Generation of a scFv Derived from an IgM-Producing Hybridoma for the Detection of REST Expression in Premalignant Lesions and Invasive Squamous Cell Carcinoma

Rodríguez-Nava, Cynthia; Cortés-Sarabia, Karen; Riaño-Umbarila, Lidia; Becerril-Luján, Baltazar; Medina-Flores, Yolanda; Mata-Ruíz, Olga; Lloret-Sánchez, Lourdes; Illades-Aguiar, Berenice; Alarcón-Romero, Luz del Carmen; Ortuño-Pineda, Carlos

doi:10.3390/ijms262411946

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Generation of a scFv Derived from an IgM-Producing Hybridoma for the Detection of REST Expression in Premalignant Lesions and Invasive Squamous Cell Carcinoma

by

Cynthia Rodríguez-Nava

¹,

Karen Cortés-Sarabia

²

,

Lidia Riaño-Umbarila

³

,

Baltazar Becerril-Luján

⁴,

Yolanda Medina-Flores

⁵,

Olga Mata-Ruíz

⁵,

Lourdes Lloret-Sánchez

⁵

,

Berenice Illades-Aguiar

⁶

,

Luz del Carmen Alarcón-Romero

^1,*

and

Carlos Ortuño-Pineda

^7,*

¹

Laboratorio de Investigación en Citopatología e Histoquímica, Universidad Autónoma de Guerrero, Chilpancingo 39086, Mexico

²

Laboratorio de Investigación en Inmunobiología y Diagnóstico Molecular, Universidad Autónoma de Guerrero, Chilpancingo 39086, Mexico

³

Investigadora por México, Secretaría de Ciencia, Humanidades, Tecnología e Innovación, Cuernavaca 62210, Mexico

⁴

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca 62210, Mexico

⁵

Laboratorio de Anticuerpos Monoclonales, Unidad de Desarrollo Tecnológico e Investigación Molecular, Instituto de Diagnóstico y Referencia Epidemiológico “Dr. Manuel Martínez Báez” (InDRE), Mexico City 01480, Mexico

⁶

Laboratorio de Investigación en Biomedicina Molecular, Universidad Autónoma de Guerrero, Chilpancingo 39086, Mexico

⁷

Laboratorio de Ácidos Nucleicos y Proteínas, Universidad Autónoma de Guerrero, Chilpancingo 39086, Mexico

^*

Authors to whom correspondence should be addressed.

Int. J. Mol. Sci. 2025, 26(24), 11946; https://doi.org/10.3390/ijms262411946

Submission received: 11 October 2025 / Revised: 27 November 2025 / Accepted: 6 December 2025 / Published: 11 December 2025

(This article belongs to the Special Issue Recent Advances in New Biomarkers for Cancers)

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Abstract

Cervical cancer (CC) can be prevented through continuous screening and the timely detection of cervical intraepithelial neoplasia (CIN) using immunohistochemistry techniques to identify biomarker expressions. In a previous study, we proposed nuclear REST loss as a biomarker in precancerous lesions and CC; however, no validated antibodies are available for detecting REST in cytology or cervical tissues. Although we have developed an IgM-type anti-REST monoclonal antibody capable of detecting REST in liquid-based cytology cells, it was not useful for the detection of REST in cervical tissues by immunohistochemistry. The main objective of this study is to generate single-chain variable fragments (scFvs) for the clinical evaluation of REST in cervical tissues from women with CIN and CC. Using RNA from an IgM-producing hybridoma anti-REST, we conducted RT-PCR and PCR to obtain the coding sequences for the variable regions of the heavy and light chains. These sequences were joined with a linker to create a single-chain antibody. The scFv was then cloned into the pSyn1 vector, expressed in E. coli TG1, and purified through chromatography. Subsequently, it was characterized using immunological methods to assess its biological activity and employed to evaluate REST expression in cytological samples and cervical tissues. The anti-REST scFv represents an innovative detection tool that retains the antigen recognition of the parental IgM while overcoming its size limitation, enabling tissue penetration and detection of REST in cervical samples. Its application facilitates the identification of REST in cervical samples, reinforcing REST’s potential as a diagnostic biomarker for CC and CIN.

Keywords: cervical cancer; immunocytochemistry; scFv; REST

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MDPI and ACS Style

Rodríguez-Nava, C.; Cortés-Sarabia, K.; Riaño-Umbarila, L.; Becerril-Luján, B.; Medina-Flores, Y.; Mata-Ruíz, O.; Lloret-Sánchez, L.; Illades-Aguiar, B.; Alarcón-Romero, L.d.C.; Ortuño-Pineda, C. Generation of a scFv Derived from an IgM-Producing Hybridoma for the Detection of REST Expression in Premalignant Lesions and Invasive Squamous Cell Carcinoma. Int. J. Mol. Sci. 2025, 26, 11946. https://doi.org/10.3390/ijms262411946

AMA Style

Rodríguez-Nava C, Cortés-Sarabia K, Riaño-Umbarila L, Becerril-Luján B, Medina-Flores Y, Mata-Ruíz O, Lloret-Sánchez L, Illades-Aguiar B, Alarcón-Romero LdC, Ortuño-Pineda C. Generation of a scFv Derived from an IgM-Producing Hybridoma for the Detection of REST Expression in Premalignant Lesions and Invasive Squamous Cell Carcinoma. International Journal of Molecular Sciences. 2025; 26(24):11946. https://doi.org/10.3390/ijms262411946

Chicago/Turabian Style

Rodríguez-Nava, Cynthia, Karen Cortés-Sarabia, Lidia Riaño-Umbarila, Baltazar Becerril-Luján, Yolanda Medina-Flores, Olga Mata-Ruíz, Lourdes Lloret-Sánchez, Berenice Illades-Aguiar, Luz del Carmen Alarcón-Romero, and Carlos Ortuño-Pineda. 2025. "Generation of a scFv Derived from an IgM-Producing Hybridoma for the Detection of REST Expression in Premalignant Lesions and Invasive Squamous Cell Carcinoma" International Journal of Molecular Sciences 26, no. 24: 11946. https://doi.org/10.3390/ijms262411946

APA Style

Rodríguez-Nava, C., Cortés-Sarabia, K., Riaño-Umbarila, L., Becerril-Luján, B., Medina-Flores, Y., Mata-Ruíz, O., Lloret-Sánchez, L., Illades-Aguiar, B., Alarcón-Romero, L. d. C., & Ortuño-Pineda, C. (2025). Generation of a scFv Derived from an IgM-Producing Hybridoma for the Detection of REST Expression in Premalignant Lesions and Invasive Squamous Cell Carcinoma. International Journal of Molecular Sciences, 26(24), 11946. https://doi.org/10.3390/ijms262411946

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Generation of a scFv Derived from an IgM-Producing Hybridoma for the Detection of REST Expression in Premalignant Lesions and Invasive Squamous Cell Carcinoma

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