Surface plasmon resonance biosensors as a tool in antibody engineering

The objective of this study was to perform intracytoplasmic sperm injection (ICSI) on in vitro matured equine oocytes and to improve in vitro embryonic development on Vero cells after activation of the microinjected oocytes with calcium ionophore. After maturation (23 or 40 h, 38.5 degrees C, 5% CO2), the cumulus-oocyte complexes were denuded, centrifuged and all oocytes exhibiting the first polar body were microinjected. ICSI was performed using fresh semen from three fertile stallions. Microinjected oocytes were activated with calcium ionophore A23187 (10 min, 10 microM) and cultured individually for 7 days on Vero cells in microdrops. In seven trials, 353 cumulus-oocyte complexes were matured and 103 oocytes were microinjected. Eight oocytes were sham microinjected. After ICSI, 85 oocytes (82.5%) survived the sperm injection procedure. Among the 76 successfully microinjected oocytes, 52 (68%) were fertilized (two pronuclei, syngamy stage and cleaved ova). Sham microinjected oocytes were not activated. After in vitro culture, 35 ova (46%) were cleaved 2 days after ICSI and early embryonic development was obtained (three embryos of 23 cells, 50 cells and more than 80 cells) 5 to 7 days after ICSI.     

Surface plasmon resonance sensor for heparin measurements in blood plasma

Surface plasmon resonance (SPR) was used as an affinity biosensor to determine absolute heparin concentrations in human blood plasma samples. Protamine and polyethylene imine (PEI) were evaluated as heparin affinity surfaces. Heparin adsorption onto protamine in blood plasma was specific with a lowest detection limit of 0.2 U/ml and a linear window of 0.2-2 U/ml. Although heparin adsorption onto PEI in buffer solution had indicated superior sensitivity to that on protamine, in blood plasma it was not specific for heparin and adsorbed plasma species to a steady-state equilibrium. By reducing the incubation time and diluting the plasma samples with buffer to 50%, the non-specific adsorption of plasma could be controlled and a PEI pre-treated with blood plasma could be used successfully for heparin determination. Heparin adsorption in 50% plasma was linear between 0.05 and 1 U/ml so that heparin plasma levels of 0.1-2 U/ml could be determined within a relative error of 11% and an accuracy of 0.05 U/ml.     

Electropolymerization of a phenol-modified peptide for use in receptor-ligand interactions studied by surface plasmon resonance

The combination of surface plasmon resonance (SPR) with an electrochemical method for surface modification is presented. The SLP1 sequence of the sodium channel protein of rat cardiac muscle cells was N-terminally modified with an electropolymerizable group and immobilized on a gold-coated glass slide by oxidative polymerization. The resulting peptide-functionalized substrate was incubated with a polyclonal-specific anti-SLP1 serum. Growth of the peptide layer and the immunological reaction between ligand and receptor were detected on-line by SPR. The applicability of this approach for the rapid and selective analysis of receptor-ligand interactions is demonstrated.     

Surface plasmon resonance for detection and measurement of antibody-antigen affinity and kinetics

Kinetic and affinity information on biospecific interaction can be generated by analysis of adsorption of analyte to immobilized ligand on a sensor based analytical system. The analysis can be performed free of label and directly using culture supernatants. It was found to be particularly valuable for analysis of antibody-antigen interactions.     

Immobilized chicken antibodies improve the detection of serum antigens with surface plasmon resonance (SPR)

BACKGROUND: There is currently no validated questionnaire that assesses both the presence and severity of dyspepsia. AIM: To develop the Leeds Dyspepsia Questionnaire (LDQ) as a measure of the presence and severity of dyspepsia, and to assess the validity, reliability and responsiveness of this instrument. METHODS: Unselected patients attending either a hospital dyspepsia clinic or a general practice surgery were interviewed by a trained gastroenterologist or a general practitioner on the presence and severity of dyspepsia. This opinion was compared with the results of the nurse-administered LDQ. Test-retest reliability was assessed by the same research nurse re-administering the LDQ 4-7 days after the initial visit in a subgroup of hospital patients. In a further subgroup of patients one researcher interviewed the patients and a second researcher re-administered the LDQ within 30 min to evaluate inter-rater reliability. The responsiveness of the LDQ was measured by repeating it in patients with endoscopically proven peptic ulcer or oesophagitis 1 month after receiving appropriate therapy. RESULTS: The LDQ was administered to 99 general practice and 215 hospital patients. In the GP population 41/98 (42%) had dyspepsia according to the GP and the LDQ had a sensitivity of 80% (95% CI: 65-91%) and a specificity of 79% (95% CI: 66-89%). The weighted kappa statistic for the agreement between the LDQ and the clinician for the severity of dyspepsia was 0.58 in the GP population and 0.49 in hospital patients. The kappa statistic for test-retest reliability was 0.83 in 107 patients. The LDQ had excellent inter-rater reliability with a kappa statistic of 0.90 in 42 patients. The median LDQ score fell from 22.5 (range 9-36) to 4.5 (range 0-27) in 12 patients 1 month after receiving appropriate therapy (Wilcoxon signed rank test, P < 0.0001). CONCLUSION: The LDQ is a valid, reliable and responsive instrument for measuring the presence and severity of dyspepsia.     

