CN1928563B - Immunodiagnosis reagent kit for breast cancer and test slip - Google Patents
Immunodiagnosis reagent kit for breast cancer and test slip Download PDFInfo
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- CN1928563B CN1928563B CN2005100295348A CN200510029534A CN1928563B CN 1928563 B CN1928563 B CN 1928563B CN 2005100295348 A CN2005100295348 A CN 2005100295348A CN 200510029534 A CN200510029534 A CN 200510029534A CN 1928563 B CN1928563 B CN 1928563B
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Abstract
The disclosed testing block for Her2 express in mammary tissue includes: the Her2 high-express area, the express contrast area, the no-express contrast area, and the testing area for target tissue. This invention can provide accurate evaluation standard for clinic diagnosis on breast cancer.
Description
Technical field
The invention belongs to field of immunology, relate to a kind of diagnosing tumor and detect articles for use, further, the present invention relates to detect test pieces that Her2 expresses in the breast tissue and the kit that contains this test pieces.
Background technology
Breast cancer is women's common malignancy, accounts for the first place of women's malignant tumour at the incidence of disease of American-European countries.Though China belongs to the low relatively area of sending out of breast cancer, the incidence of disease rises just year by year.The prognosis of most patient with breast cancer's early operations is better, but the overexpression of Her2 acceptor is arranged in the patient's of about 25-30% the cancerous tissue, these patients' prognosis obviously is worse than the low patient who expresses of Her2, be presented as recurrence after operation rate height, rapid, the chemotherapy paracmasis weak point of PD, and chemotherapeutics easily produced resistance, disease free survival and total survival rate all are lower than the breast cancer of Her2 feminine gender.Her2 extensively distributes in vivo, and the gene erbB-2 of this albumen of encoding is called the neu gene again, is one of the highest gene of frequency that changes in the human tumor.U.S. Society of Oncology once recommended " Her2/neu " as the breast cancer marker thing in 2000, and the detection that diagnosis is still recurred all has important value.
Her2 is one of desirable target spot of neoplasm targeted therapy, has prepared multiple anti-Her2 monoclonal antibody both at home and abroad, finds that they can significantly suppress external, the interior growth of body of Her2 positive tumor cell.Because mouse source antibody can produce serious heterogenetic antigen reaction after being injected to human body, people have carried out humanization modified to mouse source antibody again, as drugs approved by FDA in 1998 novel anti Her2 humanized antibody be used for the treatment of the metastatic breast cancer of the Her2 positive, this antibody is Herceptin, has obtained very definite curative effect in clinical testing and follow-up application.Domestic patent 01132225, X also discloses a kind of anti-Her2 antibody, the preclinical study in early stage verified its can the mammary tumor cells specific of external and Her2 receptor positive, reversible, combine high-affinity, the in-vitro multiplication that can suppress the tumour cell of Her2 positive expression also can suppress the tumor growth of the tumour cell bare mouse different species transplantation model of Her2 positive expression.Detection in 24～60 hours found that antibody is concentrated and is distributed in the tumor tissues after tumor-bearing mice was injected this antibody.
Because the indication of anti-Her2 monoclonal antibody drug is the patient with breast cancer of the Her2 positive, therefore when carrying out clinical research and even clinical practice from now on, need definite alternative patient tumors to organize the expression of Her2 antigen.But present existing breast cancer diagnosis reagent ubiquity running program is loaded down with trivial details, and non-specific high problem can not judge easy, rapidly, exactly that patient tumors organizes the expression of Her2 antigen.Therefore, pressing for medically at present that exploitation is a kind of can be quick, convenient and carry out the articles for use of breast cancer in-vitro diagnosis exactly.
Summary of the invention
The purpose of this invention is to provide a kind of carrying out clinical research and when diagnosis, can accurately measure the test pieces of expression of the Her2 antigen of experimenter's breast tissue.
Another object of the present invention provides a kind of immunohistochemical diagnosis kit of breast cancer.
Another object of the present invention provides the method for Her2 antigenic expression in a kind of experimenter's of detection tumor tissues.
