The bioanalysis of trastuzumab in human serum using precipitate-enhanced ellipsometry.

For the bioanalysis of therapeutic monoclonal antibodies in biological matrices, immunoassays--especially enzyme-linked immunosorbent assays (ELISAs)--are the most widely used techniques. Although ELISAs are very sensitive, the obtained sensitivity is not always sufficient. In this study, we have investigated the possibilities of performing a precipitate-enhanced immunoassay (PEIA) with ellipsometric detection for the bioanalysis of the therapeutic monoclonal antibody trastuzumab. Hydrophobic silicon slides were coated with anti-idiotype trastuzumab antibodies. Trastuzumab in serum samples could bind to this primary catcher, and biotinylated anti-idiotype antibodies were used for detection. After binding of streptavidin-poly-horseradish peroxidase (HRP), the precipitating substrate 3,3'-diaminobenzidine tetrahydrochloride (DAB) was added. Precipitation speed was analyzed using a novel prototype eight-cell ellipsometer, and calibration curves were obtained by plotting this speed versus the trastuzumab concentration. Results demonstrate that the PEIA is at least four times more sensitive than the same ELISA using the chromogenic substrate 3,5,3',5'-tetramethylbenzidine (TMB) instead of precipitating DAB. The calibration range of the assay is 11 to 700 pg/ml. Serum samples are diluted 10 times prior to incubation corresponding to 110 to 7000 pg/ml in undiluted serum. Validation results demonstrate that these low concentrations can be analyzed accurately and precisely. In addition, samples of a patient treated with trastuzumab were analyzed with both the PEIA and the ELISA. Results demonstrate excellent correlation (r=0.984) between the methods. Thus, when more sensitivity is required than in a conventional immunoassay, a PEIA with ellipsometric detection may be a useful alternative. The prototype ellipsometer is still in development, and from the data obtained in this study, improvements will be implemented.

Thrombomodulin-modified thrombin generation after in vivo recombinant factor VIII treatment in severe hemophilia A.

BACKGROUND: Thrombin generation has been shown to reflect coagulation potential and factor VIII (FVIII) levels in patients with hemophilia A. We hypothesize that thrombin generation in the presence of thrombomodulin reflects plasma FVIII levels better. DESIGN AND METHODS: Plasma FVIII levels were determined chromogenically and thrombin generation was measured with and without thrombomodulin in 12 patients with severe hemophilia A. Blood was sampled at baseline and 15 min, 1, 3, 6, 24 and 48 hours after recombinant FVIII administration. RESULTS: FVIII administration restored the decreased baseline thrombin generation (reflected by endogenous thrombin potential, peak height, slope and time to peak). Lag time did not change. All thrombin generation parameters except time to peak returned to baseline within 48 hours, while plasma FVIII concentration was increased and time to peak shortened. Endogenous thrombin potential and peak height showed wide inter-individual variation, with strong intra-individual correlations. Addition of thrombomodulin to the assay shortened time to peak and decreased endogenous thrombin potential and peak height. The decrease in peak height was almost completely offset by FVIII administration. Multiple linear regression analysis revealed thrombomodulin-modified thrombin generation to be a moderately better predictor of plasma FVIII levels than thrombin generation in the absence of thrombomodulin (adjusted R(2) 0.79 vs. 0.71). CONCLUSIONS: Addition of thrombomodulin has pronounced effects on all parameters of thrombin generation. This thrombomodulin-modified thrombin generation assay better reflects plasma FVIII levels than thrombin generation in the absence of thrombomodulin.

Correction of feline lipoprotein lipase deficiency with adeno-associated virus serotype 1-mediated gene transfer of the lipoprotein lipase S447X beneficial mutation.

