee steadily in the long term, to be conducive to improve under low concentration
The efficiency and sensitivity of detection.
It can be realized quick and precisely using the detection kit or detection reagent of the application for cell surface mesothelin egg
White detection, higher than the detection sensitivity and accuracy of similar kit on the market at present.The application is detected for immunohistochemistry
The characteristics of technology design and obtain can be suitable for immunohistochemical experiment high specificity monoclonal antibody, overcome due to
It changes through MSLN antigenic structure in the fixed paraffin-embedded tissue of formalin, the antibody of commercially available anti-MSLN is difficult to effectively combine
And realize the defect of detection.
The application adaptive immune group antibody reagent can guarantee reactivity and the validity period of anti-MSLN monoclonal antibody, and
User just can be used directly after obtaining the application Related product without carrying out any operation, improve the convenience of detection.
Detailed description of the invention
Fig. 1 shows anti-MSLN monoclonal antibody purity detecting map result；It is analyzed through gray scale scanning, anti-MSLN monoclonal
Antibody band area percentage is 95%, and purity meets the requirements.
Dyeing effect compares under the conditions of Fig. 2 .A figure shows different antigen retrievals；Wherein (1) and (2) be respectively two not
Same oophoroma pathological tissue；1-3 respectively represents antigen retrieval condition 1-3；B figure shows different dilution ratio 3-2G6 antibody
To the coloration result of OVCAR-3 cell paraffin section, wherein 1-6 is the 3-2G6 antibody of 1mg/mL: PBST ratio is respectively 1:
25,1: 100,1: 400,1: 1600,1: 6400 and 1: 25600 when to the coloration result of OVCAR-3 cell paraffin section, identical
The staining power of OVCAR-3 paraffin section is corresponding with the medium MSLN staining power of tumour cell in tissue under experiment condition；C figure
Show the coloration result of 30 minutes (1) and 60 minutes (2) of primary antibody incubation time.
Fig. 3 .A figure shows the comparison of dyeing effect under Tris buffer and PB buffer, and " pH7.4 has been respectively adopted
0.05M TRIS buffer " and " pH7.4 0.05M PB buffer " diluted 3-2G6 antibody incubation carry out immunohistochemical staining,
Picture lists the staining conditions in 4 visuals field respectively；B figure shows the coloration result of 5 ionic concentration buffer liquid.Condition 1- is slow
Total Na ion concentration is 0.020M in fliud flushing, and total Na ion concentration is 0.095M in condition 2- buffer, in condition 3- buffer
Total Na ion concentration is 0.165M, and total Na ion concentration is 0.184M in condition 4- buffer, in condition 5- buffer total Na from
Sub- concentration is 0.219M；C figure shows instant 3-2G6 antibody preparation to MSLN expression cell different degrees of in tumor tissues
Coloration result；Wherein, 1-4 is respectively the instant 3-2G6 antibody preparation of preferred buffer preparation to MSLN table in tumor tissues
Up to the coloration result for the tumour cell expressed for feminine gender, weakly positive, the positive and strong positive；D figure shows that 3-2G6 antibody preparation exists
45 DEG C of potency testing results after heat damage 7 days, 15 days and 30 days；It can be seen that 4- parameter curve is declined slightly, but still conforms to and exempt from
The requirement of epidemic disease groupization detection stability.
Fig. 4 shows the immunohistochemical staining figure to variety classes tumour cell pathological section.
Specific embodiment
Embodiment 1: the preparation of anti-MSLN monoclonal antibody
(1) foundation of hybridoma cell line
1. the preparation of experimental material:
Using MSLN-ECD (article No. FCL1589) protein fragments of G&P Biosciences company as immunogene, the segment
Containing 317 amino acid, it is located at the Glu296-Ser592 of MSLN (UniProt accession#Q13421, isoform 2) sequence
Region；Used medium includes but is not limited to HAT, HT Selective agar medium, DEME culture medium；Experimental animal includes Balb/c small
Mouse；Adjuvant used is emulsification Freund's complete adjuvant, emulsification incomplete Freund's adjuvant.
2. animal immune
Fundamental immunity: antigen is fully emulsified in equal volume with Freund's complete adjuvant, and branch subcutaneous injection, injection dosage is 50 μ
Only, 2 Balb/c mouse are immunized in g/.
Booster immunization: antigen and incomplete Freund's adjuvant emulsify；3 days before carrying out cell fusion, directly intraperitoneal injection contains
The normal saline solution of 50 μ g antigens.
Tail blood examination is surveyed: the tail blood of immune mouse being taken to carry out indirect elisa method detection.
