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PubMed1982

Determination of total IgE by ELISA in tubes and plates compared with PRIST.

IgE was measured by ELISA in tubes and in microtiter plates, the results being compared with PRIST data. The recommended readings of the tube contents in a spectrophotometer (SPM) were compared with results using a multi-channel photometer (MCP). Geometric mean values (International Units) and standard deviations of 44 normal sera examined by the 3 different methods were: PRIST 16.2 +/- 4.0; SPM 15.6 +/- 4.9 and MCP 18.4 +/- 4.4. Correlation coefficients were: PRIST-SPM r = 0.98; PRIST-MCP r = 0.98; and SPM-MCP r = 0.97. Intra- and inter-assay coefficients of variation were smaller for MCP than for SPM. In addition, reading in microtiter plates was much faster, while having little effect on sensitivity.

Adult↗
PubMed1985

Simple and rapid radioimmunoassay for the routine determination of vasopressin in plasma.

A simple, rapid and sensitive radioimmunoassay for plasma arginine-vasopressin (AVP) has been developed for routine use. AVP is first extracted from plasma with use of an octadecasilyl silica cartridge. The mean (+/- SEM) recovery is 73.1 +/- 2.1% (n = 24). The antibody and the 125I-AVP are both obtained from commercial sources. Following a 48 h incubation time, bound and free fractions of AVP are separated by dextran-charcoal. The reproducibility of the method is acceptable (between- and within-assay CV of 9.5 and 7.6%). This technique allows the detection of 0.39 pg/tube of AVP. This assay is applicable to determination of human plasma AVP levels; mean (+/- SEM) plasma AVP levels in normal human subjects in standing or sitting positions, or after an oral water load, were respectively 5.2 +/- 0.7, 3.6 +/- 0.4 and 2.7 +/- 0.4 pg/mL. This method has also been validated by determinations of plasma AVP levels in rabbits and hamsters in various conditions. The commercial availability of the antibody and radioactive AVP, and the simplicity of the method, make this technique suitable for clinical and research purposes.

Animals↗
PubMed1980

Protein A as an immunoadsorbent in radioimmunoassay: usefulness in a rapid assay for urinary luteinizing hormone.

A staphylococcal cell wall protein, designated protein A, is known specifically and rapidly to bind to immunoglobulin G. We have utilized this protein as an immunoadsorbent to separate antigen-antibody complexes from free antigen in a rapid 6-hour radioimmunoassay for urinary human luteinizing hormone (hLH). We have further documented the efficacy of this system and the optimal conditions for this assay. The ability of this assay to provide rapid and reliable measurements of hLH in timed urinary specimens provides a useful tool for determination of the preovulatory LH surge and prediction of the time of ovulation in women.

Antigen-Antibody Complex↗
PubMed1977

Complexed IgE in atopy.

Evidence of circulating high-molecular-weight forms of IgE was found in 16 patients with hayfever and/or atopic eczema. This finding of what appears to be complexed IgE in the serum of atopic patients may help to elucidate the aetiology of atopy.

Antigen-Antibody Complex↗
PubMed1998

Tear IgE concentrations in allergic conjunctivitis.

PURPOSE: Tear IgE has been considered to play an important role in allergic conjunctivitis and the measurement of tear IgE concentrations can help to diagnose this condition. Locally produced IgE levels have been shown to be the largest contributor to the severity of allergic conjunctivitis. METHODS: One hundred and thirteen allergic conjunctivitis patients (70 seasonal allergic conjunctivitis (SAC), 21 perennial allergic conjunctivitis (PAC), 22 vernal keratoconjunctivitis (VKC)), 14 bacterial conjunctivitis (BC) patients, 13 epidemic keratoconjunctivitis (EKC) patients and 18 normal controls were recruited. Tear samples were collected using the microcapillary method and tear IgE levels were measured using an immunoenzyme assay. RESULTS: Tear IgE concentrations showed significant increases in the VKC (322.2 +/- 45.7 ng/ml), SAC (194.7 +/- 21.7 ng/ml) and PAC (134.8 +/- 23.1 ng/ml) groups when compared with controls (52.1 +/- 9.7 ng/ml, p < 0.01). No significant difference was found between EKC (97.2 +/- 11.7 ng/ml) and BC (92.6 +/- 13.8 ng/ml) groups and controls (p = 0.1). CONCLUSIONS: Tear IgE concentrations showed a significant increase in allergic conjunctivitis patients when compared with controls. Our results suggest that measuring tear IgE concentrations can help to diagnose allergic conjunctivitis.