In one aspect of the invention, provide a kind of test pieces that Her2 expresses in the breast tissue that detects, had with lower area on the first type surface of described test pieces:
(a) Her2 high expressed check plot, described high expressed check plot is provided with the tumor tissue section of Her2 high expressed;
(b) express the check plot among the Her2, the described middle check plot of expressing is provided with the tumor tissue section of expressing among the Her2;
(c) Her2 does not have the expression check plot, and described nothing is expressed the check plot and is provided with the histotomy that Her2 does not have expression;
(d) Her2 detection zone, described detection zone are used to place breast tissue to be detected.
As a preference of the present invention, in the test pieces that Her2 expresses in described detection breast tissue, the tumor tissue section of described Her2 high expressed is selected from: D2F2/E2, HTB-20 or SK-BR-3 tumor tissue section.
As a preference of the present invention, in the test pieces that Her2 expresses in described detection breast tissue, the tumor tissue section of expressing among the described Her2 is selected from: KYC or LYX tumor tissue section.
As a preference of the present invention, in the test pieces that Her2 expresses in described detection breast tissue, the histotomy that described Her2 does not have expression is selected from: A431, QYC, MCF-7, RENCA tumor tissue section or Her2 do not have the normal structure section of expression.
As a preference of the present invention, in the test pieces that Her2 expresses in described detection breast tissue, described histotomy is paraffin-embedded section.
In a second aspect of the present invention, a kind of Her2 immunodiagnosis reagent kit of breast cancer is provided, it comprises the test pieces that Her2 expresses in the described detection breast tissue.
As a preference of the present invention, in the Her2 of described breast cancer immunodiagnosis reagent kit, also comprise immunohistochemical staining reagent in the described kit, described immunohistochemical staining reagent comprises: the blocking agent of nonspecific binding site, the initial antibodies that is connected with tissue antigen, the second antibody that is connected with initial antibodies, enzyme, substrate, developer.
As a preference of the present invention, in the Her2 of described breast cancer immunodiagnosis reagent kit, described initial antibodies is the anti-Her2 monoclonal antibody of anti-Her2 Humanized monoclonal antibodies or mouse source, and described second antibody is biotin labeled anti-human IgG or anti-mouse IgG.
As a preference of the present invention, in the Her2 of described breast cancer immunodiagnosis reagent kit, the blocking agent of described nonspecific binding site is selected from: animal blood serum, from albumen; Described enzyme is a horseradish peroxidase; Described substrate is H
2O
2Described developer is selected from: DAB-H
2O
2, AEC-H
2O
2, CN-H
2O
2
In a third aspect of the present invention, the detection method of Her2 antigenic expression in a kind of vitro detection sample is provided, described sample is a tumor tissues, the method comprising the steps of:
Tumor tissues is made section;
Described section is placed the Her2 detection zone of described test pieces, carry out immunohistochemical staining and detect, Her2 high expressed, middle expression on the dyeing situation of sample and the test pieces, the dyeing situation of not having a histotomy of expression are compared, the result judges as follows:
The low district of expressing of dyeing situation≤Her2 is judged to feminine gender (-);
The dyeing situation is expressed between the district between low the expression among district and the Her2 of Her2, is judged to the weak positive (+);
The dyeing situation is expressed in Her2 between district and the Her2 high expressed district, is judged to middle strong positive (++);
Dyeing situation 〉=Her2 high expressed district is judged to strong positive (+++).
More particularly, in described detection method, the tumor tissue section of described Her2 high expressed is selected from D2F2/E2, HTB-20 or SK-BR-3 tumor tissue section; The tumor tissue section of expressing among the described Her2 is selected from KYC or LYX tumor tissue section; The histotomy that described Her2 does not have expression is selected from the histotomy that A431, QYC, MCF-7, RENCA or other Her2 do not have expression.
Description of drawings
Fig. 1 has shown in the kit of the present invention, the distribution schematic diagram of histotomy on slide, and wherein, 1,2 and 3 represent Her2 high expressed, middle expression, the low check plot of expressing, 4 expression detection zones respectively.
Fig. 2 has shown that several cells present the different Flow cytometry results that express intensity fluorescent, and wherein, A represents the result of LYX cell, and its Her2 does not have expression; B represents the result of QYC cell, and its Her2 does not have expression; C represents the result of MCF-7 cell, and its Her2 does not have expression; D represents the result of RENCA cell, and its Her2 does not have expression; E represents the result of KYC, and its Her2 moderate is expressed; F represents the result of LYX cell, and its Her2 moderate is expressed; G represents the result of SK-BR-3 cell, and its Her2 highly expresses; H represents the result of HTB-20 cell, and its Her2 highly expresses; I represents the result of D2F2/E2 cell, and its Her2 highly expresses.