3. the preparation of hybridoma
Myeloma cell is merged with the splenocyte of 1 mouse, and fused cell spreads 10 piece of 96 porocyte culture plates；Use HAT
Culture solution selection culture 1 week, HT culture medium was replaced later and continues to cultivate.Positive cell clone is screened using indirect ELISA method,
To selected positive cell after expanding culture, 1 subclone is carried out using limiting dilution assay, 20 plants is obtained altogether and is subcloned through 1 time
Monoclonal hybridoma strain after operation.
4. the screening and foundation of hybridoma cell strain
Collect 20 plants of 1 subcloned cells supernatants, respectively measure antibody production and using denaturation Western-blot, indirectly
ELISA, immunohistochemistry (antigen retrieval mode is multiple for citrate hot repair), antibody test performance is analyzed.As a result it clones
Antibody secreted by cell strain number for 3-2G6 can identify that denaturation and is classified the cell of MSLN variable expression MSLN
Dyeing.The cell strain of the 3-2G6 chosen is subjected to the 2nd subclone using limiting dilution assay, it is ensured that obtain the hybridization of monoclonal
Tumor cell strain.By 2 times subclone after 3-2G6 cell strain generate antibody carry out denaturation Western-blot, indirect ELISA,
Immunohistochemical assay carries out analysis verifying to antibody test performance.2 subclone 3-2G6 cell strains after antibody verifying is qualified
I.e. amplification freezes as final hybridoma cell strain.
(2) preparation of anti-MSLN monoclonal antibody
1. the preparation of mouse ascites
Select 10 week old female BAl BIcs/c mouse, adaptive feeding 2 days, backward every mouse peritoneal inject 0.5mL liquid
Body paraffin.Cell inoculation, dosage of inoculation 1x10 are carried out to mouse after 7 days6A cell/ml, inoculation volume are 300ul/, abdomen
Chamber injection.Mouse growth state and abdomen size are observed, takes mouse abdomen when the obvious blow-up of mouse web portion (generally at 7-10 days)
Water enters mouse peritoneal along nearly groin using No. 12 syringe needles, flows out ascites naturally, and sterile centrifugation tube is collected.
2. the purifying of mouse ascites
By the ascites obtained in above-mentioned steps in 4 DEG C of high speed centrifugations (12000rpm, 20min), supernatant is micro- through 0.45 μm
Hole filter membrane filters, and the ProteinG SepharoseTM 4FF affinity column for being then 2mL with volume is purified.Balance is slow
Fliud flushing is 20mM phosphate buffer, and pH7.0, elution buffer is 0.1M glycine, pH3.4, stop buffer 1M
Tris-HCl, pH9.0.Using ultrafiltration by the buffer exchange in anti-MSLN monoclonal antibody be PBS, pH7.2, contain 0.05%
ProClin300.Ensure IgG concentration >=1mg/ml, is denaturalized non-reduced SDS-PAGE and is detected as single band.Anti- MSLN monoclonal
Antibody purity test map is shown in Fig. 1, according to display result it can be seen that anti-MSLN monoclonal antibody band area percentage
Than being 95%, purity meets the requirements.
Embodiment 2: anti-MSLN monoclonal antibody 3-2G6 experimental condition optimization
(1) antigen retrieval condition is optimized by assessing different antigen retrieval buffers.1 (Cit of repairing condition
6.0) refer to the citrate buffer Pressure method 2.5min using acid pH 6.0.Repairing condition 2 (Tris 9.0) is to instigate
With the Tris-EDTA buffer Pressure method 2.5min of alkaline pH 9.0.Repairing condition 3 (Tryp) refers to be repaired with 0.25% pancreatin
Multiple liquid repairs 10min under the conditions of 37 DEG C.It is as shown in Figure 2 A: in these proof conditions, 6.0 Pressure method after stain of Cit
Color effect is better than other several repair modes, is embodied in target cell (tumour cell) dyeing clearly, interstitial cell does not have
Unspecific staining.Therefore this method is finally selected as the applicable antigen retrieval method of this antibody.
(2) assessment has been carried out to screen suitable antibody concentration to the staining power of various concentration 3-2G6.According to 3-2G6
Initial concentration is 1mg/ml calculating, antibody is carried out a series of 25 times to 25600 times dilution ratios, with the antibody after dilution to mesh
It marks cell (OVCAR-3) paraffin section that antigen presentation intensity is 2+ and carries out immunohistochemical staining.According to fig. 2 shown in B: antibody is dilute
The positive staining result for releasing the coloration result that concentration is 1600 times and 6400 times is obvious, and non-specific background's reaction is relatively fewer,
Our final choice antibody dilute 3200 times and are used for verification test.
