Hapten
Haptens are small molecules that elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself.
Once the body has generated antibodies to a hapten-carrier adduct, the small-molecule hapten may also be able to bind to the antibody, but it will usually not initiate an immune response; usually only the hapten-carrier adduct can do this. Sometimes the small-molecule hapten can even block immune response to the hapten-carrier adduct by preventing the adduct from binding to the antibody, a process called hapten inhibition.
The mechanisms of absence of immune response may vary and involve complex immunological mechanisms, but can include absent or insufficient co-stimulatory signals from antigen-presenting cells.
Haptens have been used to study allergic contact dermatitis and the mechanisms of inflammatory bowel disease to induce autoimmune-like responses.
The concept of haptens emerged from the work of Karl Landsteiner
who also pioneered the use of synthetic haptens to study immunochemical phenomena.
Examples of haptens
The first researched haptens were aniline and its carboxyl derivatives.A well-known example of a hapten is urushiol, which is the toxin found in poison ivy. When absorbed through the skin from a poison ivy plant, urushiol undergoes oxidation in the skin cells to generate the actual hapten, a reactive quinone-type molecule, which then reacts with skin proteins to form hapten adducts. Usually, the first exposure causes only sensitization, in which there is a proliferation of effector T-cells. After a subsequent, second exposure, the proliferated T-cells can become activated, generating an immune reaction that produces typical blisters of a poison ivy exposure.
Some haptens can induce autoimmune disease. An example is hydralazine, a blood pressure-lowering drug that occasionally can produce drug-induced lupus erythematosus in certain individuals. This also appears to be the mechanism by which the anaesthetic gas halothane can cause a life-threatening hepatitis, as well as the mechanism by which penicillin-class drugs cause autoimmune hemolytic anemia.
Other haptens that are commonly used in molecular biology applications include fluorescein, biotin, digoxigenin, and dinitrophenol.
Lastly, nickel allergy is caused by nickel metal ions penetrating the skin and binding to skin proteins.
Hapten conjugation
Due to their nature and properties, hapten-carrier adducts have been essential in immunology. They have been used to evaluate the properties of specific epitopes and antibodies. They are important in the purification and production of monoclonal antibodies. They are also vital in the development of sensitive quantitative and qualitative immunoassays. However, to achieve the best and most desirable results, many factors are needed to be taken into the design of hapten conjugates. These include the method of hapten conjugation, the type of carrier used and the hapten density. Variations in these factors could lead to different strengths of immune response toward the newly formed antigenic determinant.In general, these carrier proteins should be immmunogenic and contain enough amino acid residues in the reactive side chains to conjugate with the haptens. Depending on the haptens being used, other factors in considering the carrier proteins could include their in vivo toxicity, commercial availability and cost.
The most common carriers include serum globulin, albumins, ovalbumin and many others. Although proteins are mostly employed for hapten conjugation, synthetic polypeptides such as Poly-L-glutamic acid, polysaccharides and liposomes could also be used.
Methods of Hapten Conjugation
While selecting a suitable method for hapten conjugation, functional groups on the hapten and its carrier must be identified. Depending on the groups present, one of the two main strategies could be employed:- Spontaneous Chemical Reaction: Used when hapten is a chemical reactive molecule such as anhydrides and isocyanates. This method of conjugation is spontaneous and no cross-linking agents are needed.
- Intermediary Molecules Cross-linkage: This method mainly applies to nonreactive haptens. Agents with at least two chemically reactive groups such as carbodiimide or glutaraldehyde are to aid the conjugation of haptens to their carriers. The extent of cross-linkage is dependent upon the hapten/carrier to coupling agent ratio, hapten/carrier concentration and the temperature, pH of the environment.
- *Carbodiimide: A group of compounds with a general formula of R-N=C=N-R′, where R and R′ are either aliphatic or aromatic. Conjugation using a carbodiimide requires the presence of α or ɛ-amino and a carboxyl group. The amino group usually comes from the lysyl residue of the carrier protein while the carboxyl group comes from the hapten. The exact mechanism for this reaction is still unknown. However, two pathways are proposed. The first postulates that an intermediate that can react with an amine is formed. The second stating that a rearrangement of an acyl urea, the main side product of the reacction at high temperature, has occurred.
- *Glutaraldehyde: This method works by the reaction between glutaraldehyde with amine groups to form Schiff bases or Michael-type double bond addition products. The yield of conjugates can be controlled by varying the pH of the reaction. Higher pH would give rise to more Schiff base intermediates and subsequently lead to the increase in hapten conjugates' number and size. Overall, cross-linkage involving glutaraldehyde is very stable. However, immunized animals tend to regconise glutaraldehyde's cross-linking bridges as epitopes.
- High Performance Capillary Electrophoresis: High Performance Capillary Electrophoresis is an alternative method in optimizing hapten-protein conjugation. HPCE is predominantly used in separating carbohydrates with a very high separation capacity. There are numerous advantages to using HPCE as a technique to investigate certain conjugates such as only requiring minute sample sizes. In addition, the sample used does not need to be pure and no type of radiolabeling is needed. A great benefit to this method of hapten conjugation is that there is automated analysis of sample and the testing of sample interactions can be determined in free solution. This method of hapten-protein conjugation is exceptionally effective with conjugates of low epitope densities, where it is otherwise very challenging by the use of other methods to determine their electrical or ionic mobility.
Hapten inhibition