CN113671187A

CN113671187A - IHC detection kit for detecting MYCN protein

Info

Publication number: CN113671187A
Application number: CN202110922034.6A
Authority: CN
Inventors: 李彦欣; 杨怡; 冯海忠
Original assignee: Shanghai Childrens Medical Center Affiliated to Shanghai Jiaotong University School of Medicine
Current assignee: Shanghai Childrens Medical Center Affiliated to Shanghai Jiaotong University School of Medicine
Priority date: 2021-08-12
Filing date: 2021-08-12
Publication date: 2021-11-19

Abstract

The invention relates to an IHC detection kit for detecting MYCN protein, which comprises a protein hybrid antibody of MYCN. The protein hybrid antibody to MYCN was purchased from Cell Signaling Technology, Inc. under the #84406 s. The invention finds that the antibody of MYCN with the product number of #84406s of Cell Signaling Technology company can be used for immunohistochemical Technology of neuroblastoma tissue samples; the goodness of fit of the composition to FISH is over 75 percent, and the goodness of fit to prognosis is obviously higher than that of FISH; the kit is more beneficial to the detection of clinical MYCN and the judgment of prognosis; solves the problem that the international MYCN has no commercial antibody for immunohistochemical detection. And the detection accuracy of the MYCN and the judgment of prognosis are improved.

Description

IHC detection kit for detecting MYCN protein

Technical Field

The invention relates to a detection kit, in particular to an IHC (protein immunohistochemistry) detection kit for detecting MYCN protein.

Background

Neuroblastoma is the most common extracranial solid tumor in children, accounting for approximately 12% of cancer deaths in children. The disease course has 2-step differentiation, some patients can self-heal without treatment, and some patients can not recover lives after various treatments. Finding an index that predicts prognosis is therefore of central importance in neuroblastoma in children. The MYCN amplification rate of neuroblastoma is about 22%, which is the first genetic marker for neuroblastoma with important clinical and prognostic value. MYCN amplification in the grouping indices used by the International Neuroblastoma Risk Group (INRG) and the pediatric tumor group (COG) is currently a large component.

To date, clinical and laboratory detection methods have been limited primarily to the nucleic acid level due to the lack of reliable IHC antibodies to MYCN, such as conventional Polymerase Chain Reaction (PCR), quantitative real-time quantitative PCR (qRT-PCR), semi-quantitative differential PCR (sq-PCR), digital PCR (ddpcr), Fluorescence In Situ Hybridization (FISH), Chromosome In Situ Hybridization (CISH), multiple ligation dependent probe amplification (MLPA), and the like. The MYCN genetic status of FISH detection has now been integrated into risk classification. However, several studies have demonstrated that high expression of MYCN protein is also seen in cases where no amplification of MYCN is found at DNA level, and patients with amplification of MYCN gene at DNA level also found low expression at protein level. The FISH detection of MYCN widely applied in clinic at present detects the COPY number of MYCN at a DNA level, and generally, the COPY number at the DNA level is high, and the RNA level and the protein level are high. However, there are still a number of patients who are inconsistent because of the problems of transcriptional and translational and post-translational modifications, degradation of ubiquitination. Thereby affecting its prognosis.

MYCN is a method for detecting intracellular protein levels that is widely used in biological effects of protein levels, protein level detection, and immunohistochemical detection. However, the antibody sensitivity of many MYCNs is not achieved, the FISH result and the prognosis are not consistent well, and the clinical requirement is not met. Therefore, the method for rapidly, reliably and economically detecting the expression of the MYCN protein is of great significance. Currently there is no commercial antibody to IHC of MYCN that can be used in clinical assays internationally.

Disclosure of Invention

The purpose of the invention is: provides an application of a protein hybrid antibody of MYCN as an IHC clinical detection reagent of MYCN protein of childhood neuroblastoma.

The technical scheme adopted by the invention is as follows:

the invention provides an application of a MYCN protein hybrid antibody as a clinical detection reagent of MYCN protein of childhood neuroblastoma. In this application, the reagents detect the expression level of MYCN in a sample by immunohistochemistry.

Further, the protein hybrid antibody of MYCN was purchased from Cell Signaling Technology, Inc. under the #84406 s.