(3) different primary antibody incubation times is tested to screen suitable antibody incubation condition.Selection target egg
The pathological tissue of white weak expression carries out 37 DEG C of the primary antibody immunohistochemical staining tests for being incubated for 30min and 37 DEG C of incubation 60min, according to
Shown in Fig. 2 C: two incubation time coloration results are similar, do not differ significantly, but in order to ensure incubation time sufficiently avoids
False negative result, the experiment condition of 37 DEG C of final choice primary antibody incubation 60min.
Embodiment 3: the selection of antibody diluent
We have carried out a series of assessment, including matrix to each ingredient of 3-2G6 antibody diluent, and salt ion is dense
Degree, pH value, protected protein concentration, surfactant, preservative etc..
In terms of matrix, respectively using the TRIS buffer and PB (Phosphate Buffer) conduct that pH is 7.4,0.05M
Buffer dilutes 3-2G6 antibody and carries out immunohistochemical experiment, according to the result of Fig. 3 A: Tris buffer being used to make non-spy
Anisotropic background stainings are clearly；
In terms of salt ionic concentration, sent out after adding the NaCl of 0.015-1.2M into 0.05M TRIS and PB buffer respectively
Existing, as the increase unspecific staining of ion concentration disappears, when ion concentration increases to 0.15M or more, specific stain is strong
Degree also decreases.Under the premise of other conditions are identical, excessively high salt ionic concentration will lead to tissue staining strength reduction.Cause
This, we need to consider that destination organization dyeing is clear when selecting antibodies buffer salt ionic concentration, and reduce as far as possible non-specific
Background stainings.According to the result of Fig. 3 B: the bulk dyeing effect under 0.15M NaCl concentration is best.Therefore, Wo Menyou
Select the salinity of 0.15M NaCl.
In terms of pH, compares 0.05M TRIS (NaCl containing 0.15M) and PB that pH is 6.0,7.3,8.6 and (contain 0.15M
NaCl) influence of the buffer to immunohistochemical staining result, difference pH does not show the influence of 3-2G6 antibody staining power as the result is shown
It writes.
In terms of protected protein, compare casein, bovine serum albumin(BSA), lowlenthal serum to 3-2G6 antibody dyeability and
The influence of stability, the above ingredient influences less immunohistochemical staining result as the result is shown, and accelerated stability experimental result is aobvious
Show, in above-mentioned protein protective agent when additive amount is 1%-2%, the immunohistochemical staining of 45 DEG C of 3-2G6 after heat damage 15 days
It can will not change.
In terms of surfactant, two kinds of surfactants Tween-20 and Brij-35 are compared (i.e. are as follows: the polyoxyethylene moon
Osmanthus ether), the two does not generate difference to the dyeability of 3-2G6 as the result is shown.
In terms of preservative, 45 DEG C, 15 days accelerated stability tests show that ProClin300 (Sigma) additive amount exists
When 0.05%-0.1%, the immunohistochemical staining of 3-2G6 product is functional.
To sum up, our preferred 0.05M PB, pH7.20.15M NaCl, 1%-2%BSA, 0.05%-0.1%Tween-20
And antibody diluent of the 0.05%-0.1%ProClin300 as 3-2G6, the volume ratio of dilution and 3-2G6 antibody are
3200:1.Antibody and dilution are mixed to get antibody-solutions, the antibody-solutions that this dilution finishes are as described herein
Instant 3-2G6 antibody-solutions.
The dyeability map of above-mentioned preferred buffer is shown in fig. 3 c, the results showed that 3-2G6 antibody can be special
The opposite sex combines mesothelin protein, and can effectively reflect the expression of mesothelin protein；Stability map is in fig. 3d
Display, the results showed that the thermal stability of 3-2G6 antibody preparation is fine, and can satisfy requirement of the immunohistochemistry to stability.
Embodiment 4: it is obtained using ImmunohistochemistryMethods Methods for reading the pathological section of data
Prepare tumor specimen that existing document report over-expresses there are MSLN first (such as: cancer of pancreas, oophoroma, pernicious
Celiothelioma), sample should be fixed in formalin solution in vitro 30 minutes, then carry out graded ethanol dehydration, dehydration
Tissue afterwards is placed in transparent in dimethylbenzene, immerses in the low melt point paraffin of thawing, carries out paraffin embedding later, solidifies to wax stone
Afterwards, it is cut into 3-5 μm of thickness of thin slice, uses drift to dry instrument drying after picking up tissue using the slide for being coated with poly-D-lysine.
Immunohistochemical experiment step: histotomy after roasting piece 1 hour, carries out dewaxing in 10 minutes in 65 DEG C in dimethylbenzene
Processing, then carries out graded ethanol aquation, and concrete operations are slice after dewaxing at soaked in absolute ethyl alcohol 5 minutes, 95% second
Alcohol impregnates 5 minutes, and 75% ethyl alcohol impregnates 5 minutes, is finally immersed in distilled water.
Slice after aquation carries out antigen retrieval, and the antigen retrieval buffers (citric acid of 0.01M, pH6.0 is added in pressure cooker
Salt buffer), pressure cooker is uncapped is heated to antigen retrieval buffers boiling on electromagnetic oven, ceases fire, slice is submerged into and is repaired in liquid
Cover pot cover, buckle pressure valve, high fire (1600W, 210 DEG C) is heated to jet, since jet, be adjusted to moderate heat (800W, 130
DEG C) timing 2.5 minutes, cease fire, pulls up power supply；Pressure cooker carefully, is evenly transferred on experimental bench, until being fallen from movable lock core
To original position, determines that exhaust pipe is discharged without steam or a small amount of steam, opens pressure valve, rotate counterclockwise pot cover handle, open pot cover,
Slide is taken out to antigen retrieval buffers cooled to room temperature (at least 30min), PBST impregnates.
The liquid being sliced around tissue carefully is got rid of, is drawn a circle around tissue with immunohistochemistry oil pike, in delineation region
Interior plus Fresh 3% peroxidase blocks liquid, and 37 DEG C are incubated for 10 minutes, to eliminate tissue endoperoxides object enzymatic activity,
PBST cleaning slice 2 times.
Add instant 3-2G6 antibody-solutions, 37 DEG C are incubated for 60 minutes, and PBST cleaning slice 2 times.Every batch of is tested while being arranged
Negative control, blank control and positive control.The instant 3-2G6 antibody-solutions, the antibody diluent system as described in embodiment 3
Standby, antibody concentration is 0.31 μ g/mL.Negative antibody control is mouse monoclonal antibody IgG1 Isotype control (CST, Catalog
No.5415), clone G3A1.Positive control and negative control sample can close 1 cytoplasm control wafer with 4, and Quality Control piece is by 4
The cell of a difference mesothelin expression prepares paraffin mass, and is prepared in a new stone again according to the requirement of organization chip
In wax stone, it is sliced.Wherein the cell line of MSLN feminine gender expression is 293 (0)；MSLN weakly positive expression cell is CFPAC-
1(1)；MSLN positive expression cell OVCAR-3 (2)；The cell OVCAR-3-MSLN cell (3) of MSLN strong positive expression.
1-2 drop detection reagent I covering is added dropwise and is sliced upper tissue, 37 DEG C are incubated for 10 minutes.It is embathed 2 times with PBST.Carefully get rid of
The liquid being sliced around tissue is removed, 1-2 drop detection reagent II covering is added dropwise and is sliced upper tissue, 37 DEG C are incubated for 10 minutes, use
PBST embathes 2 times.Above-mentioned detection reagent I, II is respectively polymer iodine secondary antibody system DAB Detection Kit (purchase
Step new, article No. KIT-0016 from Foochow) reaction amplification agent and Gao Min type enzyme mark anti-mouse/rabbit igg polymer.Then using new
The DAB chromogenic reagent of fresh preparation 1 minute, PBST color development stopping.Mayer ' s bush uniformly dyeing core 1 minute, is rinsed with water extra Soviet Union
Transparent after lignin, then through being dehydrated, mounting carries out the reading of coloration result with optical microscopy and takes pictures.Different pathological is sliced
Dyeing map see Fig. 4, as the result is shown: the immunohistochemistry reagent of the application be directed to cancer of pancreas, oophoroma and mesothelioma cell energy
The expression of enough clear characterization mesothelin proteins.
