Antigenic proteins in latex glove extracts: sources of variability.

Antigenic protein levels in commercially available latex medical gloves were studied. For comparison, the gloves were divided into four main groups; powdered examination, powder-free examination, powdered surgical, and powder-free surgical. Residual protein levels of 91 different glove brands were determined using the LEAP (latex ELISA for antigenic proteins) assay and found to be extremely variable. The possible sources of variability were investigated. Lot-to-lot variability was determined by testing multiple lots of five different brands of surgical gloves. The authors also determined glove-to-glove variability, variability within an individual glove, and test-method variability. It was observed that lot-to-lot variability (25-61% relative standard deviation--RSD) and glove-to-glove variability (51% RSD) were of similar magnitude and accounted for most of the variability found within a given brand of gloves. However, the greatest source of variability identified was brand-to-brand variability (143-283% RSD).

Reduction of antigenic protein levels in latex gloves after gamma irradiation.

Gamma irradiation is currently the method most commonly used to sterilize surgical gloves. In this study, the effect of gamma irradiation on antigenic proteins in latex gloves was examined. Protein extraction and quantitation were carried out using latex gloves before and after sterilization. Antigenic protein levels were determined by an ELISA assay specific for latex proteins (LEAP). LEAP analysis revealed a significant decrease after gamma-irradiation sterilization. This observation may partially explain the lower levels of extractable antigenic proteins found in sterile surgical gloves compared with nonsterile examination gloves. However, gamma irradiation was less effective than autoclave sterilization in reducing protein levels.

In vitro studies on human IgD. I. Sources and characteristics of "externalized" IgD in tonsil lymphocyte cultures.

As part of a broader analysis into the function of IgD, and especially into the role of human IgD-secreting cells, fresh human tonsil lymphocyte cultures were analyzed. The goals were to define the origins of "externalized" supernatant IgD and to examine its relationship to spontaneous Ig secretion of IgG, IgM and IgA. Assayable IgD was found by 24 h in cultures without added mitogens. "Externalized" IgD comprised a small fraction (1.6-1.8%) of supernatant Ig when compared to the fraction of IgD-containing cells (congruent to 10%): IgD appears to disappear rapidly in vitro. Initial experiments employing irradiation and chemical inhibitors of protein synthesis suggested that "externalized" IgD was produced de novo during culture rather than representing preformed or cytophilic Ig. Most significantly, tonsil cultures with higher IgD values (greater than 20 ng IgD/10(6) cells, day 7) showed convincing evidence that secreted IgD was a major source for "externalized" IgD. This evidence included the amount of "externalized" IgD depended on viable culture conditions; radioincorporated 35S appeared as a visible IgD band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels; "higher" IgD cultures showed increased numbers of IgD-Ig-containing plasma cells, and IgD, lambda in excess of IgD, kappa; both events are associated with IgD secretion. In cultures producing high amounts of IgD, IgD secretion appeared to be spontaneous rather than induced and was independent of the polyclonal spontaneous Ig secretion of other Ig isotypes. Low titers of inhibitable IgD isotype anti-phosphorylcholine antibodies but not IgD isotype anti-polyribotol phosphate antibodies were present in tonsil supernatants: both were present in matched sera. Other laboratories have directed attention to the striking and possibly selective participation of human tonsil lymphocytes in IgD synthesis using other approaches. Present results supplement and expand earlier data and support the practical value of analysis of short-term cultured tonsil lymphocytes. Differentiation to IgD-secreting cells is suggested to be an active, underestimated and putatively an important part of the immune response in the human tonsil.

In vitro studies on human IgD. II. IgD-secreting cells preferentially elaborate IgD, lambda molecules.

The "IgD paradox" describes the unexpected finding that, despite a predominance of kappa (kappa) light (L) chains on the surface of IgD+ human B cells, the majority of monoclonal IgD proteins are of lambda (lambda) type. This potentially informative phenomenon appears to be based on a preferential association between delta (delta) heavy (H) and lambda L chains of IgD-secreting cells. The current studies analyze the phenomenon in vitro. Initial assays on tonsil "spent" supernatants showed that the higher IgD-"externalizing" cultures displayed progressively elevated IgD, lambda/kappa ratios due to parallel shifts in total IgD and IgD, lambda while IgD, kappa remained stable. The cells of the same higher-IgD cultures demonstrated that individual IgD-containing cells had a predominance of lambda chains in contrast to IgM-containing cells of the same cultures using fluorochrome-antibody double staining. Certain tonsil culture IgD-containing cells remarkably appeared to have exclusively lambda chain. These same tonsils show evidence of IgD secretion (Litwin, S. D. and Zehr, B. D., Eur. J. Immunol. 1987. 17:483); thus, these data align preferential delta-lambda chain association with IgD-secreting normal tonsil B cells in vitro and emphasize the usefulness of IgD, lambda/kappa ratio in monitoring IgD secretion. In another approach the relationship between the cellular location of IgD and the preferential delta-lambda chain association was studied using Triton X-114-partitioned cell lysates. IgD, lambda/kappa ratios were one or less in the detergent phase (membrane-enriched fraction) consistent with expected IgD, kappa predominance in membrane IgD. In contrast, the aqueous phase (intracellular-enriched fraction) of IgD-secreting cultures had 2-4 times higher supernatant IgD, lambda/kappa ratios. The restriction of high IgD, lambda/kappa ratios to intracellular fractions and supernatants of IgD-secreting cultured cells parallels the predicted distribution of secretory IgD. In sera studies, the correlation between total IgD and IgD, lambda/kappa ratio values was consistent with secreted sera IgD showing preferential lambda chain expression. It was concluded that the phenomenon of delta-lambda chain preferential association is expressed in vitro as well as in vivo; a property of normal, nonmalignant human IgD-secreting B cells; and closely related to the secretory form of IgD. In certain cultures, the delta-lambda chain preference was so striking as to imply limited heterogeneity of the IgD immune response.

