A method of designing monoclonal antibodies for a sandwich assay for a small molecule having a molecular weight of 500 to 2,000 and spatially separate binding portions such that two different antibodies can bind simultaneously to the small molecule without interfering with the binding of each other to form a three-member complex, wherein the small molecule is a macrolide, said macrolide being sirolimus, the method comprising: (a) preparing a first monoclonal antibody that binds specifically to a binding domain extending approximately from ring atom 15 to ring atom 21 and includes a triene moiety from ring atom 17 to ring atom 22 of sirolimus, wherein the first antibody is prepared from an immunogen that comprises sirolimus, wherein sirolimus is linked at carbon atom 26 or 32 through an oxime functionality to an immunogenic carrier for eliciting antibodies, and (b) preparing a second monoclonal antibody that binds specifically to sirolimus in a portion other than the domain to which the first antibody binds, wherein said second antibody binds a binding domain extending approximately from the methyl group on atom 11 to the methoxy group on atom 39 or from the methyl group on ring atom 25 to atom 41, wherein the second antibody is prepared from an immunogen that comprises sirolimus that is modified in the triene area extending approximately from ring atom 15 to ring atom 21 and including a triene moiety from ring atom 17 to ring atom 22,to which the first monoclonal antibody binds, wherein sirolimus is combined with a cyclic reagent IV under conditions for carrying out a Diels-Alder-reaction to produce a modified sirolimus of formula IIA and IIB and the modified sirolimus is linked to an immunogenic carrier at ring atom 26 or ring atom 32 for eliciting antibodies, wherein IIA, IIB and reagent IV are compounds of the formula: wherein: R 8 and R 9 are each independently H, non-bulky organic radical or a bulky organic radical, or are taken together to form a double bond to 0 or CH 2; R 10 and R 11 are each independently H, non-bulky organic radical or a bulky organic radical, or are taken together to form a double bond to O or CH 2; R 12 is H, non-bulky hydrocarbyl, or a bulky organic radical; wherein, p is 1, 2 or 3; a is 0 or 1; and D is N, O, or CH, with the proviso that a is 0 when D is O. (a) preparing a first monoclonal antibody that binds specifically to a binding domain extending approximately from ring atom 15 to ring atom 21 and includes a triene moiety from ring atom 17 to ring atom 22 of sirolimus, wherein the first antibody is prepared from an immunogen that comprises sirolimus, wherein sirolimus is linked at carbon atom 26 or 32 through an oxime functionality to an immunogenic carrier for eliciting antibodies, and (b) preparing a second monoclonal antibody that binds specifically to sirolimus in a portion other than the domain to which the first antibody binds, wherein said second antibody binds a binding domain extending approximately from the methyl group on atom 11 to the methoxy group on atom 39 or from the methyl group on ring atom 25 to atom 41, wherein the second antibody is prepared from an immunogen that comprises sirolimus that is modified in the triene area extending approximately from ring atom 15 to ring atom 21 and including a triene moiety from ring atom 17 to ring atom 22,to which the first monoclonal antibody binds, wherein sirolimus is combined with a cyclic reagent IV under conditions for carrying out a Diels-Alder-reaction to produce a modified sirolimus of formula IIA and IIB and the modified sirolimus is linked to an immunogenic carrier at ring atom 26 or ring atom 32 for eliciting antibodies, wherein IIA, IIB and reagent IV are compounds of the formula: wherein: R 8 and R 9 are each independently H, non-bulky organic radical or a bulky organic radical, or are taken together to form a double bond to 0 or CH 2; R 10 and R 11 are each independently H, non-bulky organic radical or a bulky organic radical, or are taken together to form a double bond to O or CH 2; R 12 is H, non-bulky hydrocarbyl, or a bulky organic radical; wherein, p is 1, 2 or 3; a is 0 or 1; and D is N, O, or CH, with the proviso that a is 0 when D is O. R 8 and R 9 are each independently H, non-bulky organic radical or a bulky organic radical, or are taken together to form a double bond to 0 or CH 2; R 10 and R 11 are each independently H, non-bulky organic radical or a bulky organic radical, or are taken together to form a double bond to O or CH 2; R 12 is H, non-bulky hydrocarbyl, or a bulky organic radical; wherein, p is 1, 2 or 3; a is 0 or 1; and D is N, O, or CH, with the proviso that a is 0 when D is O.