Fig. 3 has shown some photos of the biopsy tissues of strong sun, weak sun and feminine gender, and wherein, A is MCF-7 (Her2 does not have expression); B is LYX (expression of Her2 moderate); C is D2F2/E2 (Her2 highly expresses); D is a sample 3 (+); E is a sample 5 (++); F is sample 7 (+++).
Embodiment
Because present existing breast cancer diagnosis reagent running program is loaded down with trivial details, non-specific height can not judge rapidly, accurately that patient tumors organizes the expression of Her2 antigen.Therefore, the invention provides a kind of Her2 of test patient easily antigen presentation situation, and then can accurately diagnose the state of an illness.The inventor selects the multiple different cell that the Her2 antigenic expression is height, moderate and does not have expression through long term studies, is prepared into to have good homogeneous tissue, makes histotomy and is placed on certain carrier.When detecting, experimenter's histotomy and contrast section are dyeed simultaneously, compare then, can judge experimenter's Her2 expression.
The inventor chooses the multiple different cell that the Her2 antigenic expression is height, moderate and does not have expression, sets up the heteroplastic transplantation model of Balb/c nude mice, and tumor formation rate is improved greatly.Because the tumor tissues that adopts the interior inoculation of the nude mouse of clone amplification in vitro and specific immune function defective to obtain, real is the result of monoclonal tumor cell proliferation, and therefore, tumor tissues has good homogenieity, be the different parts of tumour, the antigenic expression unanimity.More reliable with such tissue as criterion, also make judgment basis have better stability.
Should be understood that to exist many kinds of Her2 not have the normal structure of expression in the prior art, the normal structure that these Her2 do not have expression also can be used for the present invention.
Fixing, paraffin-embedded section that the present invention chooses, but not frozen section guaranteeing to have reduced the requirement to experimental facilities and method difficulty under the experimental result reliable premise, are easy to popularize.
In the present invention, organizing that the tissue of excision and biopsy obtain all can be used for detecting, and reduced being examined the requirement of tissue, enlarged the scope that can accept to detect the patient.And experimenter's tumor tissues is attached at same test pieces with the control tissue that Her2 antigen is different expressions, carries out IHC dyeing simultaneously, has at utmost eliminated the error that the operating process subtle change is brought coloration result.
The present invention adopt the Her2 expression to be height, moderate and the clear and definite tumor tissues that do not have an expression in contrast, can effectively monitor the IHC dyeing course, the IHC coloration result is estimated accurately, improved the accuracy of testing result, false positive and false-negative result are controlled effectively.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, unspecified technology is a routine techniques.
Embodiment 1Her2 antigen is the selection of the tumour cell of different expressions
Adopt following steps to carry out the selection of tumour cell:
1) alternative tumour cell comprises: human liver cancer cell QYC, human liver cancer cell KYC, human liver cancer cell LYX, mouse Grawitz's tumor cell RENCA, D2F2/E2 (aforementioned five kinds of cells are available from The 2nd Army Medical College international co-operation institute of oncology), people's epidermal carcinoma cell A431 (available from upright Bioisystech Co., Ltd of Shanghai noon) and MCF-7 (available from national cancer institute), SK-BR-3 (available from upright Bioisystech Co., Ltd of Shanghai noon), HTB-20 (available from Guangzhou Wei Erman company).In vitro culture is got various cells respectively and is carried out fluorescent dye to exponential phase;
2) cell count adds tumour cell to be measured in the flow cytometer dedicated test pipe 2 * 10
5/ pipe, 200g * 5 are minute centrifugal, abandon supernatant;
3) with the PBS that contains 1% calf serum rhHer2-mAb (available from The 2nd Army Medical College international co-operation institute of oncology) is diluted to 10 μ g/mL, adds in the above-mentioned streaming pipe, 100 μ L/ pipe, ice bath, lucifuge was hatched 45 minutes;
4) contain the PBS washed cell 2 times of 1% calf serum, each all 200g * 5 minutes are centrifugal, abandon supernatant;
5) with the PBS that contains 1% calf serum FITC-goat anti-human igg (available from Shanghai Vaccine and Serum Institute) is diluted to 1 μ g/mL, adds in the above-mentioned streaming pipe, 100 μ L/ pipe, ice bath, lucifuge was hatched 45 minutes;
6) contain the PBS washed cell 2 times of 1% calf serum, each all 200g * 5 minutes are centrifugal, abandon supernatant;
7) cell precipitation is resuspended with the PBS that 300 μ L contain 1% calf serum, detect with flow cytometer, the fluorescent-labeled antibody that Her2 expresses strong more cell combination is many more.
