Preparation and characterization of polyclonal antibodies against BLM DNA Helicase

Introduction

Bloom’s syndrome helicase (BLM) is a member of the RecQ family of DNA helicases. In humans, five RecQ helicases have been identified: RecQ like helicase 1 (RecQL1), BLM, Werner syndrome helicase (WRN), RecQ like helicase 4 (RecQL4), and RecQ like helicase 5 (RecQL5). Among these, WRN, BLM, and RecQL4 have been most extensively studied. Mutations in their respective genes can lead to various human diseases. Specifically, mutations in the BLM gene cause Bloom’s syndrome (BS), an autosomal recessive disorder characterized by dwarfism, intellectual disability, facial telangiectatic erythema, and photosensitivity. In cells, BLM DNA helicase is involved in DNA replication, transcription, recombination, repair, and telomere maintenance (1,2). Aberrant expression of BLM DNA helicase has been detected in various human malignancies (3-5), and researchers worldwide have begun to focus on its role in cancer progression, particularly in tumorigenesis and metastasis (6-9). This could provide a new target for cancer treatment and prognostic evaluation (10).

At present, rabbit polyclonal antibody (pAb) production technology is well-established (11,12). Compared with mouse antisera, rabbit antisera contain high-affinity antibodies and can recognize a broader range of antigenic epitopes (13). pAbs have the advantage of simpler production procedures, shorter immunization cycles, and lower cost, making them indispensable tools for the investigation of gene products and their mechanisms of action. However, preparing highly specific antibodies that can be used for Western blot, immunohistochemistry (IHC), and even immunofluorescence (IF) is not straightforward.

Our research group has long focused on the role and mechanisms of BLM DNA helicase in cancer, especially breast cancer (14). During our studies, we noted that commercially available BLM DNA helicase antibodies often exhibit batch-to-batch variability and inconsistencies in results from different manufacturers. To address these issues, we expressed BLM^642–1,290 DNA helicase (residues 642–1,290 represent the core catalytic domain of the BLM DNA helicase) in Escherichia coli and purified it for use as an immunogen to generate rabbit pAbs. This work aims to establish a reliable source of BLM pAbs, thereby providing a solid foundation for further investigation into the role of BLM DNA helicase in the pathogenesis of breast cancer and other malignancies.

Methods

Main reagents and materials

• Recombinant E. coli strain pET-15b-BLM^642–1,290-BL21-CodonPlus: kindly provided by Director Xu Guang Xi at the Curie Institute, University of Paris-Sud (Paris XI), France.
• Tryptone: Oxoid Ltd., UK.
• Yeast Extract: Oxoid Ltd., UK.
• Luria-bertani (LB) liquid medium: dissolve 5 g yeast extract, 10 g NaCl, and 10 g tryptone in ddH₂O and adjust to 1,000 mL total volume. Sterilize at 121 ℃ and 1.034×10⁵ Pa for 20 min and store at 4 ℃.
• Ampicillin (Amp), chloramphenicol (Cam), isopropyl-β-D-thiogalactopyranoside (IPTG): Solarbio Science & Technology, Beijing, China.
• Freund’s Complete Adjuvant: Sigma-Aldrich, USA.
• Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG), fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG, enhanced chemiluminescence (ECL) substrate, diaminobenzidine (DAB) substrate kit: Zhongshan Golden Bridge Biotechnology Co., Beijing, China.
• TMB substrate: Wantai Biological Pharmacy Enterprise Co., Beijing, China.
• Mouse monoclonal anti-BLM antibody: sc-376237, Santa Cruz Biotechnology, USA.
• Rabbit monoclonal anti-GAPDH antibody: GB15004-100, Servicebio, Wuhan, China.
• Bovine serum albumin (BSA): Biosharp, China.
• 4',6-diamidino-2-phenylindole (DAPI): Solarbio, Beijing, China.

Experimental animals

Male New Zealand White rabbits (n=2) were fed in the central laboratory of Beijing Jishuitan Hospital Guizhou Hospital. Experimental Animal Ethics Approval Number (LW2024110102).

Experimental instruments

Protein purification system (NGC Quest 10 plus, Bio-Rad, USA), high-pressure homogenizer (TS 0.75KW, Constant System, UK), multimode microplate reader (Synergy 4, Bio-Tek, USA), chemiluminescence imaging system (Tanon-2500, Tanon, China), fluorescence microscope (LV100ND POL-DS, Nikon, Japan).