The interpretation of 5 MSLN expression value of embodiment
Virologist's combination cell Quality Control piece carries out the immunohistochemical staining situation of tumour cell in examined tissue specimen
The integer by 0-3 of sxemiquantitative is given a mark.Before carrying out clinical research simultaneously with MSLN targeted drug first using staining power as
Standards of grading.Wherein 0 point of expression: tumour cell does not occur any type of dyeing；1 point of expression: there is cell membrane in tumour cell
Dyeing, but staining power≤CFPAC-1 cell staining power；2 points of expressions: there is cell membrane dyeing in tumour cell, dyeing
Intensity is greater than CFPAC-1, is less than OVCAR-3-MSLN；3 points of expressions: there is cell membrane dyeing in tumour cell, and staining power >=
OVCAR-3-MSLN.It should be clear that participating in scoring is tumour cell living, and marking is to be based on being located on cell membrane
MSLN (that is: the only having the cell that individually dyes of cytoplasm not to be included in count range) that carries out according to above-mentioned standard, in Fig. 4
The expression value of MSLN carries out interpretation, and interpretation result is as follows:
| Organization name | Marking result |
| Cancer of pancreas 1# | 2 |
| Oophoroma 1# | 2 |
| Celiothelioma 1# | 3 |
The preparation of 6 cytoplasm control wafer of embodiment
The preparation of 6.1 OVCAR-3-MSLN cells
The building of destination gene expression carrier
Design primer expands overall length from the carrier (pCMV3, buying stick up Divine Land from justice) comprising people's MSLN coded sequence
MSLN carries out gel extraction using plastic recovery kit, and carries out digestion with restriction enzyme, while using restriction endonuclease of the same race
Digestion Lentiviral (pHBLV-U6-MCS-CMV-ZsGreen-PGKL-PURO is purchased from Chinese Hang Seng object).By MSLN
Genetic fragment and expression vector are attached, and connection product is transformed into competent cell, spread plate after amplification.Picking list
A bacterium colony carries out bacterium colony PCR, chooses the correct bacterium colony of amplified production and is sequenced.It is to build that correct recombinant plasmid, which is sequenced,
Expression vector.
The packaging of target gene slow virus
293T cell is inoculated in T75 culture bottle, replaces serum free medium after being incubated overnight, by expression vector, packaging
(it is respectively: pHBLV-U6-MCS-CMV-ZsGreen-PGKL-PURO, paPAX2, pMD.2G, buying for carrier and envelope vector
From Chinese Hang Seng object) mixing after mix again with Lipofectamine2000 after addition 293T cell (transfected Adenovirus E1A gene
Human renal epithelial cell line) in, culture 48 as a child collect supernatant and be concentrated after dispense.
The acquisition of aim cell
Logarithmic growth phase aim cell is added in 24 orifice plates, is incubated overnight；The original culture medium of cell is sucked, is added
Viral dilution and the polybrene solution diluted, infection total volume are 250 μ L, are mixed gently；After infection 24 hours, go
Except the culture solution containing virus, fresh complete medium (Gibco RPMI1640-A10491-01 culture medium+20%FBS+ is changed
0.01mg/ml insulin) continue to cultivate；Virus for carrying puromycin resistance gene changes the fresh complete of purine-containing mycin
Full culture medium obtains the cell strain of stable transfection.
The overexpression MSLN target gene stable cell strain of above-mentioned acquisition is obtained into monoclonal using limiting dilution assay, according to
MSLN expression intensity in pathological tissue selects the consistent cell of MSLN high expression intensity in MSLN expression intensity and pathological tissue
Strain, so that obtaining OVCAR-3-MSLN is MSLN strong positive expression cell.
6.2 cell paraffin embedding
Experimental material:
MSLN four-in-one cytoplasm control wafer includes four cell strains: 293 (Number:CRL-1573TM)、
CFPAC-1(Number:CRL-1918TM)、OVCAR-3(Number:HTB-161TM) and
OVCAR-3-MSLN (is prepared) according to above method.Material used in cell culture and embedding process can pass through business way road
Purchase obtains.
Experimentation:
(1) cell culture
Cell Name: 293 (staining powers 0)；
Culture medium: Gibco RPMI1640 culture medium+10%FBS；
Cell Name: CFPAC-1 (staining power 1)；
Culture medium: Gibco IMDM culture medium+10%FBS；
Cell Name: OVCAR-3 (staining power 2)；
Culture medium: Gibco RPMI1640-A10491-01 culture medium+20%FBS+0.01mg/ml insulin；
Cell Name: OVCAR-3-MSLN (staining power 3)；
Culture medium: Gibco RPMI1640-A10491-01 culture medium+20%FBS+0.01mg/ml insulin；
Wherein the FBS is fetal calf serum, and Gibco RPMI1640 (ATCC improvement) culture medium is U.S. Gibco public affairs
Take charge of 1640 culture medium of RPMI (as Gibco RPMI1640-A10491-01) that article No. is A1049101；Gibco IMDM training
Feeding base is the IMDM culture medium that U.S.'s Gibco company article No. is 12440053；Gibco RPMI1640 ordinary culture medium is Gibco
The culture medium that company's article No. is 11875135；
Condition of culture: 37 DEG C, 5%CO2 stationary culture；2-3 days, cell Proliferation was passed on to culture dish bottom 80-90% is accounted for
Once.
