The development of an enzyme-linked immunosorbent assay for bovine lactoferrin : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
Methods were established for the estimation of bovine lactoferrin by enzyme-linked
immunosorbent assay (ELISA) on microtitre plates and nitrocellulose (dot assay).
Mfinity-puri:fied antibodies to bovine lactoferrin were prepared and conjugated to
horseradish peroxidase by the periodate method. Conjugates with enzymatic and
immunological activity had an apparent molecular weight of 65 or 95 kd.
Four methods of ELISA on microtitre plates and three dot assays were developed.
Differences between the seven assays could be attributed to the absorption capacity of the
solid phase and the types of conjugates and substrates used. The range of the two
successful quantitative assays were 3-lOOOng lactoferrin/ml (sandwich) and 60-8000ng
lactoferrin/ml (competitive), while the qualitative dot assays had a range of 1-lOOJ.lg
lactoferrin/ml. More replicates would be required to reduce variability.
Results from these assays generally corresponded to results from ROCKET
electrophoresis. Dot assay on nitrocellulose has a greater potential for reproducible and
quantitative assays than assay on microtitre plates, because of the greater adsorption
capacity of the nitrocellulose. In addition, the dot assays are faster and lend themselves to
more applications than either ROCKET electrophoresis or ELISA on microtitre plates.
The ELISA developed in this project appear to be the first alternatives to radial
immunodiffusion and ROCKET electrophoresis for the measurement of bovine lactoferrin.