Expression and purification of BLM^642–1,290 DNA helicase

Recombinant E. coli transformed with pET-15b-BLM^642–1,290-BL21-CodonPlus was inoculated into LB medium containing 50 µg/mL Amp and 30 µg/mL Cam, and cultured at 37 ℃ with shaking at 190 rpm until OD₆₀₀ reached 0.5–0.6. Expression of BLM^642–1,290 DNA helicase was induced with 0.4 mM IPTG for 20 h at 18 ℃ and 190 rpm. After induction, cells were collected by centrifugation at 4,000 g for 20 min at 4 ℃. The cells were then disrupted using a high-pressure homogenizer, and the lysate was centrifuged at 13,000 g for 40 min at 4 ℃. The supernatant was subjected to Ni²⁺-affinity chromatography [loading buffer: 20 mmol/L tris(hydroxymethyl)aminomethane (Tris), 500 mmol/L NaCl, 5 mmol/L imidazole, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), pH 7.9; elution buffer: 20 mmol/L Tris, 500 mmol/L NaCl, 500 mmol/L imidazole, pH 7.9] for purification of the recombinant BLM^642–1,290 DNA helicase, which was used for antibody production.

Preparation of rabbit pAb against human BLM DNA helicase

Purified BLM^642–1,290 DNA helicase was used as the immunogen to immunize adult male New Zealand White rabbits. For the initial immunization, equal volumes of BLM^642–1,290 DNA helicase and Freund’s complete adjuvant were thoroughly mixed and emulsified. A total of 400 µg of immunogen per rabbit was administered subcutaneously at two sites on the back (left and right). Thereafter, booster immunizations were performed every 5 days, each with 100 µg immunogen per rabbit, for a total of five boosts. Seven days after the final immunization, blood was drawn from the rabbit’s ear vein to determine antibody titer. If the titer reached at least 1:32,000, the rabbit was exsanguinated from the carotid artery, and the whole blood was collected. After centrifugation at 4,000 g for 10 min at 4 ℃, antiserum was obtained.

ELISA for determination of antibody titer

Purified BLM^642–1,290 DNA helicase was diluted to 1 µg/mL with ELISA coating buffer, and 100 µL was added to each well of a 96-well plate (in triplicate) and incubated at 4 ℃ overnight. The following day, the antigen solution was discarded, and the plate tapped dry. A commercially obtained ELISA blocking buffer was added and incubated at room temperature for 2 h. After removing the blocking solution, the plate was washed three times with phosphate buffered saline tween 20 (PBST) (300 µL/well, 5 min each) and then air-dried at 37 ℃. Subsequently, two-fold serial dilutions of rabbit antiserum were prepared (1:500–1:64,000) and 100 µL of each dilution was added. Normal rabbit serum (pre-immune serum) served as the negative control, and mouse anti-BLM monoclonal antibody (sc-376237, Santa Cruz Biotechnology) served as the positive control. After incubation at 37 ℃ for 1 h, the plate was washed three times with PBST for 5 min each. Then, 100 µL HRP-conjugated goat anti-rabbit IgG (for the rabbit antiserum) or HRP-conjugated goat anti-mouse IgG (for the positive control) was added and incubated at 37 ℃ for 45 min. After washing three times with PBST, 100 µL TMB substrate was added and allowed to react in the dark at 25 ℃ for 15 min. The reaction was terminated by adding 50 µL of 0.5 M H₂SO₄. Absorbance at 450 nm (A₄₅₀) was recorded using a Synergy 4 multimode microplate reader.

Western blot to assess the recognition ability of the pAb

Six-well plates seeded with MCF-7 breast cancer cells were washed three times with PBS. Then, 200 µL of RIPA lysis buffer containing 1 mmol/L PMSF (final concentration) was added to each well to evenly cover the cell surface. Cells were lysed on ice for 30 min to extract total protein. The total protein was mixed thoroughly with protein loading buffer, then boiled for 10 min and cooled on ice for 5 min. Proteins were separated by SDS-PAGE electrophoresis (80 V 20 min, then 120 V 90 min) and transferred onto a PVDF membrane. The membrane was blocked in TBS containing 50 g/L skim milk for 2 h and then incubated overnight at 4 ℃ with the rabbit polyclonal BLM antiserum (1:1,000 dilution). On the following day, the membrane was washed three times with TBST for 10 min each, incubated with HRP-conjugated goat anti-rabbit IgG at 25 ℃ for 1 h, and washed again. ECL substrate was added, and the signal was detected using a Tanon-2500 chemiluminescence imaging system. A mouse monoclonal anti-BLM antibody (sc-376237, Santa Cruz Biotechnology) served as the positive control.

