Development Of Enzyme Immunoassay Research Articles

Determination of slime-producing S. epidermidis specific antibodies in human immunoglobulin preparations and blood sera by an enzyme immunoassay : Correlation of antibody titers with opsonic activity and application to preterm neonates

Slime-producing Staphylococcus epidermidis is responsible for severe infections in immunocompromised patients and, particularly, in premature infants who are transiently deficient in IgG. A sulfated polysaccharide with molecular mass of 20-kDa (20-kDa PS) has been recognized as the major polysaccharide component and antigenic determinant of S. epidermidis extracellular slime layer. The presence of adequate amounts of antibodies to 20-kDa PS in patients’ sera would be of importance to prevent or treat slime-producing S. epidermidis bacteremia. Administration of intravenous immunoglobulin (IVIG) is considered to be a reasonable IgG replacement therapy and has been widely used to prevent or treat neonatal sepsis. Clinical trials have shown conflicting results on the efficacy of IVIGs and this phenomenon has been attributed to the variability of IVIG preparations in the content and opsonic activity of IgG against microorganisms of clinical importance. Monitoring of antibodies to distinct bacterial macromolecules, which are species-specific and responsible for bacterial infections, has not been performed previously. A highly precise and repeatable enzyme immunoassay was developed to determine quantitatively the levels of antibodies against the 20-kDa PS of S. epidermidis slime. The amount of 20-kDa PS specific antibodies found in 27 lots of an IVIG preparation (Sandoglobulin®) correlated well with their in vitro opsonic activity against slime-producing S. epidermidis. The majority of lots (75%) having titers higher than 200 units/ml showed significant opsonic activity (50–75%) towards slime-producing S. epidermidis. Sandoglobulin® lots with titers higher than 200 units/ml of 20-kDa PS specific IgG were administered as a prophylactic agent to low-birth weight (lower than 1700 g) preterm neonates immediately after birth. The levels of total and 20-kDa PS specific IgG in neonates’ blood sera were significantly higher than those found in the control group, even 10 days after the last infusion. The rate of slime-producing S. epidermidis bacteremia in neonates who received IVIG was also considerably lower than those in the control group. The results of this study suggest that specific IgG titers estimated by the developed enzyme immunoassay may well be indicative of the IVIG opsonic activity against slime-producing S. epidermidis. Furthermore, administration of Sandoglobulin® with titers higher than a cut-off value of 200 units/ml may significantly protect preterm neonates against slime-producing S. epidermidis bacteremia.

Measurement of urinary albumin using newly developed enzyme immunoassay in the rat

Background. Since it is well known that microalbuminuria predicts the late development of diabetic nephropathy, the measurement of urinary albumin (Ualb) is important in human diabetic nephropathy. Ualb also increases in diabetic rats and the measurement of Ualb in the rat is important to determine diabetic nephropathy. Many methods have been developed for measuring human Ualb. However, there are few methods for rat Ualb assay. Since specific antialbumin antibody for rat should be used, the aim of this study was to basically evaluate a new enzyme immunoassay for determining Ualb in rats.

Usefulness of a Novel Enzyme Immunoassay for the Detection of Helicobacter pylori in Feces

Background: In this study we assessed the reliability of a newly developed enzyme immunoassay (HpSA) kit for detecting Helicobacter pylori antigen in stool. Methods: This study included 309 patients, 147 of whom were defined as positive and 162 as negative by the 13C-urea breath test, rapid urease test, and pathologic findings. From these patients fresh stool specimens were collected for HpSA. Results: When 0.100 was adopted as the cut-off value, in accordance with the manufacturer's recommendations, the sensitivity, specificity, and accuracy of the HpSA were 98.0%, 87.0%, and 92.2%, respectively. However, these values were much improved when a cut-off value of 0.300 was adopted, which was obtained with our receiver-operator characteristics curve; with this value the sensitivity, specificity, and accuracy of HpSA were 93.9%, 95.7%, and 94.8%, respectively. Conclusion: These results indicate that HpSA is a highly reliable diagnostic method for H. pylori infection and is useful in confirming eradication.