(2) cell is collected and is fixed
When above-mentioned cell culture is to the 80-90% for being paved with Tissue Culture Dish bottom, cell is collected.Culture medium is outwelled, is used
1-2ml PBS washed once, and 0.25% pancreatin of 1-2ml is added to digest 3-5min, and microscopically observation cell rounding adds 1ml cell
Culture medium terminates digestion, and cell is blown and beaten uniform collection with pipettor and is counted, and 3-5ml PBS is added and washs cell, 1000rpm
It is centrifuged 5min, abandons supernatant, 4% paraformaldehyde of cell or 10% neutral formalin fixer are resuspended, the addition of fixer
Amount is 2 × 107Cells/1ml fixer, the fixed 16h-24h of room temperature.
(3) agarose supports cell
Ago-Gel is prepared: being weighed the solution that agarose adds PBS to be configured to 3-4% concentration, is covered masking foil in microwave
It is heated to agarose in furnace to be completely dissolved, taking-up is placed in heat preservation for standby use in 60 DEG C of water-baths.
Cell is mixed with agarose: 1-2 × 107Cells passes through fixation procedure, about 100 μ l of natural subsidence to volume, with shifting
Liquid device carefully removes supernatant.The centrifuge tube for filling cell is preheated in 60 DEG C of water-baths, is pipetted with the pipettor of preheating and cell
Isometric Ago-Gel is added in centrifuge tube and mixes that (mixing movement needs soft, and whole process is preferably controlled in 3min with cell
Within, reduce the influence of moisture evaporation), agarose, cell mixture after mixing move on to the 1ml syringe needle tube of removal syringe needle
In, room temperature is cooled and shaped.
(4) cell mass is dehydrated
The cell mass mixed with agarose is taken out, the segment of 0.5cm thickness is cut into, is placed on the dewatering basket marked
It is interior.Tissue block is successively immersed in the ethyl alcohol of various concentration gradient and carry out tissue dewatering: 70% ethyl alcohol stays overnight → 80% ethyl alcohol
The ethyl alcohol of 1.5h → 90% ethyl alcohol of the 1.5h → 95% ethyl alcohol I ethyl alcohol II of 50min → 100% of 1.5h → 100% 50min.
(5) cell mass is transparent
Carried out the cell mass of graded ethanol dehydration successively block be immersed in dimethylbenzene I and dimethylbenzene II carry out it is transparent, often
Secondary progress clearing time is 20min.
(6) cell mass waxdip
Paraffin mass is placed in waxdip cup that 2h melts completely to paraffin in 65 DEG C of baking ovens, will carry out the tissue block after transparency of organization
It is immersed in the paraffin of thawing and carries out tissue waxdip, wax is changed after waxdip 1 time and carries out 1 waxdip, each 1h again.
(7) cell mass embeds
Manual embedding process carries out in 60 DEG C of baking ovens.The paraffin of thawing is poured into organization embedding bed die, by waxdip knot
The cell mass of beam takes out from corresponding dewatering basket, is put into rapidly in the paraffin of organization embedding bed die, tries not when being put into
There is bubble generation, adjust the position of cell, the embedding frame marked up and down will be carried out and be covered on embedded box, cooling bench is transferred to
Upper cooling 30min can obtain organization embedding paraffin mass.Before using paraffin mass, by marginal lappet outside 293 cell positions
Vertical cut falls a fritter paraffin, is used as slice azimuth mark.
6.3 microsection manufacture methods
(1) fixed: the operating instruction that slicer is provided according to production firm is carried out using should be ensured that blade before use
It is sharp, then wax stone is fixed on supporter, pays attention to guaranteeing the upper right corner that corner cut position is located at wax stone.
(2) it repairs block: repairing under block mode, repair adjustable to 15-20 μm of block slice thickness, repair and cut wax stone to four embedded
Cell mass all can be cut.It should avoid repairing block number as far as possible, and should be ensured that the continuity of tissue.
(3) be sliced: adjustment slice thickness is 3-5 μm or so, is then sliced, and slice must assure that complete, uniform, thick
Degree is suitable for, does not have tool marks, corrugationless and cracking.