IHC staining to detect BLM DNA helicase expression in breast cancer tissues

Breast cancer tissues and normal breast tissues were obtained from Dejiang Ethnic Traditional Chinese Medicine Hospital (Guizhou, China) between 2016 and 2019. Two pathologists verified the diagnoses of breast tumors. None of the patients had received treatment before surgical removal of the tumor. The agreement for use of these tissue samples was approved by the Biomedical Ethics Committee of Beijing Jishuitan Hospital Guizhou Hospital (approval No. LW2024110101), and the confidentiality of patient information was maintained. Tissue samples were fixed in 4% paraformaldehyde for 48 h, dehydrated in graded alcohol (100%, 90%, 75%, 50%), cleared in xylene, and embedded in paraffin. Sections were deparaffinized, rehydrated, and washed three times with PBS. Antigen retrieval was performed in citrate buffer using a microwave oven with high power for 3 min, medium power for 5 min, and low power for 5 min. After washing three times in PBS for 3 min each, the sections were blocked with 5% BSA for 30 min. The rabbit polyclonal anti-BLM antiserum (1:200 dilution) was applied, and the sections were incubated overnight at 4 ℃. The next day, the sections were washed three times in PBS, incubated with 50 µL polymer enhancer for 20 min at room temperature, washed, and then incubated with 50 µL HRP-conjugated goat anti-rabbit IgG for 30 min at room temperature. After three washes in PBS, 50 µL DAB substrate solution was added, and color development was monitored microscopically. Sections were rinsed in water, counterstained with hematoxylin for 8 s, and differentiation was performed with 0.1% HCl-ethanol for 2 s followed by a 2 s ammonia water dip. Finally, sections were dehydrated through graded alcohols, cleared in xylene, and mounted with neutral resin. Mouse monoclonal anti-BLM antibody (sc-376237, Santa Cruz Biotechnology) served as the positive control.

IF staining to detect BLM DNA helicase expression in MCF-7 cells

MCF-7 cells grown on coverslips were washed three times in PBS for 3 min each. Cells were then fixed in 4% paraformaldehyde for 20 min and washed three times in PBS. Permeabilization was performed with 0.5% Triton X-100 for 20 min at room temperature, followed by three PBS washes (3 min each). After blotting dry with filter paper, 10% goat serum in PBS was used to block nonspecific binding for 30 min. The blocking solution was removed (without washing), and the rabbit polyclonal anti-BLM antibody (1:200 dilution) was added and incubated at 4 ℃ overnight. The next day, the coverslips were washed three times in PBST for 3 min each. FITC-conjugated goat anti-rabbit IgG (1:200 dilution) was then applied, and the coverslips were incubated at 37 ℃ for 30 min. After three PBS washes (3 min each), DAPI solution (0.5 µg/mL) was added and incubated in the dark for 5 min. Slides were washed four times with PBS (5 min each) to remove excess DAPI. After drying with filter paper, antifade mounting medium was used to mount the coverslips. Images were acquired with a fluorescence microscope. Mouse monoclonal anti-BLM antibody (sc-376237, Santa Cruz Biotechnology) served as the positive control.

Ethical declaration

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Biomedical Ethics Committee of Beijing Jishuitan Hospital, Guizhou Hospital (No. LW2024110101) and individual consent for this retrospective analysis was waived.

Results

Production and purification of highly purified BLM^642–1,290 DNA helicase

SDS-PAGE analysis revealed that following induction with 0.4 mM IPTG and purification via Ni²⁺-affinity chromatography, a distinct protein band appeared at approximately 70 kDa, consistent with the expected molecular mass of BLM^642–1,290 DNA helicase (Figure 1). After affinity chromatography, the purity of the BLM^642–1,290 DNA helicase exceeded 95%. About 20 mg of BLM^642–1,290 DNA helicase could be produced per L E. coli culture.