By the contrast fluorescence intensity, obtain Her2 antigen and be D2F2/E2, SK-BR-3, the HTB-20 that highly expresses; Her2 is LYX, the KYC that moderate is expressed, and Her2 does not have MCF-7, QYC, A431, the RENCA cell of expression.The fluidic cell testing result that above-mentioned several cell presents different expression intensities is presented among Fig. 2.
The preparation method:
1) choose D2F2/E2, LYX and three kinds of tumour cells of MCF-7 that the Her2 that obtains among the embodiment 1 presents height, moderate, nothing expression respectively, the amplification of in the RPMI-1640 complete medium that contains 10% hyclone, going down to posterity, condition of culture is 5%CO
2, 37 ℃ and saturated humidity;
2) three of exponential phase kinds of cells, PBS washing, 200g * 5 are minute centrifugal, abandon supernatant, and cell precipitation is resuspended among the PBS, and counting is adjusted cell density to 1 * 10
7/ mL;
3) it is subcutaneous the tumour cell suspension to be inoculated in Balb/c nude mice neck respectively, the generalized case of routine observation inoculation local tumor growing state and animal subject;
4) treat that tumour grows to 0.5cm
3About the size (the gross tumor volume computing formula is x
2Y/2, wherein x is the tumour major diameter, y is the tumour minor axis), the cervical vertebra dislocation method is put to death animal subject, the aseptic tumor tissues of winning;
5) the tumor tissues arrow is broken, grind by 200 order steel meshes;
6) cell suspension of crossing steel mesh washs with PBS, and 200g * 5 are minute centrifugal, abandon supernatant, and cell precipitation is resuspended in the RPMI-1640 complete medium that contains 10% hyclone, carries out the primary tumor cell in vitro culture, and condition of culture is 5%CO
2, 37 ℃ and saturated humidity;
7) tumour cell grows to exponential phase, and as above method is inoculated the Balb/c nude mice once more, the generalized case of routine observation inoculation local tumor growing state and animal subject;
8) treat that tumour grows to 0.5cm
3About the size (the gross tumor volume computing formula is x
2Y/2, wherein x is the tumour major diameter, y is the tumour minor axis), the cervical vertebra dislocation method is put to death animal subject, the aseptic tumor tissues of winning carries out formerly being commissioned to train fosterly once more, and three kinds of tumour cell D2F2/E2, LYX and the tumor formation rate of MCF-7 in the Balb/c nude mouse that the result obtains have reached 100%;
9), be inoculated in the Balb/c nude mice, the generalized case of routine observation inoculation local tumor growing state and animal subject to the logarithmic growth after date with the tumour cell cultured and amplified in vitro of aforementioned acquisition;
10) treat that tumour grows to 0.5cm
3About size (the gross tumor volume computing method as hereinbefore), the cervical vertebra dislocation method is put to death animal subject, wins tumor tissues, is fixed in paraformaldehyde.
The preparation of embodiment 3 tumor tissue pathologies section and the detection of surperficial Her2 antigen presentation
1. film-making
1) preparation of paraformaldehyde phosphate buffer: paraformaldehyde 40g, the PBS500mL of 0.1mol/L pH7.4, both are mixed and heated to 60 ℃, stir and drip 1N NaOH to solution clear till, add after the cooling PBS to cumulative volume be 1000mL.
2) win tumor tissues after, handle the tissue draw materials as early as possible, make every effort to keep organizing fresh, moist, piece of tissue can not be excessive or blocked up, especially thickness surpasses 1cm.
