CN115505040A - Immunohistochemical kit for detecting EDB protein and application thereof - Google Patents

Abstract

The invention provides a modified L19 antibody, the sensitivity of the modified L19 antibody is obviously improved by biotin modification, and a reliable and accurate identification result can be obtained in the detection of clinical samples of patients, so that the modified L19 antibody has a wide application prospect.

Description

Immunohistochemical kit for detecting EDB protein and application thereof

Technical Field

The invention relates to the field of biological detection, in particular to an immunohistochemical kit for EDB protein and application thereof.

Background

Fibronectin (Fn) is a unique macromolecular glycoprotein with a structure of two identical polypeptide chains formed by arranging and combining multiple repeating units, and is formed by connecting C-terminals of the two polypeptide chains with each other by disulfide bonds. The repeating units of the polypeptide chain are mainly divided into three types I, II and III, which respectively correspond to different binding sites, thereby playing different biological functions, due to the existence of variable shearing phenomenon in organisms, some repeating units are often inserted or deleted in the III type repeating units, the inserted or deleted repeating units are respectively called as Extra Domain A (EDA), extra Domain B (EDB) and IIICS variable domain, thereby causing the unique ecology of multiple Fn variable shearing variant in organisms, fn is also divided into two types of Plasma Fn (pFN) and Celluar Fn (cFn) according to the characteristic of the existence of the inserting unit, the existence of the inserted repeating unit is insoluble to the existence of Fn, the playing important role of biological function is influenced, such as the existence of the inserted repeating unit in the Plasma Fn, is soluble, is mainly existed in blood Plasma (0.65 mg/ml), plays an important role in the formation of thrombus, hemostasis, maintenance of normal cell morphology, cell adhesion, healing and wound, the healing process and the formation of angiogenesis and regeneration of multiple tumor cells, and the intercellular tissue regeneration process are closely related to the angiogenesis and the development of the angiogenesis.

Among the various forms of fibronectin, the expression of the EDB-domain-containing Fn is particularly remarkable, the EDB-domain is a type III Fn-repeat unit containing 91 amino acids, is inserted between the III7 unit and the III8 unit, is found in 1985, is expressed in tumor perivascular tissues and embryonic tissues, and is hardly expressed in normal tissues, and various studies show that the EDB-Fn is expressed in various solid tumors and lymphomas, and is derived from not only tumor cells, tumor-associated fibroblasts related to tumor microenvironment, neovascular endothelial cells and the like, and shows the high expression of the EDB-Fn, and the EDB-Fn is closely related to the topological structure of the tumor microenvironment and the neogenesis of tumor blood vessels. Some studies have further shown that EDB-Fn expression is positively correlated with the progression of various tumors.

Due to the high specificity and relevance of EDB-Fn existing in tumor microenvironment, researchers continuously consider whether EDB-Fn can be used as a target to be combined with the current technologies of pyrogenic immunotherapy, targeted imaging and the like, and based on the combination, a plurality of antibody proteins capable of being combined with EDB protein are developed, wherein the application of L19 antibody is the most extensive, the L19 antibody is discovered by phage display technology in 1998, and experiments prove that the EDB protein can be combined with EDB with 28 times higher affinity than a large number of functional antibodies with similar composition and performance, and the off-target effect can be effectively reduced.

Immunohistochemistry (IHC) is a commonly used technique and means in clinical pathological diagnosis. From the 70 s of the 20 th century, immunohistochemical technology has been applied to pathological diagnosis, which has great influence on tumor diagnosis, tumor classification and prognosis judgment, expands the understanding of people on various diseases and tumor formation processes, and improves the pathological diagnosis and research level. The basis of immunohistochemistry is that the immunological basic principle of antigen-antibody reaction, i.e. the combination between antibody and antigen has high specificity, and firstly, some chemical substance in tissue or cell is extracted, and then the extracted chemical substance can be used as antigen or hapten. Then, specific antibody is obtained after the animal is immunized, and the antibody is used for detecting the same kind of antigen substance in tissues or cells. Since the complex of antigen and antibody is colorless, the site of antigen-antibody binding must be displayed by histochemical methods (fluorescein, enzyme, metal ion, isotope) to achieve qualitative, localized or quantitative investigation of unknown antigen in tissues or cells.