Comparison of enzyme immunoassays for antibodies to Haemophilus ducreyi in a community outbreak of chancroid in the United States

The performance of two EIAs (adsorption EIA and lipooligosaccharide [LOS] EIA) that detect antibodies to Haemophilus ducreyi was evaluated with serum specimens obtained from 163 patients (96 with genital ulcer disease [GUD]). Paired serum specimens (initial and follow-up) were obtained from 52 of the GUD patients. By use of initial serum specimens from 82 GUD patients whose etiologic agents for their ulcers had been identified, the adsorption EIA had a sensitivity and specificity for chancroid of 53% and 71%, while the LOS EIA had a sensitivity and specificity of 48% and 89%, respectively. Sensitivity and specificity of the adsorption EIA increased to 78% and 84%, respectively, when the results of follow-up serum specimens were used to calculate optimal performance. The proportion of patients testing positive for H. ducreyi who had anti-H. ducreyi IgG antibodies, as determined by adsorption EIA, increased with the duration of infection, thus limiting the role of EIAs in the diagnosis of chancroid.     

Beyond all measures: criteria for the use and selection of a collection agency in a medical practice

The need for protecting the accounts receivable of a medical practice by using collection agencies is well known. This paper provides background material and information necessary to establish criteria for placing accounts with a collection agency.     

Therapeutic use of monoclonal antibodies: a new era?

The serum pyruvate and lactate levels were studied after exercise on a bicycle ergometer in a family of diabetes mellitus (DM) associated with a mutation at nucleotide 3243 in the mitochondrial gene. A 56-year-old Japanese woman with the mutation at a percentage of 5% in the blood had insulin-dependent DM and sensory hearing loss without muscle symptoms. Her serum lactate and pyruvate levels increased markedly during and after exercise on a bicycle ergometer. Two of her sons were found to have the same mutation at a percentage of 17% and 18%, respectively. Her 26-year-old son was found to have borderline DM after oral glucose loading, although he showed no abnormalities of the metabolism of pyruvate and lactate. Her 31-year-old son showed no abnormalities after oral glucose loading and after exercise on a bicycle ergometer. Although the same mutation causes more severe MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), little is known about whether these diabetic patients are subclinically involved with myopathy. The noninvasive ergometer exercise with determination of serum pyruvate and lactate may be useful in evaluating the severity of myopathy in these patients.     

Covalent coupling of immunoglobulin G to self-assembled monolayers as a method for immobilizing the interfacial-recognition layer of a surface plasmon resonance immunosensor

Protocols have been developed for the random and site-directed covalent coupling of immunoglobulin G [anti-hIgG] [IgG] to silver surfaces modified with a self-assembled monolayer [SAM] of thioctic acid, mercaptopropionic acid [MPA], L-cysteine or 4-aminothiophenol [PATP]. A surface plasmon resonance [SPR] immunosensor fabricated with a more ordered and hydrophilic IgG-SAM-silver interfacial layer, demonstrates an increased ability for performing sensitive and selective assay of human immunoglobulin G [hIgG] compared with a device fabricated with a physically-adsorbed IgG-silver interfacial-layer due to reduced levels of non-specific binding. Detection limits [DL] for hIgG from serum down to 6 micrograms/ml (40 nM) and assay sensitivities up to 0.24 ng hIgG/mm2/nM are reported.     

Use of colloidal gold surface plasmon resonance peak shift to infer affinity constants from the interactions between protein antigens and antibodies specific for single or multiple epitopes

The surface plasmon resonance (SPR) wavelength of colloidal gold particles coated with a monoclonal antibody is red-shifted when the antibody interacts with its specific ligand. This shift results from the change in the refractive index of the particles as induced by ligand binding. This property is used to monitor in real-time the association and dissociation kinetics of the interaction in solution. The monitoring is performed in a clinical chemistry automated analyzer during a few minutes of incubation at 37 degrees C. Data treatment allows calculation of the affinity constant of the interaction. The SPR wavelength shift does not necessarily require agglutination or aggregation of the particles to occur since particles coated with one monoclonal antibody specific for a single epitope on the ligand can be used in the procedure. The affinity constants measured by this procedure correlate with those calculated from Scatchard plots or BIAcore data.     

Interaction of heparin with fibrinogen using surface plasmon resonance technology: investigation of heparin binding site on fibrinogen

Persistence of the Lyme disease spirochete, Borrelia burgdorferi, in the presence of an active immune response has been well documented. Evidence from the past year indicates that modulation of surface antigens by the spirochete may be a major mechanism for evading the immune response.     

The use of monoclonal antibodies for detecting the influenza virus

The possibilities of using influenza A (Leningrad) 385/80 (H3N2) virus matrix protein-specific FITC-labeled D8 monoclonal antibodies in immunofluorescence assays were investigated. The virus antigen accumulation was detected in chorioallantoic cells of chick embryos. Exhibiting the type-specific properties, the fluorescent antibodies stain the perinuclear space, cytoplasmic membrane, and granular structures in the cytoplasm of infected cells. The haemagglutination test tires in the corresponding specimens were at least 1:16.     