Clinical outcome in pneumococcal meningitis correlates with CSF lipoteichoic acid concentrations.

Lipoteichoic and teichoic acids are components of the cell wall of Streptococcus pneumoniae. A recently developed enzyme immunoassay was used in patients with pneumococcal meningitis to investigate lipoteichoic and teichoic acid concentrations in CSF at the first lumbar puncture in relation to the clinical outcome determined by the Glasgow Outcome Scale. Lipoteichoic and teichoic acid concentrations in CSF were significantly associated with neurologic sequelae and mortality in S. pneumoniae meningitis.

A Comparison of Performances of Four Enzymes Used in Elisa with Special Reference to β-Lactamase

Horse radish peroxidase, alkaline phaosphatase and β-D-galactosidase are widely used as labels in the development of enzyme immunoassays (EIAs). Enzyme β-lactamase, though introduced as a label in late seventies has not yet become very popular inspite of having the necessary features of an enzyme to be used in EIAs. the present article reviews assays developed with this enzyme, highlights its salient features and brings out an argument in favour of its wide spread use in EIAs.

Detection of Chlamydia trachomatis antibodies by 2 novel tests: rELISA and peptide EIA.

The performance of 2 newly developed enzyme immunoassays (EIAs) intended for the routine serological diagnosis of chlamydial infections was evaluated. rELISA is based on a recombinant lipopolysaccharide antigen which detects chlamydia genus-specific antibodies, and Chlamydia trachomatis EIA is based on a peptide derived from major outer membrane protein and is therefore species-specific. Both tests distinguished patients with tubal factor infertility (TFI) or pelvic inflammatory disease (PID) from the controls. The prevalence of IgA antibodies was higher for the PID patients than for the TFI patients; the finding indicates a more active state of infections for the PID patients. Furthermore, C. trachomatis EIA detected more IgG antibodies in the TFI patients than in patients with non-tubal factor infertility. In conclusion, rELISA detected chlamydial antibodies in general, and C. trachomatis EIA detected species-specific antibodies. These EIA tests may be useful in the serodiagnosis of chlamydial infection.

VIPER ENVENOMING: EVALUATION OF TREATMENT BY RESTORATION OF HAEMOSTASIS AND VENOM CLEARANCE

Treatment of systemic envenoming in snakebite victims has, in the past, depended almost entirely on the individual clinician's experience in assessing the severity of envenoming. The efficacy of treatment is obviously related to the neutralising potency of the antivenom used, the route by which it is administered and the dose. The use of techniques for evaluating the efficacy of antivenoms has proved to be very useful, as an adjunct to recognised clinical observations, for a more objective evaluation of antivenom efficacy and dosage. In patients bitten by many vipers, including the Brazilian pit vipers, the reversal of the venom-induced coagulopathy provides an ideal indicator as to the efficacy of an antivenom. Likewise, the development of enzyme immunoassay has permitted us to estimate levels of circulating specific venom and antivenom levels at any time after the bite in the patient's blood; the efficacy of an antivenom can thus be objectively assessed by measuring the neutralisation and clearance of venom antigen. In Brazil, it appears that clinicians treat patients with excessive amounts of highly efficient antivenoms, which results in an unacceptably high incidence of reactions. In Sri Lanka, the use of imported Indian antivenom is relatively ineffective in neutralising the procoagulant and other effects of the venoms of Sri Lankan snakes, demonstrating the real problem of venom variability within individual species. In West Africa, the improved restoration of blood coagulability, the resolution of haemorrhagic disturbances and the increased rate of clearance of venom following treatment of Echis victims with a monospecific as opposed to a polyspecific antivenom has been demonstrated, and new smaller fragment Fab antivenoms have been developed and are now under clinical assessment. Such clinically based coagulation and immunological studies should result in more efficient and controlled use of expensive antivenoms for treatment of systemic envenoming and the accurate assessment of newly designed products. Such studies also emphasise the importance of individual countries producing their own antivenoms for treatment of systemic envenoming. Likewise, the use of such objective systems now enable us to assess the use of first aid measures such as tourniquets.

Clinical evaluation of serum prostate-specific antigen-alpha1-antichymotrypsin complex values in diagnosis of prostate cancer: a cooperative study.

We studied the clinical significance of serum prostate-specific antigen bound to alpha1-antichymotrypsin (PSA-ACT) values determined with a newly developed enzyme immunoassay. Serum PSA-ACT values were determined in a total of 652 sera. Clinical utility for the diagnosis of prostate cancer was compared to that of Tandem-R PSA and gamma-seminoprotein (gamma-Sm). The new enzyme immunoassay is based on the use of the Stanford reference as an international standard for PSA assays. Serum PSA-ACT values ranged from less than 0.10 to 1.4 ng/mL in healthy males (n = 100) while values in patients with benign prostatic hyperplasia (n = 155) averaged 3.4 +/- 3.8 ng/mL (mean +/- SD). In patients with prostate cancer, serum PSA-ACT values increased significantly with progression of the clinical stage and there were statistically significant differences between benign prostatic hyperplasia and each stage of prostate cancer except for stage A. Using BPH levels as controls (4.8 ng/mL for PSA-ACT, 7.2 ng/mL for PSA, 3.8 ng/mL for gamma-Sm, and 2.4 ng/mL for the complexed/free PSA ratio of PSA-ACT/gamma-Sm), specificity was 80%. The sensitivity of prostate cancer detection was 79% for PSA-ACT, 77% for PSA, 57% for gamma-Sm, and 46% for the ratio between PSA-ACT/gamma-Sm. Although the determination of serum PSA-ACT showed essentially the same utility as that of PSA for the diagnosis of prostate cancer, PSA-ACT may allow prediction of the clinical stage. The PSA-ACT assay may therefore replace PSA in the detection of prostate cancer.

An objective approach to antivenom therapy and assessment of first-aid measures in snake bite

Treatment of systemic envenoming in snake-bite victims has, in the past, depended almost entirely on the individual clinician's experience in assessing the severity of envenoming. The efficacy of treatment is obviously related to the neutralizing potency of the antivenom used, the route by which it is administered and the dose. The development of enzyme immunoassays has permitted a more scientific appraisal, allowing estimation of circulating specific venom and antivenom concentrations at any time after the bite in the patient's blood. It is therefore possible to measure accurately the efficacy of antivenom in the neutralization and clearance of venom antigen. In Brazil, it appears that clinicians treat patients with excessive amounts of highly efficient antivenoms and this results in an unacceptably high incidence of reactions. In Sri Lanka, the use of imported, Indian antivenom is relatively ineffective in neutralizing the venoms of Sri Lankan snakes, demonstrating the real problem of venom variability within individual species. In West Africa, the improved clearance of venom following treatment of Echis victims with a monospecific as opposed to a polyspecific antivenom has been demonstrated, and new, smaller fragment, Fab antivenoms have been developed and are now under clinical assessment. Such clinically-based immunological studies should result in more efficient and controlled use of expensive antivenoms for treatment of systemic envenoming and the accurate assessment of newly designed products. Such studies also emphasise the importance of individual countries producing their own antivenoms for treatment of systemic envenoming. Likewise, the use of such objective systems now enables the use of first-aid measures such as tourniquets to be properly assessed.

An objective approach to antivenom therapy and assessment of first-aid measures in snake bite.

Treatment of systemic envenoming in snake-bite victims has, in the past, depended almost entirely on the individual clinician's experience in assessing the severity of envenoming. The efficacy of treatment is obviously related to the neutralizing potency of the antivenom used, the route by which it is administered and the dose. The development of enzyme immunoassays has permitted a more scientific appraisal, allowing estimation of circulating specific venom and antivenom concentrations at any time after the bite in the patient's blood. It is therefore possible to measure accurately the efficacy of antivenom in the neutralization and clearance of venom antigen. In Brazil, it appears that clinicians treat patients with excessive amounts of highly efficient antivenoms and this results in an unacceptably high incidence of reactions. In Sri Lanka, the use of imported, Indian antivenom is relatively ineffective in neutralizing the venoms of Sri Lankan snakes, demonstrating the real problem of venom variability within individual species. In West Africa, the improved clearance of venom following treatment of Echis victims with a monospecific as opposed to a polyspecific antivenom has been demonstrated, and new, smaller fragment, Fab antivenoms have been developed and are now under clinical assessment. Such clinically-based immunological studies should result in more efficient and controlled use of expensive antivenoms for treatment of systemic envenoming and the accurate assessment of newly designed products. Such studies also emphasise the importance of individual countries producing their own antivenoms for treatment of systemic envenoming. Likewise, the use of such objective systems now enables the use of first-aid measures such as tourniquets to be properly assessed.

Enzyme immunoassays for specific IgG and IgE antibodies toPichia pastoris components in normal humans

We developed enzyme immunoassays for human anti-Pichia pastoris components (PPC) IgG and anti-PPC IgE antibody titers. Anti-PPC IgG antibody assay were performed using antigen-coated plate and anti-human IgG peroxidase conjugate. The intra- and interassay coefficients of variation (CV) of anti-PPC IgG antibody were 1.83-2.51% and 1.97-2.76%, respectively. The anti-PPC IgE antibody assay was performed using an anti-IgE monoclonal antibody-coated plate, biotin-labeled PPC and avidin-labeled peroxidase, which was not subject to interference by the high titer of anti-PPC IgG antibody. The intra- and interassay CV were 3.83-5.34% and 3.56-5.84%, respectively. We determined and compared anti-PPC IgG antibody titers in the 40 normal individuals. We confirmed that a high titer of anti-PPC IgG antibody is contained in all normal human sera and that these antibodies are directed primarily to mannan by immunoblotting analysis. The ratio of the maximum to minimum anti-PPC IgG antibody titers in normal individuals was > 8,000. Anti-PPC IgG antibody titers did not correlate with the age. However, we did not detect anti-PPC IgE antibody in normal individuals.

Development of an Enzyme Immunoassay for the Determination of the Insecticide Pymetrozine

Abstract Pymetrozine (CGA 215 944) is a selective insecticide against plant sucking pests. Polyclonal antibodies were raised in rabbits against two pymetrozine related compounds conjugated to BSA. The antisera produced against one of the two compounds led to development of a competitive Enzyme Immuno Assay (EIA) against pymetrozine with high precision and accuracy and a detection limit of 50 ng of pymetrozine per g of plant fresh weight. Only a weak cross-reactivity was obtained with pymetrozine metabolites. Plant extracts produced a background corresponding to 10–30 ng equivalents of pymetrozine per g fresh weight. The detection limit was set above this background noise at 50 ng/g fresh weight. In other systems or following other extraction protocols the detection may yet be below this limit.

Urinary markers of estrogen metabolism 2- and 16α-hydroxylation in premenopausal women

There is considerable scientific interest in whether measurement of the major estrogen metabolites 2- and 16α-hydroxyestrone will shed light on the role of estrogen in the risk of breast cancer. These have been difficult to measure in large numbers because of the need for radiolabeled tracers, but a new assay is able to utilize spot urine samples. The main objective of this study was to assess the reliability of a newly developed enzyme immunoassay (EIA) for the measurement of 2- and 16α-hydroxyestrone in urine samples collected from a large group of healthy premenopausal women enrolled in a clinical trial. A secondary objective was to assess the impact of several factors such as body weight on the urinary estrogen metabolite ratios. The study cohort included 174 women aged 44–50, who were enrolled in the Cardiovascular Risk Factors and Menopause Trial, also referred to as the Women's Healthy Lifestyle Project (WHLP), an ongoing 5-year clinical trial of 535 premenopausal women randomized either to an intensive dietary life-style intervention group or to an assessment-only control group. Measurements of 2- and 16α-hydroxyestrone showed a high intraclass correlation for blind duplicate urine samples ( R = 0.94 and R = 0.80), cross-sectionally and over time ( R = 0.79 and R = 0.62), in this population of healthy premenopausal women. The intervention diet (of 25% of total calories from fat) did not appear to influence the estrogen metabolite ratio. This new estrogen metabolite EIA demonstrates good reliability and thus may be appropriate for use in large epidemiologic studies of estrogen-related diseases. There was no relation between dietary fat reduction, weight loss, and increased exercise and change in the ratio among premenopausal women in this study.

Clinical significance of measurements of urinary and serum thrombomodulins in patients with non-insulin-dependent diabetes mellitus

The aim of our study was to elucidate whether serum thrombomodulin (S-TM) and urinary thrombomodulin (U-TM) levels would reflect the pathogenesis of diabetic complications. Studies were conducted in 188 patients with non-insulin-dependent diabetes mellitus (NIDDM) and 132 age-matched healthy subjects. TM was measured by a newly developed enzyme immunoassay. Both S-TM and U-TM levels in NIDDM were much higher than those in healthy controls. S-TM values in NIDDM correlated significantly with age ( P < 0.05), HbA 1c ( P < 0.05), serum 1.5 anhydroglucetol (AG) ( P < 0.05) and urinary albumin concentration (UAC) ( P < 0.01), respectively. On the other hand, fasting plasma glucose (FPG) ( P < 0.001), HbA 1c ( P < 0.01), serum fructosamine ( P < 0.05) and serum 1.5 AG ( P < 0.05) were closely correlated with U-TM values in NIDDM. Patients with clinical nephropathy showed obviously higher S-TM levels ( P < 0.05) than patients with latent nephropathy. Furthermore, S-TM values in patients with diabetic proliferative retinopathy increased significantly compared with those in patients without diabetic retinopathy ( P < 0.05). When all diabetic patients with normoalbuminuria were studied, no significant changes of S-TM were observed between the no diabetic retinopathy group and the proliferative diabetic retinopathy group. Conclusions: The present data suggest that an increase in U-TM reflects the grade of glucose metabolism, whereas an increase in S-TM appears to reveal the advance of diabetic microangiopathy, including nephropathy.

Local Increase in Polymorphonuclear Leukocyte Elastase Is Associated With Tumor Invasiveness in Non-small Cell Lung Cancer

The production of tumor cell proteases is implicated in tumor cell invasion and metastasis. To determine whether lung cancer cells can produce polymorphonuclear leukocyte elastase (PMN-E), we measured the concentration of immunoreactive (ir)-PMN-E in the conditioned medium of seven lines of non-small cell lung cancer cells, EBC-1, LC-1sq, LK-2, A-549, PC-3, RERF-LC-MS, and RERF-LC-OK, and three normal lung epithelial cell lines, CCD-8Lu, WI-1003, and LL-24, by using a recently developed enzyme immunoassay (EIA). We measured the concentration of ir-PMN-E in extracts of 40 non-small cell lung cancers, and evaluated its association with the clinicopathologic findings in these patients. The ir-PMN-E level in the culture medium increased with time in six of the seven lines of lung cancer cells; the exception was PC-3. No detectable ir-PMN-E was secreted into the culture medium of the three lines of normal lung epithelial cells. The ir-PMN-E was detected in tissue extracts from 34 to 40 specimens at concentrations ranging from 0.11 to 15.5 micrograms/100 mg of protein. When 40 specimens of lung cancer were categorized by clinical stage of disease, the ir-PMN-E concentration was significantly higher in stage IIIB vs stages I, II, or IIIA. Similarly, the ir-PMN-E concentration was significantly higher in stage IIIA than in stage I. Evaluation of correlations between the ir-PMN-E concentration and patient characteristics showed that the ir-PMN-E level was significantly higher in T3 and T4 tumors than in T1 or T2 tumors. Analysis of prognostic factors in a group of 101 patients with non-small cell lung cancer demonstrated that those with high ir-PMN-E had a significantly shorter overall survival vs those with a low ir-PMN-E at the cutoff point of 3.5 micrograms/100 mg of protein. Multivariate analysis showed that ir-PMN-E was a significant porgnostic factor for early death (hazard ratio, 4.04; 95% confidence interval, 1.65 to 9.95) (p = 0.005), suggesting it was an independent marker for prognosis. Results suggest that the local production of PMN-E may be involved in the tumor invasion associated with a poor prognosis in patients with non-small cell lung cancer.

The prognostic value of serum troponin T in unstable angina

Background: Cardiac troponin T is a regulatory contractile protein not normally found in blood. Its detection in the circulation has been shown to be a sensitive and specific marker for myocardial cell damage. We used a newly developed enzyme immunoassay for troponin T to determine whether its presence in the serum of patients with unstable angina was a prognostic indicator. Methods: We screened 72 patients with unstable angina (Class III, acute unstable angina) for serum creatine kinase activity, creatine kinase myocardial band (isoenzyme M) activity, and troponin T, every 8 h for 2 days after admission to the hospital. The outcomes of interest during the hospitalization were death and myocardial infarction. Results: Troponin T was detected in the serum of 24 of the 72 patients (34%) with acute angina at rest. Only four of these patients had elevated creatine kinase M activity. Of the 24 patients who were positive for troponin T, 12 had myocardial infarction, and 6 of these died during hospitalization. In contrast, only 2 of the 48 patients with angina at rest who were negative for troponin T had an acute myocardial infarction, and these patients died. Thus, 12 of the 14 patients with myocardial infarctions had detectable levels of troponin T; only 2 had elevated creatine kinase M activity. Conclusions: Cardiac troponin T in serum appears to be a more sensitive indicator of myocardial-cell injury than serum creatine kinase MB activity, and its detection in the circulation may be a useful prognostic indicator in patients with unstable angina.

Serological evidence for hepatitis E virus infection in Israel.

Israel is suspected to be endemic for hepatitis E virus (HEV) because of its geographic location and the large-scale immigration from endemic countries. Although no cases of local HEV infection have been diagnosed, a serological survey would provide indirect evidence for such infection. We examined sera from 1,416 healthy subjects, including 1,139 Jews from various regions of Israel and 277 Arabs, most of whom reside in the West Bank of the Jordan River. In addition, we tested 13 non-A, non-B, and non-C viral hepatitis patients. Sera were screened for antibody to hepatitis E virus (anti-HEV) by a newly developed enzyme immunoassay (EIA) and by immunoblots for both IgG and IgM anti-HEV activity. Positive samples were confirmed by neutralization. The seroprevalence found by EIA was 2.81% and 1.81% in the Jewish and Arab populations, respectively. More than a 2-fold higher prevalence in males compared to females and an increase with age were found in both populations. However, these differences were nonsignificant. The geographical distribution was even throughout the country, except for two clusters of 3 and 4 seropositive individuals possibly reflecting past foci of infection. Eight of 37 EIA-positive sera were positive for IgG, and 3 were positive for IgM by the immunoblot assay. Among hepatitis patients (9 acute and 4 chronic), one patient with chronic hepatitis was positive for both IgG and IgM. Our study provides indirect evidence that Israel is endemic for HEV.(ABSTRACT TRUNCATED AT 250 WORDS)

Preoperative Myocardial Cell Damage in Patients with Unstable Angina Undergoing Coronary Artery Bypass Graft Surgery

Troponin-T is one of the contractile proteins of the myocardium. Its release into the circulation indicates various degrees of myocardial cell damage. Troponin-T may be measured in serum with a recently developed enzyme immunoassay. This immunoassay was used to evaluate the preoperative myocardial cell damage in patients with stable and unstable angina undergoing elective coronary artery bypass graft surgery, and it was compared with conventional assays of creatine kinase (CK) MB isoenzyme activity and mass. Twenty-one patients with unstable angina and 31 with stable angina were studied. Troponin-T, CK-MB activity, and CK-MB mass were measured 24 h before anesthesia and surgery, immediately before induction of anesthesia, before and after cardiopulmonary bypass, at the end of surgery, and 24 h afterward. In 90% (19 of 21) of the patients with unstable angina, troponin-T was increased 24 h before anesthesia (median 0.33 microgram/l, range 0.15-5.2 micrograms/l), whereas only 3% (1 of 31) of the patients with stable angina had increased values (median 0.0 microgram/l, range 0.0-0.53 microgram/l). The difference was statistically significant (P < 0.001). The same profile was found in patients with and without unstable angina immediately before induction of anesthesia (86% [18 of 21] and 0%, respectively) and before cardiopulmonary bypass (62% [13 of 21] and 0%, respectively). In contrast to troponin-T, CK-MB activity was increased in only 0-14% of patients with unstable angina, and CK-MB mass was increased in only 9-24%. After bypass troponin-T increased in both groups (P < 0.01), but there was no longer a statistically significant difference between the groups. Twelve percent (4 of 31) of the patients in the stable angina group and 28% (6 of 21) in the unstable group had major cardiac events (P not significant). The study data suggest that many patients with unstable angina undergoing elective coronary artery bypass graft surgery have already increased troponin-T levels preoperatively, although conventional biochemical markers such as CK-MB activity and mass are at a normal range. Increased troponin-T and normal CK-MB concentrations may reflect some degree of ischemic myocardial cell damage. Because of the small number of patients in the study, the influence of preoperative myocardial injury on perioperative outcome could not be clarified.

Platelet adhesion and activation under static and flow conditions

An investigation of platelet deposition and activation was carried out using E.C. reference materials (EUROBIOMAT program), applying static and flow test systems. The following materials were investigated: polyethylene (PE), polypropylene (PP), poly(vinyl chloride with di(ethylhexyl) phthalate as plasticizer (PVC-D) and pellethane (PEL). Platelet adhesion and activation were studied under non-flow conditions, applying a recently developed enzyme immunoassay (EIA) for the detection of the platelet proteins GP Ib and GMP 140, also known as PADGEM, after a 30 min incubation. Furthermore, how platelets adhere under laminar flow conditions was examined. These experiments were carried out with use of a capillary perfusion model in order to estimate the adhesion and activation of platelets in open and closed loop systems at a shear rate of 2000 s −1. The contact times between anti-coagulated whole blood and the test materials were from about 2 min for the open loop system to 20 rain for the closed loop system. As parameters, the retention of platelets and the release of β-thromboglobulin were measured. A modified EIA for GMP 140 was applied to obtain more insight into the distribution of platelets over the length of the tubes. It was shown that testing under non-flow conditions yielded comparable data to results obtained under flow conditions in the closed loop system. PE and PP exhibited low haemocompatibility compared with PVC-D and PEL, as indicated by the measurement of GP Ib and GMP 140 under static conditions. The investigation under flow conditions demonstrated generally the same sequence concerning platelet attachment and activation. However, it was shown that platelet retention and activation increased with contact time.

Rochalimaea antibodies in HIV‐associated neurologic disease

Rochalimaea henselae, a recently described pathogen thought to cause syndromes as varied as bacillary angiomatosis, parenchymal bacillary peliosis, fever with bacteremia, and cat-scratch disease, is associated with CNS diseases including cerebral and retinal bacillary angiomatosis, as well as cat-scratch-related encephalitis, myelitis, cerebral arteritis, and retinitis. We used a newly developed enzyme immunoassay and the polymerase chain reaction to investigate the association of R henselae infection with HIV-related CNS disease and found that whereas seroprevalence rates in HIV-positive patients unselected for neurologic disease were 4% to 5.5%, those with neurologic disease had seroprevalence rates of 32%. The ratio of organism-specific antibodies in CSF compared with serum suggested intra-blood-brain-barrier synthesis of these antibodies. CSF specimens containing only R henselae IgM had 16S rDNA specific for R henselae. Stored serum from one of these patients indicated he had developed R henselae-reactive IgM antibodies 10 months prior to the onset of neurologic disease. In the 14 patients for whom clinical data were available, evidence of CNS invasion by R henselae was accompanied by acute and subacute mental status changes including hallucinations, disorientation, and rapidly progressive dementia.