Further, in the application, the sample is a tissue sample.

The invention also provides an IHC kit for predicting the prognosis of the neuroblastoma in children, which comprises a protein hybrid antibody of MYCN.

Further, the IHC kit also comprises a secondary antibody anti-rabbitt-IgG, HRP linked, purchased from Cell Signaling company and having a product number of # 7074.

The invention also provides a kit for predicting the prognosis of the neuroblastoma in children, which comprises the IHC detection kit and a FISH detection kit of MYCN.

The method for judging the prognosis prediction of the neuroblastoma of the children comprises the following steps:

expression of MYCN protein was detected by immunohistochemical methods with rabbit anti-MYCN antibodies (#84406s, Cell Signaling Technology). The MYCN dyeing intensity is graded as 0-3 (0 is negative, 1 is weak, 2 is medium, 3 is strong), the positive proportion is graded as 0-4 (0 is 0%, 1 is less than 25%, 25% is more than or equal to 2 and less than 50%, 50% is more than or equal to 3 and less than 75%, 75% is more than or equal to 4 and less than or equal to 100%). Immunohistochemical grading was scored independently by two pathologists, averaged, and finally scored as the positive proportion of staining intensity. Grade 0 is negative, grade 1-4 is low expression, grade 5-8 is medium expression, grade 9-12 is high expression.

Diagnostic criteria if FISH is used in conjunction with immunohistochemistry:

when the FISH of the patient is positive and the IHC is more than 9 (more than or equal to 9), the prognosis is indicated to be poor, and if the IHC is between 0 and 8, the prognosis is indicated to be good; if the patient is negative for FISH, the IHC value is equal to 0, the prognosis is good, the IHC is greater than 0, and the prognosis is poor.

The immunohistochemical rating of the invention is more than 0 and equal to 0, and the guidance prognosis is more obvious and accurate than that of FISH; if FISH is used in combination with immunohistochemistry: the IHC of FISH + is less than 9 and the IHC of FISH-is equal to 0, the prognosis is good, the IHC of FISH + is greater than or equal to 9, and the IHC of FISH-is greater than 0, the prognosis is poor. More relevant to prognosis than using FISH and immunohistochemistry alone.

The invention has the following beneficial effects:

the invention finds an antibody of MYCN (the commercial application Technology field of which is disclosed as protein imprinting, immunoprecipitation, immunofluorescence, flow Cell and chromatin immunoprecipitation Technology) with the serial number #84406 of Cell Signaling Technology company, and the invention finds that the antibody can also be used for immunohistochemical Technology of neuroblastoma tissue samples. The goodness of fit of the fusion protein with FISH is more than 75%, and the goodness of fit with prognosis is obviously higher than that of FISH. It is more beneficial to clinical MYCN detection and prognosis judgment. Solves the problem that the international MYCN has no commercial antibody for immunohistochemical detection. And the detection accuracy of the MYCN and the judgment of prognosis are improved.

Drawings

FIG. 1 comparison of the consistency of IHC and FISH results, wherein a) MYCN protein expression is detected by IHC and the results are scored synthetically; b) analyzing the consistency of MYCN expression detected by IHC and FISH; c-e) categorizing and grouping IHC results according to FISH detection results (c, d), INSS stages ((c, d) and clinical outcome (e).

FIG. 2 is a graph of a better predictive prognostic assay combining IHC and FISH detection methods; wherein a-b) performing further grouping and Kaplan-Meier survival analysis on the MYCN-amp (a) and MYCN-non (b) populations respectively according to the IHC result; c) simultaneously, the FISH and IHC results are taken as grouping standards, and the difference between groups is compared by using Kaplan-Meier survival analysis; d) the relationship between each clinical factor and the patient's prognosis was summarized using a chromatogram (none, P >0.05, differences not statistically significant, P <0.05, P <0.0001, differences statistically significant).

