Immunogen Strategies for Antibody Generation

The overall analytical sensitivity and specificity of an immunoassay is, to a significant extent, related to the characteristics of the antibody used in the assay. Because drugs such as cannabinoids are small molecular weight haptens, a carrier protein is needed to produce an effective Immunogen. The site of linkage on the drug molecule to the protein carrier can determine the reactivity of the resulting antibodies. The specificity of an antibody is usually directed toward those structures on the hapten that are distal to the linkage group. Thus, the linkage site allows haptens to be coupled to the carrier in such a way that characteristic functional groups are exposed for antibody generation (20,21,87-89).

Figure 3 shows the published linkage sites for coupling cannabinoid haptens to a carrier protein. These linker groups include those out of the Cl-position, the C2-posi-tion, the C9-position, and the C5'-position of the THC-COOH compound or a very closely related compound. Various immunogen design structures were described in the National Institute on Drug Abuse Research Monographs 7 and 42 (20,21). Most of these antibodies were used for the development of RIAs with the exceptions of immunogen structures depicted for developing EMIT assay with the enzyme "pig heart malate dehydrogenase." There are a few major families of US/European/World patents for cannabinoid immunoassays along with claims for the structures of drug derivatives and/or immunogens. The patent families include those for Abbott's FPIA and those for Roche's RIA, enzyme immunoassay, FPIA, and KIMS cannabinoid assays (88,89).

Salamone et al. (87) comprehensively reviewed the selectivity of different immunogen structures and also described an approach to generate antibodies with a broader spectrum of cross-reactivities towards THC metabolites by "sequential immunization" and by designing a noncannabinoid, benzpyran core, immunogen. Taken together, the antibody generation approaches can be summarized as follows:

1. In general, antibodies generated from immunogens with the linkage position out of the C1-, C2-, or C5'-positions are more selective for the cyclohexyl ring, hence they usually display high selectivity for the unconjugated form of THC-COOH. The cross-reactivities for the 8-, 9-, and 11-substituted metabolites is lower because of the high recognition of the antibodies for this part of the molecule. Likewise, the cross-reactivities with the glucuronidated compounds are lower because the ether bond forms between

Chidlren Adrenochrome
Fig. 3. Immunogen strategies for the generation of anticannabinoid antibodies: common sites of linkage of cannabinoid haptens to a carrier protein. (From refs. 4,19,20,83-85.)

glucuronic acid and the hydroxyl moiety at C-11 for 11-OH-THC, and the ester bond forms between the glucuronide and the carboxyl moiety at C-11 for THC-COOH.

2. On the other hand, antibodies generated by immunogens with the C-9 position linkage are less selective for the cyclohexyl ring. Nevertheless, these antibodies typically show better binding to the 8-, 9-, and 11-substituted metabolites, as well as improved binding to their corresponding glucuronides. The antibodies also exhibit some selectivity for the cannabinoid nucleus in this region. These types of antibodies can be selected for high cross-reactivities for some, but not all, of the 8-, 9-, and 11-hydroxylated metabolites.

3. To increase the spectrum and degree of cross-reactivities for THC metabolites, a noncannabinoid immunogen was designed not to hold the antigenic determinants of the cyclohexyl ring, and hence the resulting antibodies will be indifferent to the cyclohexyl portion of the cannabinoid nucleus. Such a bicyclic immunogen contained only the structure of the benzpyran core. By eliminating the portion of the molecule that undergoes extensive metabolism from the immunogen and by preserving the core structure, antibodies with higher cross-reactive values with positive clinical samples can be generated. The resulting antibodies from the benzpyran core immunogens all showed broader cross-reactivities towards the 8-, 9-, and 11-hydroxylated metabolites.

The broad-spectrum antibodies can be utilized beyond the development of immunoassays. Feng et al. (80) immobilized THC antibody that was generated from the benzpyran core immunogen to prepare immunoaffinity chromatography for developing a simpler extraction procedure for A9-THC and its metabolites from various biological specimens. Good recovery was achieved by simultaneous extraction of A9-THC and its major metabolites, including THC-COOH, 11-OH-THC, and 8-|3,11-diOH-A9-THC, from plasma or urine after enzyme hydrolysis. A similar approach was also used for meconium analysis and confirmed that 11-OH-THC (80) is indeed an important metabolite in meconium.

The evolution of assay specificity can also be observed from the review of three decades of publications regarding cannabinoid immunoassays. In the earlier stages of drug immunoassay development, immunogens were used to produce polyclonal antibodies from selected animals. Naturally, polyclonal antibodies have broader cross-reactivities that are collectively contributed by a range of antibody affinity, avidity, and binding characteristics. The overall cross-reactivity manifestation can vary a bit from animal to animal and may change slightly over different time periods. Thus, it is not unusual for large pools of polyclonal antibodies to be validated and sequestered. Most current DAT immunoassays use monoclonal antibodies that are much more selective and specific and possess consistent quality. High specificity toward the target THC-COOH may increase overall immunoassay specificity at the expense of sensitivity. Thus, high antibody specificity may have the disadvantage of lower detection rate for clinical samples that contain THC-COOH near the screen cutoff concentration. Broad-spectrum monoclonal antibodies can possess the advantages of both monoclonal antibody consistency and the broader cross-reactivity profile. Nevertheless, the increased immunoassay sensitivity resulting from the higher values of THC-COOH equivalents might have the disadvantage of producing unconfirmed positives and might need a lower GC/MS cutoff (87).

Bearing in mind the variations in the relative percentages and forms of A9-THC metabolites present in the testing samples, both the detection and confirmation rates can have trade-offs, especially for near-cutoff samples. The ultimate goal for a can-nabinoid immunoassay design is to balance the assay sensitivity and specificity for its comparative performance to the GC/MS analysis according to their respective cutoff guidelines and regulations.

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