Candidate epitopes for measurement of hCG and related molecules: the second ISOBM TD-7 workshop.

Participants of the Second International Workshop (WS) on human chorionic gonadotropin (hCG) of the International Society of Oncology and Biomarkers Tissue Differentiation 7 (ISOBM TD-7) have characterized in detail a panel of 69 antibodies (Abs) directed against hCG and hCG-related variants that were submitted by eight companies and research groups. Specificities of the Abs were determined using the First WHO International Reference Reagents for six hCG variants, i.e., hCG, hCGn, hCGß, hCGßn, hCGßcf, and hCGα, which are calibrated in SI units, and hLH. Molecular epitope localizations were assigned to the ISOBM-mAbs by comparing ISOBM-Ab specificity, sandwich compatibility, and mutual inhibition profiles, to those of 17 reference monoclonal (m)Abs of known molecular epitope specificities. It appeared that 48 Abs recognized hCGß-, 8 hCGα-, and 13 αß-heterodimer-specific epitopes. Twenty-seven mAbs were of pan hCG specificity, two thereof with no (<0.1%; epitope ß1), 12 with low (<1.0%; epitopes ß2/4), and 13 with high (>>1%; epitopes ß3/5) hLH cross-reactivity. The majority of hCGß epitopes recognized were located in two major antigenic domains, one on the peptide chain of the tips of ß-sheet loops 1 and 3 (epitopes ß2-6; 27 mAbs) and the second around the cystine knot (e.g., epitopes ß1, ß7, and ß10; 9 mAbs). Four mAbs recognized epitopes on hCGßcf-only (e.g., epitopes ß11 and ß13) and six mAbs epitopes on the remote hCGß-carboxyl-terminal peptide (epitopes ß8 and ß9 corresponding to amino acids 135-144 and 111-116, respectively). For routine diagnostic measurements, methods are used that either detect hCG-only, hCGß-only, or hCG together with hCGß or hCG together with hCGß and hCGßcf. Sandwich assays that measure hCG plus hCGß and eventually hCGßcf should recognize the protein backbone of the analytes preferably on an equimolar basis, should not cross-react with hLH and not be susceptible to blunting of signal by nonmeasured variants like hCGßcf. Such assays can be constructed using pairs of mAbs directed against the cystine knot-associated epitope ß1 (Asp10, Asp60, and Gln89) in combination with epitopes ß2 or ß4 located at the top of ß-sheet loops 1 + 3 of hCGß involving aa hCGß20-25 + 68-77. In summary, the results of the First and Second ISOBM TD-7 WSs on hCG provide the basis for harmonization of specificities and epitopes of mAbs to be used in multifunctional and selective diagnostic hCG methods for different clinical purposes.

Variations in levels of care within a hospital provided to acute trauma patients.

INTRODUCTION: Caring for trauma patients is a dynamic process, and it is often necessary to move the trauma patient around the hospital to different locations. This study attempted to document the quality of observations performed on acute trauma patients as they moved through the hospital during the first 24 hours of care. METHODOLOGY: This study was a student elective and was undertaken at Grey's Hospital, Pietermaritzburg. A third-year medical student was assigned to follow acute trauma patients throughout the hospital during the first 24 hours after admission. This single independent observer recorded the frequency with which vital signs were recorded at each geographical location in the hospital for each patient. A scoring system was devised to classify the quality of the observations that each patient received in the different departments. The observer recorded all the geographical movements each patient made during the first 24 hours after admission. RESULTS: Fifteen patients were recruited into this study over a 4-week period. There were 14 adult males (average age 28 years, range 18 - 56 years) and a 7-year-old girl in the cohort. There were significant differences in the quality of the observations, depending on the geographical location in the hospital. These variations and differences were consistent in certain locations and highly variable in others. Observations in the intensive care unit (ICU) and operating theatre were uniformly excellent. In the radiology suite the level of observations was universally poor. In casualty and the wards there was great variability in the level of observation. A total of 45 distinct geographical visits were made by the study cohort. Each patient made an average of 3 (range 2 - 5) visits during their first 24 hours after admission. All patients attended casualty, and there were 11 patient visits to the ward, 10 to radiology, 4 to ICU and 5 to theatre. CONCLUSION: Significant variations exist in the level of observations of vital signs between different geographical locations within the hospital. This is problematic, as acute trauma patients need to be moved around the hospital as part of their routine care. If observations are not done and acted upon, subtle clinical deterioration may be overlooked and overt deterioration may be heralded by a catastrophic event.

Cleavage of the hormone human chorionic gonadotropin, by the Type 1 IgA1 protease of Neisseria gonorrhoeae, and its implications.