Figure 1 SDS-PAGE analysis of purified BLM^642–1,290 DNA helicase. BLM, Bloom’s syndrome helicase; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Determination of the titer of BLM DNA helicase pAb

ELISA was used to determine the titer of the rabbit antiserum raised against BLM DNA helicase. The results indicated that the immunized rabbits produced a robust immune response, and the resulting pAbs against BLM DNA helicase reached a titer greater than 1:64,000 (Figure 2).

Figure 2 The titer of the BLM DNA helicase polyclonal antibody by ELISA. BLM, Bloom’s syndrome helicase; ELISA, enzyme-linked immunosorbent assay.

The BLM DNA helicase pAb specifically binds to BLM DNA helicase in breast cancer tissues and MCF-7 cells

Western blot analysis demonstrated that the prepared BLM DNA helicase pAb recognized BLM DNA helicase in MCF-7 breast cancer cells (Figure 3). To further validate the suitability of our pAb for experimental applications, we employed IHC and IF to detect BLM DNA helicase in breast cancer tissues and MCF-7 cells. The results showed that the positive staining for BLM DNA helicase was localized to both the nucleus and cytoplasm of breast cancer cells in tissues (Figure 4). Similarly, BLM DNA helicase was detected in both the nucleus and cytoplasm of MCF-7 cells (Figure 5). Collectively, these findings confirm that our rabbit pAb against BLM DNA helicase is both effective and specific, making it suitable for use in localization studies of BLM DNA helicase in tissues and cells.

Figure 3 The BLM polyclonal antibody specifically recognizes BLM DNA helicase in MCF-7 breast cancer cells. BLM, Bloom’s syndrome helicase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4 Detection of BLM DNA helicase expression in breast tissue using the BLM polyclonal antibody (immunohistochemical staining, ×400). BLM, Bloom’s syndrome helicase.

Figure 5 Detection of BLM DNA helicase expression in MCF-7 breast cancer cells using the BLM polyclonal antibody (immunofluorescence staining, ×400). BLM, Bloom’s syndrome helicase; DAPI, 4',6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.

Discussion

DNA helicases play crucial roles in DNA metabolism, including genome replication, repair, recombination, chromosome segregation, and transcription initiation (15-17). Through DNA replication, genetic information is passed on to the next generation, and via mRNA transcription and ribosomal translation, proteins are synthesized. During DNA replication, helicases use the energy from NTP hydrolysis to unwind double-stranded DNA (dsDNA), providing a single-stranded DNA (ssDNA) template for complementary DNA synthesis. Furthermore, helicases interact with other repair proteins to regulate the structural rearrangements that lead to genetic recombination, thereby preventing aberrant and harmful crossover events (18). Investigating the function of BLM DNA helicase and its specific roles in breast cancer progression holds significant value for improving diagnostic approaches and developing novel therapeutic strategies.

In this study, we confirmed the experimental utility of our prepared pAb against BLM DNA helicase by comparing it with a commercially available monoclonal antibody. First, we employed ELISA to determine the antibody titer. The results showed a titer exceeding 1:64,000, which may be attributed to the immunization regimen and dosage. Typically, four to five immunizations yield high-titer antibodies; an additional immunization beyond five times might further increase the titer. Next, we used Western blot to verify the specificity of the prepared antibody for BLM DNA helicase. Both the prepared polyclonal and the commercial monoclonal antibody successfully recognized BLM DNA helicase. We then applied IHC and IF to detect BLM DNA helicase in breast cancer tissues and MCF-7 cells. The results confirmed that the prepared pAb specifically bound to BLM DNA helicase in both systems, demonstrating its applicability in the localization of BLM DNA helicase within tissues and cells.

In summary, our study successfully induced and purified high-quality BLM^642–1,290 DNA helicase and generated a corresponding pAb capable of being employed in ELISA, Western blot, IHC, and IF analyses. This antibody serves as a valuable research tool, facilitating future investigations into the mechanisms by which BLM DNA helicase contributes to the development and progression of breast cancer.

Conclusions

A high-purity preparation of BLM^642–1,290 DNA helicase was successfully obtained and used as an immunogen to produce a rabbit pAb against BLM DNA helicase. The resulting antibody can be applied in a variety of detection techniques, including Western blot, IHC, and IF assays.

Acknowledgments

None.