Although EDB related targeted drugs are continuously developed, clinical sample detection modes required by products moving to clinics are quite limited, and clinical and laboratory detection methods of EDB proteins are mainly limited to nucleic acid levels, such as conventional Polymerase Chain Reaction (PCR), quantitative real-time quantitative PCR (qRT-PCR), semi-quantitative differential PCR (sq-PCR), digital PCR (ddPCR) and the like, but detection of the nucleic acid levels has the defects of incapability of visually displaying antigen distribution in tissues, high requirements on sampling and experimental operation, easiness in misjudgment and the like, immunohistochemistry can relatively accurately and qualitatively display EDB protein distribution conditions in clinical samples of patients, and more accurate pathological interpretation can be given by combining with the nucleic acid detection modes. At present, no immunohistochemical detection kit capable of verifying EDB protein in clinical samples in a standardized, positioned and quantitative manner is available in China, the reason for the detection is probably that EDB protein in tissue slice samples is distributed in a trace manner, and the sensitivity of EDB related antibodies at present cannot reach the immunohistochemical detection level.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a modified L19 antibody, the sensitivity of the modified L19 antibody is obviously improved, and a reliable and accurate identification result can be obtained in the detection of a clinical sample of a patient. In order to achieve the technical effects, the invention provides the following technical scheme.

In a first aspect of the invention, a modified L19 antibody is provided, wherein the modification is biotin labeling, fluorescein labeling or HRP enzyme labeling.

In a preferred embodiment, the modification is a biotin label.

In a second aspect of the present invention, there is provided a use of the modified L19 antibody in the preparation of a product for detecting EDB protein in a biological sample.

In one embodiment, the sample is a tissue sample.

In a third aspect of the present invention, there is provided the use of the modified L19 antibody as described above in the manufacture of a product for use in diagnosis of tumors.

In a fourth aspect of the invention, there is provided the use of the modified L19 antibody as described above in the manufacture of a product for use in the classification of tumours.

In a fifth aspect of the present invention, there is provided a use of the modified L19 antibody as described above for the preparation of a product for prognosis evaluation of tumors.

In one embodiment, the product is a reagent or a kit.

In a preferred embodiment, the kit is an immunohistochemical kit.

The invention has the beneficial effects that: the invention provides a method for detecting EDB protein through immunohistochemistry for the first time, and makes up the deficiency of an EDB protein immunohistochemical detection means.

The detection sensitivity of the modified L19 antibody provided by the invention is greatly improved, and the problem that the detection result cannot be obtained due to low sensitivity in immunohistochemical detection is solved.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

FIG. 1 is a purification map of L19 antibody.

FIG. 2 shows a comparison between before and after purification of Protein A column (SDS gel).

Fig. 3 shows WB verification of the antigen binding activity of the purified L19 antibody.

Fig. 4 is a graph of purified peaks of Biotin-modified L19 antibody.

FIG. 5 shows the WB validation of biotin modification of purified L19-biotin antibody.

FIG. 6 shows WB confirmation of the antigen binding activity of the purified L19-biotin antibody.

FIG. 7 is the results of immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma tissue using L19 antibody.

FIG. 8 shows the results of immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma tissue using Biotin-modified L19 antibody.

Fig. 9 shows the results of immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma tissue using Biotin-modified L19 antibody.

FIG. 10 shows the results of immunohistochemical analysis of paraffin-embedded human paracolon adenocarcinoma tissues using Biotin-modified L19 antibody.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Example 1: and (3) construction, purification and modification of the L19 antibody.

1. Expression of L19 antibody: an L19 heavy chain and light chain fragment sequence (SEQ ID NO. 1-2) is connected into pCDNA3.1 plasmid through enzyme, fermentation extraction is carried out, PEI reagent is used for transforming into 293F cells after sequencing verification, the density of the 293F cells is 100w/mL during transformation, after 96 hours of transformation, the cell viability is reduced to about 50%, 1300rpm of the cells is taken for centrifugation for 10mins, and the supernatant is taken for purification and detection of an L19 antibody.

2. Purification of L19 antibody: the L19 antibody was purified by Protein A column, according to the following procedure.

1. The cell suspension cultured until day 3 was 100ml,6000g was centrifuged for 10mins, and the supernatant was collected and passed through a 0.45 μm filter and stored at 4 ℃.

2. The liquid phase was thoroughly washed with purified water at 5mL/min, then the flow rate of the liquid phase was adjusted to 0.2mL/min, the Protein A column was packed, and the ethanol in the column was further washed completely at a flow rate of 1mL/min.

