Analytical
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MALDI-TOFMS of Recombinant Hepatitis MALDI-TOFMS makes it possible to obtain timely, accurate information on the identity of the reagents produced for immunoassays. Hepatitis B virus (HBV) is the most prevalent of all the viral hepatitis agents that can cause death or significant debilitation. The infection is transmitted primarily through exposure tq blood or blood products; congenital and sexual routes of infection are common.{1). Estimates indicate more than 200 million carriers of HBV worldwide, and they are the primary reservoir of new infections (2). People with active liver infections or who are asymptomatic chronic carriers have varying concentrations of viral particles of different forms. The most common forms of particles are spherical or filamentous with a mean diameter of 22 nm (3) and are known as hepatitis B surface antigen (HBsAg) particles. They lack the nucleocapsid (nucleic acid-protein complexes) antigens and viral DNA. The less common 42-nm particles are the intact infectious virions which contain multiple subunits of the HBsAg the nucleocapsid lipids viral proteins and a single of the viral DNA (4) (Figure la) The nucleocapsid contains 180 copies of the hepatitis B core anticren H-TRcAtr! in an icosohedral
Martin A. Winkler Naxing Xu Huaiqin Wu Hoda Aboleneen Abbott Laboratories 664 A
arrangement. A DNA polymerase is also associated with the virus (2). The HBsAg is composed mostly of multiple copies of a protein subunit called the S antigen (25,400 Da), the glycosylated form of the S antigen (27,000 Da), and a lipid membrane (4) (Figure lb). The exact numbers and stoichiometry of the S and glycosylated S antigens are not known. The S subunits are extremely hydrophobic transmembrane proteins, which snake through the cellular membrane four times and are tightly associated with each other through hydrophobic interactions. Disulfide bonds are essential in maintaining the structure of the HBsAg and they play a role in its antigenicity and secretion (5 6) These intermolecular disulfide bonds stabilize the HBsAg structure Free unbonded sulfhydryls also exist (7) To identify viral subtypes by serological methods, an antigenic specificity associated with all HBsAg particles, known as «, has been identified; in addition, there are two sets of exclusive serological determinants, designated dory and worr (3). Thus, the four main subtypes of HBsAg are adw, adr, ayw, and ayr. .n HBV-infected individuals who have sufficient cellular and humoral responses viral replication is aborted and antibodies to HBsAg prevent the reinfection of liver cells; otherwise the infection persists Currently available HBV vaccines are
Analytical Chemistry News & Features, October 1, 1999
based on recombinant HBsAg as an antigen for producing antibodies to HBsAg. This Analytical Approach focuses on the analysis of chemically modified recombinant HBsAg, which is used in diagnostics to detect antibodies to HBsAg and which monitors either the recovery from infection or the vaccination response. In particular, we describe matrix-assisted laser desorption/ionization (MALDI)time-of-flight(TOF) MS techniques for determining the extent of biotinylation of recombinant HBsAg.