Adult↗
PubMed1984

Development of a non-extracted 'two-site' immunoradiometric assay for corticotropin utilizing extreme amino- and carboxy-terminally directed antibodies.

The development of a 'two-site' immunoradiometric assay (i.r.m.a.) for the direct estimation of human corticotropin-(1-39)-peptide in plasma is described. The assay is based on the simultaneous addition of 125I-labelled sheep anti-(N-terminal corticotropin) IgG (immunoglobulin G) antibodies and rabbit anti-(C-terminal corticotropin) antiserum to standards and unknowns (0.5 ml) followed by 18h incubation. The use of solid-phase reagents was avoided in order to minimize non-specific effects and the time required for reactants to reach equilibrium. Instead, the separation of corticotropin-bound from free labelled antibody is achieved by the addition of sheep anti-(rabbit IgG) antiserum, which precipitates bound labelled antibody by complex-formation with rabbit anti-corticotropin antibodies, which are also hormone-bound. Several 125I-labelled sheep anti-(N-terminal corticotropin) IgG preparations were assessed in the i.r.m.a. Although each was derived from antisera raised to a thyroglobulin conjugate of synthetic corticotropin-(1-24)-peptide (Synacthen), purification of immunoglobulins before iodination by selective immunoadsorption resulted in preparations with distinct specificities which demonstrated marked differences in binding to intact human corticotropin-(1-39)-peptide. These preparations are compared in combination with two rabbit anti-(C-terminal corticotropin) antisera. A 'two-site' assay based on the use of 125I-labelled sheep anti-[ corticotropin-(2-16)-peptide] IgG and rabbit anti-[corticotropin-(34-39)-peptide] antiserum was optimized, since steric inhibition of antibody binding was avoided with this combination and because the measurement of only intact human corticotropin-(1-39)-peptide and not fragments was assured by the use of terminal antibodies. This i.r.m.a. is characterized by rapid equilibration of reactants, a wide 'operating range' (the precision of dose estimates was less than 4% over the range 30-2200 pg/ml) and high sensitivity [8 pg of corticotropin/ml (95% confidence interval 3.7-12.0) (4 pg minimal detectable mass) can be detected directly in plasma].

Adrenocorticotropic Hormone↗
PubMed1985

Synthesis of an antigenic site of native acetylcholine receptor peptide 159-169 of Torpedo acetylcholine receptor alpha-chain.

A region of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of the Torpedo electric organ, containing residues 161-166, has been proposed to be a major antigenic site in the native AChR protein. We report the synthesis of a peptide corresponding to residues 159-169, which contains the proposed antigenic region. In quantitative radiometric titrations, radiolabelled anti-(native AChR) antibodies from three different species, rabbit, rat and dog, exhibited considerable binding (approx. 15% relative to native AChR) to Sepharose-immobilized peptide 159-169, but did not bind significantly to Sepharose-immobilized unrelated proteins or peptides. Specificity was further confirmed by the finding that no rabbit anti-AChR antibodies bound to the peptide after absorption with native AChR. These data indicate that the region 159-169 contains an antigenic site that is readily accessible in solubilized native Torpedo AChR.

Animals↗
PubMed1977

The development of a radioimmunoassay procedure for the estimation of Tamm-Horsfall glycoprotein in human serum.