Detailed Description

The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

The experimental methods in the following examples, which do not indicate specific conditions, all employ conventional techniques in the art, or follow the conditions suggested by the manufacturers; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Sample collection

In 2010 to 2019, pathological examination confirmed 332 patients with NB initial diagnosis in diagnosis and treatment in our hospital, and 41 patients had MYCN-amp. Because of factors such as serious destruction of tumor tissues or too few tumor cells, adverse protein detection and the like caused by too long storage time of tumor samples, only the tumor tissues of 28 cases of MYCN-amp patients participate in the test. As a control, we additionally selected tumor samples from 28 MYCN-non patients for IHC testing, 7 of which had disease-related deaths and 19 of which were in good condition at the last follow-up. All patients were diagnosed at an age of less than 12 years, with 30 males and 26 females. Table 3 lists the patient's age, gender, clinical stage, risk grouping, primary site, FISH test results, MYCN immunohistochemical score, prognosis, current status, follow-up time, etc. Each patient signed an informed consent prior to sampling, according to the rules prescribed by the ethical review board.

Preparing a tissue sample:

tumor specimens were surgically excised, cut into small pieces half an hour, fixed in 10% formalin, standard paraffin embedded, and sliced into 2-3 μm thick sections for subsequent analysis.

Test method

Experimental methods for IHC

Preprocessing paraffin sections:

a. incubating the paraffin sections stored at room temperature at 65 ℃ for 1h to melt the paraffin;

b. sequentially soaking the slices in 3 dye vats filled with xylene for dewaxing for 15min each time;

c. soaking the slices in gradient alcohol in sequence for rehydration: anhydrous ethanol for 2 times, 3-5 min/time; 90% ethanol for 2 times, 3-5 min/time; 70% ethanol for 2 times, 3-5 min/time; 1 time of tap water and 3 min/time;

place the sections in 3% H₂O₂Soaking the inner chamber in warm and dark place for 10min, and soaking in tap water at room temperature for 5 min;

placing the slices in 1 × citric acid antigen repairing solution, heating with high fire (98 deg.C-100 deg.C) microwave to boil (paper can be placed under a dye vat, if the paper is soaked, it is boiling, generally about 3min-4min), standing in microwave oven for 10min after power failure;

the dye vat was removed from the microwave oven and cooled to room temperature on ice (Note: without opening the dye vat during the period);

after the citric acid antigen repairing solution is cooled to room temperature, taking out the slices, and washing the slices for 3 times and 3-5 min/time by using 1 XPBS;

wiping off the excess liquid with a paper towel, drawing a hydrophobic isolation area on the target area by using an immunohistochemical pen, dropwise adding primary antibody (anti-MYCN, Cell Signaling, 84406s, 1:200 dilution), and incubating overnight at 4 ℃ in a humid cassette; taking out the wet box from 4 deg.C, standing at room temperature for 10min, and washing with 1 × PBS for 3 times, 3-5 min/time;

wiping off excessive liquid with paper towel, adding secondary antibody (anti-rabbitt-IgG, HRP linked, Cell Signaling corporation, #7074, without dilution), and incubating at room temperature for 20-25 min;

washing with 1 × PBS for 3 times, 3-5 min/time;

wiping off redundant liquid by using a paper towel, dripping a proper amount of DAB color developing agent prepared in proportion, reacting at room temperature, observing color development change under a microscope, and stopping color development reaction in tap water at proper time;

counterstaining with hematoxylin for about 1-2min, differentiating with hydrochloric acid alcohol for 1-2s, and soaking in tap water to turn blue for 10-15 min;

soaking and dehydrating the slices in gradient alcohol in sequence: 70% ethanol for 2 times, 3-5 min/time; 90% ethanol for 2 times, 3-5 min/time; anhydrous ethanol for 2 times, 3-5 min/time;

soaking the slices in 3 dye vats filled with xylene for 5min each time;

the gum was mixed with purified xylene according to 2: diluting the mixture according to the proportion of 1, sealing the mixture as a sealing agent, naturally drying the slide at room temperature, and performing microscopic examination after xylene is completely volatilized.

