The Multiple Antigen Blot Assay: A Simple, Versatile and Multipurpose Immunoenzymatic Technique

Abstract

This technique is based on the sensitization of different antigens in a single nitrocellulose strip, which react when exposed to an immune serum and thereafter with the appropriate peroxidase conjugate and the corresponding substrate. Signals in those reactive spots are recorded as black squares in a negative photographic film, using a chemiluminiscent substrate or as blue spots when a precipitable colorimetric substrate is used. This technique allows the simultaneous demonstration of antigenicity of different antigens (peptides, recombinant molecules, and crude preparations), with a high sensitivity and specificity. Its major value is based on its versatility, since it is possible to rapidly evaluate and to compare various antigenic preparations and to use it for diagnosis of different infectious, allergic and autoimmune diseases, at a low cost.

High diagnostic efficiency of IgM-ELISA with the use of multiple antigen peptides (MAP1) from T. gondii ESA (SAG-1, GRA-1 and GRA-7), in acute toxoplasmosis

“…In contrast, the non-acute/recent toxoplasmosis group, in spite of presenting some residual IgM antibodies detectable by a commercial kit, IgM antibodies to MAP1 were demonstrated. NOYA et al 27 investigated the use of MAP in an assay for the diagnosis of unrelated pathologies and they found similar specificity and sensitivity as we did with MAP1. In study of PIETKIEWICZ et al 28 using recombinant (SAG-1, GRA-1 and GRA-7) antigens, the acute/recent and non-acute/recent phase of toxoplasmosis could not be discriminated.…”

“…However, some problems can be observed in laboratory practice, such as false negative results after treatment with anti-toxoplasma drugs, low and focal distribution of parasites in the tissues or the presence of non-viable parasites in autolyzed tissue. Excreted and secreted antigen (ESA) of T. gondii has been used in detection of specific antibodies for the diagnosis of acute toxoplasmosis 1,2,3,4,6,7,15,27 . As previously mentioned, circulating antigens in infected humans are constituted by T. gondii ESA, and are one of the first targets of the host immune response 26 .…”

“…In a recent study, NOYA et al 27 compared various antigenic preparations with MAP for the diagnosis of different infections and reported high sensitivity and specificity of assays at low cost.…”

“…These facts imply that the reactivity among antigens may vary greatly, contributing to inaccurate provision of diagnostic efficiency by assay systems. Several reports 1,2,4,6,7,10,16,23,27,29 have emphasized the value of the detection of specific antibodies raised to excreted-secreted antigens (ESA) of T. gondii for the diagnosis of acute toxoplasmosis. The T. gondii ESA constitutes 90% of the circulating antigens in infected humans, thus is one of the first targets of the host immune response 6. Recombinant antigens and synthetic peptides have been prepared from ESA antigens, however, the exact composition and association of recombinant antigens and peptides to be used in immunoassays to detect toxoplasma antibodies is still an open question.…”