1. A quantitative radioimmunoassay was developed for the measurement of nanogram amounts of Tamm-Horsfall (TH) glycoprotein in the presence of serum proteins at concentrations above 30 mg/ml. 2. Specific anti-(TH-glycoprotein) antibodies were labelled with 125I and these were usable for a period of 8 weeks. 3. Agarose beads (Sepharose 4B), to which TH-glycoprotein had been coupled via cyanogen bromide activation of the Sepharose, were used as the solid phase in the assay. This proved stable upon storage at 4 degrees C for periods in excess of 4 months. 4. The dissociation of the glycoprotein in the presence of serum proteins that was necessary for quantification was achieved by subjecting the sample to ultrasonication. 5. Assays conducted on a small sample of normal serum produced evidence that normal serum contained amounts (50-180 ng/ml) of a substance that reacted similarly to TH-glycoprotein in the assay procedure and in a series of experiments conducted to confirm the presence of this substance in human serum.

Antibodies↗
PubMed1979

A radioimmunosorbent technique for the assay of B- and C-types of carbonic anhydrase in human tissues.

A radioimmunosorbent technique for the assay of the human carbonic anhydrase isoenzymes HCA B and HCA C in tissue fluids was developed. The sensitivity of the method was 0.2 ng/ml and the precision was 5% in duplicate determinations for both enzymes. The presence in a tissue of up to 20 times higher concentrations of one isoenzyme will not interfere with the assay of the other. Haemolysates contained (mean +/- SE, n = 11) 12.1 +/- 0.52 and 1.5 +/- 0.06 mg enzyme/g Hb, and serum 0.63 +/- 0.12 and 0.2 +/- 0.02 microgram/ml of HCA B and HCA C, respectively. Pilot experiments indicated that the isoenzymes can be determined also in tissues, i.e. urine, saliva and cerebrospinal fluid, where catalytic methods previously have indicated absence of or only weak carbonic anhydrase activity. N-terminals of both enzymes were not antigenic.

Antibodies↗
PubMed1976

Sequential adsorption analysis of antibodies to neuroectodermal cells by an indirect quantitative method.

An indirect radioimmunoassay was developed for the sequential adsorption analysis of rabbit antibodies raised against rat neuroectodermal cells. The quantitativee relationship between primary adsorbed rabbit antitumor antibodies and secondary adsorbed goat antibodies to rabbit IgG was explored by paired radioiodine analysis. In the final indirect method, the amount of unlabeled rabbit antibody removed by cultured monolayers of cells at each step in a sequence of cells could be determined from an equation relating the unlabeled amount to values of bound 125I-labeled goat antirabbit IgG. To obtain the total quantity of rabbit antibody in a particular antiserum reagent, a sequential adsorption analysis was done with successive steps of homologous cells. To obtain the amount of antibody that was cross-reactive for other cell lines, we included those lines in the first several steps of the sequence. The sequential adsorption profile was considered as a more important indicator of the quality of a particular antiserum reagent than was the total amount of antibody contained in it. Neuroectodermal cell lines used as illustrative examples included subclones of the C6 astrocytoma and of the RN-2 schwannoma.

Animals↗
PubMed2003

Defective regulation of the protective IgE response against intestinal helminth Ascaris lumbricoides in malnourished children.

It is well established that malnutrition affects the immune response and increases the susceptibility to parasitic infection. In the present study we evaluated some aspects of the cellular and cytokine network that regulate the IgE responses, which are important components of host defence mechanisms against helminthic parasites in children infected with the intestinal helminth Ascaris lumbricoides, and with differing degrees of malnutrition. We found a defective T cell response in malnourished children, as indicated by diminished levels of circulating total (CD3+), helper (CD4+), IL-2-receptor-bearing (CD4+CD25+) and memory helper T cell responses (CD4+CD45RO+) in keeping with the decreased specific IgE levels against Ascaris lumbricoides. In contrast, the proportions of total B cells (CD20+), and those bearing the low-affinity IgE receptor (CD23+) were increased in the moderated malnourished children. Moreover, serum IL-4 levels and total IgE were also increased in these children. We suggest that malnutrition can cause an imbalance in T cell subpopulations that may lead to a defective T cell maturation and a decreased specific anti-Ascaris IgE response thus increasing the susceptibility to such infections. The high levels of total IgE observed may be related to a non-specific stimulation of the proliferation of activated B cells, probably caused by helminthic parasites and other infectious agents that are frequent in malnourished children.

Analysis of Variance↗
PubMed1989

A new method for IgE detection in nasal mucosa.

A new method for determining IgE antibodies on mucosal surfaces has been developed with the purpose of overcoming the main problems of nasal secretion sampling and standardization. The method is based on the principle of performing the incubation of the solid-phase coupled allergen with IgE antibody directly on the mucosal surface by means of a proper applicator. About two times higher values of specific IgE have been obtained with 5 min incubation on septal mucosa, behind the internal ostium, than with 3 hr in-vitro incubation with native secretion. In a study of 53 children with allergic rhinitis and asthma and 10 healthy non-atopic controls the sensitivity and specificity of this new method were evident. Because of its reliability and easy execution the new method could be widely used in diagnosis of allergic disease. Further, it seems to offer a good opportunity to study the IgE-mediated reaction in the target organ more extensively.

Child↗
PubMed1982

Total serum IgE concentrations in adolescents and adults using the phadebas IgE PRIST technique.

Total serum IgE measured with the Phadebas PRIST technique was titrated in 117 normal non-allergic subjects, 237 allergic adolescents or adults and 89 non-allergic patients who suffered from asthma, rhinitis, or conjunctivitis. All subjects were of Caucasian origin. In normal subjects, mean total serum IgE was 38 +/- 43 kU/l. This value is exactly the same as that found in a study of Caucasian New Zealanders and very similar to the values found in most U.S. studies. This suggests that the mean total serum IgE concentration is remarkably constant in normal non-allergic Caucasians. The upper limit of the normal range is considered to be 150 kU/l. 38% of allergic patients have total IgE concentrations within the normal range. Some pollen or hymenoptera venom-sensitive patients have a total serum IgE concentration below 20 kU/l. The non-allergic patients had a mean IgE concentration of 94 +/- 93 kU/l, and 25% of them had a total serum IgE above the normal range. Asthmatic patients had higher mean IgE levels than those who were suffering from either rhinitis or conjunctivitis.

Adolescent↗
PubMed1984

Purification of patient-related allergens by means of bioaffinity chromatography on a sepharose anti-IgE patient IgE immunosorbent.

A method is described for bioaffinity chromatographic purification of patient-specific IgE reactive allergens by using sepharose coupled rabbit anti-human IgE antibodies, patient serum and an allergen extract. The conditions for this bioaffinity chromatographic procedure have been optimised, and it was shown that the nonspecific binding of serum proteins and antigens can be kept at very low levels by a high ionic strength. The specificity of the method has been determined by showing that allergens to which the patient did not react were not bound to the affinity matrix. We suggest that the isolated allergens could be used for more specific immunotherapy in the future.

Allergens↗
PubMed1990

Stinging insect allergy: sensitization to vespids in Madrid and surroundings. Cross-reactivity study.

The study comprised 28 patients from the centre of Spain (Madrid and surroundings) who had suffered systemic reactions when stung by vespids. Specific IgE antibodies to Vespula spp. and Polistes spp. venoms were measured by RAST. All patients had positive RAST to Vespula venom and half of them also had positive RAST to Polistes venom. A patient can be sensitive to both venoms due to either a clinical sensitization to both venoms, or common antigenic determinants. To differentiate these states we used a RAST inhibition assay. We could inhibit Polistes RAST with either Polistes or Vespula venom to a similar degree. Inhibition of Vespula RAST was possible with Vespula venom, but only to a limited degree with Polistes venom. Direct RAST and RAST inhibition studies indicate that in our geographic region sensitization to Vespula venom is more common than to Polistes venom and Polistes might have cross-reactivity in our patients.

Adolescent↗
PubMed1990

Hyposensitization in asthmatics with mPEG-modified and unmodified house dust mite extract. III. Effect on mite-specific immunological parameters and correlation to changes in mite-sensitivity and symptoms.

Forty-six adult asthmatics allergic to D. pteronyssinus (Dp) participated in a 2-year study. Thirty-one underwent hyposensitization (HS-group). Fifteen were treated with Dp-extract (Dp-group), and 16 with a similar extract modified by monomethoxypolyethylene glycol with reduced allergenicity (mPEG-Dp-group). Fifteen patients served as controls. Dp-specific antibodies and histamine release from blood basophils were determined and compared with Dp-sensitivity in lungs and skin. In addition, IgG and IgE against the major allergen Der p I were followed in a subgroup. Dp-specific IgG, IgG1, and IgG4 increased significantly in both HS-treated groups after 1 and 2 years (median: 2.5- to 11.6-fold). IgG4 was not induced if maintenance dose during the first year was less than 20,000 BU. Median skin sensitivity decreased 4.4- to 8.2-fold after 1 year and 7.4- to 21.4-fold after 2 years. Der p I specific IgG response was unrelated to the occurrence or change in IgE with the same specificity. The mPEG-Dp-extract tended to have less effect on skin sensitivity and immunological parameters, differences reaching statistical significance for skin sensitivity only. In the HS-group, the decrease in bronchial sensitivity was significantly correlated to a decrease in IgE (r = 0.36), IgG1/IgG4 (r = 0.49), Dp-specific histamine release (r = 0.58), and to an increase in Dp-specific IgG4 (r = -0.36) and IgG4/IgE (r = -0.48). In patients improving clinically, Dp-specific IgG4/IgE increased, and median Dp-specific IgE was reduced to 80% compared with an increase to 150-160% seen in the unchanged or deteriorated group (P less than 0.05). Findings indicate an improvement of effect, if the allergen dose is sufficient to reduce specific IgE and/or induce an IgG and especially IgG4 response.

Adult↗
PubMed1997

Inflammation markers and symptom activity in children with bronchial asthma. Influence of atopy and eczema.

BACKGROUND: Eosinophil cationic protein (ECP) has been reported to reflect the eosinophil inflammatory activity in asthma. However, the relative impact of asthma symptoms and atopic eczema upon serum (s)-ECP in asthmatic children has not been established. OBJECTIVES: To examine s-ECP levels and s-myeloperoxidase (MPO) in relation to asthma symptoms and atopic eczema in asthmatic children. METHODS: S-ECP and s-MPO were assessed in relation to symptom activity, lung function, exercise-induced bronchoconstriction and bronchial responsiveness in 101 children; median age 9 years, range 1-16 years; with moderate to severe asthma, admitted to Voksentoppen Center. RESULTS: S-ECP was significantly higher in children with persistent compared to episodic or no asthma symptoms in the past four weeks. S-ECP was also higher in children with atopic compared to non-atopic asthma, as well as in those with active compared to past history or no history of atopic eczema. S-MPO was higher in children with persistent asthma symptoms, but did not differ in relation to atopy or eczema state. Persistent asthma symptoms had the greatest impact upon s-ECP levels, followed by atopy and active eczema. CONCLUSION: S-ECP may be used in assessing symptom activity in asthmatic children, but with the realisation that active eczema and the presence of atopy may also influence levels.

Adolescent↗
PubMed1978

Effects of tricyclic antidepressants on human plasma levels of TSH, GH and prolactin.

Six healthy male volunteers were given chlorimipramine 25 mg t.i.d. or nortriptyline 25 mg t.i.d. in a randomized order of 7 days. Plasma samples were assayed for TSH, GH and prolactin before and after stimulation with TRH 200 microgram i.v. It was found that the tricyclic antidepressants did not exert any influence on plasma hormonal levels compared with no treatment conditions. Diminished TSH-responses following daily TRH injections were demonstrated in endogenously depressed and chronic schizophrenic patients. A decreased TSH-response was observed in healthy volunteers after a second TRH injection with an interval of 2 days between the TRH injections. A complete restoration of the TSH-response was obtained after an interval of 4 days.

Adult↗