The　authors　describe　a　colorimetric　immunoassay　for　the　model　nalyte　aflatoxin　B1　(AFB1).　It　is　based　on　the　just-in-time　generation　of　an　MnO2　nanocatalyst.　Unlike　previously　developed　immunoassay,　the　chromogenic　reaction　relies　on　the　just-in-time　formation　of　an　oxidase　mimic　without　the　aid　of　the　substrate.　Potassium　permanganate　(KMnO4)　is　converted　into　manganese　dioxide　(MnO2)　which　acts　as　an　oxidase　mimic　that　catalyzes　the　oxidation　3,3′,5,5′-tetramethylbenzidine　(TMB)　by　oxygen　to　give　a　blue　colored　product.　In　the　presence　of　ascorbic　acid　(AA),　KMnO4　is　reduced　to　Mn(II)　ions.　This　results　in　a　decrease　in　the　amount　of　MnO2　nanocatalyst.　Hence,　the　oxidation　of　TMB　does　not　take　place.　By　adding　ascorbate　oxidase,　AA　is　converted　into　dehydroascorbic　acid　which　cannot　reduce　KMnO4.　Based　on　these　observations,　a　colorimetric　competitive　enzyme　immunoassay　was　developed　where　ascorbate　oxidase　and　gold　nanoparticle-labeled　antibody　against　AFB1　and　magnetic　beads　carrying　bovine　serum　albumin　conjugated　to　AFB1　are　used　for　the　determination　of　AFB1.　In　presence　of　AFB1,　it　will　compete　with　the　BSA-conjugated　AFB1　(on　the　magnetic　beads)　for　the　labeled　antibody　against　AFB1　on　the　gold　nanoparticles.　This　makes　the　amount　of　ascorbate　oxidase/anti-AFB1　antibody-labeled　gold　nanoparticles,　which　conjugated　on　magnetic　beads,　reduce,　and　resulted　in　an　increase　of　ascorbic　acid.　Under　optimal　conditions,　the　absorbance　(measured　at　652 nm)　decreases　with　increasing　AFB1　concentrations　in　the　range　from　0.1　to　100 ng　mL−1,　with　a　0.1 ng　mL−1　detection　limit　(at　the　3Sblank　level).　The　accuracy　of　the　assay　was　validated　by　analyzing　spiked　peanut　samples.　The　results　matched　well　with　those　obtained　with　a　commercial　ELISA　kit.　Conceivably,　the　method　is　not　limited　to　aflatoxins　but　has　a　wide　scope　in　that　it　may　be　applied　to　many　other　analytes　for　which　respective　antibodies　are　available.　[Figure　not　available:　see　fulltext.].　©　2018,　Springer-Verlag　GmbH　Austria,　part　of　Springer　Nature.