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Anti-hapten antibody problems

Haptens are small molecules bound to a larger carrier protein to enable antibody discovery. This often yields antibodies with poor affinity and selectivity. Here we discuss why this is and present Optimer discovery as a solution to support you with small molecule detection and monitoring.

Why are haptens such a hard-to-solve target class for antibody development?

Small molecules are ubiquitous in our environment, from hormones and toxins to drugs and vitamins. There is an obvious need to detect and monitor this valuable target class for human health. However, generating antibodies to small molecule targets is not a simple feat and assays for key targets are still lacking.

Anti-hapten antibody production – how is it done?

To generate antibodies to a target, an animal is typically immunized with the target and the anti-target antibodies that develop as part of the resulting immune response are used directly in the collected serum or isolated and used to generate recombinant monoclonal binders.

Haptens are small molecule targets, which when injected into an animal do not result in antibody production. Due to the small size of haptens, they are not taken up, processed and presented on MHC clas II complexes by antigen-presenting cells, and so can’t be presented to T cells to induce a sufficient immune response for antibody generation. Instead, to create antibodies to these small molecule targets, the hapten molecule must be conjugated to a larger carrier protein. The term hapten comes from half-antigen, as they are only immunogenic when conjugated to a larger molecule. Routine carrier proteins include BSA and keyhole limpet hemocyanin (KLH) that are themselves inert, eliciting no immune response. Attaching a hapten molecule to a larger carrier allows recognition by the immune system and the production of antibodies. From these carrier-hapten antibodies, a proportion of those produced will be anti-hapten antibodies.

anti hapten antibody production aptamers

Small molecules require conjugation to carrier proteins for the development of antibodies in immunised animals. 

Why use anti-hapten antibodies?

Anti-hapten antibodies are essential in the development of diagnostics to improve human health. They are used as tool reagents to detect and quantify small molecules across a range of assay types, such as ELISA, lateral flow tests, biosensors and IHC. Haptens of interest include amino acid metabolites, lipids, drugs, toxins and pesticides. Even post-translationally modified peptides have been used to generate anti-hapten antibodies targeting specific PTMs.

Problems with anti-hapten antibody production

Despite their obvious uses in monitoring small molecule targets, the production of anti-hapten antibodies remains challenging for a number of reasons.

  1. Anti-hapten antibody production often results in reagents with limited target affinity.
  2. As these targets are so small they offer limited numbers of epitopes for antibodies to bind.
  3. Conjugation of the hapten to a carrier molecule or solid support for screening of libraries can often utilize a key chemical moiety further limiting the number of reactive epitopes available for antibody binding.
  4. Commonly, anti-hapten antibody production results in the many antibodies that bind to the linker region used to conjugate the hapten to the carrier or immobilize the hapten, rather than being specific for the small molecule target.
  5. To overcome the lack of sensitivity with traditional competition assays, sandwich-based assays require antibody pairs for the capture and detection of a target. Developing two antibodies to such small molecules doubles the problem and may not be possible in many cases.

To improve the performance of the anti-hapten antibodies produced extensive affinity maturation is often required, resulting in many months of further development time and cost.

A need for new anti-hapten antibodies

As hapten molecules make up such a diverse range of molecules in our environment and biology, the ability to detect and monitor these small molecules is important. Many assays that could improve patient outcomes and health monitoring are currently unavailable or do not provide sufficient sensitivity due to the difficulties in raising sensitive and specific antibodies to hapten targets. The development of adequate affinity reagents in this field could yield huge potential for improved patient outcomes and reduced healthcare costs.

Therapeutic drug monitoring with anti-hapten antibodies

With the advent of personalized medicine, many are pursuing the potential of monitoring key treatments, such as chemotherapeutics and antibiotics. This could allow patients to maintain their dose within the therapeutic window, ensuring treatment efficacy whilst lowering the potential for side effects that reduce compliance. The ability to offer rapid, point-of-care tests to detect and monitor these medicines could revolutionize patient treatment. Current practices for small molecule analysis are largely based on chromatography linked to mass spectrometry. This requires high cost, specialised equipment with large footprints and skilled staff to operate, meaning most clinics rely on centralized facilities for these assays. The ability to deliver point-of-care diagnostics for therapeutic drug monitoring of small molecule hapten drugs, based on standard assays, would offer faster and more targeted treatment responses, potentially reduce antibiotic resistance, and move towards patients being able to monitor their treatment within their own homes.

The ability to monitor patient plasma concentrations at point of care rather than through high cost, laborious processes currently required offers significant patient and clinician advantages.

Dr Federico Polo, Università Ca’ Foscari, Venice, Italy.

Improving assay sensitivity and selectivity with anti-hapten antibodies

Assays that do not offer the required sensitivity can prevent adequate monitoring of health.

17β-Estradiol (E2) is the primary female sex hormone, controlling the development and maintenance of female sexual characteristics, and plays a critical role in spermatogenesis, modulating libido and erectile function in males. Accurate measurements of serum estradiol are thus essential to understand physiology, development and the health of reproductive processes in both sexes, as well as the cause of diseases related to estrogens.  However, the available commercial immunoassays are not sensitive or accurate enough to quantify E2 at the low levels seen in children, menopause or males. This lack of assay sensitivity is due to problems with anti-hapten antibody production for the E2 target.

Vitamin D testing is becoming increasingly important with recent research demonstrating correlations between vitamin D insufficiency and metabolic diseases, immunodeficiencies and other diseases. Existing 25-hydroxyvitamin D (25OHD) assays lack adequate performance for accurate monitoring of serum levels due to problems with the cross-reactivity of the available anti-hapten antibodies for vitamin D2 with other vitamin D metabolites, matrix interference and a lack of standardization of the assays from poor antibody performance.

Developing new solutions for anti-hapten antibody production

As the life science industry has moved towards the use of recombinant affinity reagents for reliable and reproducible performance, many have started to explore alternative affinity reagents. As these are generated from libraries that are screened in vitro, they offer the potential to target novel molecules, including haptens, to generate anti-hapten antibody alternatives for use in diagnostic assays for improved patient health. While Fab fragments, scFVs and other antibody mimetic platforms have shown some successes in binding hapten targets, problems remain in the necessity to immobilize the hapten for library screening and the generation of matched pair reagents for highly sensitive immunoassays.

We have developed an in vitro Optimer selection platform specifically for hapten targets, which expands the target range and overcomes problems of limitation by the immune system, allows selection for the hapten target in solution and has a success rate of over 80% in developing high affinity, high selectivity Optimers to hapten targets to replace anti-hapten antibody production requirements in your assays. For more information on how our Optimers for small molecule targets could benefit your discovery and development get in touch.

Download the small molecule application note


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