3. Balancing: the column was equilibrated by washing 10 column volumes with 20mM phosphate buffer pH7.0 at a flow rate of 1ml/min.

4. Loading: the sample was slowly loaded at a flow rate of 0.2 mL/min.

5. And (3) flushing: the column was washed with 20mM phosphate buffer pH7.0 at 1mL/min for 10 column volumes to elute contaminating proteins.

6. And (3) elution: eluting L19 protein with 0.1M glycine hydrochloric acid buffer solution with pH of 2.7 at an elution speed of 1mL/min, collecting protein with 4mL centrifuge tubes, collecting 1mL eluate in each tube, adding 150uL 1M Tris-HCl buffer solution with pH of 9.0 into the collection tubes to neutralize pH value, and eluting for 15 column volumes.

7. Column regeneration: 10 column volumes were washed with 20mM phosphate buffer at a rate of 1mL/min.

8. Desalting: the 10 column volumes were washed with purified water at a rate of 1mL/min.

9. The protein A column was filled with 20% ethanol at a rate of 0.2 mL/min.

10. And (4) recovering the liquid in the corresponding collecting pipe according to the liquid phase peak-out diagram, and storing at 4 ℃.

The purification pattern of the L19 antibody is shown in FIG. 1, and the comparison (SDS gel) before and after the purification on the Protein A column is shown in FIG. 2.

3. Verification of binding specificity of the purified L19 antibody to EDB protein: EDB protein 10ug expressed by laboratory fermentation is taken for gradient dilution, and the dilution concentration is 10 ng/mu L,1 ng/mu L and 0.1 ng/mu L respectively. Loading 4-12% of prefabricated gel, carrying out membrane conversion after electrophoresis with the loading amount of 10 mu L,160v,40mins under the conditions of 100v,90mins, carrying out room-temperature sealing with 5% of skimmed milk powder for 1h after membrane conversion is finished, 1:1000 dilutions of L19 antibody were incubated overnight, TBST washed 5mins x 6 times with shaking, and incubated 1:20000 diluted HRP goat anti-human IgG (H + L) antibody 1h, tbst washes 5mins x 6 times followed by exposure, WB confirmed the antigen binding activity of the purified L19 antibody.

WB results are shown in FIG. 3, and the prepared L19 antibody has good binding specificity and dose-effect relationship with EDB protein. Can be used for the immunodetection of EDB protein.

4. Biotin (biotin) labeling of the L19 antibody:

1. since the Biotin labeling of about 200ul, i.e., about 1.3mg protein samples is performed, the addition of glycerol to the antibody has an effect on the following labeling, it is necessary to first perform ultrafiltration and centrifugation on the antibody to remove glycerol, to obtain 200ul of the antibody stock solution, to add 10ml of DPBS to dissolve the antibody stock solution, to centrifuge the antibody stock solution in a 10kda ultrafiltration tube at 3000g for 20mins, to repeat 4 times to obtain about 200ul of the final solution, and to detect the concentration of the concentrated antibody by the BCA detection kit.

2. Performing biotin labeling according to the concentration of the antibody, wherein the kit is a super biotin labeling kit of frdbio, and according to the condition that the size of an L19 antibody is about 150kda, the labeling quantity is 1mg, and 13.33ul of 10mM biotin is needed, a 10kda ultrafiltration tube is adopted, 1mg of antibody stock solution and 5mL of antibody labeling buffer solution are added, centrifugation is performed for 10mins at 12000g, and the steps are repeated for 5 times, so that system replacement is completed.

3. And after the final ultrafiltration is finished, adding a proper amount of marking buffer solution to adjust the concentration of the antibody to be about 2mg/mL, adding 13.3 muL of dissolved active super biotin into the ultrafiltration tube, and lightly blowing, beating and uniformly mixing. Placing into a 37 ℃ incubator and incubating for 30min in the dark.

4. 12000g is centrifuged for 10mins, 5mL of marking buffer solution is added into the ultrafiltration tube, the ultrafiltration tube is gently blown and evenly mixed, centrifugation is carried out for 10min at 12,000g, the step is repeated for 8 times, and the mixture is concentrated until the final liquid volume is about 1mL, and the purified sample is prepared.

5. The liquid phase was fully washed with purified water at 5mL/min, then the flow rate of the liquid phase was adjusted to 0.2mL/min, the Protein A column was packed, and the ethanol in the column was continuously washed completely at a flow rate of 1mL/min.