The fixation of tissue of 3) drawing materials is in the paraformaldehyde phosphate buffer, fixedly liquid measure wants sufficient, and at least 20 times to the amount of tissue, otherwise can cause concentration of fixation fluid to reduce, set time is organized as suitable (48 hours) to soak into fully, and the set time is after influencing Color for a long time.
4) dehydration of tissue, transparent, waxdip: the principle of GPRS is dehydration transparent will reaching and but in the whole operation process, and the temperature of waxdip should not surpass 64 ℃, otherwise antigen active is descended, reduce recall rate, or cause organize brown and crisp, section difficulty, specific procedure such as following table 1.
5) take out the back from the wax cylinder and carry out embedding with paraffin.
6) pre-service of microslide: the processing of microslide is an important step of carrying out immunohistochemical staining, and the present invention adopts poly-l-lysine to handle microslide.
Section: with paraffin slicing machine the organized wax stone of embedding is thinly sliced, slice thickness 4 μ m, 45 ℃ of water temperature exhibition sheets will flatten, otherwise fold place are prone to non-specific staining as far as possible.55 ℃ of incubators were baked sheet 2 hours, waterproof sealing, and cryopreservation is standby.
Table 1 tissue dewatering, transparent, waxdip program
7) dewaxing to water of paraffin section: paraffin section de-waxing to water is the reverse process of tissue dewatering, transparent, waxdip and embedding, and the concrete operations flow process is as follows:
A) 4 μ m paraffin sections, 55 ℃ of roasting sheets of incubator begin dewaxing after 18 hours;
B) dimethylbenzene I, II and III level are each 10 minutes;
C) absolute ethyl alcohol, 90% ethanol, 80% ethanol and 70% ethanol are each 2 minutes;
D) wash 2-3 minute from the beginning, enter IHC dyeing.
2. SABC (IHC) dyeing
(1) thermoinducible antigen retrieval
1) hot repair compound method: 100 ℃ of water-baths, 10 minutes * 2;
2) the multiple medium of hot repair: 0.01mol/L pH6.0 citrate buffer solution.
Handle by antigen retrieval, can improve antigen-antibody positive detection rate.
(2) ABC method dyeing
The characteristics of ABC method are to utilize Avidin to connect biotin labeled second antibody and biotin labeled enzyme respectively.Staining procedure is as follows:
1) the section routine dewaxes to water, and PBS washed 3 * 3 minutes;
2) 3% hydrogen peroxide (H
2O
2) hatched 5 minutes, in order to remove the endogenous peroxydase reaction;
3) distillation washing, PBS soaked 5 minutes;
4) Xi Shi normal calf serum (blocking agent), incubated at room 20 minutes;
5) unnecessary calf serum is removed in suction;
6) the anti-Her2 monoclonal antibody in anti-Her2 Humanized monoclonal antibodies or mouse source (available from The 2nd Army Medical College international co-operation institute of oncology) is diluted to 10 μ g/mL, hatches for 37 ℃ to spend the night in 60 minutes or 4 ℃;
7) PBS washed 3 * 3 minutes;
8) biotin labeled anti-human IgG or anti-mouse IgG (available from crystalline substance U.S. company) were hatched 30-60 minute for 37 ℃;
9) PBS washed 3 * 3 minutes;
10) ABC compound (available from crystalline substance U.S. company) was hatched 30-60 minute for 37 ℃;
11) PBS washed 3 * 3 minutes;
12) DAB-H
2O
2Developed the color washing 5-10 minute;
13) haematoxylin redye, dehydration, sealing.
Histopathologic slide's sampling Detection with preparation, coloration result confirms: D2F2/E2, LYX and MCF-7 tumor tissues detect through aforesaid ICH decoration method, that the expression of its Her2 antigen is respectively is high, in and invariably with level, i.e. colour developing is strong positive, the weak positive and feminine gender.The tissue staining result of different slice positions is even, shows that the tumor tissues that tumour cell forms in the inoculation animal body has good homogenieity.Therefore, this group section can be used for detecting the expression of tumor patient tumor tissues Her2 antigen.
The detection of embodiment 4 tumor patient tumor tissues Her2 antigenic expressions
Obtain the tumor tissues of 10 tumor patient excisions or the tumor tissues that biopsy obtains, through method as hereinbefore fix, after the dehydration, transparent, waxdip, embedding, section, section is attached at the other end that contrast section slide glass is used in the detection that has prepared, and the dyeing that then utilizes identical method to carry out ICH detects.
