Evaluation of a commercial enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody in diagnosis of human leptospiral infection

The PanBio Leptospira immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) is a commercially available screening test for the diagnosis of acute leptospiral infection. The ability of the test to diagnose early or recent Leptospira interrogans infection was assessed by testing sera with known microagglutination test (MAT) titers to serovars pomona, hardjo, copenhageni, and australis. The IgM ELISA detected all 41 cases of early or recent leptospiral infection (sensitivity, 100%), with a positive ELISA result seen in many cases before MAT antibody titers reached 1:50. Thirty-eight of 41 patients showed seroconversion (fourfold or greater increase in titer by MAT, 2 of 41 patients had a single sample with elevated titer, and 1 patient from whom leptospires were isolated from a blood sample failed to show MAT titers, despite a seroconversion (negative to positive result) in the ELISA. Follow-up sera obtained from 8 of 12 patients (67%) for 3 to 48 months after the acute stage of illness showed persisting IgM antibody. However, the range of levels detected in these samples (maximum ELISA ratio, 2.0) was lower than the range seen when infection was recent. Reactivity in the IgM ELISA was observed for only 1 of 59 serum samples from asymptomatic donors (specificity, 98%) and 16 of 233 serum samples from patients with Ross River virus, brucella, Epstein-Barr virus, cytomegalovirus, mycoplasma, Q-fever, toxoplasma, hepatitis A virus, Treponema pallidum, or Borrelia burgdorferi infection (specificity, 93%), with the majority of these patients showing lower levels of IgM in comparison to those in patients with leptospiral infection. We conclude that this ELISA is sufficiently sensitive for use as an initial screen for leptospiral infections, with subsequent confirmation of positive test results by MAT.     

Neuropeptides are potent modulators of human in vitro immunoglobulin E synthesis

We determined the effect of adrenocorticotropin hormone (ACTH) on the regulation of IgE synthesis. Depending on the concentration, ACTH enhanced or inhibited IgE synthesis in a culture system where IgE synthesis was induced with interleukin-4 (IL-4) and anti-CD40 monoclonal antibody in peripheral blood mononuclear cells. Similar effects on IgE synthesis were observed by adding ACTH-related peptides, e.g. corticotropin-releasing factor (CRF), the inducer of ACTH, or alpha-melanocyte stimulating hormone (alpha-MSH), a cleavage product of ACTH. However, ACTH had no effect on IgG or IgM synthesis in this culture system. ACTH did not act directly on either B or T cells as there was no influence on IgE synthesis in a system using purified B cells alone or co-cultured with T cells. The effect of ACTH on IgE synthesis was mediated by accessory cells. This was shown by priming purified CD14-positive monocytes with ACTH and reconstitution experiments. Therefore, these findings suggest that ACTH and the related peptides CRF and alpha-MSH can influence the microenvironment modulating an IL-4 and anti-CD40 monoclonal antibody driven class switching to IgE via accessory cells.     

Identification of the high affinity receptor binding region in human immunoglobulin E

We have investigated the capacity of N- and C-terminally truncated and chimeric human (h) IgE-derived peptides to inhibit the binding of 125I-labeled hIgE, and to engage cell lines expressing high and low affinity receptors (Fc-epsilon-RI/II). The peptide sequence Pro343-Ser353 of the hC-epsilon-3 domain is common to all h-epsilon-chain peptides that recognize hFc-epsilon-RI. This region in IgE is homologous to the A loop in C-gamma-2 that engages the rat neonatal IgG receptor. Optimum Fc-epsilon-RI occupancy by hIgE occurs at pH 6.4, with a second peak at 7.4. N- or C-terminal truncation has little effect on the association rate of the ligands with this receptor. Dissociation markedly increases following C-terminal deletion, and hFc-epsilon-RI occupancy at pH 6.4 is diminished. His residue(s) in the C-terminal region of the epsilon-chain may thus contribute to the high affinity of interaction. Grafting the homologus rat epsilon-chain sequence into hIgE maintains hFc-epsilon-RI interaction without conferring binding to rat Fc-epsilon-RI. hFc-epsilon-RII interaction is lost, suggesting that these residues also contribute to hFc-epsilon RII binding. h-epsilon-chain peptides comprising only this sequence do not block hIgE/hFc-epsilon-RI interaction or engage the receptor. Therefore, sequences N- or C-terminal to this core peptide provide structures necessary for receptor recognition.     

Enzyme-linked immunosorbent assay for measuring ileal symbiont intracellularis-specific immunoglobulin G response in sera of pigs

Proliferative enteritis (PE) is a common intestinal disease on pig farms. The disease is caused by ileal symbiont (IS) intracellularis (Campylobacter-like organisms) bacteria. An enzyme-linked immunosorbent assay (ELISA) was developed to measure IS intracellularis-specific immunoglobulin G (IgG) response in the sera of pigs. The antigen used in the ELISA was filtered, percoll gradient-purified IS intracellularis extracted from the intestines of pigs affected with proliferative hemorrhagic enteropathy. The antibody responses of pigs challenged with intestinal homogenates from pigs affected with proliferative hemorrhagic enteropathy containing IS intracellularis or percoll-gradient purified IS intracellularis were low and variable. The low IgG titers measured in challenged pigs support previous findings that IgG plays a minor role in the immune response of pigs to IS intracellularis. On a farm in which infection was endemic, pigs seroconverted at between 7 and 24 weeks of age. High IgG titers, indicative of maternally acquired antibody, were present in 3-week-old pigs. The IgG titers in piglets were lowest at 6 weeks of age, which approximates the age of onset of clinical disease. These results suggest that IgG plays a role in determining the susceptibilities of pigs to natural infection. Measurements of seroconversion by the ELISA might aid in epidemiological investigations of PE in naturally infected herds. However, the variable antibody responses in experimentally challenged pigs would seem to limit its usefulness as an antemortem diagnostic test for PE.     

A microagglutination test for human Brucella canis antibodies

A microagglutination test for the detection of Brucella canis antibodies in humans is described. The use of safranin-dyed B. canis organisms as antigen gave easily interpretable settling patterns and reproducible endpoints. The finding of 18 of 1,147 sera with titers greater than or equal to 1:160 resulted in a presumed false-positive rate of 1.6%. Repeat testing of these positive sera in diluent to which 0.6 m NaCl had been added showed a decrease in titer to less than or equal to 1:40 in eight. Conversely, the increased salt concentration permitted detection of blocking antibodies in two sera which were originally thought to be negative. The test provided serologic evidence for B. canis infection in four patients with otherwise undiagnosed febrile illness. The disease is underdiagnosed due to general lack of serologic testing facilities and misconceptions concerning its prevalence.     

A regional programme to collect plasma for preparation of human immunoglobulin anti-rabies

We identified over 100 recipients of rabies vaccine (human diploid cell vaccine) and recruited them as plasma donors. Some were plasmapheresed at static centres, others by mobile teams. The operation of these teams is described in detail. No plasma was sent to the Fractionation Centre until the date of vaccination had been checked. Assay results showed that the majority of plasma collected between four and 20 weeks after the second dose of vaccine contained 6 or more IU/ml.     

Amino acid residues that influence Fc epsilon RI-mediated effector functions of human immunoglobulin E

Immunoglobulin E (IgE) mediates its effector functions via the Fc region of the molecule. IgE binding to and subsequent aggregation of the high-affinity receptor (Fc epsilon RI) by allergen plays a pivotal role in type I hypersensitivity responses. Earlier studies implicated the C epsilon 2 and 3 interface and the A-B loop in C epsilon 3 in the IgE-Fc epsilon RI interaction. These regions and glycosylation sites in C epsilon 3 were now targeted by site-specific mutagenesis. IgE binding to Fc epsilon RI was compared with surface plasmon resonance (SPR) measurements, which assessed the binding of the soluble extracellular domain of Fc epsilon RI to IgE. Kinetic analysis based on a pseudo-first-order model agrees with previous determinations. A more refined SPR-based kinetic analysis suggests a biphasic interaction. A model-free empirical analysis, comparing the binding strength and kinetics of native and mutant forms of IgE, identified changes in the kinetics of IgE-Fc epsilon RI interaction. Conservative substitutions introduced into the A-B loop have a small effect on binding, suggesting that the overall conformation of the loop is important for the complementary interaction, but multiple sites across the C epsilon 3 domain may influence IgE-Fc epsilon RI interactions. Asn394 is essential for the generation of a functional IgE molecule in mammalian cells. A role of Pro333 in the maintenance of a constrained conformation at the interface between C epsilon 2-3 emerged by studying the functional consequences of replacing this residue by Ala and Gly. These substitutions cause a dramatic decrease in the ability of the ligand to mediate stimulus secretion coupling, although only small changes in the association and dissociation rates are observed. Understanding the molecular basis of this phenomenon may provide important information for the design of inhibitors of mast cell degranulation.     

Performance characteristics of an enzyme-linked immunosorbent assay for determining salivary immunoglobulin G response to Helicobacter pylori

We evaluated the salivary immunoglobulin G (IgG) immune response to Helicobacter pylori in 70 subjects by enzyme-linked immunosorbent assay (ELISA). Subjects with a positive H. pylori culture showed significantly higher titers of antibodies than subjects with no detectable H. pylori: the overall sensitivity and specificity of the test were 84 and 90%, respectively. The detection of salivary anti-H. pylori IgG antibodies may be considered as an alternative to serum IgG detection for ease of sample collection or when blood samples are not available in screening of patients with dyspepsia.     

Enzyme-linked immunosorbent assay for TA-2005-glucuronide in human plasma

Although White Carneau (WC) pigeons are known to be more susceptible to atherosclerosis than Show Racer (SR) pigeons, the reasons for this difference are not fully understood. While no major differences are known in the lipoprotein composition, a difference in the cholesteryl ester (CE) composition was reported. However, there is little information on the activity or specificity of lecithin:cholesterol acyltransferase (LCAT), the major source of plasma CE. In order to determine whether the esterification of cholesterol or other functions of LCAT are compromised in WC pigeons, we studied the various reactions catalyzed by LCAT in the two groups. The cholesterol esterification was found to be significantly lower in WC pigeons, whether assayed with exogenous or endogenous substrates. Furthermore, lyso phosphatidylcholine (PC) esterification and oxidized PC hydrolysis, two other reactions carried out by LCAT, were also lower in WC. We found evidence for the presence of an active lysophospholipase in pigeon plasma, and this activity was also lower in WC compared to SR. A significant increase in the FC/PC ratio, another reported atherogenic risk factor, was found in WC. plasma. Because of the absence of other hydrolytic enzymes in pigeon plasma, LCAT may play an important role in the metabolism of oxidized PC generated during lipoprotein oxidation, and therefore a decrease in its activity in White Carneau pigeons may contribute to increased risk of atherosclerosis.     

Raised serum immunoglobulin E in Wegener's granulomatosis

Five patients with Wegner's granulomatosis were found to have significantly raised serum immunoglobulin E (IgE) levels. The rise in IgE was not related to the extent of clinical involvement, was not part of a generalized serum immunoglobulin rise, and was not associated with eosinophilia. Raised serum IgE may be a clue to the pathogenesis of this disease.     

Thermodynamics of the interaction of human immunoglobulin E with its high-affinity receptor Fc epsilon RI

We have employed isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy to characterize the binding of soluble fragments of IgE (IgE-Fc and Fc epsilon 3-4) to a soluble fragment of the high-affinity receptor Fc epsilon RI alpha-chain (sFc epsilon RI alpha). The thermodynamic parameters for the interaction of IgE-Fc and Fc epsilon 3-4 with sFc epsilon RI alpha, determined using ITC, confirm the earlier conclusion that the C epsilon 2 domain is not involved in the interaction and that the stoichiometry of both complexes is 1:1. For both IgE-Fc and Fc epsilon 3-4, the value of Delta H degrees is -36.9 +/- 4.6 kcal mol-1 at 37.3 degreesC and Delta Cp degrees is -820 +/- 120 cal mol-1 K-1. The temperature at which DeltaS degrees is zero is 284 +/- 1 K, indicating that the entropy contribution to the thermodynamics of association is unfavorable at physiological temperature. Of particular interest is the large value of Delta Cp degrees. The large surface area of IgE and Fc epsilon RI alpha that is implicated in complex formation from previous mutagenesis studies on the two proteins may account in part for the magnitude of Delta Cp degrees. Additional contributions may arise from hydration within the binding site and changes in tertiary structure of the individual components of the complex. However, the CD spectra of IgE, IgE-Fc, and Fc epsilon 3-4 complexes with sFc epsilon RI alpha are merely the sum of the spectra of their individual components, indicating that the secondary structure of the immunoglobulin domain folds are preserved on complex formation. Thus, any change in tertiary structure must be limited to the relative disposition of the immunoglobulin domains C epsilon 3 and C epsilon 4 in IgE and the two immunoglobulin-like domains in the alpha-chain of Fc epsilon RI.     

Receptors for human immunoglobulin on acute myeloid leukaemic leucocytes

Immunoglobulin (Fc) receptors were detected on leucocytes from patients with acute myeloid leukaemia (AML) by rosette formation with human cDE/cDE erthyrocytes (HE) sensitized with Rhesus (Rh) antisera (HEA). Of 7 Rh antisera tested, erythrocytes sensitized with anti-d (Gm10) detected the highest numbers of rosette-forming cells (HEA-RFC) in normal and AML leucocyte preparations. Using this assay, HEA-RFC was studied in 22 untreated AML patients and ce assay detected 11-6% lymphocyte and 2-1% granulocyte HEA-RFC in normal peripheral blood. Leucocytes from 16 to 22 AML patients had a similar or lower percentage than normal lymphocyte HEA-RFC, which could be explained by the dilution of peripheral blood leucocytes by poorly or non-rosetting leukaemic blasts. Ten of these 16 patients were diagnosed as having acute myeloblasts leukaemia. Six of the 22 AML patients had high HEA-RFC values of which 5 were diagnosed as having myelomonocytic leukaemia. Cytocentrifuge preparations of HEA-RFC showed that the proportion able to form rosettes was lower in myeloblasts than in monoblasts. Enzyme treatment (pronase), inhibition or simultaneous labelling of surface Ig and Fc receptors showed that the characteristic surface Ig found to AML cells is, at least in part, bound to Fc receptors. The HEA-RFC test described in this paper could be useful in the immuno-diagnosis of myelomonocytic leukaemia.     

Protein tyrosine kinases in activation signal of human basophils through the immunoglobulin E receptor type l

Human basophils activated through high-affinity immunoglobulin E (IgE) receptors (Fc epsilon RI) are involved in the late phase of the allergic reaction. To investigate the possible involvement of protein-tyrosine kinases in this activation we used human acute basophilic leukemia (ABL) cells in culture as well as a pure population of normal basophils in vitro-derived from human bone marrow precursor cells (HBMB). ABL cells were 50-80% basophils at various stages of maturation as assessed by staining, morphology, ultrastructure, and flow cytometry analysis, and only basophils in ABL cells expressed Fc epsilon RI. Aggregation of Fc epsilon RI by IgE and anti-IgE, IgE and antigen, or anti-Fc epsilon RI monoclonal antibodies on ABL cells or on HBMB, led to increased tyrosine phosphorylation of 120-, 100-, 80-, 72-, 50- to 65-, and 38-kDa substrates. Tyrosine phosphorylations in ABL cells were in basophils because 1) they were detected after a 5-s stimulation, 2) they were observed under conditions where mediator release is minimal, i.e., in the absence of extracellular calcium, 3) hapten addition during antigen stimulation resulted in almost total disappearance of tyrosine phosphorylations within 30 s. There was correlation between histamine release and tyrosine phosphorylation in anti-IgE dose-responses and in dose-responses of the tyrosine kinase inhibitor genistein. The tyrosine kinase p72syk was detected in the cells. Stimulation of ABL cells for 1 min resulted in extracellular calcium-independent tyrosine phosphorylation and activation of p72syk. Therefore, tyrosine kinases are involved in the early steps of human Fc epsilon RI signaling in basophils. Tyrosine kinases and their substrates could represent new potential therapeutic targets to prevent the development of the allergic reaction.     

Sensitive enzyme-linked immunosorbent assay for human serum amyloid A (SAA) and application to clearance study

Serum amyloid A (SAA), an apolipoprotein of high density lipoprotein (HDL), is a sensitive acute phase reactant. We established a sandwich type enzyme-linked immunosorbent assay for human SAA utilizing a monoclonal antibody and polyclonal antibodies. This assay was sensitive enough to detect SAA at 40 pg/ml. The use of nonionic detergent, Tween-20, in reaction buffer was essential to enhance specific binding of SAA to antibodies and reduce nonspecific binding to plastic. The values by this assay showed a good agreement with those by previously established latex agglutination immunoassay. Plasma clearance of human SAA was studied in mice by injection with SAA-rich human HDL and serial SAA measurement by the present assay. Half-life of injected SAA was 55 minutes, similar to mean of the reported value of murine SAA isotypes.     

Modified allergen immunotherapy: effect on immunoglobulin E production

Mycobacterium xenopi and Mycobacterium avium complex (MAC) are biochemically similar. To define the laboratory characteristics of M. xenopi that distinguish it from MAC, 53 M. xenopi isolates from different areas in the United States and 47 isolates recovered at one hospital were evaluated by 13 biochemical tests, AccuProbe MAC (Gen-Probe, Inc., San Diego, CA, USA), colony morphology, formation of X-colonies, pigmentation in response to light, growth on MacConkey agar without crystal violet, and relative growth rates at 25 degrees C, 36 degrees C, and 45 degrees C on solid media. Relative growth rates of 10 M. xenopi and 11 MAC isolates were measured at 25 degrees C, 36 degrees C, and 42 degrees C in Middlebrook broth processed using the BACTEC TB System. Ten M. xenopi were tested for p-nitro-alpha-acetylamino-beta-hydroxypropiophenone inhibition at 36 degrees C and 42 degrees C. Reevaluation of 81 isolates previously identified as MAC by biochemical tests alone revealed that two were M. xenopi. The most reliable characteristics distinguishing M. xenopi from MAC were the presence of X-colonies (M. xenopi 97% vs MAC 1%), positive 3-day arylsulfatase (M. xenopi 88% vs MAC 1%), growth at 25 degrees C (M. xenopi 0% vs MAC 100%), and AccuProbe MAC test results (M. xenopi 0% hybridized). Retrospective chart review of 37 patients using American Thoracic Society criteria revealed that six (16%) patients had clinically important isolates. At one of our hospitals M. xenopi was the second most common mycobacterial species isolated for 1990-1992, accounting for 27% of all isolates, whereas at our other hospital it accounted for 1% of isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

PCR enzyme-linked immunosorbent assay for diagnosis of leishmaniasis in human immunodeficiency virus-infected patients

A PCR enzyme-linked immunosorbent assay (ELISA) involving the use of bone marrow aspirates (BMA) and blood samples (BS) for the diagnosis of visceral leishmaniasis (VL) in human immunodeficiency virus-infected patients was developed with primers selected from the sequence of the small-subunit rRNA gene and compared with direct examination and in vitro cultivation. The PCR was optimized for routine diagnosis: processing of samples with lysis of erythrocytes without isolation of leukocytes, enzymatic prevention of contamination, internal control of the reaction, and ELISA testing in a microtitration plate hybridization. Of 79 samples (33 BMA and 46 BS) from 77 patients without VL, all the results were negative. Fifty-three samples (9 BMA and 44 BS) were obtained from 13 patients with VL: 6 samples drawn during anti-Leishmania treatment were negative whatever the technique used, and 47 samples (9 BMA and 38 BS) were positive with at least one technique. The sensitivities were 51% (24 of 47), 81% (38 of 47), and 98% (46 of 47) for direct examination, culture, and PCR, respectively. Thus, PCR ELISA is reliable for diagnosing VL in human immunodeficiency virus-infected patients, and blood sampling should be sufficient for the follow-up.     

A method of estimating the numbers of human and mouse immunoglobulin V-genes

Mutations in immunoglobulin V-genes can be due to gene multiplication, allelic variations, mutations induced by antigens or somatic mutations, etc., and various combinations of these. Since the number of different mouse lambda light V-gene nucleotide sequences is relatively small, a pairwise comparison between these sequences can provide a rough idea as to the contributions of the above mechanisms to the number of nucleotide differences between sequences. A plot of occurrences against the number of differences suggests that differences between one to five can be attributed to somatic mutations. Six to 12 differences can be allelic. Thirteen to 17 may be due to allelic variations together with somatic mutations. Differences > 17 appear to be derived from gene multiplication. Although these numbers are most likely somewhat different in humans, they can nevertheless provide a rough guide to sort out the effect of gene multiplication. Estimations of human heavy, kappa and lambda light chain immunoglobulin V-genes are in reasonably good agreement with recent experimental studies. For mouse kappa light and heavy chains, our estimations can provide some insight to future analyses by direct sequencing of these gene segments.     

Anti-CD4 activity of normal human immunoglobulin G for therapeutic use. (Intravenous immunoglobulin, IVIg)

The effects of intravenously administered normal immunoglobulin G (IVIg) in autoimmune diseases are dependent on the ability of IVIg to interact with surface molecules of lymphocytes. In the present study, we demonstrate the presence of anti-CD4 activity in IVIg by showing the ability of IVIg to bind to CD4 and to inhibit CD4-dependent cellular functions. Binding of IVIg to recombinant soluble human CD4 was assessed by ELISA, immunoblotting and real time analysis of complex formation. Anti-CD4 antibodies isolated from IVIg by affinity-chromatography bound to human CD4+ T cells. These anti-CD4 antibodies inhibited proliferative responses in MLR and infection of CD4+ human T cells with HIV. These results indicate that IVIg contains antibodies reactive with human CD4 and that these anti-CD4 antibodies exhibit biological functions. The presence of anti-CD4 antibodies in IVIg may be relevant to the immunoregulatory effects of normal polyspecific immunoglobulin G.