We previously demonstrated that a deficiency of natural antibodies against CD25, Mucin 1 (MUC1) and vascular endothelial growth factor receptor 1 (VEGFR1) could contribute to high risk of non-small cell lung cancer (NSCLC). This study was designed to investigate if natural IgG antibodies against POU domain class 5 transcription factor 1 (POU5F1), tumor necrosis factor-α (TNF-α) and the combination of CD25, VEGFR1 and MUC1 could play an anti-tumorigenic role against developing NSCLC. An ELISA was developed in-house to examine plasma IgG against peptide antigens derived from POU5F1, TNF-α and a combination of peptide antigens derived from CD25, MUC1 and VEGFR1 in 211 patients with NSCLC and 200 healthy controls. Mann-Whitney U test demonstrated that plasma IgG levels for the combination of peptide antigens derived from CD25, MUC1 and VEGFR1 were significantly lower in NSCLC patients than control subjects (Z=-12.978, P＜0.001) although plasma levels of IgG antibodies for POU5F1 and TNFα were not significantly changed. The in-house ELISA made with the CD25-MUC1-VEGFR1 combination had a sensitivity of 49.6% against a specificity of 95% to detect early stage NSCLC. In conclusion, natural antibodies against the combination of CD25, VEGFR1 and MUC1 may be an effective biomarker for early diagnosis of NSCLC.This article is protected by copyright. All rights reserved.