Analytische Chemie
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In 2019 over 30 000 people were killed or injured by explosions caused by explosives like TNT, PETN, HMX and RDX. Therefore, highly sensitive assays for the detection of TNT are needed. In this study we compared two commercially available TNT antibodies: A1.1.1 and EW75C with a highly optimized indirect competitive ELISA based on a BSA-TNA conjugate. As a result, a precision profile for both antibodies was determined with a LOD of 170 pmol L-1 for the clone A1.1.1 and a LOD of 3,2 nmol L-1 for the clone EW75C. The measurements showed that the clone A1.1.1 is a highly sensitive antibody for the detection of TNT while the clone EW75C does show medium performance at most.
In the cross-reactivity characterization of both antibodies many substances, closely related to the structure of TNT were tested. Both antibodies showed strong cross reactivity with trinitroaniline and trinitrobenzene. For the clone A1.1.1, which is known to originate from immunization of mice with an TNP-glycine-KLH conjugate, this has to be expected. Interestingly the clone EW75C, which was not characterized yet, showed similar behavior. This suggests a TNA-conjugate as immunogen for the EW75C antibody as well. None of both antibodies showed cross-reactions to the high explosives PETN, HMX and RDX. Also, the cross-reactions of nitro musks with the antibodies were investigated. Despite their prohibition, nitro musks are still used in Asia especially and are particularly popular in India. The overall superior clone A1.1.1 showed a significant cross-reactivity to musk ambrette. For practical reasons the influence of musk ambrette on this assay when used in natural environment should be investigated.
In further experiments, the highly sensitive TNT antibody A1.1.1 was digested with papain to obtain monovalent Fab-fragments. Due to its high stability against the digestion, a custom protocol for the IgG1 subclass of mice, to which the clone A1.1.1 belongs, was developed, resulting in a quantitative digestion of the intact antibody to Fab fragments. The success of the digestion was determined with MALDI-TOF-MS and SDS-PAGE. It was shown that this protocol worked for many different antibodies of IgG1 subclass as well.
Thousands of antibodies for diagnostic and other analytical purposes are on the market. However, it is often difficult to identify duplicates, reagent changes, and to assign the correct original publications to an antibody. This slows down scientific progress and might even be a cause of irreproducible research and a waste of resources. Recently, activities were started to suggest the sole use of recombinant antibodies in combination with the open communication of their sequence. In this case, such uncertainties should be eliminated. Unfortunately, this approach seems to be rather a long-term vision since the development and manufacturing of recombinant antibodies
remain quite expensive in the foreseeable future. Also, nearly all commercial antibody suppliers may be reluctant to publish the sequence of their antibodies, since they fear counterfeiting. De-novo sequencing of antibodies is also not feasible today for a reagent user without access to the hybridoma clone. Nevertheless, it seems to be crucial for any scientist to have the opportunity to identify an antibody undoubtedly to guarantee the traceability of any research activity using antibodies from a third party as a tool. For this purpose, we developed a method for the identification of antibodies based on a MALDI-TOF-MS fingerprint. To circumvent lengthy denaturation, reduction, alkylation, and enzymatic digestion steps, the fragmentation was performed with a simple formic acid hydrolysis step. Eighty-nine unknown monoclonal antibodies were used for this study to examine the feasibility of this approach. Although the molecular assignment of peaks was rarely possible, antibodies could be easily recognized in a blinded test, simply from their mass-spectral fingerprint. A general protocol is given, which could be used without any optimization to generate fingerprints for a database. We want to propose that in most scientific projects relying critically on antibody reagents, such a fingerprint should be established to prove and document the identity of the used antibodies and to assign a specific reagent to a datasheet of a commercial supplier, a public database record or an antibody ID.
The primary screening of hybridoma cells is a time-critical and laborious step during the development of monoclonal antibodies. Often, critical errors occur in this phase, which supports the notion that the generation of monoclonal antibodies with hybridoma technology is difficult to control and hence, a risky venture. We think that it is crucial to improve the screening process to eliminate most of the critical deficits of the conventional approach. With this new microarray-based procedure, several advances could be achieved: Selectivity for excellent binders, high-throughput, reproducible signals, avoidance of misleading avidity (multivalency) effects, and performance of simultaneous competition experiments. The latter can also be used to select clones of desired cross-reactivity properties. In this paper, a model system with two excellent clones against carbamazepine, two weak clones, and blank supernatant containing fetal bovine serum was designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the immunoglobulin G (IgG) concentration, which is usually unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration are not feasible in most cases. Furthermore, in this approach, the enrichment, isolation, and purification of IgG for characterization is not necessary. Raw cell culture supernatant can be used directly, even when fetal calf serum (FCS) or other complex media is used. In addition, an improved method for the oriented antibody-immobilization on epoxy-silanized slides is presented. Based on the results of this model system with simulated hybridoma supernatants, we conclude that this approach should be preferable to most other protocols leading to many false positives, causing expensive and lengthy elimination steps to weed out the poor clones.