Characterization by serial deletion competition ELISAs of HIV-1 V3 loop epitopes recognized by monoclonal antibodies.

Characterization of the sites recognized by antibody on the V3 loop of the envelope glycoprotein gp120 of HIV-1 was done by competition ELISAs on a series of four mouse mAbs, a human mAb and a human Fab. The solid-phase antigen consisted of biotin-YNKRK-RIHIGPGRAFYTTKN, a sequence from the center of the V3 loop of gp120MN, applied to streptavidin-coated wells. Competing antigens were two series of peptides with the HIV-1MN sequence each serially deleted at either the N or C terminus but kept constant at the other terminus. For each series, the amino acid at the deleting end needed to give a minimum KD was identified. The epitope was defined as the sequence including both of the identified amino acids as terminal amino acids. For the six antibodies reported, the epitope length ranged from seven to 14 amino acids. Use of a cyclic peptide as competing fluid-phase antigen suggested the influence of conformational constraints on presumed "linear" epitopes. The operationally-defined epitope was longer than the contact residues in one of two instances in which the X-ray crystallographic structure had been determined. The longer estimates of epitope length in the current study based on competition ELISAs with serial deletions suggest that non-contact residues are significant both in epitope definition and in functional applications including immunogen design.

Influence of solid-phase antigen in competition enzyme-linked immunosorbent assays (ELISAs) on calculated antigen-antibody dissociation constants.

The competition ELISA method described in 1985 by Friguet and colleagues has frequently been used to determine dissociation constants (KD) of antigen-antibody reactions. Subsequently Stevens suggested a correction for the bivalency of IgG. In either case, the KD is assumed to vary only with the composition of competing fluid-phase antigen and consequently should not be affected by the solid-phase antigen. However, during the course of experiments defining epitopes to the V3 loop of human immunodeficiency virus type 1, both the composition and density of solid-phase antigen were capable of significantly influencing the calculated values. With one solid-phase antigen, the calculated KD was a function of antigen density. With a second solid-phase antigen, the calculated KD did not vary with the density. With the latter antigen or with low densities of the former, KDs calculated using the Stevens correction for bivalency were close to the estimate obtained by a radiolabeled peptide precipitation assay. Accordingly, since the density and composition of solid-phase antigen may alter the KD calculated from competition ELISAs, such estimates should be confirmed by a more readily interpretable immunological method.

Reduction in antibiotic costs by restricting use of an oral cephalosporin.

Antibiotic cost control programs are important; however, they may be difficult to implement if they include intensive involvement of infectious diseases specialists. In a large municipal hospital, review of antibiotic cost data indicated that 31 percent of the total antibiotic expenditure was for an oral cephalosporin, cephalexin. The requirement that an antibiotic justification form be completed did not decrease use of the drug. However, the requirement that the prescribing physician telephone an infectious diseases specialist resulted in marked restriction of the oral cephalosporin and was accompanied by a 29 percent reduction (adjusted for inflation) in total antibiotic costs. Since comparatively few telephone requests were made and since the decision process to use an oral cephalosporin is comparatively simple, marked reduction in antibiotic costs was achieved with relatively little effort by the infectious disease expert.

Serial deletion mapping by competition ELISA assay: characterization of a linear epitope in the V3 loop of HIV-1.

Precise epitope mapping and characterization is important for development of a subunit vaccine. To identify epitopes in the principal neutralizing determinant (PND) within the V3 loop of human immunodeficiency virus type 1 (HIV-1), sera were screened in a direct ELISA assay with a coating peptide consisting of IHIGPGRAF, a specific sequence commonly found in the loop, linked at the C terminus to GAGAAK, a nonspecific hexapeptide. Epitope mapping experiments revealed that a competition ELISA assay using IGPGRAFGAGAAK as coating peptide was superior to a direct ELISA assay for epitope definition and characterization. The competing peptides contained only specific sequences and were serially deleted of single amino acids first at the N terminus and then at the C terminus. Study of the most highly reactive serum identified in the initial screening identified the epitope (the shortest peptide with the most potent inhibitory activity) as IGPGRAF. Deletion of a single amino acid from the C terminus of the epitope resulted in complete loss of activity as competing peptide. In contrast, single amino acid deletions of three N-terminal amino acids resulted in a stepwise 2700-fold reduction in affinity. RAF was the shortest peptide with inhibitory activity. Additional studies are needed, especially with regard to choice of coating peptide, to establish the general utility of the described epitope mapping procedure. However, the above method, termed serial deletion mapping, may be useful for defining and characterizing linear epitopes and thus may be particularly informative in investigating the multiple overlapping epitopes of the PND.

Skin staples in potentially contaminated wounds.