Membrane IgD-positive B cells of "low-IgD serum phenotype" individuals fail to secrete IgD and fail to shift to preferential lambda light-chain expression in vitro.

IgD production by short-term human peripheral blood mononuclear cell (PBM) cultures was studied to establish the in vitro correlates of low serum IgD expression. Cells of persons with less than 3 micrograms/ml IgD in the serum, referred to as the low-serum IgD phenotype (LISP), were analyzed. Advantage was taken of recently developed data on spontaneous IgD biosynthesis by human B cells and the observation that lambda light chains are preferentially expressed by IgD-secreting cells in vitro. Initial analysis of an IgD serum distribution showed that all LISP sera contained low but detectable amounts of IgD, with a mean value of 0.85 microgram/ml; this figure was 30- to 35-fold lower than the mean of the majority of the population. LISP PBM contained normal numbers of IgD-positive B cells which displayed a normal intensity of IgD per cell using comparative analysis of mean channel fluorescence by cell flow cytometry. Several lines of evidence suggested that IgD-secreting cells could not be generated from LISP lymphocytes in vitro. Namely, it was found that no IgD immunoglobulin-containing cells were found among PBM of LISP persons; cell lysates enriched for the intracellular fraction by Triton X-114 phase separation showed low IgD in LISP cells despite "normal" amounts of IgD in membrane-enriched fraction preparations; there was no spontaneous IgD secretion by any LISP PBM cultures; and neither LISP sera nor cellular IgD preparations showed IgD lambda/kappa ratios greater than 1.0, indicative of the absence of the preferential lambda light-chain expression associated with secretion of IgD.(ABSTRACT TRUNCATED AT 250 WORDS)

Human IgD and IgA1 compete for D-galactose-related binding sites on the lectin jacalin.

Lectin selectivity for human Ig classes is based on carbohydrate differences. Earlier reports that the lectin jacalin precipitated human IgA were confirmed and supplemented by the current study, which demonstrates that jacalin also binds human IgD as evaluated by micro-ELISA and SDS-PAGE. Experimental findings indicated that: (i) Monoclonal and polyclonal (sera) IgD, IgA1, but not IgA2, IgM, or IgG1-4 reacted with jacalin. (ii) Six tested monoclonal IgD proteins each bound approximately equally to jacalin when antigenicity rather than protein concentration was measured: the results weigh against the presence of jacalin-detectable IgD subclasses or genetic variants. (iii) IgD and IgA1 both associated maximally in 4-8 h at 4 degrees C. There was no dissociation at 4 degrees C but limited dissociation occurred at 37 degrees C after 24 h. (iv) Both IgD and IgA1 were eluted from jacalin by galactose-related sugars. (v) IgD and IgA1 bind competitively to jacalin. The results suggested that jacalin reacts with O-linked oligosaccharide N-acetyl-galactosamine (GalN) residues found on the hinge region of both IgD and IgA1. Jacalin also interacted with one major and several minor unidentified sera proteins. The findings offer an approach to the isolation of serum polyclonal IgD and to the characterization of the unusual carbohydrates of the human delta heavy chain with respect to their function.

In vitro studies on human IgD. III. Immunologic features of individuals with high sera IgD and spontaneous IgD biosynthesis.

The immunologic characteristics of normal persons with high sera IgD values were analyzed. Elevated sera IgD appeared to be the consequence of increased biosynthesis as reflected by an increased number of IgD-Ig-containing cells, an elevated sera IgD, lambda/kappa ratio, and increased spontaneous IgD secretion in vitro. These same findings have been previously linked to increased IgD production (S.D. Litwin and B.D. Zehr, Eur. J. Immunol. 17, 483, 491, 1987). Elevated sera IgD proved relatively stable over 21 months, in five selected individuals, favoring a genetic vs acquired explanation. The failure of sera IgG, IgA, and IgM to positively correlate with IgD weighed against polyclonal Ig synthesis. However, high IgD was inversely correlated to IgM class values and a high sera IgD subset of the population (greater than 31 micrograms/ml) had lower levels of certain IgM antibodies. Analysis of spontaneous IgD secretion, an event frequently encountered in high sera IgD persons, discloses 2/29 cultures with rising supernatant IgD implying in vitro induction. The results emphasize a role for active IgD biosynthesis in the immune responses of certain individuals.

