A peroxidase-linked enzyme immunoassay for tumour necrosis factor alpha utilising alternative colorimetric or chemilumimetric substrates.

We present a double antibody immunoassay for tumour necrosis factor alpha (TNF alpha) with a peroxidase dependent endpoint which can be detected by absorbance or chemiluminescence depending on the choice of substrate. The chemilumimetric and colorimetric assays have a detection threshold in human serum of 3.9 pg/ml and 7.8 pg/ml respectively and are able to recognise both rTNF alpha and natural TNF alpha. Concentrations of TNF beta, interleukin-1 alpha (IL-1 alpha), IL-beta, IL-2, IL-3, IL-6 or interferon-gamma (IFN-gamma) up to 5 ng/ml failed to show any cross-reactivity. The monoclonal antibody clone 5-2, used in the assays, did not neutralise rTNF alpha in the L929 bioassay. The assay was able to detect rTNF alpha in the presence of excess concentrations of both TNF alpha receptors (p55 and p75). Removal of interference by rheumatoid factor was achieved by the absorbance of the polyclonal antiserum with mouse serum and the inclusion of 10(-2) M dithiothreitol in the buffer containing the TNF alpha polyclonal antiserum. The assay will be useful for the quantitation of endogenous human TNF alpha in serum, other body fluids and culture supernatants, and can also be used to monitor levels of rTNF alpha in clinical trials.

Investigation of IgG4 levels in atopic patients using a competitive inhibition assay employing biotinylated IgG4 myeloma and avidin peroxidase.

Modification of a 'sandwich' ELISA assay developed for the determination of serum IgE levels proved to be unsatisfactory for the measurement of IgG4. This was attributed to the limited capacity of the microtitre plate solid phase which required high serum dilutions in order to measure IgG4 levels. To overcome this problem a competitive inhibition assay was developed with monoclonal anti-IgG4 attached to the plate. In this system biotinylated IgG4 myeloma and sample IgG4 compete for the limited antibody binding sites present on the solid phase. The attached biotinylated myeloma is detected by addition of avidin conjugated with peroxidase and following development with substrate, IgG4 levels are calculated by reference to a calibrated inhibition curve. The inhibition ELISA assay has been used clinically to measure IgG4 levels in atopic and normal individuals and the values obtained correlated closely (r = 0.99) with the IgG4 levels determined by radial immunodiffusion. For 43 atopic dermatitis patients investigated the median IgG4 level was 1.1 g/l which was significantly elevated when compared to a median of 0.385 g/l for 60 blood donors (P less than 0.0001, Mann-Whitney U). Among the 47 hay fever patients investigated the median was 0.6 g/l which, although lower than in atopic dermatitis, was again significantly increased (P less than 0.025). Within this latter group, 25 patients were investigated for the effects of desensitization with commercial grass pollen injections. The total IgG4 showed a variable but significant rise between the start and finish of treatment (P less than 0.01 Wilcoxon signed ranks test).

Postmortem findings after fatal anaphylactic reactions.

AIMS: To determine the frequency at which classic manifestations of anaphylaxis are present at necropsy after fatal anaphylactic reactions. METHODS: A register has been established of fatal anaphylactic reactions in the UK since 1992, traced from the certified cause of death and other sources. Details of the previous medical history and the reaction suggest anaphylaxis as the cause of death for 130 cases; a postmortem report was available for 56. RESULTS: The 56 deaths studied included 19 reactions to bee or wasp venom, 16 to foods, and 21 to drugs or contrast media. Death occurred within one hour of anaphylaxis in 39 cases. Macroscopic findings included signs of asthma (mucous plugging and/or hyper-inflated lungs) (15 of 56), petechial haemorrhages (10 of 56), pharyngeal/laryngeal oedema (23 of 56), but for 23 of 56 there was nothing indicative of an allergic death. Mast cell tryptase was raised in 14 of 16 cases tested; three of three tested had detectable IgE specific for the suspected allergen. CONCLUSIONS: In many cases of fatal anaphylaxis no specific macroscopic findings are present at postmortem examination. This reflects the rapidity and mode of death, which is often the result of shock rather than asphyxia. Investigations that might help determine whether anaphylaxis was the cause of death had rarely been performed. In the presence of a typical clinical history, absence of postmortem findings does not exclude the diagnosis of anaphylaxis.

An unusual association of Felty syndrome and TCR gamma delta lymphocytosis.