Human lipoprotein lipase (hLPL) deficiency, for which there currently exists no adequate treatment, leads to excessive plasma triglycerides (TGs), recurrent abdominal pain, and life-threatening pancreatitis. We have shown that a single intramuscular administration of adeno-associated virus (AAV) serotype 1 vector, encoding the human LPL(S447X) variant, results in complete, long-term normalization of dyslipidemia in LPL(/) mice. As a prelude to gene therapy for human LPL deficiency, we tested the efficacy of AAV1-LPL(S447X) in LPL(/) cats, which demonstrate hypertriglyceridemia (plasma TGs, >10,000 mg/dl) and clinical symptoms similar to LPL deficiency in humans, including pancreatitis. Male LPL(/) cats were injected intramuscularly with saline or AAV1-LPL(S447X) (1 x 10(11)-1.7 x 10(12) genome copies [GC]/kg), combined with oral doses of cyclophosphamide (0-200 mg/m(2) per week) to inhibit an immune response against hLPL. Within 3-7 days after administration of >or=5 x 10(11) GC of AAV1-LPL(S447X) per kilogram, the visible plasma lipemia was completely resolved and plasma TG levels were reduced by >99% to normal levels (10-20 mg/dl); intermediate efficacy (95% reduction) was achieved with 1 x 10(11) GC/kg. Injection in two sites, greatly limiting the amount of transduced muscle, was sufficient to completely correct the dyslipidemia. By varying the dose per site, linear LPL expression was demonstrated over a wide range of local doses (4 x 10(10)-1 x 10(12) GC/site). However, efficacy was transient, because of an anti-hLPL immune response blunting LPL expression. The level and duration of efficacy were significantly improved with cyclophosphamide immunosuppression. We conclude that AAV1-mediated delivery of LPL(S447X) in muscle is an effective means to correct the hypertriglyceridemia associated with feline LPL deficiency.

Plasma markers of activated hemostasis in the early diagnosis of acute coronary syndromes.

BACKGROUND: Because acute coronary syndromes (ACS) are caused by intracoronary thrombosis, plasma markers of coagulation have relevance for early diagnosis. AIMS AND OBJECTIVES: To provide a critical review of these studies and specific attempts to close the diagnostic time gap left by traditional plasma markers of heart injury. METHODS: Studies of ACS patients, with at least one control group, were included when blood samples were taken within 24 h after first symptoms prior to medication or intervention. Special attention was paid to studies reporting diagnostic performance, or combination of several markers into a single diagnostic index. RESULTS: Markers with short plasma half-life (FPA, TAT, etc.) reflect ongoing thrombosis and may identify patients at increased risk. Markers with longer half-life (F1+2, D-Dimer, etc.) may be more useful to indicate a single acute thrombotic event. However, results are highly variable and depend on sampling time, clot property, degree of coronary obstruction and physiological condition. Early diagnostic performance of hemostatic markers was poor even when combined with heart injury markers. CONCLUSIONS: Early measurement of hemostatic plasma markers in ACS patients provides pathophysiological information and may be helpful in risk stratification or to monitor anticoagulant therapy, but does not seem useful in routine clinical diagnosis of ACS.

Gene therapy for lipoprotein lipase deficiency: working toward clinical application.

Lipoprotein lipase (LPL) deficiency causes hypertriglyceridemia and recurrent, potentially life-threatening pancreatitis. There currently is no adequate treatment for this disease. Previously, we showed that intramuscular administration of an adeno-associated virus serotype 1 (AAV1) vector encoding the human LPL(S447X) variant cDNA (AAV1-LPL(S447X)) normalized the dyslipidemia of LPL-/- mice for more than 1 year. In preparation for a clinical trial, we evaluated the safety and biodistribution of AAV1-LPL(S447X) in wild-type mice and fully characterized six LPL-deficient patients. Toxicological analysis in mice showed that intramuscular administration was well tolerated. Acute inflammatory response markers were transiently increased, and anti- AAV1 antibodies were generated. Histological analyses indicated a dose-dependent reversible spleen hyperplasia, and myositis at the injection sites. Biodistribution data showed short-term vector leakage from injection sites into the circulation, followed by liver-mediated clearance. Persistence of vector DNA was limited to the injected muscle and draining lymph nodes, and spread to reproductive organs was limited. Characterization of LPL-deficient patients showed that all patients presented with hypertriglyceridemia and recurrent pancreatitis. LPL catalytic activity was absent, but LPL protein levels were 20-100% of normal. Myoblasts derived from skeletal muscle biopsies of these patients were efficiently transduced by AAV1-LPL(S447X) and secreted active LPL. These data support the initiation of a clinical trial in LPL-deficient patients, for which regulatory approval has been granted.

Impaired renal clearance explains elevated troponin T fragments in hemodialysis patients.