Selective concentration of IgD class-specific antibodies in human milk.

The participation of human IgD class antibody in local immune responses of breast tissue was studied by analysing the sera-to-milk ratios of total IgD, IgM, IgA, IgG isotypes and albumin found in matched samples, and by analysing the sera-to-milk (S/M) ratios of IgD, IgM, IgA, IgG antibodies against Haemophilus influenzae capsular polysaccharide (PRP), phosphorylcholine, tetanus and in some cases diphtheria antigens. The study group consisted of eight women immunized during pregnancy with PRP, and control, unimmunized women. Albumin, and total IgG showed high S/M ratios. IgA had a low S/M ratio as expected, consistent with reports that IgA is locally concentrated. Total IgD and IgM isotype ratio values were intermediate between IgG and IgA suggesting they were selectively concentrated in breast fluids due to local production or transport mechanisms, or both. Ratios for specific antibodies of IgA and IgM isotypes and for total IgA and IgM isotype showed parallel data. Among the IgD antibodies, those specific for PRP and phosphorylcholine suggested a higher degree of selective concentration as compared with tetanus antigen. In the group of unimmunized women, although selective concentration of total IgD was observed, specific antibody studies were inconclusive due to the low milk IgD antibody levels encountered. The results indicate that IgD (and also IgM) may participate in local immune responses of human breast tissues and fluids; possibly influenced by the nature of the antigen, the state of immunization and the hormonal environment (pregnancy).

Measurement of natural rubber proteins in latex glove extracts: comparison of the methods.

BACKGROUND: Health care workers and individuals with frequent contact with latex are at risk for latex protein allergy. OBJECTIVE: The purpose of this study was to compare several established methods for measuring protein in extracts from latex-containing medical devices. METHODS: Extracts from latex gloves were analyzed for natural rubber proteins using a modified Lowry assay and two different immunochemical assays. The immunochemical methods were competitive inhibition assays that employed either immune rabbit serum or human serum with antibodies directed against natural rubber proteins. RESULTS: Seventy extracts representing five different brands of gloves from four manufacturers were analyzed. A good linear correlation (R = 0.88) was found between the immunoassay methods. Correlation to the modified Lowry method was not possible because many of the samples were below the limit of detection for the Lowry assay. Reference extracts and antisera were further characterized by Western blot analysis. The data demonstrate that the proteins recognized by rabbit antisera and the proteins recognized by human IgE are similar. The greatest difference in the immunochemical assays appears to be the relative binding of the antibody sources to high and low molecular weight natural rubber proteins in the reference extracts. CONCLUSIONS: The immunochemical assays are specific for latex proteins and provide a more sensitive and biologically relevant method for determining protein levels in latex medical products.

A one-step, low background coomassie staining procedure for polyacrylamide gels.

Present Coomassie staining procedures require hours of destaining and/or have high backgrounds. This one-step staining procedure is easier, gives lower background with no loss in sensitivity, uses less chemicals, requires less time, and can be followed by silver stain if increased sensitivity is desired after analyzing the results.

Serum lipase and amylase ratio in acute alcoholic and nonalcoholic pancreatitis by using Dupont ACA discrete clinical analyzer.

This work involves a retrospective analysis of serum amylase, lipase, and lipase/amylase ratio in alcoholic and nonalcoholic patients diagnosed with acute pancreatitis. The purpose of this study was to test the reliability of the Dupont ACA method with respect to the lipase/amylase ratio as a discriminator, for the etiology of pancreatitis. Thirty-six consecutive patients with the diagnosis of acute pancreatitis were studied. These patients were divided in two groups. Group I consisted of 11 patients who had presumed acute alcoholic pancreatitis. In group II, 19 patients had acute biliary pancreatitis, including two with necrcotizing pancreatitis and abscess formation secondary to cholilathiasis, five cases were idiopathic in nature, and one was thought to be medication induced (hydrochlorothiazide). In all cases, the Dupont ACA discrete clinical analyzer was used to determine serum levels of amylase and lipase. Concerning the lipase/amylase ratio, the geometric mean ratio for group I was 0.32 (range: 0.11-0.86) and for group II the mean ratio was 0.22 (range: 0.04-0.93). With P > 0.1, the difference between geometric mean ratios was not statistically significant. This study reveals that the lipase/amylase ratio would not have been a good indicator of alcoholic vs nonalcoholic acute pancreatitis. Although there was no significant statistical difference between geometric means, this study does show a significant difference in the number of individuals with serum amylase > 2000 IU/dl in nonalcoholic acute pancreatitis patients (8/25 showed levels above 2000 IU/dl) when compared to alcoholic acute pancreatitis patients (0/11 showed levels above 2000 IU/dl). Chi-square analysis between < 2000 IU/dl and > 2000 IU/dl for the nonalcoholic vs the alcoholic groups yielded a P value of 0.03.