Felty syndrome, comprised of neutropenia, rheumatoid arthritis and splenomegaly, occurs in approximately 1% of patients with rheumatoid arthritis. Up to one third of these patients have an increased number of large granular lymphocytes. The usual immunophenotype of these cells is CD3+, CD8+, CD57+, T cell receptor (TCR) alpha beta. A patient with Felty syndrome and large granular lymphocytosis, who had an unusual immunophenotype CD3+, CD4-, CD8-, TCR gamma delta, is described. Her neutropenia responded to treatment with granulocyte colony stimulating factor (G-CSF), which was given in order to raise her neutrophil count prior to bilateral knee replacement surgery. Thus, Felty syndrome with large granular lymphocytosis is a heterogeneous condition, one in which TCR gamma delta large granular lymphocytosis may be found, and also shows a response to treatment with G-CSF.

Pure red cell aplasia associated with malignant thymoma, myasthenia gravis, polyclonal large granular lymphocytosis and clonal thymic T cell expansion.

A case with the triad of pure red cell aplasia (PRCA), myasthenia gravis, and malignant thymoma is reported. There was a clonal proliferation of T cells within the thymoma, as demonstrated by a T cell antigen receptor (TCR) delta chain gene rearrangement. However, despite a large granular lymphocytosis, clonality could not be shown in the peripheral blood either before or after thymectomy. There was no evidence of human T cell lymphotrophic virus type 7 (HTLV1) infection. It is postulated that the clonal thymic T cell population secreted cytokine(s), which stimulated the polyclonal proliferation of large granular lymphocytes, which in turn suppressed erythropoiesis. Thymectomy removed the stimulus to the large granular lymphocytes and hence there was a resurgence of erythropoiesis.

IgM ganglioside GM1 antibodies in patients with autoimmune disease or neuropathy, and controls.

AIMS: To compare the titre of anti-ganglioside antibodies (AGA) to GM1 ganglioside in patients with central and peripheral neurological disease and pure motor and sensorimotor neuropathy, in patients with classic autoimmune diseases, and controls. METHODS: AGA to GM1 were measured using an enzyme linked immunosorbent assay (ELISA) technique, highly purified bovine GM1 ganglioside, and sequential dilution of control and test sera. Antibody titre was calculated using the optical density readings of three consecutive serum dilutions multiplied by the dilution factor. RESULTS: A considerable overlap was evident in the titre of AGA to GM1 in control and test sera. High antibody titres were most frequent in patients with multifocal motor neuropathy with conduction block (MMNCB). Low AGA titre were observed in several patient groups. Compared with the controls, the median titre of AGA to GM1 was significantly higher in patients with multiple sclerosis, rheumatoid arthritis, primary Sjögren's syndrome and systemic lupus erythematosus. In contrast, the median titre in patients with diabetic peripheral neuropathy, motor neurone disease, sensorimotor neuropathy and chronic inflammatory demyelinating polyneuropathy was no different from that in normal control subjects. CONCLUSIONS: Estimation of AGA to GM1 may be helpful in the diagnosis of MMNCB in patients with a pure motor neuropathy but in few other conditions. Low titre AGA to GM1 are evident in several autoimmune conditions. The pathogenetic importance of AGA to GM1 in patients with neuropathy is not clear.

Peritoneal endometriosis in women requesting reversal of sterilization.

Seventy-six women requesting reversal of sterilization underwent at least 1 operative procedure during a 27-month period, and 14 (18.4%) were found to have pelvic endometriosis. The endometriosis patients were noted to have had significantly fewer pregnancies (1.8 versus 2.9, P less than 0.01) before sterilization than those without endometriosis, but the two groups did not differ significantly in mean age (30.8 versus 30.3 years), type of sterilization or in mean number of years since sterilization (5.0 versus 5.5 years). In only two individuals were proximal tubal segment fistulas found at the time of reversal, and neither had endometriosis. We conclude that pelvic endometriosis is more common in patients with bilateral tubal occlusion than previously suspected and that its presence indicates that endometriosis implants can persist for prolonged periods of time, can give rise to new implants, or do not require the tubal reflux of menstrual debris to form.

Increased levels of sCD23 in rheumatoid arthritis are related to disease status.