BACKGROUND: Patients with severe renal dysfunction often have unexplained elevated serum concentrations of cardiac troponin T (cTnT). We investigated whether in vivo fragmentation of cTnT could explain these increases. METHODS AND RESULTS: cTnT, creatine kinase isoenzyme MB, and myoglobin serum concentrations were measured in all 63 dialysis patients of our in-hospital dialysis department. A highly sensitive immunoprecipitation assay, followed by electrophoresis and Western blotting, was used to extract and concentrate cTnT and its possible fragments from serum of these 63 hemodialysis patients. Although creatine kinase isoenzyme MB values excluded recent ischemic myocardial events in 55 of the 63 cases, cTnT fragments ranging in size from 8 to 25 kDa were present in the serum samples of all dialysis patients. CONCLUSIONS: cTnT is fragmented into molecules small enough to be cleared by the kidneys of healthy subjects. Impaired renal function causes accumulation of these cTnT fragments and is very likely the cause of the unexplained elevations of serum cTnT found in patients with severe renal failure.

Cells in human postmortem brain tissue slices remain alive for several weeks in culture.

Animal models for human neurological and psychiatric diseases only partially mimic the underlying pathogenic processes. Therefore, we investigated the potential use of cultured postmortem brain tissue from adult neurological patients and controls. The present study shows that human brain tissue slices obtained by autopsy within 8 h after death can be maintained in vitro for extended periods (up to 78 days) and can be manipulated experimentally. We report for the first time that 1) neurons and glia in such cultures could be induced to express the reporter gene LacZ after transduction with adeno-associated viral vectors and 2) cytochrome oxidase activity could be enhanced by the addition of pyruvate to the medium. These slice cultures offer new opportunities to study the cellular and molecular mechanisms of neurological and psychiatric diseases and new therapeutic strategies.

Fatty acid-binding proteins as plasma markers of tissue injury.

BACKGROUND: One of the novel and promising plasma markers for detection of tissue injury is the family of 15 kDa cytoplasmic fatty acid-binding proteins of which various tissue-specific types occur. AIMS AND OBJECTIVES: The present status of heart-type fatty acid-binding protein (H-FABP) as a diagnostic and prognostic marker for acute and chronic cardiac injury, as well as the preliminary diagnostic use of other types of FABP for detecting injury in other organs, is reviewed. METHODS: This review is based on an overview of the literature on clinical diagnostics of various forms of organ injury, and uses additional literature on physiological aspects relevant for the interpretation of plasma marker concentrations. RESULTS: H-FABP not only proves to be an excellent early marker for cardiac injury in acute coronary syndromes, but also allows detection of minor myocardial injury in heart failure and unstable angina. Preliminary results indicate that sensitivity, rule-out power and prognostic value of H-FABP in cardiac injury surpass the performance of the standard early marker myoglobin. The liver only contains liver-type FABP (L-FABP), but co-expression of H-FABP and L-FABP occurs in the kidney. Similarly, intestinal-type FABP (I-FABP) and L-FABP are found in intestines, and brain-type FABP (B-FABP) and H-FABP occur in the brain. Preliminary but promising applications of these proteins have been demonstrated for liver rejection, viability selection of kidneys from non-heart-beating donors (NHBD), inflammatory and ischemic bowel disease, traumatic brain injury and in the prevention of muscle injury in trained athletes. CONCLUSIONS: Further study of the diagnostic and prognostic use of various FABP types is warranted, but their clinical application will require further commercialization of automated and rapid assays.

Secretory type II phospholipase A(2) binds to ischemic myocardium during myocardial infarction in humans.

OBJECTIVE: An increase of circulating secretory Phospholipase A(2) (sPLA(2)) is a risk factor for coronary artery disease. We hypothesized that this reflects participation of sPLA(2) in local inflammatory reactions ensuing in ischemic myocardium. Therefore, we studied the course of circulating sPLA(2), in patients with acute myocardial infarction (AMI) or unstable angina pectoris (UAP), and investigated the presence of sPLA(2) in infarcted myocardial tissue. METHODS: Plasma samples of 107 patients with AMI or UAP, collected on admission and at varying intervals thereafter, were tested for the presence of sPLA(2) and C-reactive protein (CRP). Cumulative release values of these parameters were calculated, which allowed for comparison of the results rearranged in time according to the onset of symptoms. By immunohistochemistry we studied the presence of sPLA(2) and CRP in myocardial tissue of 30 patients who died subsequent to AMI. RESULTS: Levels of sPLA(2) became elevated during the disease course in 66 of the 87 patients with AMI, and were higher than those of the patients with UAP of whom 8 of the 20 had elevated levels. By immunohistochemistry sPLA(2) was found to be localized in the infarcted myocardium, particularly in its borderzone, from 12 h after the onset of AMI. Positive staining for sPLA(2) was more extensive than that for CRP. CONCLUSIONS: The localization pattern of sPLA(2) in infarcted myocardium as well as its plasma course, in relation to those of CRP, are in line with a supposed pro-inflammatory role during AMI for sPLA(2) as a generator of lysophospholipids serving as ligands for CRP.

