Cut-off on demand: adjustment of the threshold level of an immunochromatographic assay for chloramphenicol

Abstract

15Immunochromatographic assay of small molecules is carried out in a competitive format and is based on the 16 disappearance of analytical zone colouration, as the analyte concentration exceeds a certain threshold level. 17Therefore, the assay of toxic contaminants has the highest information content if this threshold level (cut-off 18 between negative and positive samples) corresponds to the maximum residue limit of the analyte. In this study, 19adjusting the threshold level was investigated for an immunochromatographic assay for chloramphenicol, which is a 20 veterinary drug of concern in the food industry because of its toxic effects. Test strips were produced using hapten-21 (protein carrier) and antibody-(gold nanoparticle) conjugates, and the effects of the compositions and concentrations 22 of these conjugates on the threshold level of the assay were investigated. Changing all of these parameters at once 23 shifted the threshold level to values that could reach more than two orders of magnitude. Thus, varying the 24 composition for chloramphenicol-bovine serum albumin conjugates causes a shift of the threshold level from 1,500 25 to 2 ng mL -1 , and varying the composition for chloramphenicol-soybean trypsin inhibitor conjugates causes a shift 26 from 930 to 9 ng mL -1 . Besides, the threshold shift is accomplished by the shift of the working range for quantitative 27 immunochromatography based on measurements of the analytical zone colouration. This way, the limit of 28 instrumental detection could also be varied by more than two orders of magnitude. The proposed approach can be 29 applied to different low-molecular-weight compounds and it allows the adoption of an immunochromatographic test 30 of the desired levels to control the target analyte without screening of a series of antibodies. 31 32 33 chloramphenicol 34 35 36 The contamination of food is an important public health issue and, therefore, immediate feedback after 37 exceeding the maximum residue limits is of high demand [1, 2]. Efficient tools for these purposes are 38 immunochromatographic test strips that provide rapid (5-15 min) assay results with a simple qualitative («yes» or 39 «no») interpretation [1, 3-6]. Toxic low-molecular-weight compounds are controlled by the competitive format of 40 immunochromatography. Antigen (contaminant) that is potentially present in the sample competes with the antigen-41 corresponding to OD 520 = 2.0. It enables the formation of heavily stained lines in the course of the assay, combined 34 with the completeness of the conjugate removed from the starting zone and the absence of a non-specific colouration 35 of the working membrane. 36The obtained data allows one to select the test strip completion to reach any desired threshold level (as well 37 as the limit of detection) in a wide range of CAP concentrations. This way, the tests may be easily adjusted to the 38 actual needs of the contaminant control.

Towards Lateral Flow Quantitative Assays: Detection Approaches

“…Limits of detection of the competing immunoassay (from [55], with additions and amendments). Visual detection can determine whether or not there is staining; detection limit corresponds to point A dividing these options.…”

A Spellbinding Interplay Between Biological Barcoding and Nanotechnology

“…Barcoded-nanotechnology is used to detect viruses using “barcode lateral flow immune-assay.” A barcode lateral flow immunoassay based on magnetic nanoparticle is used to optimize antibody affinity for a specific antigen. To increase sensitivity and broaden the applications of lateral flow immunoassay, modifications have been made by controlling the cut-off level and insertion of MNPs ( Zvereva et al, 2015 ; Orlov et al, 2016 ). In this way, this assay can be used to diagnose hormones, cardiac and cancer biomarkers, and to detect plant diseases ( Wang L. et al, 2014 ).…”

Ways to Reach Lower Detection Limits of Lateral Flow Immunoassays

“…In Zvereva et al [63], the possibility to change the cut-off level by varying the composition of the hapten-protein and the antibody-(gold nanoparticles) conjugates is considered. Using an example of competitive LFIA of chloramphenicol, it was shown that by reducing the load of immunoreagents on carriers, it was possible to shift the detection limit by two orders of magnitude.…”

“…In some cases, the developer does not need to achieve maximum sensitivity but to fix the threshold that separates the positive and negative results in accordance with the regulatory requirements for MRLs. This allows the composition of conjugates used in the analysis discussed above to be varied [63]. A qualitative "yes-no" analysis can be transformed into a semiquantitative one with a change in the number of colored bands corresponding to several threshold levels.…”