(4) it opens up piece: gently provoking slice with writing brush, (temperature 37-40 is unfolded in the water surface moved in drift baking instrument with tweezers
DEG C, during slice is transferred to the drift baking instrument water surface, slice can be rotated but cannot be overturn).
(5) it fishes out piece: the slice of expansion is affixed on glass slide, the right hand holds glass slide frosted area when fishing out piece, and paraffin is kept to lack
Mouth is located at upper left side, avoids generating bubble between slice and glass slide as far as possible.Glass slide is tilted to 45.To the upper extra water of slice
It flows down.
(6) bake piece: glass slide, which is placed in 60 DEG C of baking 30min in baking oven, can be made tissue paraffin section de, storage in case of
Subsequent experimental.
Claims (15)
1. for detecting the immunohistochemistry antibody compositions of MSLN, containing anti-MSLN monoclonal antibody 3-2G6, the antibody by
It is thin with the hybridoma that deposit number CCTCC C201811 is preserved in China typical culture collection center on December 20th, 2017
Born of the same parents generate.
2. immunohistochemistry antibody compositions described in claim 1, the antibody compositions pass through the antibody and antibody is dilute
It releases liquid phase to be mixed to get, wherein the antibody diluent includes the phosphate buffer of PH 7.2, it is preferable that the phosphate buffer
Concentration is 0.005-0.5M, more preferable 0.01-0.1M.
3. immunohistochemistry antibody compositions as claimed in claim 2, the antibody diluent also includes: the albumen of 0.5%-5%
The preservative of matter protective agent, the surfactant of 0.05%-0.5% and 0.01%-0.2%；Preferably, the albumen of 1%-3%
The preservative of matter protective agent, the surfactant of 0.05%-0.2% and 0.02%-0.1%；It is highly preferred that 1% protein
Protective agent, 0.05% surfactant and 0.05% preservative.
4. immunohistochemistry antibody compositions as claimed in claim 3, wherein the protein protectant includes bovine serum albumin(BSA),
The surfactant includes polysorbas20, and the preservative includes 2-methyl-4-isothiazolin-3-one and 5- chloro-2-methyl -4-
Isothiazoline -3- ketone.
5. immunohistochemistry antibody compositions as claimed in claim 2, wherein the antibody diluent also includes 0.05-0.5M
NaCl, preferably 0.1-0.3M NaCl, more preferable 0.15M NaCl.
6. immunohistochemistry antibody compositions as claimed in claim 2, wherein the antibody diluent includes: 0.05M phosphoric acid buffer
Liquid, 0.15M NaCl, 1% bovine serum albumin(BSA), 0.05% polysorbas20 and 0.05% ProClin300.
7. immunohistochemistry antibody compositions of any of claims 1 or 2, wherein the anti-MSLN monoclonal antibody is with antibody-solutions
Form exist, wherein antibody concentration be 0.05-20 μ g/ml, more preferable 0.2-10 μ g/ml, most preferably 0.3-5 μ g/ml.
8. immunohistochemistry antibody compositions as claimed in claim 7, wherein the antibody-solutions (initial concentration 1mg/ml) and institute
The volume ratio for stating antibody diluent is 1: 200-1: 18000, preferably 1: 1600-1: 6400, more preferable 1: 3200.
9. immunohistochemistry antibody compositions of any of claims 1-8 are preparing mesothelin detection immunohistochemistry reagent
Application in box.
10. the immunohistochemical kit for detecting MSLN, it includes immunohistochemistry of any of claims 1-11
Antibody compositions.
11. immunohistochemical kit described in any one of claim 10, also includes: antigen retrieval buffers, immunohistochemistry pen and cell
Quality Control piece.
12. immunohistochemical kit described in claim 11, the antigen retrieval buffers include the citric acid or its salt of 0.01M,
Its pH is 6.0.
13. immunohistochemical kit described in claim 11, the cytoplasm control wafer includes 4 kinds of cells as control,
It is respectively as follows: 293 cells of mesothelin-negative expression, CFPAC-1 cell, the mesothelin positive expression of the expression of mesothelin weakly positive
The OVCAR-3-MSLN cell that OVCAR-3 cell, mesothelin strong positive are expressed, wherein the OVCAR-3-MSLN cell is through losing
Modification is passed to express the OVCAR-3 cell of external source MSLN.
