Forensic significance of ELISA technique for hormone assessment: a comprehensive review

Enzyme-linked immunosorbent assay (ELISA) is a rapid, sensitive, and economical tool for detecting hormones in diverse biological matrices, making it valuable in forensic endocrinology. This review summarizes ELISA's principles, applications, advantages, and limitations in medico-legal practice. ELISA enables quantification of hormones such as cortisol, testosterone, estrogen, melatonin, thyroid hormones, progesterone, and human chorionic gonadotropin (hCG). It supports postmortem toxicology, sexual assault investigations, doping control, and forensic psychiatry. Cortisol may help reconstruct perimortem stress, while hCG detection assists in pregnancy confirmation in assault or maternal death cases. In sports, ELISA screens for anabolic steroids, erythropoietin, and growth hormone, with LC-MS/MS required for confirmation. Its compatibility with blood, saliva, urine, and hair enhances versatility. Key challenges include antibody cross-reactivity, matrix interference, degradation, variability among commercial kits, and limited multiplexing. False positives, hook effects, and inconsistent validation affect admissibility, necessitating strict quality assurance, ISO/IEC 17025 compliance, and confirmatory testing. ELISA is unsuitable for paternity determination; DNA profiling remains the legal standard. Emerging advances - digital ELISA, nanotechnology, AI, and biosensors - promise greater sensitivity and automation but face regulatory, cost, and training barriers. ELISA should be regarded as a complementary, high-throughput screening method integrated into validated, multimodal forensic workflows.