Nanobodies Based on a Sandwich Immunoassay for the Detection of Staphylococcal Enterotoxin B Free from Interference by Protein A

Abstract

As one of the leading causes of food poisoning, staphylococcal enterotoxins (SEs) secreted by Staphylococcus aureus pose a serious threat to human health. The immunoassay has become the dominant tool used for the rapid detection of harmful bacteria and toxins as a result of its excellent specificity. However, with regard to SEs, staphylococcal protein A (SpA) is likely to bind with the fragment crystallizable (Fc) terminal of the traditional antibody and result in a false positive, limiting the practical application of this method. Therefore, to eliminate the bottleneck problem, the sandwich immunoassay was development by replacing the traditional antibody with a nanobody (Nb) that lacked a Fc terminal. Using 0.5 × 107 colony-forming units, the Nb library was constructed using Bactrian camels immunized with staphylococcal enterotoxin B (SEB) to obtain a paired Nb against SEB with good affinity. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed using one Nb as the capture antibody and a phage-displayed Nb with signal-amplifying properties as the detection antibody. In optimal conditions, the current immunoassay displayed a broad quantitative range from 1 to 512 ng/mL and a 0.3 ng/mL limit of detection. The recovery of spiked milk, milk powder, cheese, and beef ranged from 87.66 to 114.2%. The Nbs-ELISA was not influenced by SpA during the detection of SEB in S. aureus food poisoning. Therefore, the Nb developed here presented the perfect candidates for immunoassay application during SE determination as a result of the complete absence of SpA interference.

Aptasensors for Staphylococcus aureus Risk Assessment in Food

“…In another sandwich ELISA assay, nano-antibodies acted as capturing antibodies, and the detection antibodies were acted by phage nanoantibodies with amplified signal properties. Under the optimal cases, the quantitative range of this method was 1-512 ng/ml, and the detection limit was 0.3 ng/ml (Ji et al, 2020). In actual detection, this method is highly dependent on equipment; secondly, antibody preparation is difficult and it is prone to false positives; finally, the experiment has certain requirements such as the professional level of the experimenter.…”

Phage‐based technologies for highly sensitive luminescent detection of foodborne pathogens and microbial toxins: A review

“…The favorable characteristics, such as high selectivity, high affinity, unique complementary sites, and small volume, make them an ideal tool to detect some antigens with recessive conformational epitopes that cannot be reached and recognized by traditional antibodies (Meyer et al., 2014; Gonzalez‐Sapienza et al., 2017; Stijlemans et al., 2004). A sandwich ELISA can be developed with one Nb as the capture antibody and a phage‐displayed Nb with signal‐amplifying properties as the detection antibody (Ji et al., 2020).…”

Recombinant antibodies and their use for food immunoanalysis

“…As described in the literature, immunodetection of S. aureus has been hampered by the unspecific and undesirable interaction between staphylococcal protein A (SpA), an immunoglobulin-binding protein of the surface of the bacteria, and the Fc region of most mammalians immunoglobulins that results in false positive signals [99,103]. The use of nanobodies has been proposed as an alternative to avoid such interaction with the advantages of high sensitivity, stability in extreme conditions, and easy genetic manipulation, for example, for further tagging.…”

“…Staphylococcal enterotoxin B (SEB) is the most potent toxin produced by S. aureus that shows high stability and high toxicity even at very low concentrations. Several analytical techniques have been implemented for the analysis of SEB in food, including commercial ELISA kits which are commonly applied in routine analysis [103]. However, as discussed previously, the presence of SpA in the sample may result in false positives that will have a negative impact on the analysis of SEB.…”

“…The construction of immunized libraries requires the availability of patients or immunized/infected animals to isolate affinity matured antibodies, and a new library must be produced for every target of interest. Such libraries are widely applied not only in medical research, but also for the isolation of highly sensitive antibodies for food analysis [75,[101][102][103]. On the other hand, the use of naïve libraries allows circumventing the ethical problems and limitations associated to the use of animal-derived antibodies.…”