In potentially contaminated surgical procedures, wound infection is more likely when percutaneous sutures are used rather than skin tapes. Our reluctance to use tapes routinely because of variability in their adhesive properties prompted this evaluation of the ability of skin staples to resist abscess formation after contamination of the subcutaneous space. In each of 180 mice, a predetermined quantity of Staphylococcus aureus was injected into the subcutaneous space of a fresh skin incision. Closure with the skin stapler was most resistant to abscess formation. Presumably, percutaneous sutures provide a nidus for bacterial growth in the relatively avascular subcutaneous space. This problem is avoided by the use of skin tapes or staples. For those who are insecure about the strength of a closure with skin tapes, the skin stapler should provide an alternative in potentially contaminated cases where delayed primary closure is not elected.

Skin wound closure. The effect of various wound closure methods on susceptibility to infection.

A continuous subcuticular polyglycolic acid suture with buried knots protected by a paint-on plastic dressing is shown to be resistant to surface contamination with Staphylococcus aureus in mice and is suggested to be superior to percutaneous skin sutures in infectability and to a tape closure in security.

Cell division in staphylococci: a clue to the three-dimensional structure of peptidoglycan.

The composition of the monomeric unit of staphylococcal peptidoglycan has been known since the late 1960s. However, the three-dimensional structure of this macromolecule has been unclear. Staphylococci divide with expansion of a central cross wall into a peripheral hemisphere under conditions that suggest that additional monomers are not introduced, but rather that the shape of existing monomers is changed. The monomeric unit can be divided into a glycan chain piece, a connecting peptide, and a peptide chain piece, which define a solid parallelogram. In the proposed model, appropriate change in the angle of the glycan chain with respect to the peptide chain doubles the surface, as required to deform cross wall into peripheral wall. Furthermore, cross wall peptidoglycan is synthesized in a spiral, which becomes deformed into a spiral to form peripheral wall, and the glycan chain is twisted with two disaccharide units per turn. Insights from the model can be applied to other peptidoglycans and can help explain the mechanism for start of cell division, the functions of various penicillin-binding proteins, the method of resistance to methicillin, and the occurrence of osmotically growth-dependent bacteria.

Frequency of resistance to kanamycin, tobramycin, netilmicin, and amikacin in gentamicin-resistant gram-negative bacteria.

In vitro evaluation of 66 epidemiologically distinct, gentamicin-resistant, gram-negative isolates from four hospitals revealed that 92% were kanamycin resistant, 44% were netilmicin resistant, 41% were tobramycin resistant, and 6% were amikacin resistant. Combined resistance to gentamicin, tobramycin, and netilmicin occurred in 30% of the strains. Although the resistance percentage to amikacin was the lowest of the three newer agents, two strains were resistant to all of the aminoglycosides tested.

Peptococcus magnus endocarditis.

An 18-year-old man had an eventually fatal case of Peptococcus magnus endocarditis. Multiple emboli and continued valve destruction occurred during appropriate therapy. Penicillin therapy was associated with fever and neutropenia, thought to be due to an immunologic mechanism.

A model for the three-dimensional structure of peptidoglycan in staphylococci.

Although the monomeric units of peptidoglycan in Staphylococcus aureus and other staphylococci are well known, the complete structure of the peptidoglycan has not been elucidated. The peptidoglycan monomeric unit may be divided into three parts: (1) glycan chain piece, consisting of N-acetylglucosaminyl-N-acetylmuramic acid; (2) connecting peptide extending from L-alanine to the alpha-amino group of L-lysine; (3) peptide chain piece, consisting of D-alanine, the remainder of L-lysine not included in the connecting peptide, and pentaglycine (S. aureus) or mixed glycine and serine residues (other staphylococci) attached to the epsilon amino group of lysine. The deformation of cross wall into hemisphere in the course of cell division, the distensibility of peptidoglycan, and the appearance of circular (? spiral) lines in the cross wall and on the surface of the newly-formed hemisphere are clues to the structure of peptidoglycan. In the proposed model, cross wall is formed as a linear spiral with 20 turns extending in a plane from periphery to center of the cell. During cell division, the cross wall is bisected. The cross wall spiral becomes a spiral forming the peripheral wall of a new hemisphere. The width of the spiral on the cell surface is maintained by rigid glycan chains and by covalent bonds linking turns of the spiral. The length of the spiral is about 30 times the diameter of the cell. Flexible polypeptide sheets consisting of parallel polypeptide chains run along the length of the spiral. Individual polypeptides contain an average of ten peptide chain pieces. The glycan chain is a helix with two disaccharide residues per turn; consequently consecutive connecting peptides project in opposite directions and are perpendicular both to the glycan chain and to the peptide chain. In cross wall, hydrogen bonding between polypeptide chains enables the polypeptide sheet to transmit changes in tension. The deformation of cross wall into peripheral wall requires doubling of the external surface area of the peptidoglycan. A change in the angle of the glycan chain with respect to the peptide chain results in an increase of the distance between peptide chains, causing the doubling of surface area. Implications of the model include explanations for the initiation of cell division and for the existence of osmotically growth-dependent staphylococci.

