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Micromethod for quantification of SH groups generated after reduction of monoclonal antibodies.

Abstract：

:A simple, rapid, and reproducible micromethod for quantification of sulfhydryl (SH) groups generated after reduction of monoclonal antibody (MAb) disulfide bonds with 2-mercaptoethanol (2-ME) is described. The number of SH groups per molecule of antibody in the 2-ME and in the other reducing agents was calculated from the cysteine standard curve using Ellman's reagent to develop the yellow color. Results were plotted as absorbance at 405 nm vs. cysteine concentration (microgram/mL). After subtraction of the background due to Ellman's reagent, a straight-line relationship passing through the origin was obtained. Absorption spectrum of the yellow products was controlled, and no significative differences were found between optical density at 412 nm and 405 nm. Using a small quantity of antibody in the order of 37 micrograms, the lowest detection limit for cysteine quantification was 0.03 microgram. An excellent linear correlation was found between both cysteine concentration and absorbance (r = 0.999), and the mean value of the relative error in the quantification of cysteine from samples was 2.8%. A statistical Student t-test showed an excellent linearity and parallelism between cysteine standard and samples.

journal_name

Nucl Med Biol

journal_title

Nuclear medicine and biology

authors

Iznaga Escobar N,Morales A,Núñez G

doi

10.1016/0969-8051(96)00028-5

subject

Has Abstract

pub_date

1996-07-01 00:00:00

pages

641-4

issue

5

eissn

0969-8051

issn

1872-9614

pii

0969805196000285

journal_volume

23

pub_type

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