The low affinity receptor for IgE (Fc epsilon RII, CD23) is involved in many aspects of T and B cell regulation. In the current study, serum levels of sCD23 were measured in monozygotic (MZ) twins discordant for rheumatoid arthritis (RA) to examine whether an increased level of sCD23 in RA is, at least in part, genetically determined. Paired analysis showed significantly elevated sCD23 levels in affected twins when compared with their unaffected co-twins (p < 0.01). There was no significant difference in sCD23 in the unaffected twins compared with normal controls. Higher levels of sCD23 were found in males compared to females in both affected and unaffected twins. Soluble CD23 showed a significant increase with age in RA affected twins (p = 0.013), but no association with disease duration (p = 0.87). There was no significant variation in sCD23 level with HLA-DR phenotype. We conclude that elevations in serum sCD23 in patients with RA are primarily disease related.

Computer models of the human immunoglobulins Binding sites and molecular interactions.

In last month's issue(1) Richard Pumphrey showed how low resolution computer models could be used to investigate the shape and segmental flexibility of human immunoglobulin molecules. In this article, he extends the use of these models to the study of interactions between immunoglobulins and other molecules, such as their receptors, J chain, secretory component and complement fragments. Although precise studies of molecular interaction require computation in atomic detail, there is still much to be learnt from low resolution modelling, in which atomic details are glossed over but general principles become more obvious.

Lessons for management of anaphylaxis from a study of fatal reactions.

BACKGROUND: The unpredictability of anaphylactic reactions and the need for immediate, often improvised treatment will make controlled trials impracticable; other means must therefore be used to determine optimal management. OBJECTIVES: This study aimed to investigate the circumstances leading to fatal anaphylaxis. METHODS: A register was established including all fatal anaphylactic reactions in the UK since 1992 that could be traced from the certified cause of death. Data obtained from other sources suggested that deaths certified as due to anaphylaxis underestimate the true incidence. Details of the previous medical history, the reaction and necropsy were sought for all cases. RESULTS: Approximately half the 20 fatal reactions recorded each year in the UK were iatrogenic, and a quarter each due to food or insect venom. All fatal reactions thought to have been due to food caused difficulty breathing that in 86% led to respiratory arrest; shock was more common in iatrogenic and venom reactions. The median time to respiratory or cardiac arrest was 30 min for foods, 15 min for venom and 5 min for iatrogenic reactions. Twenty-eight per cent of fatal cases were resuscitated but died 3 h-30 days later, mostly from hypoxic brain damage. Adrenaline (epinephrine) was used in treatment of 62% of fatal reactions but before arrest in only 14%. CONCLUSIONS: Immediate recognition of anaphylaxis, early use of adrenaline, inhaled beta agonists and other measures are crucial for successful treatment. Nevertheless, a few reactions will be fatal whatever treatment is given; optimal management of anaphylaxis is therefore avoidance of the cause whenever this is possible. Predictable cross-reactivity between the cause of the fatal reaction and that of previous reactions had been overlooked. Adrenaline overdose caused at least three deaths and must be avoided. Kit for self-treatment had proved unhelpful for a variety of reasons; its success depends on selection of appropriate medication, ease of use and good training.

The clinical spectrum of anaphylaxis in north-west England.

OBJECTIVE: To assess the clinical spectrum of anaphylaxis with a view to developing management guidelines and as a foundation for an epidemiological study. METHODS: Study of the reaction histories and investigations of 172 patients, including children and adults, referred because of anaphylactic reactions. RESULTS: Over 700 reactions occurred in 172 patients from age 5 months to 69 years. There were equal numbers of males and females; when ranked by age at worst reaction, the youngest quartile (0-4 years) was 75% male and the oldest quartile (40+ years) was 74% female. The severity of reactions graded continuously from fatal to mild One hundred and twenty of 172 had two or more reactions; the worst reaction was the first in 33, midsequence in 35 and the most recent in 52. Suspected causes of each patient's worst reaction associated with positive allergy tests include peanuts (42), tree nuts (23), other foods (25, in five associated with exercise), venoms (six bee, 22 wasp), muscle relaxants (seven) and latex (six). Twenty were classified as idiopathic and, in a further 13, investigation of the suspected cause proved negative. There was doubt about the nature of some of the 'reactions' reported even though these had been treated as for anaphylactic reactions. CONCLUSION: The clinical spectrum of anaphylaxis has been defined for the area served by our unit. Management guidelines and future epidemiological studies will have to address the continuous distribution of severity of reactions, the wide age range and the multiplicity of causes.

