A computer program for quantification of SH groups generated after reduction of monoclonal antibodies.
Reduction of disulfide bonds to sulfhydryl (SH) groups for direct radiolabeling of antibodies for immunoscintigraphic studies of colorectal and other cancers continues to be of considerable research interest. We have developed a general strategy and a versatile computer program for the quantification of the number of SH per molecule of antibody (Ab) generated after the treatment of monoclonal antibodies (MAbs) with reducing agents such as 2-mercaptoethanol (2-ME), stannous chloride (SnCl2), dithiothreitol (DTT), dithioerythritol (DTE), ascorbic acid (AA), and the like. The program we describe here performs an unweighted least-squares regression analysis of the cysteine standard curve and interpolates the cysteine concentration of the samples. The number of SH groups per molecule of antibody in the 2-mercaptoethanol and in the other reducing agents was calculated from the cysteine standard curve using Ellman's reagent to develop the yellow color. The linear least-squares method fit the standard data with a high degree of accuracy and with the correlation coefficient r of 0.999. A program has been written for the IBM PC compatible computer utilizing a friendly menu to interact with the users. The package allows the user to change parameters of the assay, to calculate regression coefficients slope, intercept and its standard errors, to perform statistical analysis, together with detailed analysis of variance, and to produce an output of the results in a printed format.
Center of Molecular Immunology, Havana, Cuba. normando@ict.sld.cu
N
N
Iznaga Escobar
A
A
Morales
G
G
Núñez
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001205:Ascorbic Acid; D002272:Carcinoembryonic Antigen / Q000276:immunology; D003545:Cysteine / Q000032:analysis; D004226:Dithioerythritol; D004229:Dithiothreitol; D066246:ErbB Receptors / Q000276:immunology; D005753:Gastric Mucosa / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D007413:Intestinal Mucosa / Q000276:immunology; D016018:Least-Squares Analysis; D008623:Mercaptoethanol; D051379:Mice; D010084:Oxidation-Reduction; D012044:Regression Analysis; D012984:Software; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013439:Sulfhydryl Reagents; D017971:Tin Compounds
D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D007074:Immunoglobulin G; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D017971:Tin Compounds; C023599:stannous chloride; D008623:Mercaptoethanol; D004226:Dithioerythritol; D066246:ErbB Receptors; D003545:Cysteine; D001205:Ascorbic Acid; D004229:Dithiothreitol