Creative Diagnostics - Food & Feed Analysis


Antigens are usually macromolecules or macromolecular complexes that contain distinct antigenic sites or “epitopes,” which can be recognized and interact with the various components of the immune system. They can exist as individual molecules composed of synthetic organic chemicals, proteins, lipoproteins, glycoproteins, RNA, DNA, and polysaccharides—or they may be parts of cellular structures (bacteria or fungi) or viruses.

Small molecules like short peptides, although normally able to interact with the products of an immune response, often cannot cause a response on their own. These “haptens,” as they are called, are actually incomplete antigens, and while not able by themselves to cause immunogenicity or to elicit antibody production, they can be made immunogenic by coupling them to a suitable carrier molecule. Carriers are typically antigens of higher molecular weight that are able to cause an immunological response when administered in vivo. Development of a good immunoassay for small molecules depends greatly on the affinity and selectivity of the antibody for the analyte. The affinities and selectivities of the coating/enzyme labeled haptens for the antibody, the choice of protein carriers.


Immunoassay performance is a function of the affinity and selectivity of the antibody. The immunizing hapten should represent a near perfect mimic of the target molecule in structure, electronic and hydrophobic properties. These haptens are tethered with an antigenically inert handle distal to the determinant group(s) and do not mask or alter any functional group. Optimal hapten design criteria are based on extending an existing carbon chain, or replacing a C-H moiety of the target molecule with a CH2 chain terminated by a functional group for conjugation to proteins. Careful selection of immunizing hapten can lead to the production of compound or class selective antibodies. A multiple hapten approach, based on handle location, length, and composition, results in assays with sub-ppb levels of detection and improved selectivity. Examination of cross-reactivity data of the haptens led to the identification of the best coating/enzyme-labeled haptens for improved heterologous assays.

Guidelines for Immunizing Hapten Synthesis

  1. 1. of Handle on Target Molecule
       • Distal to important haptenic determinants
       • Avoid attachment to functional groups
  2. 2. Handle Selection
       • Length of handle
       • Avoid functional groups in handle ~ use alkyl or aralkyl handles
  3. 3. Functional Group for Coupling
       • Type of reaction of coupling
       • Compatibility of reaction with target molecule functional groups
  4. 4. Solubility of Hapten and/or Conjugates
  5. 5. Stability of Hapten Under Coupling Conditions and Subsequent Use
  6. 6. Ease of Synthesis
  7. 7. Determination of Hapten: Protein Ratio

Guidelines for Coating/Enzyme Labeled Haptens

  1. 1. Heterology of Hapten Structure
       • Position of handle
       • Composition of handle
       • Length of handle
       • Conjugation chemistry
  2. 2. Alterations in Target Molecule Structure
       • Use of partial structure
       • Change of key determinants, i.e. sulfur for chlorine
  3. 4. Cross-Reactivity Data of Hapten Structures (or Derivatives)
  4. 5. Determination of Haptem: Protein Ratio


Protein Carriers

The most common carrier proteins in use today are keyhole limpet hemocyanin (KLH; MW 4.5 × 105 to 1.3 × 107), bovine serum albumin (BSA; MW 67,000), aminoethylated (or cationized) BSA (cBSA), thyroglobulin (MW 660,000), ovalbumin (OVA; MW 43,000), and various toxoid proteins, including tetanus toxoid and diphtheria toxoid. Other proteins occasionally used include myoglobin, rabbit serum albumin, immunoglobulin molecules (particularly IgG) from bovine or mouse sera, tuberculin purified protein derivative, and synthetic polypeptides such as poly-l-lysine and polyl-glutamic acid.

Liposome Carriers

The overall composition of a liposome—its morphology, composition (including a variety of potential phospholipids and the degree of its cholesterol content), charge, and any attached functional groups—can affect the antigenicity of the vesicle in vivo. When liposomes are used as carriers for immunization purposes, the haptens or antigens are usually attached covalently to the head groups using various phospholipid derivatives and crosslinking strategies.

Synthetic Carriers

Synthetic molecules may be used as immunogen carriers if they are designed with the appropriate functional groups to facilitate the coupling of hapten molecules. These carriers may consist of simple polymers such as poly-l-lysine, poly-l-glutamic acid, Ficoll, dextran, polyethylene glycol (PEG), dendrimers, or other synthetic organic constructs. The advantage of a synthetic vaccine carrier is the potential for having little to no immune response to the carrier itself.


Carbodiimide-mediated hapten-carrier conjugation
NHS ester-mediated hapten-carrier conjugation
NHS ester-maleimide heterobifunctional cross linker-mediated hapten-carrier conjugation
Active-hydrogen-mediated hapten-carrier conjugation
Glutaraldehyde-mediated hapten-carrier conjugation
Reductive Amination-mediated hapten-carrier conjugation

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