**5. Immunological and antibody-based assays**

Enzyme-linked immunosorbent assay (ELISA) is a common assay used to detect BoNTs. This method utilizes anti-BoNT capture and detector antibodies arranged in a "sandwich" format. The detection formats are most commonly luminescent- or colorimetric-based. Prior genera‐ tions of BoNT immunoassays were approximately 10 times less sensitive than the mouse bioassay described in the previous section. Although not as sensitive, ELISA methods are relatively fast, inexpensive, and simple to perform. They are also less subject to inhibitory matrix effects. An amplified ELISA for detecting toxins in food matrices has also been described [23]. Toxins for serotypes A, B, E, and F could be detected in liquids, solid food, and semisolid food. Assay performance was evaluated in a range of food matrices, such as broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meats, and dairy foods. The assay sensitivity varied for each botulinum serotype. The tests readily detected 2 ng/mL of serotypes A, B, E, and F in various foods tested. Recently, traditionally formatted, very sensitive sandwich ELISAs used high affinity mAbs against BoNT/A and BoNT/B to detect BoNT/A as low as 5 and 25 pg/mL in buffer and in a milk matrix, respectively; and BoNT/B at 100 fg/mL and 39 pg/mL in buffer and milk matrix, respectively [24–26].

These mAbs were used in an electrochemiluminesence (ECL) immunosorbent assay using the Sector 2400 Imager (Meso Scale Discovery [MSD], Rockville, MD, USA) instrument [27, 28]. Detection sensitivities for BoNT/A in this system were similar to traditional ELISAs in buffer but were markedly improved in liquid food matrices because of the reduced background signal. The higher sensitivity and reduced time required for these new immunosorbent methods make them potential alternatives to the mouse bioassay. Sharma et al. recently developed another ECL assay for simultaneous detection of several biothreat agents (including clostridial neurotoxins) in milk products, with limit of detection (LOD) of 40 pg/mL for BoNT/ A complex [28]. The ECL assay was also successfully applied to screen *C. botulinum* serotype A outbreak strains. The study also showed that this sensitive ECL assay is rapid (it can be completed in less than 6 hours). The ECL assay also has potential for using as an *in vitro* screening method, complementing or replacing other immunoassays.

Cheng and Stanker [27] evaluated the performance of antitoxin mAbs using the same electro‐ chemiluminescence immunoassay platform (Sector 2400 Imager, MSD). The ELISA and ECL methods were observed to be more sensitive than the mouse bioassay. In fact, the ECL assay was able to outperform ELISA in terms of detection sensitivity—including food matrices spiked with BoNT/A and in some food matrices spiked with BoNT/B. The ELISA and ECL methods are fast and simple alternatives to the mouse bioassay and can be used for detecting BoNTs in food matrices and serum samples.

One example of mAb development using a recombinant immunogen was the work of Liu et al. [29], who expressed the recombinant H(C) subunit of BoNT type A (rAH(C)). Two out of 56 mAbs were selected to establish a highly sensitive sandwich chemiluminescence enzyme immunoassay (CLEIA) with LOD for both rAH(C) and BoNT/A of 0.45 pg/mL. This CLEIA can be used to detect BoNT/A in matrices, such as milk and beef extract. This method is 20– 40-fold more sensitive than the mouse bioassay and takes only 3 hours to complete, making it a useful method to detect and quantify BoNT/A.

The multiplex technology discussed above to detect nucleic acid has also been applied to the development of methods to analyze multiple epitopes on a single antigen and multiple targets in a single sample. This approach uses multiple mAbs as well as polyclonal antibodies to reduce false-positive and false-negative results. A commercialized system, Luminex xMAP technology, utilizes microsphere beads conjugated with antibodies. It employs paramagnetic beads instead of non-magnetic polystyrene beads and is very useful for the analysis of food matrices. The antibody-bead complexes detect multiple epitopes in a single sample. This technology was used to detect abrin, ricin, BoNTs, and staphylococcal enterotoxins in spiked food samples [30].

Zhang et al. [31] developed ELISA-based protein antibody microarrays to simultaneously detect six serotypes of BoNTs. Using numerous different food and other matrices, the micro‐ array is capable of detecting BoNT serotypes A through F. Using engineered, high-affinity antibodies, these serotypes were detected to similar levels in various matrices and were comparable to detection in buffer.

Accurate and sensitive detection of contaminated food and other biological samples in the environment is critical. Brunt et al. [32] have developed an affinity column-based assay for detecting neurotoxin in food matrices—specifically serotypes A, B, E, and F. The detection limit for BoNT/A was reported as 0.5 ng, which is two-fold more sensitive than lateral flow methods (also see Section 6) [32]. For serotypes B, E, and F, the minimum detection limit ranged from 5 to 50 ng. Although not as sensitive as ELISA or mouse bioassay, rapid immunochro‐ matographic methods generally require only 15–30 minutes to complete. They do not require enrichment steps and are amenable to use in the field.

Koh et al. have presented a new technology called SpinDx [33]. This method utilizes a centrifugal microfluidic platform to detect BoNTs based on a sedimentation immunoassay. A reagent mixture is prepared, consisting of capture beads conjugated with target-specific antibodies and fluorescent detection antibodies. The reagents are mixed with the sample and forced through a channel containing dense medium, a process that washes the sample and removes interfering substances. The beads that collect at the end of the channel are queried to determine the amount of antigen bound. SpinDx was used to quantify BoNTs with sensitivity that surpassed the mouse assay.