2 Paraffin section fluorescence immunoassay in situ hybridization (FISH), kit name LSI N-MYC Spectrum orange Probe:

1) preprocessing paraffin sections:

c. soaking the slices in gradient alcohol in sequence for rehydration: anhydrous ethanol for 2 times, 3-5 min/time; 90% ethanol for 2 times, 3-5 min/time; 70% ethanol for 2 times, 3-5 min/time;

d. placing the slices in 0.2M HCl, and incubating at room temperature for 10 min;

e. washing with 1 × PBS for 3 times, 3-5 min/time;

f. the sections were incubated in 0.01M citrate buffer (pH 6.0) at 80 ℃ for 1 h;

g. washing with 2 XSSC buffer solution for 2 times, 3-5 min/time;

h. washing with DDW for 3-5 min/time;

i. preheating 0.5mg/ml pepsin and 0.02M HCl solution at 37 deg.C for 5min, mixing at equal ratio of 1:1 to obtain 0.25mg pepsin/ml 0.01M HCl solution;

j. dropping 300 μ l of pepsin/HCL solution on the slices, covering with a cover glass, and incubating at 37 ℃ for 10 min;

k. washing with 2 XSSC buffer solution for 2 times, 3-5 min/time;

l. fixing the slices with 0.4% paraformaldehyde at room temperature for 10 min;

washing with 1 × PBS for 3 times (3-5 min/time);

and n, soaking and dehydrating the slices in gradient alcohol in sequence: 70% ethanol for 2 times, 3-5 min/time; 90% ethanol for 2 times, 3-5 min/time; anhydrous ethanol for 2 times, 3-5 min/time

Naturally airing the slide at room temperature;

2) pretreatment of the probe:

incubating the probe at 75 deg.C for 5min, immediately placing at 0 deg.C for 5-10min to denature the double-stranded DNA probe;

3) and (3) hybridization:

dropping 10 μ l of probe mixture solution on the denatured and dehydrated section, covering with a cover slip, sealing the section, and hybridizing overnight (about 15-17h) at 37 ℃ in a moist cassette;

4) and (3) elution:

a. the next day, the sections were removed and the coverslips gently removed, placed in 4 × SSPE and incubated at 47 ℃ for 5 min;

b. placing the slide in 4 × SSPE again, and incubating at 55-72 deg.C for 10 min;

c. soaking and dehydrating the slices in gradient alcohol in sequence: 70% ethanol for 2 times, 3-5 min/time; 90% ethanol for 2 times, 3-5 min/time; anhydrous ethanol for 2 times, 3-5 min/time;

d. soaking the slices in hexane/isopropanol mixture at room temperature for 10min (hexane: isopropanol are mixed at a ratio of 6: 4);

e. soaking the slices in isopropanol at room temperature for 5 min;

f. soaking the slices in anhydrous ethanol at room temperature for 5 min;

g. naturally airing the glass slide at room temperature until the ethanol is completely volatilized;

h. covering a cover glass after DAPI counterstaining, slightly extruding redundant liquid, and storing at 4 ℃ in a dark place after mounting;

2 determination of MYCN expression levels

1) MYCN Immunohistochemistry (IHC) score:

the MYCN dyeing intensity is divided into 0-3 points, wherein 0 is negative, 1 is weak, 2 is medium, and 3 is strong;

the positive proportion of MYCN dyeing is divided into 0 to 4 parts, wherein 0 percent is 0 part, 25 percent is 1 part, 2 percent to 2 percent is more than or equal to 25 percent and less than 50 percent, 3 percent to 3 percent is more than or equal to 50 percent and less than 75 percent, and 4 percent to 75 percent is more than or equal to 4 percent and less than or equal to 100 percent;

and c, final scoring, staining intensity x positive proportion. 0 is classified as "negative", 1-4 is classified as "low expression", 5-8 is classified as "medium expression", and 9-12 is classified as "high expression".

2) FISH result determination of MYCN

A MYCN/PAX3 ratio >5 is positive, i.e. MYCN amplification; the ratio of MYCN/PAX3 is less than or equal to 1, namely the MYCN is not amplified