Anaphylactoid reaction to oxytocin in pregnancy.

A case of an allergic reaction to Syntocinon (synthetic oxytocin) administered during Caesarean section is reported.

Palacos and peanut allergy.

Palacos cement contains peanut oil. The manufacturers instruction states that its use is contraindicated in patients allergic to peanuts. Awareness of this fact by orthopaedic surgeons was evaluated by postal questionnaire, which showed that 73% of those responding were not aware. However, on the basis of the available evidence in the literature, the clinical relevance of the manufacturers advice appears dubious. (Hip International 2004; 14: 254-7).

Antiphospholipid antibodies and Mycoplasma pneumoniae infection.

Anticardiolipin antibody levels were measured in 57 patients with Mycoplasma pneumoniae infection and 21 patients with other infections. Significantly more patients in the mycoplasma group had increased IgM and IgG anticardiolipin. Within the mycoplasma group significantly higher titres were found in patients with severe infection (assessed by need for hospital admission) and in patients with cold agglutinins. A tendency for particularly high titres to occur in patients with extra-pulmonary complications was identified.

C-reactive protein levels in serum: a rapid enzyme immunoassay.

A simple and rapid competitive-inhibition enzyme linked immunoassay for serum C-reactive protein (C-RP) is described. The assay has a working range of 4.4-700 mg/l of C-RP, uses two serum dilutions and requires only 90 min to perform. Comparisons are presented between this assay and three commercially-available kits. The assay performs as well as the best of these kits but with added advantages of speed, ease of use and increased working range.

Evidence for circulating complexes containing IgE in patients with atopic dermatitis.

18 out of 34 patients with atopic dermatitis were shown to have 5-32% of their serum IgE present in a high molecular weight (HMW) form (470,000 daltons). There was no simple correlation between the amount of HMW and the total serum IgE. The HMW peak was not affected by freezing and thawing the serum, but the complexes could be split by lowering the pH of serum fractionation. Some patients were tested sequentially on different occasions and although their clinical status may have altered during this time, there was no marked alteration of the HMW fraction. Results are compared with patients with raised IgE due to parasitic infections. Anti-IgE antibodies have been demonstrated in the HMW fraction.

Cyclical eosinophilia: a manifestation of periodic haemopoiesis?

A patient is described who has had a marked eosinophilia of unknown cause for 9 years in association with episodic facial swelling. Weekly blood counts for 8 months showed cyclical variations in eosinophils, neutrophils and monocytes (mean cycle length 35 d). Marrow culture studies showed fluctuation in the incidence of granulocyte-macrophage colonies (GM-CFC), and the proportion of eosinophil colonies was higher than the values reported for normals. The blood lymphocyte T4/T8 ratio was reversed, due to an increase of T8+ cells. It is suggested that this condition is a rare form of periodic haemopoiesis.

Specific immunoglobulin E to peanut, hazelnut and brazil nut in 731 patients: similar patterns found at all ages.

BACKGROUND: Previous studies have reported reactions to an increasing range of nuts as patients with nut allergy grow older. Most patients with symptoms suggesting nut allergy have specific IgE to more than one nut. Furthermore, fatal reactions have followed eating nuts different from any causing the deceased's previous reactions. OBJECTIVE: To explore the pattern of specific IgE to three distantly related nuts in patients of all ages with nut allergy. METHODS: This study includes all patients referred to our laboratory for nut allergy testing from January 1994 to August 1998 who were tested for peanut, hazelnut and brazil nut, and had specific IgE to at least one of these nuts. All tests were performed using the Pharmacia Unicap system. RESULTS: Seven hundred and thirty-one patients (age 7 months to 65 years, median 6.6 years) had specific IgE >0.35 kU(A)/L to at least one of these three nuts: 282 had IgE to one nut, 130 to two nuts, and 319 to all three nuts. When analysed by gender and age quartile, very similar patterns were found in all subgroups though significant age trends and age interactions were found for IgE to individual nuts and combinations of nuts. CONCLUSIONS: The probability of a patient with nut allergy having specific IgE to a particular combination of peanut, hazelnut and brazil nut is similar, whatever their age or sex. The apparent increase in multiple nut reactivity with increasing age may therefore be due to exposure of previously unchallenged sensitivity. The frequency of multiple-nut specificity is sufficiently high that patients should always be tested for allergy to a range on nuts if they have a history of reacting to any nut.