Use of horseradish peroxidase-labelled antiglobulin for the colorimetric quantitation of erythrocyte antibodies. 1975

T J Greenwalt, and E M Swierk, and E A Steane

Horseradish peroxidase (HRP)-labelled antiglobulin has been used extensively as a histochemical marker. In the method described the stable reaction product formed by using 3,3'-diaminobenzidine (DAB) as the hydrogen donor in the peroxidase reaction was dissolved in a fluoro alcohol, 1,1,1,3,3,3-hexafluoroisopropanol (HPF). Reproducible standardisation curves resulted with HRP. Based on these observations a colorimetric assay procedure was developed to quantitate the amount of antibody coating red cells. This was achieved by treating the coated red blood cells with HRP-labelled rabbit antihuman IgG followed by hypotonic lysis separating the ghosts for colour development with the DAB reagent, solubilisation in HFP, and reading at 450 nm. Preliminary estimates of the number of anti-D molecules on Rhesus positive red cells were found to approximate the results reported using radioiodinated antibodies. The smallest number of anti-D molecules detected was 350 per red cell. Qualitative studies indicate that this procedure can be applied to other blood group systems.

UI MeSH Term Description Entries
D011817 Rabbits A burrowing plant-eating mammal with hind limbs that are longer than its fore limbs. It belongs to the family Leporidae of the order Lagomorpha, and in contrast to hares, possesses 22 instead of 24 pairs of chromosomes. Belgian Hare,New Zealand Rabbit,New Zealand Rabbits,New Zealand White Rabbit,Rabbit,Rabbit, Domestic,Chinchilla Rabbits,NZW Rabbits,New Zealand White Rabbits,Oryctolagus cuniculus,Chinchilla Rabbit,Domestic Rabbit,Domestic Rabbits,Hare, Belgian,NZW Rabbit,Rabbit, Chinchilla,Rabbit, NZW,Rabbit, New Zealand,Rabbits, Chinchilla,Rabbits, Domestic,Rabbits, NZW,Rabbits, New Zealand,Zealand Rabbit, New,Zealand Rabbits, New,cuniculus, Oryctolagus
D003124 Colorimetry Any technique by which an unknown color is evaluated in terms of standard colors. The technique may be visual, photoelectric, or indirect by means of spectrophotometry. It is used in chemistry and physics. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)
D004912 Erythrocytes Red blood cells. Mature erythrocytes are non-nucleated, biconcave disks containing HEMOGLOBIN whose function is to transport OXYGEN. Blood Cells, Red,Blood Corpuscles, Red,Red Blood Cells,Red Blood Corpuscles,Blood Cell, Red,Blood Corpuscle, Red,Erythrocyte,Red Blood Cell,Red Blood Corpuscle
D006735 Horseradish Peroxidase An enzyme isolated from horseradish which is able to act as an antigen. It is frequently used as a histochemical tracer for light and electron microscopy. Its antigenicity has permitted its use as a combined antigen and marker in experimental immunology. Alpha-Peroxidase,Ferrihorseradish Peroxidase,Horseradish Peroxidase II,Horseradish Peroxidase III,Alpha Peroxidase,II, Horseradish Peroxidase,III, Horseradish Peroxidase,Peroxidase II, Horseradish,Peroxidase III, Horseradish,Peroxidase, Ferrihorseradish,Peroxidase, Horseradish
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D000888 Antibodies, Anti-Idiotypic Antibodies which react with the individual structural determinants (idiotopes) on the variable region of other antibodies. Anti-Antibodies,Anti-Idiotype Antibodies,Antibodies, Internal Image,Antigamma Globulin Antibodies,Antiglobulins,Anti Antibodies,Anti-gamma Globulin Antibodies,Anti Idiotype Antibodies,Anti gamma Globulin Antibodies,Anti-Idiotypic Antibodies,Antibodies, Anti,Antibodies, Anti Idiotypic,Antibodies, Anti-Idiotype,Antibodies, Anti-gamma Globulin,Antibodies, Antigamma Globulin,Globulin Antibodies, Anti-gamma,Globulin Antibodies, Antigamma,Image Antibodies, Internal,Internal Image Antibodies
D000906 Antibodies Immunoglobulin molecules having a specific amino acid sequence by virtue of which they interact only with the ANTIGEN (or a very similar shape) that induced their synthesis in cells of the lymphoid series (especially PLASMA CELLS).
D000937 Antigen-Antibody Reactions The processes triggered by interactions of ANTIBODIES with their ANTIGENS. Antigen Antibody Reactions,Antigen-Antibody Reaction,Reaction, Antigen-Antibody,Reactions, Antigen-Antibody
D012204 Rh-Hr Blood-Group System Erythrocyte isoantigens of the Rh (Rhesus) blood group system, the most complex of all human blood groups. The major antigen Rh or D is the most common cause of erythroblastosis fetalis. Rh Factors,Rhesus Blood-Group System,Antigen D, Rh Blood Group,Blood-Group System, Rh-Hr,Blood-Group System, Rhesus,Blood-Group Systems, Rh-Hr,Blood-Group Systems, Rhesus,Factor, Rh,Factors, Rh,Rh Factor,Rh Hr Blood Group System,Rh-Hr Blood-Group Systems,Rhesus Blood Group System,Rhesus Blood-Group Systems,System, Rh-Hr Blood-Group,System, Rhesus Blood-Group,Systems, Rh-Hr Blood-Group,Systems, Rhesus Blood-Group

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