Results of the experiment

1 testing the effectiveness of the antibodies used in the IHC assay of the invention:

to verify the effectiveness of the antibodies used in the present invention (MYCN #84406s, Cell Signaling), we included more samples (n ═ 56) for IHC detection. All tumor samples used in the experiment were FISH-detected, with 1/2 identifying MYCN-amp (Table 1). The results show that there are large differences in the positive proportion of malignant cells and the staining intensity among different tumor tissues. For the convenience of subsequent analysis, we scored the staining results comprehensively based on the above two points (fig. 1 a). On this basis, we analyzed the consistency of the FISH and IHC results (Table 2). The results show that ≧ 75% IHC results are consistent with FISH results (Table 4, FIG. 1b), regardless of MYCN-amp. For patients with inconsistent results, we included clinical information from patients for comprehensive analysis to determine which approach was more relevant to prognosis (to ensure the accuracy of individual prognosis, we included only 2016 and previously diagnosed patients when the follow-up time was involved). The results show that IHC scores increase with increasing ins staging and are highly correlated with the clinical prognosis of the patient (fig. 1 c-e). Among the MYCN-amp patients, 7 of 11 MYCN protein high expression (IHC score ≧ 9) patients had poor prognosis, while 5 patients with no expression or low expression had no adverse events; of the MYCN-non patients, all (4) patients in which MYCN protein expression was detected had a poor prognosis, while 17 patients with a good prognosis had no MYCN protein expression (Table 3). All the data show that the MYCN antibody has good sensitivity and specificity, has higher consistency with the FISH result, has a complementary effect on IHC of patients who are not detected by FISH, can improve the MYCN detection rate, and has great clinical application value.

TABLE 1 patient clinical information

Gender 0 ═ female; 1 ═ male;

grading the danger, namely 1, low-risk; 2 is medium-risk; 3, high risk; 4, being extremely high-risk;

the primary part is 1 as the neck; 2 is chest; 3, abdomen; 4, the pelvic cavity;

CR complete remission (complete remission)

TABLE 2 comparison of the consistency of the IHC and FISH results in this experiment

TABLE 3 correlation analysis of MYCN protein expression with clinical prognosis

2 the IHC and FISH combined detection method can better predict prognosis

According to the previous results, IHC can more accurately detect the expression of MYCN in the tumor and predict the prognosis. To further demonstrate this, the MYCN-amp and MYCN-non populations were further grouped using IHC results and statistically analyzed using Kaplan-Meier survival analysis. The IHC results were found to further differentiate the prognosis of the MYCN-amp and MYCN-non populations (FIGS. 2 a-b). Furthermore, if we included only FISH results as a grouping basis, the event-free survival rates for MYCN-amp and MYCN-non were 56.2% and 63.0%, respectively, and the survival analysis showed no statistical difference between the two groups. While IHC results were included only as a basis for grouping, the difference in survival between groups was statistically significant (fig. 2 c). By combining two approaches, i.e. FISH + IHC <9, FISH-IHC ═ 0 as one group, FISH + IHC ≥ 9, FISH-IHC >0 as the other group, the best clinical prediction effect can be achieved (fig. 2 c). At the same time, we found that if the FISH assay suggested MYCN-non, but if the IHC could detect MYCN protein expression, this patient had many with poor prognosis; in contrast, FISH detection suggested MYCN-amp, whereas IHC failed to detect MYCN protein expression or only low expression, which was much better prognosis for this patient (FIG. 2 d). In conclusion, IHC can make up the defects of FISH in the aspects of evaluating prognosis and guiding treatment, and the combined analysis of two detection results can achieve the optimal clinical prediction effect.

Claims

1. An application of a MYCN protein hybrid antibody as an IHC clinical detection reagent of MYCN protein of childhood neuroblastoma.

2. Use according to claim 1, characterized in that: the protein hybrid antibody to MYCN was purchased from Cell Signaling Technology, Inc. under the #84406 s.

3. Use according to claim 1 or 2, characterized in that: in the application, the sample is a tissue sample.

4. An IHC kit for predicting the prognosis of neuroblastoma in children, which is characterized in that: protein hybrid antibodies including MYCN.

5. The IHC kit for predicting the prognosis of neuroblastoma in children according to claim 4, wherein: the protein hybrid antibody to MYCN was purchased from Cell Signaling Technology, Inc. under the #84406 s.

6. The IHC kit for predicting the prognosis of neuroblastoma in children according to claim 5, wherein: the IHC kit also comprises a secondary antibody anti-rabbitt-IgG, HRP linked, purchased from Cell Signaling company and having a cargo number of # 7074.

7. A kit for predicting the prognosis of neuroblastoma in a child, comprising: a FISH assay kit comprising MYCN and the IHC assay kit of claims 5-6.