Research paper Immunofiltration assay for aflatoxin B 1 based on the separation of pre-immune complexes Debjani Saha, Dipika Roy, Tarun K. Dhar ⁎ Drug Development, Diagnostics and Biotechnology Division, Council of Scientific and Industrial Research, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700 032, India article info abstract Article history: Received 30 January 2013 Received in revised form 22 February 2013 Accepted 4 March 2013 Available online 13 March 2013 A new approach for quantitative determination of AFB 1 based on the separation of pre-immune complexes in the same immunoassay system has been developed. No additional step for the separation of pre-immune complexes is required. The method uses a test device for separation of pre-immune complexes from the free AFB 1 -enzyme conjugate by filtration through the membrane strips spotted with anti-AFB 1 antibody. The bound enzyme conjugate was visualized by super-catalyzed reporter deposition (Super-CARD) signal amplification method. The measured signal intensity is directly proportional to the amount of AFB 1 present in the sample. The detection limit obtained by the present method was 15 pg/ml. The data on the analytical parameters indicate that the new format of AFB 1 detection in foodstuffs is reproducible, accurate and specific. The method is user friendly and does not require any costly equipment or a well-equipped laboratory. © 2013 Elsevier B.V. All rights reserved. Keywords: Aflatoxin B 1 Immunofiltration assay Pre-immune complexes Separation Super-CARD amplification 1. Introduction Membrane-based immunoassays are now widely used for the detection of small molecules like mycotoxins, pesticides, drugs etc. (Pal and Dhar, 2004; Paepens et al., 2004; Wang et al., 2005). The most favored format presently used for their measurement is based on competitive reaction between an unlabeled analyte and a labeled analyte in the presence of limiting amounts of anti-analyte antibody. In these assays, the measured signal intensity decreases with increasing analyte concentration. On the other hand, macromolecules such as proteins having more than one antigenic determinant are measured by noncompetitive immunoassay. The signals obtained are directly related to the amount of the analyte present in the sample. These assays have the potential for improved sensitivity and working range compared with corresponding competitive assays. However, these types of assays are not applicable to small molecules because their low molecular masses preclude simultaneous binding of two antibody molecules. During the last decade, several alterna- tive strategies have been reported for small molecules, but they are limited to microtiter plate technology (Acharya and Dhar, 2008; Anfossi et al., 2004; Giraudi et al., 1999). We recently reported a cost-effective analytical device for performing an immunofiltration-based immunoassay without using a pump (Pal and Dhar, 2004). It has also been successfully applied for rapid estimation of mycotoxins by immunoassay (Pal and Dhar, 2004; Saha et al., 2007), homogeneous spotting of antibody over the membrane surface (Saha et al., 2006), the estimation of protein (Acharya et al., 2008) and for improved catalyzed reporter deposition (Super-CARD) method of signal amplification (Saha et al., 2007). The device is simple in construction and consists of two layers: nitrocellulose mem- brane strips and a rectangular piece of filter paper placed over a semirigid polyethylene card. The membrane strips were placed Journal of Immunological Methods 392 (2013) 24–28 Abbreviations: AFB 1 , aflatoxin B 1 ; AFB 1 -HRP, AFB 1 -horseradish peroxidase conjugate; B-T, biotinylated tyramide; p-OH-PPA-casein, 3-(p-hydroxyphenyl) propionic acid-casein conjugate; BSA, bovine serum albumin; Super-CARD, Super-catalyzed reporter deposition method; 4CN, 4-chloro-1-naphthol; DAB, 3,3′-diaminobenzidine; ELISA, enzyme-linked immunosorbent assay; Tween 20, polyoxyethylenesorbitan monolaurate. ⁎ Corresponding author. Tel.: + 91 33 2473 5112; fax: + 91 33 2473 0284/ 5197. E-mail address: tkdhar@iicb.res.in (T.K. Dhar). 0022-1759/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jim.2013.03.003 Contents lists available at SciVerse ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim