Modelling cost effectiveness of horse antithymocyte globulin for treating severe aplastic anaemia in Germany.

Acquired severe aplastic anaemia (AA) is a serious condition caused by immune-triggered bone marrow failure. For patients not eligible for bone marrow transplantation, treatment of choice is immunosuppression by a combined treatment with antithymocyte globulin (ATG) and cyclosporine. The debate on treatment optimization in AA is focused on conflicting data regarding ATG preparations from horse (h-ATG) versus rabbit (r-ATG), recently favouring h-ATG. H-ATG has been withdrawn from the European market in 2007. Reimbursement for imported preparations from outside Europe is frequently denied in negotiations with statutory health insurance companies. This raises the question of whether h-ATG is cost effective and a sensible investment with regard to healthcare budgets as well as patient health. We modelled the cost effectiveness of r-ATG versus h-ATG based on a recent randomized trial and cost data provided by the hospital pharmacy of Jena University Hospital. We calculated the amount of life years gained and the average incremental costs per life year gained when comparing h-ATG and r-ATG. Our calculations revealed average incremental costs per life year gained of 11,033.80 for the examined patient population treated with h-ATG when compared to r-ATG. Assuming a cost effectiveness threshold of 25,000-35,000 per life year gained, our calculations demonstrate cost effectiveness of h-ATG as compared to r-ATG.

Assessment of pregnancy-associated glycoprotein (PAG) concentrations in swamp buffalo samples from fetal and maternal origins by using interspecies antisera.

Pregnancy-associated glycoproteins (PAG) constitute a large family of glycoproteins found in the outer placental epithelial cell layer of the placenta in Eutherian species. In ruminants, they are noted to be structurally closely related among the different species. This study was designed to determine PAG concentrations in maternal and fetal plasma, allantoic and amniotic fluids in buffalo species. Antisera (AS) generated in rabbits against distinct PAG molecules were used in three radioimmunoassay (RIA)-PAG systems: RIA-1 (antiserum raised against bovine PAG67kDa; AS#497), RIA-2 (antiserum raised against caprine PAG55 + 62 kDa; AS#706) or RIA-3 (antiserum raised against buffalo PAG; AS#859). Samples were collected at a slaughterhouse (n = 67). PAG concentrations determined by RIA-2 gave significantly higher results in both allantoic and amniotic fluids (12.7 ± 2.1 ng/mL and 24.0 ± 7.3 ng/mL, respectively). Regarding maternal and fetal plasma, PAG concentrations obtained by RIA-2 (21.8 ± 2.4 ng/mL and 20.2 ± 2.5 ng/mL, respectively) and RIA-3 (25.0 ± 2.2 ng/mL and 21.9 ± 3.2 ng/mL, respectively) were higher than those obtained by RIA-1 (15.5 ± 1.4 ng/mL and 16.1 ± 1.8 ng/mL, respectively). The correlation among the three systems was very high. The study clearly reveals the ability of different PAG-RIA systems to measure PAG concentration in swamp buffalo samples.

Assessment of the IgG index in dogs by indirect immunoenzimatic assays as diagnostic tool for inflammatory diseases of central nervous system.

The IgG index measures the intrathecal immunoglobulin production and it is a useful tool for diagnosis of inflammatory diseases involving the central nervous system. This index is based on the precise quantification of albumin and IgG in canine cerebrospinal fluid and serum. Here, we report the development of an indirect competitive ELISAs for the detection of both antigens. Thirty-two dogs were included in this study, divided into three experimental groups. Group A was composed of 22 healthy animals, as determined by standard clinical examination. In group B, six animals, presented neurological pathologies associated with endogenous IgG production and, in group C four animals presented neurological diseases or symptoms not associated with intrathecal IgG production. Cerebrospinal fluid and serum samples were obtained from these animals. As expected, by using the indirect ELISAs proposed here, the IgG indexes obtained in healthy animals (A) were 0.371+/-0.252 (SD). In B and C, the values (3.002+/-1.897; 0.36+/-0.306, respectively), were in agreement with the pathologic conditions of the individuals in each group. Thus, the immunometric competition ELISA methods proposed here allow the discrimination of abnormal intrathecal IgG production, in a variety of inflammatory pathologic conditions of the central nervous system.

Estimación del costo de producción de dos técnicas de radioinmunoanálisis en fase líquida para medir progesterona en plasma sanguíneo / Estimation of production costs of two radioimmunoanalysis in liquid stage to measure progesterone in blood plasma

Introducción. La mediación de progesterona de origen ovárico, es un prodecidimiento que permite determinar la funcionalidad reproductiva de las hembras de todos los mamíferos domésticos, inclusive en la mujer. En México, la tecnología del radioinmunoanálisis (RIA) es la más utilizada para medir progesterona sanguínea. Sin embargo, no existe alguna industria mexicana que se dedique a producir estuches comerciales. En el presente trabajo se estimó el costo de producción de dos técnicas de RIA en fase líquida para medir progesterona sanguínea. Material y métodos. A partir de los insumos primarios (anticuerpo, calibrador o analito y trazador) se establecieron y validaron dos técncias de RIA para medir progesterona sanguínea. La técnica A utilizó anticuerpos antipropgesterona inducidos en conejo y extraídos del suero sanguíneo. La técnica B utilizó anticuerpos antiprogesterona inducidos en gallina de postura y extraídos de la yema del huevo. Resultados. La sensibilidad de ambas técnicas de RIA permitió detectar valores relativamente bajos de la hormona, lo suficiente para poder detectar actividad luteal. El costo de producción por tubo (de USD 0.14 a 0.20) disminuyó conforme aumentó el rendimiento del anticuerpo. Discusión. En nuestro medio la producción de anticuerpos contra progesterona para establecer un RIA es una alternativa viable y disminuiría el costo de utilización de esta técnica

An immunological assessment of group-housed rabbits.

Laboratory rabbits kept in barren 'traditional' cages tend to develop stereotypic behaviours and bone deformities. We have used an alternative regime, housing adult does as groups of four or five in floor pens (2.5-3 m2) supplied with hiding places and bedding. High- and low-ranking members of each group were identified, and their immunological status compared in terms of blood leucocyte function (chemiluminescence and mitogen tests), complement activity, and antibody production to soluble and cellular antigens. We found no evidence of immunosuppression, either in groups of a 'docile' breed (New Zealand White) or Dutch crosses. These results, together with the animals' general health and ease of handling, lead us to conclude that group-housed does are suitable for raising antisera and other purposes, provided that they are adequately monitored.

Serum cytotoxicity to oligodendrocytes in multiple sclerosis and controls: assessment by 51Cr release assay.

In order to quantitate oligodendrocyte cytotoxicity induced by sera from patients with multiple sclerosis (MS) and control sera, we established a 51Cr release assay system using cultured oligodendrocytes as target cells. With this assay system, we detected serum-induced cytotoxicity in isolated oligodendrocytes in sera from patients with MS, other neurological diseases (OND) and normal individuals. Oligodendrocytes were much more susceptible than astrocytes. The serum cytotoxicity was abolished by heating sera at either 56 degrees C or 50 degrees C for 30 min, and disappeared in storage at 23 degrees C for 1 month or at 4 degrees C for 2 months. Addition of fresh guinea pig serum as a source of complement did not restore the cytotoxic effect. Although the serum factor cytotoxic to oligodendrocytes is not specific for sera from patients with MS, it is possible that it may damage oligodendrocytes if the blood-brain barrier is impaired, as occurs in MS.

The effect of antiserum quality on strain specificity assessment of foot and mouth disease virus by the neutralization reaction.

The factors affecting the virus strain specificity of antibody to foot an mouth disease virus prepared by a variety of protocols in several species were evaluated by neutralization tests. The time at which the serum was taken, the antigen dose given, whether or not revaccination had occurred and the animal species in which the sera were prepared, did not appear to affect the strain specificity of serum prepared to inactivated antigens when measured in neutralization tests, probably because of the restricted nature of the antigenic site involved. However, variation was observed with convalescent animal sera or sera from animals which had received trypsin cleaved virus were used. For these reasons banks of reference antisera are prepared as pooled sera using one or two inoculations of inactivated antigen.

Assessment of myocardial infarct size by serial changes in serum cardiac myosin light chain II in dogs.

The relationship between myocardial infarct size and serum levels of cardiac myosin light chain II (LC II; 20000 daltons) was studied in 24 dogs with left anterior descending coronary artery occlusion. LC II in the serum was measured by the radioimmunoassay which we have recently developed. In our assay, 0.1--5.0 ng of LC II were effectively measurable. Serum LC II levels rose rapidly and stayed elevated long after coronary occlusion. Infarct size was determined by gross inspection. In 24 dogs, infarct size ranged from 0.3 to 41.7 per cent of left ventricular weight. LC II release was calculated by the formula of Shell and associates. Regression analysis showed good correlation between infarct size and LC II release (r = 0.78). Infarct size also correlated with maximal LC II level (r = 0.77), and LC II level 24 hours after coronary occlusion (r = 0.69). Detection of circulating LC II is a useful method since it can be applied to the diagnosis of acute myocardial infarction at the early as well as late stage and infarct size can be assessed by analysis of serum LC II levels.

Latex agglutination inhibition card test for gentamicin assay: clinical evaluation and comparison with radioimmunoassay and bioassay.

