Hapten synthesis

Small molecules, such as most food contaminants, with molecular weights lower than 5,000-10,000 do not elicit immune responses. In those cases, the molecules are named haptens because it is necessary to attach them to a large carrier protein in order to stimulate the immune response. Sometimes, the hapten is used as detector, then it is attached to an enzyme.

In both cases, as immunogen, or as detector, the attachment to the protein is through a covalent bond, and a linker or spacer arm is often used to enable greater antibody recognition. Antibody specificity is directed to sites on the hapten that are distal to the point of linker attachment [2,3]. A good displacement of hapten by the spacer arm generally produce antibodies with high titer, but sometimes fails to provide the required sensitivity in an immunoassay.

A wide variety of proteins are available for the synthesis of immunogens or antigens including bovine serum albumin (BSA), and human serum albumin, ovalbumin, conalbumin, thyroglobulin, keyhole limpet hemocyanin (KLH) or horseshoe crab hemocyanin, and the synthetic polypeptides poly-l-lysine and polyglutamic acid. Among them, KLH is often the first choice as an immunogen carrier protein because it is large (approximately 106 kDa), highly immunogenic and easy to conjugate because their high abundance of functional available groups. Thyroglobulin has been increasingly used as an immunogenic carrier protein owing to its high water solubility. Another frequently used protein in immunoassay is BSA, specially as a coating antigen carrier. Advantages of BSA include its wide availability in pure form, low cost, stability, relatively resistant to denaturation and is suitability for some conjugation procedures that involve organic solvents. BSA has a molecular weight of 64,000 Da.

A spacer arm is preferable with an alkyl chain of three to six carbons. The use of bulky functionalities, such as aromatic rings, conjugated double bonds in spacer arms should be avoided to minimize the recognition of this region by the antibodies [3]. Propionic, succinic and caproic acid are the most commonly used spacer arms for contaminant immunoassay development.

The selection of conjugation method is dependent on the functional group on the hapten (e.g., carboxylic acid, amine, aldehyde). A hapten with a carboxylic acid group can conjugate with a primary amino group of a protein using the carbodiimide, activated N-hydroxysuccinimide (NHS) ester or mixed anhydride methods. Haptens with free amino groups can be coupled to proteins using glutaraldehyde condensation or diazotization. Haptens that have been designed to contain spacers may be linked directly to the protein with methods such as the mixed anhydride, whereas haptens lacking a spacer should be coupled using methods that insert a linker between the hapten and the protein such as with glutaraldehyde.

Typical procedures are:

• Anhydride mixed method

• Carbodiimide method

• Carboxylic methods

• Hydroxyl groups.

Methods for linking hapten carboxyl groups to amine groups of antigenic proteins include activation by carbodiimides, isobutyl chloroformate or carbonyldiimidazole. In the widely used carbodiimide method, the carbodiimide activates the carboxylic acid to speed up its reaction with the amine. Acidic conditions catalyze the formation of the active O-acylurea intermediate while the protein is more reactive at higher pH, when the lysine amino groups are unprotonated. Therefore, as a compromise, a pH near 6 is used.

The choice of the carbodiimide is dependent on the reaction conditions. For example, dicyclohexylcarbodiimide (DCC) is used in nonaqueous media with nonpolar, water insoluble haptens where the carrier protein, in aqueous solution, is added to the activated hapten in a two-step reaction. For more water-soluble haptens, water-soluble derivatives of DCC such as 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide or 1-cyclohexyl-3-(2-morpholino ethyl) carbodiimide metho-p-toluenesulfonate (CMC or Morpho CDI) are used in one-step reactions. However, the 1-ethyl-3-(3-dimethylaminopropyl)-3-ethylcarbodiimide react directly with the protein, and some antibodies are generated against the resulting highly immunogenic protein-urea complex. Formation of these antibodies is not a drawback as long as a different coupling chemistry is used to prepare the plate coating antigens.

Activated NHS esters of carboxylic acids are prepared by reacting the acid with NHS in the presence of DCC. NHS esters are stable when kept under anhydrous and slightly acidic conditions, and they react rapidly with amino groups to form an amide in high yield. Like the carbodiimide method, the mixed anhydride method [4] results in an amide complex. The acid-containing hapten is dissolved in a dry, inert, dipolar, aprotic solvent such as p-dioxane, and isobutyl chloroformate is added with an amine catalyst. The activated mixed anhydride is chemically stable and can be isolated and characterized. The aqueous protein solution is added to the activated acid and the pH is maintained at around 8.5. A low temperature (around 10°C) is necessary during the reaction to minimize side reactions.

Amine groups in haptens, carrier proteins or both can be modified for conjugation through homo- or heterobifunctional crosslinkers such as succinic anhydride, succinyl chloride, or glutaraldehyde. Glutaraldehyde condensation has been used widely to produce protein-protein and hapten-protein conjugates. The glutaraldehyde reagent should not have undergone polymerization.

Aromatic amine-containing haptens are converted to diazonium salts with ice-cold nitrous acid. Diazonium salts can then react with a protein in alkali through electrophilic attack of the diazonium salt at histidine, tyrosine and (or) tryptophan residues of the carrier protein.

Other reactions can also be used to couple haptens to proteins, e.g., the periodate oxidation is suitable for compounds possessing vicinal hydroxyl groups such as some sugars.

The conjugates characterization is based on the determination of the haptens density, which is important for both immunization and assay performance. The degree of conjugation can be determined by established methods, such as characteristic ultraviolet (UV) or visible absorbance spectrum that distinguishes the hapten from the carrier protein or by the use of a radio labeled hapten. If the hapten has a similar lmax to the protein, the extent of incorporation can still be estimated when the concentration of the protein, and the spectral characteristics of the hapten and protein are known. The difference in absorbance between the conjugate and the starting protein is proportional to the amount of hapten conjugated [5].

Hapten density can also be determined indirectly by measuring the difference in free amino groups between conjugated and unconjugated protein using trinitrobenzenesulfonic acid [6].

These methods can be used as a rough estimation because the process of conjugation usually alters the apparent number of amine or sulfhydryl groups on the protein. On the other hand, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been applied to estimate the number of covalently bound haptens [7]. Although these methods offer an accurate approach, large proteins cannot be analyzed.

Hapten density, and also the common positions where haptens are bound, can also be estimated by cyanogen bromide or enzymatic cleavage of the protein and either MALDI-MS or separation of the components by reversed-phase ion-pair chromatography and electrospray or electrospray time-of-flight (TOF) analysis.

The optimum hapten ratio may depend on the study objectives, the nature of the antigen, immunization protocol, etc. A general rule of thumb is to target high hapten ratios for immunogens and low hapten ratios for coating antigens or enzyme tracers. For immunogens, a high hapten ratio implies greater exposure of the immune system to the hapten; for coating antigens or enzyme tracers, a lower hapten density implies fewer haptens to compete with the analyte in the assay. Optimum hapten density is often determined empirically with checkerboard titration procedures. Such procedures are very rapid and are normally adequate to optimize enzyme-linked immunosorbent assays (ELISAs) without knowing the exact hapten density. However, for the development of new immunosensor devices the determination of exact hapten densities may become increasingly important.

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  • esa-pekka
    How heptens are estimated by chromatographic methods?
    2 years ago

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