The result judges:
D2F2/E2, LYX and MCF-7 tumor tissues dyeing situation must be strong positive, the weak positive and feminine gender, and experimental result is effective, judge by following step, otherwise test invalid;
Patient tumors tissue staining situation and D2F2/E2, LYX and MCF-7 tumor tissues dyeing situation are compared, and dyeing situation≤MCF-7 is judged to feminine gender (-); The dyeing situation is judged to the weak positive (+) between MCF-7, LYX; The dyeing situation is judged to middle strong positive (++) between LYX, D2F2/E2; Dyeing situation 〉=D2F2/E2 is judged to strong positive (+++), the results are shown in Table 2.
Table 2
| Coloration result | Judge | |
| Given the |
＞D2F2/E2 | (+++) |
| Given the |
＞D2F2/E2 | (+++) |
| Given the |
MCF-7～LYX | (+) |
| Given the |
＜MCF-7 | (-) |
| Given the test agent 5 | LYX～D2F2/E2 | (++) |
| Given the test agent 6 | LYX～D2F2/E2 | (++) |
| Coloration result | Judge | |
| Given the test agent 7 | ＞D2F2/E2 | (+++) |
| Given the test agent 8 | LYX～D2F2/E2 | (++) |
| Given the test agent 9 | ＜MCF-7 | (-) |
| Given the test agent 10 | LYX～D2F2/E2 | (++) |
The dyeing situation of the section of part given the test agent is seen Fig. 3, and as seen, A is MCF-7 among Fig. 3, and Her2 does not have expression; B is LYX, and the Her2 moderate is expressed; C is D2F2/E2, and Her2 highly expresses; D is a sample 3 (+); E is a sample 5 (++); F is sample 7 (+++).
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned disclosure of the present invention, these equivalent form of values fall within claims institute restricted portion of the application equally.
Claims (6)
1. one kind is detected the test pieces that Her2 expresses in the breast tissue, it is characterized in that having with lower area on the first type surface of described test pieces:
(a) Her2 high expressed check plot, described high expressed check plot is provided with the tumor tissue section of Her2 high expressed; The tumor tissue section of described Her2 high expressed is selected from: D2F2/E2, HTB-20 or SK-BR-3 tumor tissue section;
(b) express the check plot among the Her2, the described middle check plot of expressing is provided with the tumor tissue section of expressing among the Her2; The tumor tissue section of expressing among the described Her2 is selected from: KYC or LYX tumor tissue section;
(c) Her2 does not have the expression check plot, and described nothing is expressed the check plot and is provided with the histotomy that Her2 does not have expression; The histotomy that described Her2 does not have expression is selected from: A431, QYC, MCF-7, RENCA tumor tissue section or Her2 do not have the normal structure section of expression;
(d) Her2 detection zone, described detection zone are used to place breast tissue to be detected.
2. the test pieces that Her2 expresses in the detection breast tissue according to claim 1 is characterized in that described histotomy is paraffin-embedded section.
3. the Her2 immunodiagnosis reagent kit of a breast cancer is characterized in that, comprises the test pieces that Her2 expresses in the described detection breast tissue of claim 1.
4. the Her2 immunodiagnosis reagent kit of breast cancer according to claim 3, it is characterized in that, also comprise immunohistochemical staining reagent in the described kit, described immunohistochemical staining reagent comprises: the blocking agent of nonspecific binding site, the initial antibodies that is connected with tissue antigen, the second antibody that is connected with initial antibodies, enzyme, substrate, developer.
5. the Her2 immunodiagnosis reagent kit of breast cancer according to claim 4, it is characterized in that, described initial antibodies is the anti-Her2 monoclonal antibody of anti-Her2 Humanized monoclonal antibodies or mouse source, and described second antibody is biotin labeled anti-human IgG or anti-mouse IgG.
6. the Her2 immunodiagnosis reagent kit of breast cancer according to claim 4 is characterized in that, the blocking agent of nonspecific binding site is selected from: animal blood serum, albumin; Described enzyme is a horseradish peroxidase; Described substrate is H
2O
2Perhaps described developer is selected from: DAB-H
2O
2, AEC-H
2O
2, CN-H
2O
2
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