14. immunohistochemical kit described in claim 13, the OVCAR-3-MSLN cell obtains by the following method:
(1) building of destination gene expression carrier
Design primer expands overall length MSLN from the carrier comprising people's MSLN coded sequence, carries out cutting glue using plastic recovery kit
Recycling, and digestion is carried out with restriction enzyme, while using endonuclease digestion Lentiviral of the same race；By MSLN gene
Segment and expression vector are attached, and connection product is transformed into competent cell, spread plate after amplification, the single bacterium of picking
Row bacterium colony PCR is dropped into, the correct bacterium colony of amplified production is chosen and is sequenced；It is the expression built that correct recombinant plasmid, which is sequenced,
Plasmid；
(2) packaging of target gene slow virus
293T cell is inoculated in culture bottle, serum free medium is replaced after being incubated overnight, by expression vector, package carrier and packet
293T cell is added after mixing again with Lipofectamine2000 after membrane carrier mixing and (has transfected people's kidney of Adenovirus E1A gene
Epithelial cell line) in, culture 48 was as a child collected supernatant and was dispensed after being concentrated；
(3) acquisition of aim cell
Logarithmic growth phase aim cell is added in 24 orifice plates, is incubated overnight；The original culture medium of cell is sucked, virus is added
Dilution and the polybrene solution diluted, infection total volume are 250 μ L, are mixed gently；After infection 24 hours, removal contains
The culture solution of virus, changes fresh complete medium and continues to cultivate；Virus for carrying puromycin resistance gene is changed
The fresh complete medium of purine-containing mycin obtains the cell strain of stable transfection；By the base of the overexpression MSLN mesh of above-mentioned acquisition
Because stable cell strain obtains monoclonal using limiting dilution assay, according to MSLN expression intensity in pathological tissue, MSLN expression is selected
The consistent cell strain of MSLN high expression intensity in intensity and pathological tissue, so that obtaining OVCAR-3-MSLN is MSLN strong positive table
Up to cell.
15. a kind of be directed in vitro paraffin group using immunohistochemistry antibody compositions according to claim 1 to 8
Knit the detection method that MSLN expression in sample carries out non-diagnostic purpose, the step are as follows:
(1) tumor tissues sample is fixed in formalin solution, then carries out graded ethanol dehydration, dewatered tissue is set
It is transparent in dimethylbenzene, it is rear to immerse in the low melt point paraffin melted, paraffin embedding is carried out, after wax stone solidification, is cut into thickness 3-5
μm thin slice, picked up using the slide for being coated with poly-D-lysine and dry instrument with drift after tissue and dry；
(2) histotomy after roasting piece 1 hour, carries out dewaxing treatment in 65 DEG C in dimethylbenzene, then carries out graded ethanol aquation；
(3) antigen retrieval buffers are added in pressure cooker, carries out high pressure or hot repair process, naturally cools to room to antigen retrieval buffers
Temperature takes out slide, and PBST impregnates；
(4) liquid being sliced around tissue is got rid of, is drawn a circle with immunohistochemistry oil pike in tissue periphery, in delineation region Nei Jiaxin
3% peroxidase of fresh preparation blocks liquid, 37 DEG C of incubations, PBST cleaning slice；Addition according to claim 1 any one of -8
The immunohistochemistry antibody compositions, while negative control, blank control and positive control are set, 37 DEG C are incubated for, and PBST is clear
Wash slice；
(5) 1-2 drop detection reagent I covering is added dropwise and is sliced upper tissue, 37 DEG C of incubations are embathed with PBST；It carefully gets rid of and is sliced group
The liquid of surrounding is knitted, detection reagent II covering is added dropwise and is sliced upper tissue, 37 DEG C of incubations are embathed with PBST；Above-mentioned detection reagent I,
II be respectively reaction amplification agent in polymer iodine secondary antibody system DAB detection kit and Gao Min type enzyme mark anti-mouse/
Rabbit igg polymer；Then the DAB chromogenic reagent of Fresh is used, PBST impregnates color development stopping；Mayer ' s bush uniformly dyeing
Core, transparent after being rinsed with water extra haematoxylin, then through being dehydrated, mounting carry out the reading and bat of coloration result with optical microscopy
According to.
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| CN113238038A (en) * | 2021-05-11 | 2021-08-10 | 图凌(杭州)生物医药有限公司 | Antibody diluent for immunohistochemical detection and preparation method and application thereof |
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| CN111896744B (en) * | 2020-07-28 | 2022-02-22 | 重庆澳龙生物制品有限公司 | Goat echinococcus antibody ELISA detection kit and preparation method and application thereof |
| CN113238038A (en) * | 2021-05-11 | 2021-08-10 | 图凌(杭州)生物医药有限公司 | Antibody diluent for immunohistochemical detection and preparation method and application thereof |
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