Long-term correction of murine lipoprotein lipase deficiency with AAV1-mediated gene transfer of the naturally occurring LPL(S447X) beneficial mutation.

Human lipoprotein lipase (LPL) deficiency causes profound hypertriglyceridemia and life-threatening pancreatitis. We recently developed an adult murine model for LPL deficiency: LPL -/- mice display grossly elevated plasma triglyceride (TG) levels (>200-fold) and very low high-density lipoprotein cholesterol (HDL-C < 10% of normal). We used this animal model to test the efficacy of adeno-associated virus-mediated expression of hLPL(S447X) (AAV1-LPL(S447X)) in muscle for the treatment of LPL deficiency. Intramuscular administration of AAV1-LPL(S447X) resulted in dose-dependent expression of hLPL protein and LPL activity (up to 33% of normal murine levels) in postheparin plasma. Remarkably, visible hyperlipidemia was resolved within 1 week; plasma TG was reduced to near-normal levels (from 99.0 to 1.8 mmol/L), and plasma HDL-C was increased 6-fold (from 0.2 to 1.1 mmol/L). At 8 months after administration of AAV1-LPL(S447X), an intravenous lipid challenge showed efficient, near-normal clearance of plasma TG. Histologic analyses of injected muscle further indicated that abnormal muscle morphology observed in LPL -/- mice was reversed after treatment. Expression of therapeutic levels of LPL(S447X), and the subsequent beneficial effect on plasma lipid levels, has lasted for more than 1 year. We therefore conclude that AAV1-mediated transfer of LPL(S447X) into murine skeletal muscle results in long-term near-correction of dyslipidemia associated with LPL deficiency.

Pharmacokinetics of C1-inhibitor protein in patients with acute myocardial infarction.

OBJECTIVES: C1-inhibitor protein (C1-INH) purified from pooled human plasma is used for the treatment of patients with hereditary angioedema. Recently, the beneficial effects of high-dose C1-INH treatment on myocardial ischemia or reperfusion injury have been reported in various animal models and in humans. We investigated the pharmacokinetic behavior of C1-INH in patients with acute myocardial infarction to calculate the amount of C1-INH required for optimal efficacy. METHODS: Twenty-two patients received an intravenous loading dose, followed by 48 hours of continuous infusion of C1-INH. Changes in the endogenous production of C1-INH were evaluated in 16 control patients with acute myocardial infarction. A 2-compartment model was used to estimate the fractional catabolic rate constant (FCR), transcapillary escape rate constant (TER), and extravascular return rate constant (ERR) of C1-INH. Software designed to analyze and fit measured data to unknown parameters in a system of differential equations was used to fit the experimental data against the 3-parameter model. RESULTS: With fixed TER and ERR values (0.014 h(-1) and 0.018 h(-1), respectively), 20 of the 22 cases yielded well-determined FCR values, and simultaneous fitting resulted in a median FCR of 0.011 h(-1) (95% confidence interval, 0.010 to 0.012 h(-1)) versus 0.025 h(-1) as reported in healthy control patients. Simultaneous estimation of TER, ERR, and FCR demonstrated weakly defined TER and ERR values, whereas the median FCR value remained unchanged. The use of a 2-compartment model resulted in a significantly better fit compared with the 1-compartment model. Physiologic explanations are offered for discrepancies in the literature. CONCLUSIONS: Dose calculation of C1-INH in patients treated with massive doses of C1-INH requires turnover parameters that differ from those found in healthy subjects, possibly because of suppression of continuous C1-INH consumption by target proteases.

Anticoagulant and membrane-degrading effects of secretory (non-pancreatic) phospholipase A2 are inhibited in plasma.