Footnote

Data Sharing Statement: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-2/dss

Peer Review File: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-2/prf

Funding: This work was supported by the Science and Technology Department of Guizhou Province (QKHJC-ZK[2023]115, QKHCG-LC[2024]002); and the project of Guizhou Provincial Health Commission (gzwkj2024-201).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-2/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Biomedical Ethics Committee of Beijing Jishuitan Hospital, Guizhou Hospital (No. LW2024110101) and individual consent for this retrospective analysis was waived.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Bythell-Douglas R, Deans AJ. A Structural Guide to the Bloom Syndrome Complex. Structure 2021;29:99-113. [Crossref] [PubMed]
2. Tan J, Wang X, Phoon L, et al. Resolution of ROS-induced G-quadruplexes and R-loops at transcriptionally active sites is dependent on BLM helicase. FEBS Lett 2020;594:1359-67. [Crossref] [PubMed]
3. Du X, Zhang C, Yin C, et al. High BLM Expression Predicts Poor Clinical Outcome and Contributes to Malignant Progression in Human Cholangiocarcinoma. Front Oncol 2021;11:633899. [Crossref] [PubMed]
4. Lan C, Yamashita YI, Hayashi H, et al. High Expression of Bloom Syndrome Helicase is a Key Factor for Poor Prognosis and Advanced Malignancy in Patients with Pancreatic Cancer: A Retrospective Study. Ann Surg Oncol 2022;29:3551-64. [Crossref] [PubMed]
5. Ledet EM, Antonarakis ES, Isaacs WB, et al. Germline BLM mutations and metastatic prostate cancer. Prostate 2020;80:235-7. [Crossref] [PubMed]
6. Mojumdar A. Mutations in conserved functional domains of human RecQ helicases are associated with diseases and cancer: A review. Biophys Chem 2020;265:106433. [Crossref] [PubMed]
7. Trizuljak J, Petruchová T, Blaháková I, et al. Diagnosis of Bloom Syndrome in a Patient with Short Stature, Recurrence of Malignant Lymphoma, and Consanguineous Origin. Mol Syndromol 2020;11:73-82. [Crossref] [PubMed]
8. Rosenthal AS, Dexheimer TS, Gileadi O, et al. Synthesis and SAR studies of 5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine derivatives as potent inhibitors of Bloom helicase. Bioorg Med Chem Lett 2013;23:5660-6. [Crossref] [PubMed]
9. Maity J, Horibata S, Zurcher G, et al. Targeting of RecQ Helicases as a Novel Therapeutic Strategy for Ovarian Cancer. Cancers (Basel) 2022;14:1219. [Crossref] [PubMed]
10. Thakkar MK, Lee J, Meyer S, et al. RecQ Helicase Somatic Alterations in Cancer. Front Mol Biosci 2022;9:887758. [Crossref] [PubMed]
11. Huang A, Deng C, Yang S, et al. Preparation and application of rabbit polyclonal antibody against human lactate dehydrogenase C4(LDHC4). Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 2023;39:1118-24.
12. Khalaf HE, Al-Bouqaee H, Hwijeh M, et al. Characterization of rabbit polyclonal antibody against camel recombinant nanobodies. Open Life Sci 2022;17:659-75. [Crossref] [PubMed]
13. Kelley JT, Kroll-Wheeler L, Hrycaj S, et al. Nonspecificity of Immunohistochemistry for Mycobacteria Species Using a Rabbit Polyclonal Antibody. Arch Pathol Lab Med 2024;148:e367-73. [Crossref] [PubMed]
14. Zhang W, Yu X, Bao L, et al. Bisbenzylisoquinoline alkaloid fangchinoline derivative HY-2 inhibits breast cancer cells by suppressing BLM DNA helicase. Biomed Pharmacother 2023;169:115908. [Crossref] [PubMed]
15. Tuteja N, Tuteja R. Unraveling DNA helicases. Motif, structure, mechanism and function. Eur J Biochem 2004;271:1849-63. [Crossref] [PubMed]
16. Phan TN, Ehtesham NZ, Tuteja R, et al. A novel nuclear DNA helicase with high specific activity from Pisum sativum catalytically translocates in the 3'-->5' direction. Eur J Biochem 2003;270:1735-45. [Crossref] [PubMed]
17. Bachrati CZ, Hickson ID. RecQ helicases: suppressors of tumorigenesis and premature aging. Biochem J 2003;374:577-606. [Crossref] [PubMed]
18. Moder M, Velimezi G, Owusu M, et al. Parallel genome-wide screens identify synthetic viable interactions between the BLM helicase complex and Fanconi anemia. Nat Commun 2017;8:1238. [Crossref] [PubMed]