The hormone human chorionic gonadotropin (hCG) serves to maintain the fetus during early pregnancy and regulate the onset of labor in late pregnancy. hCG also prevents Neisseria gonorrhoeae from developing invasive characteristics. Part of the beta subunit of hCG has an amino acid sequence similar to that of the hinge of human IgA1, which is the site of action of IgA1 proteases. This study examined the sensitivity of hCG to gonococcal IgA1 proteases, by means of autoradiography, immunoblotting, and RIA. hCG was cleaved in the beta subunit by the type 1 but not the type 2 IgA1 proteases of N. gonorrhoeae. hCG cleavage by gonococcal IgA1 proteases in vivo may increase the invasiveness of the pathogen and destroy its natural biologic activity, with major consequences for the fetus and the pregnancy.

Unaggregated serum IgA binds to neutrophil Fc alpha R at physiological concentrations and is endocytosed but cross-linking is necessary to elicit a respiratory burst.

My43 is a monoclonal antibody directed against Fc alpha Rs on monocytes that also recognizes neutrophil (PMN) Fc alpha Rs and is able to elicit a respiratory burst in purified cells. It appears to be directed against the immunoglobulin A (IgA) binding site of Fc alpha Rs or an epitope in close proximity, since IgA and My43 compete for binding to PMNs. My43 immunoblotted Fc alpha R that had been affinity purified from PMN membranes and immunoprecipitated a 50-70 kDa protein from radiolabeled PMN membranes, apparently identical to that purified on IgA-Sepharose. These results suggest the presence of a single class of Fc alpha R on PMNs. Binding of unaggregated monomeric or dimeric serum IgA to Fc alpha Rs on PMNs occurs at physiological concentrations, suggesting that in vivo Fc alpha Rs are saturated with ligand. This binding does not elicit a respiratory burst. Purified PMNs do not express surface IgA, since receptor-bound IgA is lost from the cell surface by endocytosis at room temperature although not at 4 degree C. Rapid endocytosis of receptor-bound IgA has been demonstrated by flow cytometry and confocal microscopy. Unoccupied receptor that is able to bind IgA or My43 is subsequently reexpressed. Cross-linking of surface-bound IgA using F(ab')2 anti-alpha chain antibodies triggers an oxidative burst. After internalization of the surface IgA the same F(ab')2 anti-alpha chain antibodies do not trigger the burst.

The measurement of respiratory burst induced in polymorphonuclear neutrophils by IgA and IgG anti-gliadin antibodies isolated from coeliac serum.

The properties of IgA and IgG anti-gliadin antibodies from the serum of patients with coeliac disease have been compared. The antibodies were quantified by ELISA using microtitre plates coated with crude gliadin fractions. Their specificity was confirmed by immunoblotting. Heat-treated sera containing IgA antibodies stimulated chemiluminescence when added together with neutrophils to microtitre plates coated with crude gliadin. Sera containing only IgG antibodies were less efficient. When IgA and IgG were purified from a serum containing both classes of anti-gliadin antibodies, each of the preparations was able to stimulate neutrophil chemiluminescence in plates coated with gliadin. Although the yield of anti-gliadin antibody determined by ELISA was high, the ability of the purified immunoglobulins to stimulate neutrophil chemiluminescence was much less than that of the unfractionated serum. This loss of activity was shown to be due to the ability of each class of antibody to potentiate the activity of the other in the whole serum.

Purification and characterization of human immunoglobulin IgA1 and IgA2 isotypes from serum.

A method is described for the simultaneous purification of IgA1 and IgA2 from human serum. Ammonium sulphate precipitation, gel filtration and ion-exchange chromatography on DEAE-Sephacel yielded a partially purified IgA preparation which was separated quantitatively into IgA1 and IgA2 by affinity chromatography on jacalin-Sepharose. The IgA1 which bound to the jacalin was eluted with 0.8 M D-galactose. The IgA1 preparation was apparently homogeneous by SDS-PAGE but contained a trace of C1-inhibitor and a second protein detected by immunoelectrophoresis. The IgA2 which did not bind to the jacalin was purified to apparent homogeneity by chromatography on columns of Protein G-Sepharose, Fastflow-S Sepharose and Superose 6. Typical yields were 95% and 58% for IgA1 and IgA2 respectively or 253 mg and 24 mg per 100 ml serum. The IgA1 and IgA2 were characterised by their reactivity with isotype specific monoclonal antibodies and sensitivity to bacterial proteinases. The IgA2 preparation apparently contained both allotypes, IgA2m(1) and IgA2m(2).

The specificity of the human neutrophil IgA receptor (Fc alpha R) determined by measurement of chemiluminescence induced by serum or secretory IgA1 or IgA2.