Plasma concentrations of secretory (non-pancreatic) phospholipase A2 (sPLA2) may rise 1000-fold during inflammation, and this acute phase response has been related to anticoagulant effects. In the present study this hypothesis was further investigated. Prothrombinase activity was measured for model membranes mimicking the phospholipid composition of the outer membrane of resting and activated blood platelets. Using ellipsometry, membrane degradation by sPLA2 could be measured simultaneously with inhibition of thrombin production. The same technique was used to study clotting, by the sudden appearance of fibrin strands on the membrane. Results were compared with the effects of sPLA2 on the activation of washed platelets and platelets in plasma. In buffer solution, model membranes were degraded by (patho)physiological concentrations of sPLA2. Even when only partially degraded, membranes rapidly lost their prothrombinase activity, indicating preferential degradation of phosphatidylserine. Addition of diluted plasma interfered with membrane degradation, and also with inhibition of prothrombinase activity. In agreement with these observations, sPLA2 inhibited thrombin production and annexin V-binding of activated washed platelets, but had no effects on platelet activation or clotting in plasma. These findings indicate that the elevated plasma sPLA2 concentrations observed in inflammatory disease will not reduce hypercoagulability in such patients.

Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors.

BACKGROUND: Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV) and lentiviral (LV) vectors into discrete regions of the forebrain. RESULTS: Recombinant AAV-Cre, AAV-GFP (green fluorescent protein) and LV-Cre-EGFP (enhanced GFP) were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. CONCLUSION: AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.

Evaluation of "new" cardiac markers for ruling out myocardial infarction after coronary artery bypass grafting.

STUDY OBJECTIVES: This study was conducted to evaluate the value of serum troponin T, myoglobin, and creatine kinase (CK)-MB mass concentrations for ruling out perioperative myocardial infarction (poMI) early after cardiac surgery. DESIGN: Retrospective study. SETTING: Cardiothoracic surgery department in a university hospital. PATIENTS: One hundred eighty-one patients undergoing coronary artery bypass grafting (CABG) with cardiopulmonary bypass were included. METHODS: Serum concentrations of troponin T, myoglobin, and CK-MB mass were measured preoperatively (baseline), on arrival at the cardiosurgical ICU (CICU), and at 2, 4, 8, 12, 16, and 20 h after arrival at the CICU. The strength of markers studied for ruling out poMI was studied using receiver operating characteristics curves. Based on these curves, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for each marker at every time point were calculated. RESULTS: poMI developed in 14 patients. On arrival at the CICU, all markers were significantly increased from baseline concentrations in both patient groups. In patients with poMI, serum concentrations of troponin T, myoglobin, and CK-MB mass were significantly higher than in control patients from 8, 2, and 0 h after arrival on the CICU, respectively. CK-MB mass was the earliest marker, and its NPV reached 98.6% 12 h after arrival at the CICU. On arrival at the CICU, the NPV for CK-MB mass already reached 96.7%. The NPV for myoglobin reached 98.4% 12 h after arrival at the CICU. Troponin T was not an early marker for ruling out poMI, with an NPV reaching 98.6% 12 h after arrival on the CICU. During the first 8 h after arrival at the CICU, sensitivity, specificity, PPV, and NPV of CK-MB mass exceeded those of myoglobin and troponin T. In later measurements (until 20 h after arrival at the CICU), troponin T gave the most sensitive definition of poMI. CONCLUSIONS: For ruling out poMI on the CICU after CABG, CK-MB mass is a better marker than myoglobin and troponin T during the first 12 h after arrival on the CICU. Using these markers, postoperative treatment of cardiac surgical patients might be further improved.

Intestinal-type and liver-type fatty acid-binding protein in the intestine. Tissue distribution and clinical utility.

OBJECTIVES: Intestinal-type fatty acid-binding protein (I-FABP) has been proposed as plasma marker for the detection of acute intestinal injury. However, intestinal mucosa also expresses liver-type FABP (L-FABP). We have investigated the tissue distribution of I-FABP and L-FABP in segments of the human intestine along the duodenal to colonal axis and the potential of both proteins to serve as plasma marker for the diagnosis of intestinal injury. DESIGN AND METHODS: I-FABP and L-FABP were measured with specific immunoassays in autopsy samples of the intestine (duodenum, jejunum, ileum and colon) of 23 subjects and in plasma samples from patients (n = 51) with intestinal and/or hepatic disease. Plasma reference values were established in normal healthy individuals (n = 92). RESULTS: The I-FABP tissue contents in duodenum, jejunum, ileum, proximal colon and distal colon amounted to 2.22, 4.79, 1.04, 0.27 and 0.25 mug/g ww, respectively. L-FABP tissue contents were markedly higher, amounting to 124 and 198 mug/g ww in duodenum and jejunum, and to 58, 26 and 44 mug/g ww in ileum, proximal colon and distal colon, respectively. Elevated plasma levels of both I-FABP and L-FABP were found in patients suffering from intestinal diseases, while only L-FABP was increased in cases of purely hepatocellular injury. CONCLUSIONS: I-FABP and L-FABP show a similar pattern of tissue distribution along the duodenal to colonal axis with highest tissue contents found in the jejunum but in each intestinal segment a >40-fold higher content of L-FABP than of I-FABP. Accordingly, besides I-FABP, also L-FABP is a useful plasma marker for the detection of intestinal injury, especially in patients undergoing intestinal surgery.

