HYBRIDOMA Volume 9, Number 4, 1990 Mary Ann Liebert, Inc., Publishers
Microplate Method for the Determination of Amino Groups in Monoclonal Antibodies
KEVIN LAW,12 and
'Biolherapeulks, Inc., Franklin, TN 2Current address: Lilly Research Laboratories, Indianapolis, IN 3Current address: Medarex, Inc., West Lebanon, NH
ABSTRACT The total number of amino groups in monoclonal antibodies can be estimated via a colorimetric assay using trinitrobenzene sulfonic acid (A.F.S.A. Habeeb (1966) Anal. Biochem. 14, 328-336). This work describes the adaptation of this method to a microtiter plate in order to rapidly and reliably assay large numbers of samples.
Introduction of linker molecules, dyes or affinity ligands onto involves chemical reactions with free amino groups If the substituent of interest is located on amino acid side chains. difficult to assay, then one can assess the degree of substitution indirectly by determining the total number of amino groups before and An excellent method for doing this after modification of the protein. In this assay the protein is reacted has been described by Habeeb . with 2,4,6-trinitrobenzenesulfonic acid (TNBSA), and the concentration of free amino groups is calculated using the extinction coefficient of There are aspects of the standard assay the resulting chromophore. protocol, however, which can make this assay cumbersome and unreliable when dealing with large numbers of samples. By working in the small volume of a microtiter plate, the amount of sample necessary was reduced and the addition of reagents was facilitated with the use of a multichannel pipetter. Because the absorbance values change over time due to hydrolysis of the chromophore, calculation of the sample concentration values using the extinction coefficient yielded both intra We employed a microplate reader which and interassay differences. We also allowed for rapid measurement of sample absorbance. incorporated a series of standards into the microplate assay which greatly enhanced the reproducibility of the sample concentration values. Modified and control antibodies (50 (lg) and glycine standards (0.2-2.0 p.g) were added in 50 |ll of PBS to triplicate wells of a microtiter plate (Costar, Cambridge, MA). Using a multichannel pipetter, every well received 50 \ll each of 4% NaHCC>3, pH 8.5 and 0.1%
The plate was mixed on a TNBSA (Pierce Chemical, Rockford, IL). clinical rotator (Fisher Scientific, Springfield, NJ) for 2 min and then incubated at 40OC for 1 h in a dry bath incubator (Fisher Scientific, Springfield, NJ) using a ground aluminum block (Whittaker Bioproducts, Inc., Walkersville, MD) to insure even heating of the plate and to protect the samples from light. Immediately after incubation, 50 ul of 10% SDS and 25 ul of 1 N HC1 were added and the plate was mixed for 10 min in the dark. The absorbance maximum of the chromophore is 335 nm but use of a microplate reader (Bio-Tek Instruments, Inc., Burlington, VT) necessitated reading the absorbance of the wells with a 405 nm cutoff filter. The data was analyzed using the Immunosoft software program (Dynatech Laboratories, Inc., Chantilly, VA) for the Macintosh computer. TABLE 1
51 ± 0.8 44 ± 2.8 17 ± 1.3
48 ± 2.8 40 ± 1.8 13 ± 1.4
44 ± 0.6 34 ± 4.0 10 ± 0.3
48 ± 1.4 38 ± 1.5 20 ± 2.4
49 ± 0.1 36 ± 1.7 20 ± 1.2
47 ± 1.9 35 ± 0.9 13 ± 0.6
Cis-Aconitylated 9.6X 48X 480X
(n=2) (n=4) (n=3)
Monoclonal antibodies BrNumber of Amino Groups per Mole of Antibody. 1, Co-6 and Me-10  were purified by Protein A affinity chromatography Treatment of antibodies (5 mg samples) with from mouse ascites. succinic anhydride or cis-aconitic anhydride (Sigma Chemical Co., St. Louis, MO) was carried out in 0.1 M potassium phosphate buffer, pH 8.5. Samples were incubated for 1 h at room temperature and subsequently dialyzed for 18 h against several changes of PBS. an refers to the number of experiments ^numbers followed by an X refer to the molar excess of anhydride compared to antibody in the incubation mixture Table 1 lists the mean and standard deviation of values obtained for both control and modified antibodies tested by the microplate method. For comparison, the mean and standard deviation for Br-1 using the standard assay protocol in five different assays was 77 ± All standard curves (Fig. 1) had correlation coefficients greater 5.7. The number of amino groups estimated by the microplate than 0.995. method for three control mouse monoclonal antibodies were consistent with previously reported literature values of 59 for polyclonal rabbit antibodies  and 35-82 for four different murine monoclonal antibodies . All modified antibodies displayed consistent reductions in the number of amino groups after treatment with increasing amounts of succinic anhydride or cis-aconitic anhydride (Table 1). The same results were obtained when the amount of protein in the sample wells was increased from 50 Ug to 100 Ug. The microplate assay for estimation of protein amino groups provides a rapid and reproducible method which only requires 0.3 nmol of protein per sample. Its greatest advantage over the standard method is a significant reduction in the number of manipulations when processing
Coefficient of determination
= 0.996 Coefficient of correlation = 0.998 Standard error of estimate = 0.011
CONCENTRATION OF AMINO GROUPS (uM) FIGURE 1. Representative Linear Used in TNBSA Microplate Assay.
large numbers of samples. These improvements are achieved through the of a microtiter plate, a multichannel pipetter, a set of standards and a microplate reader. use
ACKNOWLEDGEMENTS The authors would like to thank Dr. Kiu Leung and Mr. Frank Klein for their generous help in purifying and supplying monoclonal antibodies. REFERENCES 1.
Cohen, S., and Porter, of
(1966) Determination of free amino groups in trinitrobenzenesulfonic acid. Anal. Biochem. 14, 328-
R. (1964) Structure and Adv. Immunol. 4, 287-349.
Mueller, B.M., Wrasidlo, W.A., and Reisfeld, R.A. (1988) Determination of the number of e-amino groups available for
conjugation of effector Hybridoma 7, 453-456.
molecules to monoclonal antibodies.
Liao, S.K., Meranda, C, Avner, B.P., Romano, T., Husseini, S., Kimbro, B., and Oldham, R.K. (1989) Immunohistochemical
phenotyping of human solid tumors with monoclonal antibodies in devising biotherapeutic strategies. Cancer Immunol. Immunother. 28, 77-86.
Ateeq Ahmad Medarex, Inc.
Received for publication January 11, 1990 Accepted after revisions May 29, 1990
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