6. Balancing: the column was equilibrated by washing 10 column volumes with 20mM phosphate buffer pH7.0 at a flow rate of 1ml/min.

7. Sampling: the sample was slowly loaded at a flow rate of 0.2 mL/min.

8. And (3) flushing: the column was washed with 20mM phosphate buffer pH7.0 at 1mL/min for 10 column volumes to elute contaminating proteins.

9. And (3) elution: eluting L19-biotin protein with 0.1M glycine hydrochloric acid buffer solution with pH2.7 at an elution speed of 1mL/min, collecting protein with 4mL centrifuge tubes, collecting 1mL eluate per tube, adding 150uL 1M Tris-HCl buffer solution into the tubes to neutralize pH value, and eluting for 15 column volumes.

10. Column regeneration: 10 column volumes were washed with 20mM phosphate buffer at a rate of 1mL/min.

11. Desalting: 10 column volumes were washed with purified water at a rate of 1mL/min.

12. The proteinA column was flushed with 20% ethanol at a rate of 0.2 mL/min.

13. And (4) recovering the liquid in the corresponding collecting pipe according to the liquid phase peak diagram, and storing at 4 ℃.

The purification peak of the biotin-modified L19 protein (hereinafter described as L19-biotin antibody) is shown in FIG. 4.

5. Validation of biotin modification of purified L19-biotin antibody binding specificity to EDB protein: taking 20 mu L of purified and concentrated L19-biotin antibody samples in two tubes, respectively adding 5ul of reducing and non-reducing electrophoresis buffers, boiling and heating the sample tubes added with the reducing electrophoresis buffers for 10mins, cooling to room temperature, then loading 4% -12% of prefabricated gel, simultaneously loading the L19 antibody with the same concentration as a negative control, carrying out membrane transfer after electrophoresis of 5 mu L,160v and 40mins in loading amount, carrying out membrane transfer under the membrane transfer conditions of 100v and 90mins, after membrane transfer is finished, sealing for 1h at room temperature by using 5% of skimmed milk powder, and incubating at room temperature for 1: HRP-Streptavidin (SA) antibody 1h diluted 10000, exposed after shaking 5mins 6 times TBST, WB validated the activity of purified L19-biotin antibody. Meanwhile, 10ug of EDB protein expressed by laboratory fermentation is subjected to gradient dilution, and the dilution concentrations are respectively 10 ng/mu L,1 ng/mu L and 0.1 ng/mu L. Loading 4-12% of prefabricated gel, carrying out membrane conversion after electrophoresis with the loading amount of 10 mu L,160v,40mins under the conditions of 100v,90mins, carrying out room-temperature sealing with 5% of skimmed milk powder for 1h after membrane conversion is finished, 1:1000 dilution of L19-biotin antibody were incubated overnight, TBST washed 5mins 6 times with shaking, and incubated 1:20000 diluted HRP goat anti-human IgG (H + L) antibody 1h, tbst shake wash 5mins x 6 times before exposure.

As a result, as shown in fig. 5 and 6, it was confirmed that biotin was successfully modified on the L19 antibody, and the L19-biotin antibody was able to bind to a secondary HRP antibody having streptavidin, and also had good binding specificity as the EDB protein.

Example 2: and (5) verifying a sample immunohistochemistry experiment.

1. Preparing a solution:

1.1 TBST, specifically TBS buffer containing 0.025% TritonX-100% by volume; TBS prefabricated powder 1 bag; H2O 2L; tritonX-100. Mu.L.

1.2 antigen retrieval solution, in particular Tris-EDTA buffer containing 0.05% by volume of Tween-20: tris 1.21g; 0.37g of EDTA disodium; H2O 1L; adjusting the pH to 9.0; tween-20. Mu.L.

1.3, L19 and L19-biotin antibodies were 30% BSA-50% glycerol-DPBS formulated antibody stock.

2. The experimental process comprises the following steps:

2.1, preparing a section, and providing the tumor tissue section of the patient with the colon adenocarcinoma and the lung adenocarcinoma by a subsidiary hospital of the medical academy of sciences in Shandong province.

2.2, baking the slices, putting the slices into an adjustable thermostat, adjusting the temperature to 60 ℃, and standing for about 2 hours.