Prevention of cardiac cell injury during acute myocardial infarction: possible role for complement inhibition.

The purpose of this article is to describe mechanisms of cell death in patients with acute myocardial infarction, particularly the activation of the complement system. Various pro-inflammatory cytokines, released by the inflamed tissue, play a role in the activation of the complement system. Several complement inhibitors have been developed to reduce tissue damage following ischemia. According to animal studies the deleterious effects of activators of the complement system can be diminished by complement inhibition. Several clinical studies have been conducted for the potential treatment of cell injury during acute myocardial infarction. C1 inhibitor dose-dependently inhibited complement activation and appeared to reduce myocardial injury after reperfusion therapy in patients with acute myocardial infarction. C1 inhibitor dose-dependently reduced plasma levels of C4 activation fragments. In addition, cardiac enzymes (troponin T and creatine kinase-MB) returned to baseline levels more rapidly among patients treated with C1 inhibitor, compared with controls. Furthermore, preliminary results from a placebo-controlled trial indicate that treatment with intravenous pexelizumab (anti-C5 antibody) was well tolerated in a large number of patients undergoing coronary artery bypass graft surgery. Further, more randomized trials are necessary to clarify the clinical significance of this new and innovative treatment with complement inhibition.

Label-free assessment of high-affinity antibody-antigen binding constants. Comparison of bioassay, SPR, and PEIA-ellipsometry.

Assessment of high-affinity antibody-antigen binding parameters is important in such diverse areas as selection of therapeutic antibodies, detection of unwanted hormones in cattle and sensitive immunoassays in clinical chemistry. Label-free assessment of binding affinities is often carried out by immobilization of one of the binding partners on a biosensor chip, followed by monitoring the binding equilibrium of the other partner. However, for the measurement of high-affinity binding, with dissociation constants in the picomolar range or lower, equilibration times exceed practical limits and one has to resort to the measurement of sorption kinetics. Here we evaluate a new technique, using PEIA(1)-ellipsometry and establishment of equilibrium in solution. Binding parameters are determined for two high-affinity anti-interleukin 6 antibodies, anti-IL6.16 and anti-IL6.8, and compared with values obtained by a bioassay, based on IL6-dependent cell growth, and with values obtained by a standard technique based on SPR.(2) The high affinities of both antibodies as found with the bioassay (5 and 50pM for anti-IL6.8 and anti-IL6.16, respectively), could be conveniently measured by PEIA-ellipsometry. Using SPR, equilibrium measurements indeed proved too time-consuming and analysis of adsorption/desorption kinetics revealed that the binding of the antibodies on the chip caused the appearance of different populations of antibodies with different affinities.

Real-time monitoring of melittin-induced pore and tubule formation from supported lipid bilayers and its physiological relevance.

The temporal evolution of effects of antimicrobial peptide melittin on supported phospholipid bilayers (SPBs) containing negatively charged phospholipids was monitored by ellipsometry and laser scanning microscopy together with measurements of lipid mobility by Z-scan fluorescence correlation spectroscopy. Under all conditions used in our study, we observed reproducibly two effects. The first one is formation of pores in the SPB, which occupy approximately 40% of the bilayer. The formation of pores was accompanied by a decrease in lateral diffusion coefficient of the lipids to approximately 60% of its initial value. The second, simultaneous, effect is the formation of tubules of approximately 30nm radius and length of the order of 10mum. Flushing of the sample with excess of buffer removes most of the tubules, but it does not affect the pores. Further experiments performed under various conditions demonstrated reproducibility of both phenomena.