Comparison of Horseradish Peroxidase and Alkaline Phosphatase-Labelled Antibodies in Enzyme Immunoassays

Abstract

The periodate method was found to be most effective for preparing horseradish peroxidase-sheep anti-human and horseradish peroxidase-donkey anti-mouse immunoglobulin (IgG) conjugates. The conjugates were improved by carrying out the oxidation of the enzyme at low pH. Anti-human and anti-mouse IgG-peroxidase conjugates (0·5 mg/mL IgG and 0.7 mg/mL 19G, respectively) were used at 1:15 000 and 1:8000 dilutions, respectively, in a sandwich ELISA to detect human and mouse IgG in buffer or in a growth medium containing 20% foetal calf serum. Using the peroxidase conjugates, it was possible to detect human and mouse IgG at concentrations as low as I ng/mL. The glutaraldehyde method was found to be much more effective than the periodate method for conjugating alkaline phosphatase to the antibodies. The optimum dilutions for anti/human and anti-mouse IgG-alkaline phosphatase conjugates (0.18 mg/mL IgG and 0.3 mg/mL IgG, respectively) in ELISA were 1:500 and 1:1000, respectively. The detection limit with alkaline phosphatase conjugates was 7 ng/ml for human IgG and 4 ng/ml for mouse IgG.

Quantitative comparison of MiTF, Melan-A, HMB-45 and Mel-5 in solar lentigines and melanoma in situ

“…A benefit of using a red chromogen is the ability to readily distinguish the cytoplasmic staining of melanocytes from pigmented keratinocytes and melanophages. However, enzyme‐linked immunosorbent assay studies have shown a decreased ability to detect low abundance antigen using alkaline phosphatase enzymatic assay (red), as compared to a horseradish peroxidase assay (brown); 21 therefore, we used a brown chromogen in our studies.…”

Dietary Nucleotides Might Influence the Humoral Immune Response against Cow’s Milk Proteins in Preterm Neonates

“…Standard scrum with a known amount of specific anti body and the test scrum samples were added to wells and incubated overnight at 4°C. After washing the plates 3 times with PBS-Twcen, affinity-purified rab bit anti-p-lactoglobulin IgG, previously conjugated with biotin [21], was added. The development of color was performed with streptavidin-biotinylated horse radish peroxidase complex, using tetramethylenebenzidine as substrate.…”

Chemical modifications of proteins: history and applications

“…1 University of California. lecular and intermolecular cross-links, both to couple already associated species (36,37); and to join various proteins, which might or might not otherwise associate, in order to combine the properties of both into a single molecule, e.g., to make protein-protein conjugates (38,39), enzyme-linked antibodies (40,41), immunotoxins (42,43), and drug-protein conjugates (44). A large number of reagents that have been developed to serve these and a variety of other purposes are commercially available.…”