Gentamicin levels were determined in 100 serum specimens by a new latex agglutination inhibition card test, a radioimmunoassay (RIA), and a bioassay. Correlation coefficients determined by linear regression analysis demonstrated that the levels obtained by the latex agglutination inhibition card test had a high degree of correlation with the RIA and could be performed much faster and more economically when processing small numbers of specimens. The bioassay had a slightly lower degree of correlation with both the RIA and the latex test and was adversely influenced by concurrently administered antibiotics which could not be eliminated by beta-lactamase. When measuring gentamicin concentrations above 2 micrograms/ml, the coefficient of variation was less than 14% for the latex agglutination assay compared with 15% for the bioassay and 12% for RIA. The latex agglutination inhibition card test is a rapid, accurate, specific, and reproducible method for monitoring gentamicin levels in patients and is particularly applicable for laboratories processing small numbers of specimens.

Clinical laboratory: enzyme immunoassay for the rapid clinical identification of snake venom.

The treatment of snakebite could be simplified if the identity of the offending snake was more frequently known. A positive identification, which allows the use of a specific monovalent antivenom, probably occurs in less than 20% of cases. Recently published methods of venom detection (RIA and ELISA) take at least three hours to complete. We have developed a sandwich enzyme immunoassay (EIA) which is capable of detecting 0.5 ng of crude snake venom in about 90 minutes or 2 ng of crude venom in about 30 minutes. This substantial reduction in incubation times, while still retaining the sensitivity required, was due to the use of protein A purified rabbit IgG antivenom from hyperimmune serum and the enzyme horseradish peroxidase (HRPO). A rapid identification of the offending snake by this method may reduce the use of large-volume polyvent antivenoms, thus avoiding the clinical and economic disadvantages of such preparations. Other advantages would be an increased understanding of the clinical syndrome produced by the individual species of snake, and accumulation of data about the incidence of envenoming attributed to specific snakes.

[Immunochemical assessment of the antigenic relationship between the pancreatic kallikrein of rodents and carnivores]. / Immunokhimicheskaia otsenka antigennogo rodstva pankreaticheskogo kallikreina gryzunov i khishchnykh.

Using quantitative and qualitative immunochemical methods, in combination with isoelectrical focusing, it was shown that antibodies to commercial kallikrein are involved into precipitation reaction with kallikrein in extracts from the pancreas of dogs, cats, guinea pigs, rats and mice. Complexes antigen--antibody exhibit similar physico-chemical properties; during isoelectric focusing, they are found in the same pH range as the immunoactive portion of commercial kallikrein. These data indicate antigenic similarity of pancreatic kallikrein in different mammalian species.

Preparation and assessment of antisera to ACTH.

Antisera to ACTH were produced in rabbits injected repeatedly at multiple intradermal sites with synthetic [Asp25, Ala26, Gly27]alphah-corticotropin-(1-28)-octacosapeptide-bovine gamma globulin conjugate (octacosapeptide is a sequence analogue of alphah1-28-ACTH). Antibodies to extracted human or porcine ACTH were detected in all of the sera 1 month after immunization. A considerable proportion of the antisera obtained from a single final bleeding 5 months after the primary immunization were suitable for sensitive radioimmunoassay. The antisera were shown to neutralize the steroidogenic activity of ACTH in an isolated rat adrenal cell bioassay system. Titres estimated from antiserum dilution curves and relative avidities from the standard curves were compared. It was possible to detect picogram amounts of ACTH in plasma-free medium with the best antisera. The method described is an effective means of producing anti-sera to the weakly immunogenic N-terminal fragment of the ACTH molecule.

A sensitive, rapid, and economic radioimmunoassay of human growth hormone using ethanol-ammonium acetate.

An aqueous solution of 66 per cent ethanol and 6.6 per cent ammonium acetate has been used in the development of a relatively simpler and rapid as well as highly reproducible method of separation of antibody-bound and free hormones in the radioimmunoassay of human growth hormone (HGH). The present assay is sensitive to detect 20 pg. of hormone with a precision of plus or minus 5 per cent, hence, as little as 20 mul of plasma is required to perform the radioimmunoassay. Serum levels of HGH have been found to be approximately 70 per cent of the plasma concentrations in the same subjects.

Comparison of radioimmunoassay methods for human luteinising hormone.

1. Four radioimmunoassay methods for the determination of luteinising hormone in serum or urine are described. 2. Evaluation of these methods (one employing laboratory prepared reagents, three utilizing commercial kits) was made to assess their routine laboratory value. Comparable precision and accuracy were found. Great economy is achieved by pruchasing LH and anti-LH preparations and by labelling one's own hormone. 3. Suggestions for future improvements in kit LH assay control and unitage are made.

Quantitative assessment of thymus-dependent (t)-cell function in human peripheral blood.

A new micromethod for evaluating lymphocyte responses to phytohemagglutinin (PHA) has been developed and used for the survey of T-cell function in human peripheral blood. The results of lymphocyte responses to PHA stimulation in whole blood culture were expressed in terms of unit volume of blood (50 mul). This new method has eliminated the major uncontrollable factors of conventional methods, and appears to be more correlated with the cell-mediated immunity in vivo.