Simplified assay for gentamicin in the presence of other antibiotics.

Gentamicin concentration was estimated by using a clinical isolate of a multiple antibiotic-resistant strain of Staphylococcus epidermidis in an agar well diffusion assay. The presence of clinically attainable concentrations of cephalothin, ampicillin, carbenicillin, clindamycin, or chloramphenicol did not affect the assay.

Lumbar puncture-induced meningitis.

A retrospective study was done to evaluate the risk of lumbar puncture-induced meningitis. Fourteen percent (23/165) of patients with bacteremia caused by Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitis, and groups A and B streptococci had spontaneous meningitis (without a preceding lumbar puncture). In contrast, only 0.8% (7/924) of patients with blood culture containing other organisms had spontaneous meningitis and 2.1% (3/140) of these patients had clinical courses consistent with lumbar puncture-induced meningitis. However, the 2.1% incidence in the latter group is not significantly different from 0.8%, the expected incidence of spontaneous meningitis. It is suggested that if lumbar puncture-induced meningitis does occur, it is rare enough to be clinically insignificant.

Quantitative culture of intravenous catheters and other intravascular inserts.

Quantitative cultures were done on 149 intravenous catheters and 40 additional intravascular inserts. Intradermal and intravascular segments of the insert were cultured separately. The inserts were immersed in broth and flushed. The number of colony-forming units (cfu) per insert was estimated by surface culture of serial dilution of the broth. Nonquantitative culture of undiluted broth was also done. Since all inserts associated with bacteremia had at least 10(3) cfu, inserts greater than 10(3) cfu were considered infected. Staphylococcus epidermidis was more likely than more virulent organisms to colonize an insert without causing bacteremia. The inserts in one bacteremic patient were infected from a distant bloodstream focus; however, in the majority of patients, quantitative intradermal cultures suggested that the insertion site was the portal of entry. In bacteremic patients, either a positive quantitative or a nonquantitative culture identified an infected insert. However, only 33% of positive nonquantitative insert cultures from nonbacteremic patients were confirmed by quantitative insert culture.

Molecular location of a species-specific epitope on the hamster scrapie agent protein.

Scrapie is a transmissible neurodegenerative disease of sheep and goats. An abnormal host protein, Sp33-37, is the major protein component of the scrapie agent and the only known disease- or agent-specific macromolecule. Two monoclonal antibodies (MAbs), 4H8 (immunoglobulin G2b [IgG2b]) and 6B11 (IgG1), produced by immunizing mice with the intact hamster 263K scrapie agent protein, Sp33-37Ha, were found to have species specificity similar to that reported previously for MAb 3F4 (IgG2a), which was produced by using PrP-27-30 as the immunogen (R. J. Kascsak, R. Rubenstein, P. A. Merz, M. Tonna-DeMasi, R. Fersko, R. I. Carp, H. M. Wisniewski, and H. Diringer, J. Virol. 61:3688-3693, 1987). These antibodies all bound to Sp33-37 derived from hamster but not from mouse cells. Competitive binding assays demonstrated that all three MAbs bound to the same or overlapping sites on Sp33-37Ha. The molecular location of the epitope for these antibodies was determined to within 10 residues by using an antigen competition enzyme-linked immunosorbent assay in which synthetic peptides spanning Sp33-37Ha residues 79 to 93 or 84 to 93 specifically inhibited binding of these antibodies to plates coated with purified Sp33-37Ha. A synthetic peptide with the mouse-specific sequence (83 to 92) that differed from the hamster sequence by substitution at two positions (MetHa-87----LeuMo-86 and MetHa-90----ValMo-89) did not inhibit antibody binding to Sp33-37Ha. MAb 3F4 binding to hamster Sp33-37 was eliminated by chemical modification of Sp33-37Ha with diethylpyrocarbonate or succinic anhydride and by cleavage with CNBr or trypsin. The effect of diethylpyrocarbonate on MAb 3F4 binding was not reversed by hydroxylamine treatment. MAb 3F4 binding was not affected by prolonged exposure of Sp33-37Ha to 70% formic acid or by boiling in sodium dodecyl sulfate. We conclude that the epitope for these MAbs is a linear determinant that includes Met-87, Lys-88, and Met-90 and that Met-90 is probably the major species-specific determinant.