Polyclonal antibodies to lincomycin (LIN) were developed in rabbit as a result of immunization with BSA-LIN conjugate. Periodate oxidizing of hapten was the common step of both immunogen synthesis and preparation of conjugated antigens for coating plates (homologous and heterologous). Several ELISA variants on a base of the different antigens immobilized on polystyrene were compared. Heterology of solid-phase antigens was provided with relative hapten clindamycin (CLIN) and ethylene- or hexanediamine as spacer arm between hapten and carrier. The spacer insertion yielded no desirable effect, whereas gelatin-CLIN assay variant showed better test characteristics in comparison with the homologous one, although insignificant (IC(50) was 9.15 vs 18.3 ng mL(-1)). The detection limits of the developed test, being estimated as 0.43 ng mL(-1) (milk) and 0.65 ng mL(-1) (eggs), were sufficient to measure maximum residue levels for LIN in examined matrices. This value for honey was 1.9 ng mL(-1) (1.3 μg kg(-1)). The assay sensitivity was enough to dilute milk, egg, and honey samples by 10-100 times to minimize matrix effect. The examination of matrix effect and simple ways of its overcoming are detailed in the paper. The developed assay showed 111% cross-reactivity with CLIN; therefore, it is suitable for the determination of both lincosamides.

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The recognition spectrum of immunoassays developed on the basis of class-specific antibodies can include the several nearest analytes but rarely all of the desired representatives of the group. The situation may be sufficiently improved using a hybrid assay combining two antibodies with specificities that complement each other. Two monoclonal antibodies (mAb) with broad but different specificities toward sulfonamides were examined for their binding to a panel of hapten conjugates. mAb-hapten pairs without mutual cross-reactions were identified, and classical direct antigen-coated and mAb-coated ELISAs were developed as formats with referent specificities. Both interactions were combined in a single hybrid assay, which was designed as a one-step double-competitive sandwich-ELISA. For this assay, the intermediate bifunctional reagent mAb(1)-hapten(2) conjugate was synthesized and able to simultaneously bind to hapten(1) and be bound by mAb(2). Formation of a two-mAbs sandwich complex was inhibited by competitors of interaction(1) as well as by competitors of interaction(2). Thus, due to the summation effect, simultaneous determination of analytes recognized by both mAbs was achieved. The hybrid assay can be performed in two reversed arrangements using a coating antigen or coating antibody, the characteristics of which were compared and found to be similar in sensitivity and extended specificity. The suitability of the developed test for the determination of 14 sulfonamides at their maximum residue limit (MRL) concentration was demonstrated using the examples of turkey muscle and milk samples.

Seven tetracycline (TC) antibiotics were used as haptens for synthesis of conjugated antigens and as standards in indirect competitive enzyme-linked immunosorbent assay (ELISA). Seven conjugates were prepared by formaldehyde condensation (f) and another seven antigens were synthesised by linking TCs with periodate-oxidised transferrin (TF(pi)). Two gelatineÁlymecycline (LC) conjugates were the products of glutaraldehyde and activated esters reactions. To estimate the influence of solid-phase antigen on assay specificity, all the conjugates were absorbed on polystyrene plates and using anti-bovine serum albuminÁTC(f) sera, the ELISAs were developed and compared. The variant with immobilised TFÁ chlortetracycline (CTC(pi)) showed the best group specificity: recognition of seven TCs differed only in 5.3 times. This assay displayed sensitivity 0.1 ng/ml and crossreactivity indexes: TC (100%), CTC (105%), oxytetracycline (19.8%), methacycline (23.6%), minocycline (70%), doxycycline (25%) and LC (50%). The dependence of ELISA specificity on immobilised antigen spatial structure was demonstrated. Solid-phase conjugate selection is a useful tool for modifying assay specificity.

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