Sandwich assay design for small molecules

A method of designing monoclonal antibodies for a sandwich assay for sirolimus, wherein sirolimus has spatially separate binding portions such that two different antibodies can bind simultaneously to the same sirolimus molecule without interfering with the binding of each other to form a three-member complex, the method comprising:

• immunizing a first antibody-producing animal with a first immunogen comprising sirolimus linked at carbon atom 26 and/or 32 through an oxime functionality to an immunogenic carrier, wherein the immunogenic carrier of the first immunogen is selected from the group consisting of bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), egg ovalbumin, bovine gamma globulin (BGG), thyroglobulin, ovalbumin, fibrinogen, and a polysaccharide;
• isolating antibody-producing spleen cells from the first antibody-producing animal;
• selecting at least one antibody-producing spleen cell that secretes a first monoclonal antibody;
• preparing a first hybrid cell line by fusing the selected antibody-producing spleen cell(s) from the first antibody-producing animal with an appropriate fusion partner, whereby the first hybrid cell line secretes the first monoclonal antibody;
• immunizing a second antibody-producing animal with a second immunogen comprising a modified sirolimus of at least one of formula IIA and IIB, and wherein the modified sirolimus is further linked to an immunogenic carrier at ring atom 26 and/or ring atom 32 for eliciting antibodies, and wherein the immunogenic carrier of the second immunogen is selected from the group consisting of bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), egg ovalbumin, bovine gamma globulin (BGG), thyroglobulin, ovalbumin, fibrinogen, and a polysaccharide;
• wherein IIA and IIB are compounds of the formulas:
• wherein:
• R8 and R9 are taken together to form a double bond to O, and p is 1;
• R10 and R11 are taken together to form a double bond to O;
• R12 is a phenyl group, and a is 1;
• and
• D is N;
• isolating antibody-producing spleen cells from the second antibody-producing animal;
• selecting at least one antibody-producing spleen cell that secretes a second monoclonal antibody that binds specifically to sirolimus in a portion other than the domain to which the first monoclonal antibody binds;
• and
• preparing a second hybrid cell line by fusing the selected antibody-producing spleen cell(s) from the second antibody-producing animal with an appropriate fusion partner, whereby the second hybrid cell line secretes the second monoclonal antibody.