Direct determination of forskolin–bovine serum albumin conjugate by matrix‐assisted laser desorption ionization mass spectrometry

Abstract

Forskolin, a labdane diterpenoid, was isolated from the roots of Coleus forskohlii Briq. (Lamiaceae)' as the activator of adenylate cyclase,2 loading to an increase of c-AMP, used widely as an important reagent. Recently, immunoassay systems using monoclonal antibodies (MAbs) against drugs and naturally occurring biologically active compounds having smaller molecular masses like forskolin have become an important tool3 For production of monoclonal antibodies, the synthesis of hapten which is derived from immuno antigen and linker bridge, and carrier protein conjugates is necessary. Hapten is 7-deacetylforskolin 7-semisuccinyl residue. The specificity of an immunoassay method is generally dependent on the site of linkage between hapten and protein moieties, and also enumeration of hapten in the immunogen conjugate. There are no direct and appropriate methods for the analysis of hapten conjugated with carrier protein without differential UV analysis, radiochemical or chemical methods. Recently, Wengatz et al. determined the hapten density of immunoconjugates by matrix-assisted UV laser desorption/ionization mass ~pectrometry.~ Herein we wish to present an improvement for the direct analytical method of hapten and carrier protein conjugates by matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry using an internal standard, and by the production of antibodies against forskolin. For the first experiment, a 33 PM 7-deacetyl forskolin 7-~emisuccinate~ was conjugated with 0.65 p~ bovine serum albumin (BSA) as explained inf Ref. 6.A small amount (1-10 pmol) of the antigen conjugate was mixed with a 103-fold molar excess of sinapinic acid in an aqueous solution containing 10% trifluoroacetic acid. The mixture was placed inside a JMS-LDI 1700 time-of-flight (TOF) mass monitor and irradiated with a N, laser (337 nm, 3 ns pulse). The ions formed by each pulse were accelerated by a 30 kV potential into a 1.7 m evacuated tube and detected using a microchannel plate detector with an IBM PC compatible computer. This method allows for rapid analysis of protein samples as small as 1 p-M. Figure 1 shows the co-mass spectra of the antigen conjugate of forskolin and BSA which is added as an internal standard. The relative molecular mass of BSA is 66 928 (average for triplicates, C.V. 0.09%) in agreement with a previous report' of which the calculated relative molecular mass of BSA was 66267. Figure 1 also shows only the singly, doubly, and triply protonated molecules of the intact conjugate which can be used for determination of the molecular mass of the hapten conjugate. Fragmentation is not an artefact of the ionization technique. A sharp peak coinciding with the conjugate appeared at m/z 72 160 (average for triplicates, C.V. 0.049%) resulting in the calculated relative molecular mass of 71 447. Using a relative molecular mass of 66 267 for BSA, the calculated mass of hapten, the 7-deacetyl forskolin 7-semisuccinyl residues (RMM 451), is 5 180 indicating 11.5 molecules of forskolin conjugated with BSA.In order to c...

Preparation of Knockout Extract by Immunoaffinity Column and Its Application

“…To determine the hapten density of immunoconjugates, Wengatez et al used matrix-assisted UV laser desorption/ionization (MALDI) mass spectrometry [29]. We also performed direct analysis of hapten-carrier protein conjugates by MALDI-TOF mass spectrometry [1][2][3][4][5][6][7][8][9][10][11][12][13][14][30][31][32]. Figure 1b indicates the MALDI-TOF mass spectra of the G-Rb 1 -BSA conjugate.…”

Development of monoclonal antibody against isoquinoline alkaloid coptisine and its application for the screening of medicinal plants

“…The hapten ratio in the hapten-carrier protein conjugates were determined by MALDI-TOF MS as previously described (Shoyama et al 1993). A small amount (1-10 pmol) of hapten-conjugate was mixed with a 10 3 -fold molar excess of sinapinic acid in an aqueous solution containing 0.15% trifluoroacetic acid.…”

Preparation of anti-2,4-dichlorophenol and 2,4-dichlorophenoxyacetic acid monoclonal antibodies

“…Analysis of haptencarrier protein conjugate by MALDI-TOF mass spectrometry directly determined the suitability of hapten number for immunization, as reported previously (Shoyama et al 1993;Goto et al 1994). Figure 2 shows the MALDI-TOF mass spectrum of the antigen, 2,4-D-BSA conjugate.…”