Functional studies using freshly isolated tumor-infiltrating B lymphocytes(TIB) are difficult to perform and interpret. Here we document a novel function of TIB using fresh human lung cancer tissues engrafted in SCID mice; they are at activated state and produce tumor-specific antibodies in tumor microenvironment: (a) TIB engrafted in SCID mice produced human IgG; (b) IgG derived from TIB highly bound intracellular and membrane-bound antigens of autologous cancer cells; and (c) less recognition of autoantigens expressed on normal lymphocytes by IgG derived from TIB compared with IgG from the serum of the patient. On the basis of the novel findings presented in this study, we modified the original serological analysis of antigens by recombinant cDNA expression cloning design in a patient with lung cancer who expressed unusually favorable clinical evolution and analyzed humoral immunity against identified mutated p53 antigen. This study provides the first demonstration that antibodies derived from TIB recognize tumor antigens by serological analysis of antigens by recombinant cDNA expression cloning methodology and circulating anti-p53 antibodies in sera derived from TIB in tumor microenvironment. Our approach using TIB may allow the identification of key antigens in the humoral cancer-related immune system.