Isolated Polynucleotide encoding modified fab' antibody, expression vector, and transformed host cell

An isolated polynucleotide encoding a modified Fab′ antibody, which comprises an Fd chain and an L chain with the Fd chain comprising a CH1 region and the L chain comprising a CL region, wherein the modified Fab′ antibody further comprises a cysteine for binding to a fluorescent dye;

• the polynucleotide comprising an Fd chain coding region and an L chain coding region linked therebetween in a manner that will allow expression of the modified Fab′ antibody, wherein
• (a) said modified Fab′ antibody is specific for an antigen and capable of forming an immune complex with said antigen in an analytical sample;
• and
• (b) said modified Fab′ antibody being recombinantly modified from a first Fab′ antibody specific for said antigen, wherein said modified Fab′ antibody comprises modifications to provide:
• (i) a substitution of asparagine at Kabat position 162 by aspartic acid to thereby produce an H—N162D modified Fab′ antibody and
• (ii) an addition adjacent to the CH1 region C-terminus, wherein the addition comprises a single cysteine residue which is not involved in binding with the L chain, wherein the single cysteine is for binding to a fluorescent dye;
• wherein the polynucleotide encodes an amino acid sequence comprising the sequence set forth in SEQ ID NO:
• 9 which comprises said (i) substitution of asparagine at Kabat position 162 by aspartic acid and said (ii) addition adjacent to the CH1 region C-terminus.