Heat or chemically aggregated IgA or IgG stimulated degranulation of neutrophils with comparable efficiency. The same aggregates induced a neutrophil respiratory burst which could be measured by lucigenin-enhanced chemiluminescence. Serum IgA1 or IgA2 coated onto microtitre plates were both capable of inducing a respiratory burst in neutrophils, as was secretory IgA1 or secretory IgA2. All bursts were of similar size for a given concentration of IgA and were greater than the burst elicited by an equivalent concentration of IgG. For each subclass of IgA the respiratory bursts were dependent on their density on the opsonized surface. Since monomeric and dimeric forms present in secretory IgA preparations both elicit a respiratory burst in neutrophils, secretory component and J chain cannot block the receptor binding site on the Fc region. The potential of secretory IgA to act as an opsonin might have important consequences on mucosal surfaces where the availability of complement components is limited.

The effect of antibody isotype on the activation of C3 and C4 by immune complexes formed in the presence of serum: correlation with the prevention of immune precipitation.

Radioimmunoassays for C3a and C4a have been used to measure the activation of complement during the formation of immune complexes in human serum by the interaction of DNP-BSA and each of 11 mouse anti-DNP monoclonal antibodies of varied isotype and affinity. Those containing IgG2 or IgM were potent activators of C4, whilst IgG1 containing complexes were less efficient. C3 activation in normal serum was similar for complexes containing IgG1, IgG2a, IgG2b or IgM. IgA complexes did not activate C3 or C4. All complexes except those containing IgA precipitated more slowly in serum than in buffer. IgG2 antibodies were potent activators despite being very slow to precipitate in buffer. In serum containing EGTA activation of C4 was abolished and precipitation of complexes occurred at the same rate as in buffer. Nevertheless, C3 activation still occurred by the alternative pathway for all IgG and IgM complexes. Antibodies of the same isotype did not necessarily activate complement to the same extent. The ranking of the ability to activate complement was the same as that observed when performed complexes containing the same antibodies were added to serum. The levels of C4a generated were similar under both conditions but for most antibodies more C3a was generated by preformed complexes.

Colloquium on scientific authorship: rights and responsibilities.

The Colloquium on Scientific Authorship was held at the National Institutes of Health (NIH) at a time of extraordinary scrutiny by the public of the ethics of scientists, as represented by intense interest of the press and the Congress of the United States. Indeed, several regulations dealing with scientific misconduct have been proposed during the last year in the Federal Register, and new legislation has been proposed in the Congress. As a result of these concerns, conferences have been organized by the Institute of Medicine, the American Association for the Advancement of Science/American Bar Association, the Council of Biology Editors, and other groups. The colloquium at NIH, which was held May 31, 1988, and sponsored by the Intramural Scientists, focused on publication practices, especially multiple authorship, as contributing to perceived difficulties. The participants suggested various changes in conventions related to authorship that might help prevent future problems.

The activation of C3 and C4 in human serum by immune complexes containing mouse monoclonal antibodies of different isotype and affinity: effects on solubilisation.

Radioimmunoassays for C3a and C4a have been used to measure the activation of complement in human serum by immune complexes containing DNP-BSA and each of 11 mouse anti-DNP monoclonal antibodies of varied isotype and affinity. When preformed complexes were added to serum, those containing IgG2 or IgM were potent activators of C4, whilst IgG1 complexes were less efficient. C3 activation in normal serum was similar for complexes containing IgG1, IgG2a, IgG2b or IgM. IgA complexes did not activate C3 or C4. Solubilisation of complexes was greatest for IgM and IgG2b and least for IgG2a and IgA. In serum containing Mg2+ EGTA C4 activation was abolished and the amount of C3 activation was lower for all IgG and IgM complexes. Antibodies of the same isotype did not necessarily activate complement to the same extent. Unexpectedly, three of the four IgMs activated C3 in EGTA. For IgMs, neither complement activation nor solubilisation correlated with affinity. For IgG1 antibodies, solubilisation was inversely proportional to affinity. C3 or C4 activation did not correlate with affinity.

The integrity of the scientific literature.

A case of admitted scientific fraud has shed new light on the system that ensures the integrity of the scientific literature. Certain lapses from generally accepted standards of research may be more frequent than is commonly believed.

KIE: John Darsee was found to have fabricated much of the data that formed the basis of over 100 articles that he coauthored with 47 researchers at the medical schools of Harvard and Emory universities. Stewart and Feder analyzed these publications to investigate the vigilance of scientific referees, journal editors, and Darsee's coauthors in meeting accepted publication standards. They characterize their findings as revealing two types of frequently occurring lapses. Type A lapses--such as the presence of errors or inconsistencies, failure to obtain relevant data, and honorary authorship--may simply reflect carelessness. More serious Type B lapses include misleading statements or citations and failure to acknowledge sources of data or to respond appropriately to charges of fraud. The authors discuss reasons for these lapses and recommend a random study of published papers to ascertain the extent of such poor publishing practices.