2.3, dewaxing and rehydration: and after the glass slide is baked, dewaxing by adopting a dimethylbenzene solution and carrying out dehydration treatment by adopting alcohol with different gradients, and replacing paraffin in the sample specimen. The specific process is as follows: xylene (10min × 2) -absolute ethanol (5min × 2) -95% ethanol (5 min) -75% ethanol (5 min) -55% ethanol (5 min), and then immersing into tap water to wash for 3min × 3.

2.4, blocking and inactivating endogenous peroxidase: immersed in a 3% hydrogen peroxide solution and left for 15min, washed 5min x 3 with TBST (TBS +0.025% triton x-100).

2.5, repairing the antigen, heating the prepared antigen repairing liquid to boiling in a microwave oven with high fire, soaking the slices into the antigen repairing liquid, continuing heating in the microwave oven with high fire for 25min, standing at room temperature for more than half an hour, cooling the liquid to room temperature, and washing with TBST for 5min x 3.

2.6, blocking biotin, namely firstly, using an immunostaining blocking solution to carry out mass transfer on endogenous biotin blocking solution A and endogenous biotin blocking solution B, using an immunostaining blocking solution 1:100 to working concentration, carefully wiping the glass slide, drawing a circle around the specimen by using a hydrophobic pen, dripping 200ul of A working solution into the circle, sealing 30min at room temperature, washing 5min x 3 by TBST, dripping B working solution, sealing 30mins at room temperature, washing 5min x 3 by TBST.

2.7, incubate primary antibody, mix L19 and L19-biotin antibody according to 1:100 was diluted with TBS to primary antibody working solution, added dropwise to the sections to infiltrate the tissue sections, incubated overnight in a wet box at 4 ℃ and set as a control to exclude the effect of other reagents and processes other than primary antibody on the results.

2.8, incubation secondary antibody: the overnight sections were rewarmed at 30min at rt, washed 5min x 3 with tbst, and secondary antibodies HRP goat anti-human IgG (H + L) and HRP-SA, respectively, in TBS at 1:1000 and 1:10000 of the reagent solution is diluted into a secondary antibody working solution, each specimen is ensured to be soaked in an antibody solution, the specimen is incubated in a wet box for 1h at room temperature, and TBS washing is performed for 5min x 3 after the incubation is finished.

And 2.9, performing DAB color development, dripping 100ul of DAB working solution into each piece, incubating for 5min at room temperature in a dark place, and washing for 5min by using running water to terminate the reaction.

2.10, hematoxylin counterstaining: the sections were soaked in hematoxylin solution for 5min, and washed with running water for 15min to turn blue.

2.11, dehydration and transparency: the sections were each left standing for 2min with a gradient of ethanol from low to high (55%, 75%, 95%, 100%), xylene 5min x 2 to clear and finally sealed with neutral gum.

3. The experimental results are as follows: the results of immunohistochemical analysis of paraffin-embedded human paracolon adenocarcinoma tissue, human colon adenocarcinoma tissue, and human lung adenocarcinoma tissue using the L19 antibody and the L19-biotin antibody are shown in FIGS. 7 to 10. The results show that the L19-biotin antibody can be applied to immunohistochemical assay for detecting EDB protein in human tissues in the present invention (FIGS. 8-10), while the detection of EDB protein using L19 antibody cannot obtain the detection results (FIG. 7). Immunohistochemistry results show that EDB is expressed on human colon adenocarcinoma, particularly on cancerous intestinal gland cells, on human lung adenocarcinoma, particularly on cancerous alveolar wall endothelial cells, and not significantly in human colon adenocarcinoma paraneoplastic tissues.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (9)

1. A modified L19 antibody, wherein the modification is a biotin label, a fluorescein label, or an HRP enzyme label.

2. The modified L19 antibody of claim 1, wherein the modification is a biotin label.

3. Use of the modified L19 antibody of claim 1 or 2 for the preparation of a product for the detection of EDB protein in a biological sample.

4. The use of claim 3, wherein the sample is a tissue sample.

5. Use of a modified L19 antibody according to claim 1 or 2 for the preparation of a product for tumor diagnosis.

6. Use of a modified L19 antibody according to claim 1 or 2 for the preparation of a product for tumour classification.

7. Use of a modified L19 antibody according to claim 1 or 2 for the preparation of a product for the prognosis of a tumor.

8. Use according to any one of claims 5 to 7, wherein the product is a reagent or kit.

9. The use of claim 8, wherein the kit is an immunohistochemical kit.

Images