HBsAg immunoassay Immunoassays have been designed to detect the antibody to HBsAg. The most common are enzyme immunoassays, which use enzymes conjugated to antibodies or antigens. The enzymes, such as horseradish peroxidase (HRPO) and alkaline phosphatase, hydrolyze substrates to compounds detectable by light spectrophotometry. The assays are typically performed in several steps. One sensitive assay configuration for measuring antibodies to HBsAg in the blood takes advantage of the two antigen-binding sites of the antibody to form a sandwich of HBsAg anti-HBsAg antibody and biotinylated HBsAg which can then bind to antibiotin antibody carrying HRPO (Figure 2) An automated immunoassay proceeds in the following steps: First microparticles
Chemically Modified B Surface Antigen coated with recombinant HBsAg are incubated with the diluted serum test sample, then washed to remove nonspecifically bound serum proteins and unbound components. Second, the microparticles are incubated with biotinylated HBsAg. Third, the beads are washed again and exposed to anti-biotin antibody conjugated to the enzyme HRPO. Finally, the microparticles are washed a third time, and the substrate o-phenylenediamine is added along with hydrogen peroxide. The absorbance at 492 nm is proportional to the amount of human anti-HBsAg in the original Q^l*l ITU
sample An important question in the design, development, and manufacture of the antiHBsAg immunoassays is the extent to which the HBsAg particles are modified in the labeling process. If particles are modified with too many biotins—which are linked to lysines or the N-termini of the S subunits—the assay might not detect some viral-specific antibodies because antigenic sites are lost on the modified HBsAg. If the particles are not sufficiently modified with biotin, however, the assay will be insensitive because most antibodies will be bound to particles with no biotin label. To produce a uniform antigen for immunoassay, recombinant HBsAg is made by cultured human cells transfected with SV40 virus containing the DNA sequences for the S antigen component of HBsAg (8). The
recombinant S antigen forms the HBsAg particles, which are purified by using immunoaffinity columns containing monoclonal antibodies specific for HBsAg. The antibodies are immobilized on polymer beads in the column, and the antibodies bind HBsAg in spent culture medium from cells expressing the recombinant protein. The column is washed, and the HBsAg is eluted with a low-pH buffer. Several analytical procedures are needed to verify the purity and identity of the recombinant protein. The intact HBsAg particles can be directly analyzed by HPLC gel filtration, which resolves a protein peak of more than one million daltons. Also, the
subunit proteins can be resolved after dissociation in high concentrations of guanidine-hydrochloric acid, containing 417 mM dithiothreitol (DTT)) 4.2% ssodum dodecyl sulfate (SDS), and 16% |3-mercaptoethanol (9). SDS polyacrylamide gel electrophoresis under reducing conditions resolves the 25 and 27 kDa S subunits s10). The N-terminal amino acid sequences of several subtypes were determined by automated amino acid sequencing after isolating the protein from SDS-polyacrylamide gels (11). These sequences agreed with the sequences coded in the gene used to transfect the cells. Hemling et al. corroborated 85% of the HBsAg protein sequence with the DNA
Figure 1 . (a) Infectious hepatitis B particle and (b) S and glycosylated S subunits of the HBsAg particle. Analytical Chemistry News & Features, October 1, 1999 6 6 5 A
Analytical
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Figure 2. Immunoassay for human antibody to HBsAg showing interactions of mieroparticles (coated with recombinant HBsAg), human antibody to HBsAg, biotinylated HBsAg, and anti-biotin HRPO conjugated antibody.
sequence of the S antigen component of the HBsAg by using fast-atom bombardment (FAB) MS (12). In Hemling et al.'s analysis, the recombinant HBsAg (in this case, expressed from yeast cells instead of cultured human cells) was reduced with thiol, alkylated with iodoacetic acid, separated on SDS-polyacrylamide gels, electroeluted from the gels, and digested with chymotrypsin, which makes a large number of nonspecific cuts. The peptide fragments were then identified with FAB MS by aligning the sequences of the fragments with the S antigen amino acid
Analysis of the biotinylated HBsAg
Because of their insolubility in aqueous nondetergent solutions, the S and glycosylated S antigens are difficult to characterize by methods such as peptide mapping. (The mapping technique uses proteases to cleave the reduced and alkylated molecule into fragments, which can be resolved by reversed-phase HPLC.) Therefore, to determine the extent of biotinylation of HBsAg, we turned to MALDI-TOFMS. JV1AUJI-TOF Jvlo has been increasingly useful since it was nrst developed by riillenkamp and Karas (13). In this technique (14), ion mixtures can be analyzed simultaneously in the presence of low levels of salts and detergents, which can be prob666 A
lematic in other mass spectrometric techniques. One limitation of the technique, however, is that molecules larger than -500 kDa are difficult to analyze. These higher-mass macromolecules do not ionize or desorb as readily from the matrix because of their size Also the sensitivity of the instrument de-
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