Limitations on the use of Strong sales keys for selection and counseling.

"Two custom-built Strong sales keys were validated on two different types of salesmen (N = 578) in the same company. Each key was valid only for the type of salesman used in the item analysis to construct the key. The absence of significant correlation between the two keys indicated the independence of the two sales interest patterns related to success in different sales positions in one company… . These findings suggested that valid sales interest patterns can be quite specific, and that the available Strong sales keys might give misleading results if used for selection and counseling purposes in some sales areas." (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Racial bias in the use of work samples for personnel selection.

Examined the role of evaluation specificity and task relevance in explaining racial bias in the use of work samples. 56 White maintenance mechanics evaluated a videotaped performance of a Black job applicant and a White job applicant performing a relevant task (laying out, drilling, and tapping) and an irrelevant task (indexing drill bits). The applicants were evaluated by using a highly specific behavioral recording form, a global rating scale, or both. Race-linked bias was found only when Ss were asked to make global evaluations after observing an applicant's performance on a task representing irrelevant job behavior. Race-linked bias was not found when Ss used the behavioral recording form or in global evaluations made following the behavioral recordings. Race-linked bias was not evident when evaluations were based on observations of relevant job behavior. It is concluded that by using careful work sample development procedures and by assisting Ss in focusing on and recording relevant behavior, the potential for bias in the use of work samples appears small. (15 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Phosphorus nuclear magnetic resonance: a non-invasive technique for the study of muscle bioenergetics during exercise

Phosphorus nuclear magnetic resonance (31P NMR) spectroscopy is a non-destructive analytical laboratory technique that, due to recent technical advances, has become applicable to the study of high-energy phosphate metabolism in both animal and human extremity muscles (in vivo). 31P NMR can assay cellular phosphocreatine, ATP, inorganic phosphate, the phosphorylated glycolytic intermediates, and intra-cellular pH in either resting or exercising muscle, in a non-invasive manner. NMR uses non-perturbing levels of radio-frequency energy as its biophysical probe and can therefore safely study intact muscle in a repeated fashion while exerting no artifactual influence on ongoing metabolic processes. Compared with standard tissue biopsy and biochemical assay techniques, NMR possesses the advantages of being non-invasive, allowing serial in situ studies of the same tissue sample, and providing measurements of only active (unbound) metabolites. NMR studies of exercising muscle have yielded information regarding fatigue mechanisms at the cellular level and are helping resolve long-standing questions regarding the metabolic control of glycolysis, oxidative phosphorylation, and post-exercise phosphocreatine re-synthesis. NMR is also being utilized to measure enzymatic reaction rates in vivo. In the near future, other forms of NMR spectroscopy may also permit the non-invasive measurement of tissue glycogen and lactate content.     

Prevalence and a drug use development model for the study of adolescent drug use in Japan

The purposes of this study were to estimate the prevalence of drug use, to examine the correlation between drug use and drinking/smoking in Japan, and to test a developmental model of adolescents' drug use based on three basic theories of deviance: Strain, Social control, and Differential association. The survey was conducted from April to May, 1996, in 14 public high schools in Tokyo, using anonymous self-reporting. A total of 4,171 (99.45%) students responded to our questionnaire. It included central concept items in the three deviant theories, beliefs about the ill effects of drugs, sex, grades, etc. The following findings were obtained: 1) Among the subjects, 255 students (6.1%) reported drug use within the past year. Of these drug users, 73.3% used drugs soon after being "tempted" by friends. Responding to "When was it.", as a first year junior high school was the most common first experience, and the next most common was during the sixth year of elementary school. 2) Drinking and smoking appeared to be gateway drugs for adolescents in Japan as has been shown in the United States. 3) Multiple regression and logistic multiple regression analyses suggested that differential association variables were far more powerful predictors of adolescent drinking, smoking and drug use than either the control or strain variables. Results provided modest support for the Differential Association Theory as an explanation of drug use. 4) LISREL's goodness-of-fit statistic indicated a much better fit between the model and the data. (CN: 282, GFI: 0.967, AGFI: 0.941). These findings show two processes by which adolescents become involved in drug use. Strain and Social Control do not directly affect drug use. However, Social Control is important because it works indirectly, through Differential Association resulting in drug use. Second, though weaker, Differential Association appears to lead to dangerous beliefs in drug use followed by actual drug use.     

Prevalence of hepatitis A antibodies in southeastern Spain: a population-based study

OBJECTIVES: To identify variables, other than legal definitions, that may have influenced North Cheshire's Coroner in returning a verdict of 'suicide' or an 'open verdict' on unexpected deaths of the elderly. DESIGN: Retrospective review of all coroner's verdicts in North Cheshire during a 5-year period 1989-1993. MATERIAL: Forty-seven 'suicide' and 'open verdict' deaths in North Cheshire between 1989 and 1993 as defined in ICD classification 'E' codes E950-E959 and E980-E989, respectively, that were returned on the deceased aged 65 and above who died within North Cheshire. FINDINGS: Logistic regression analysis suggests that the Coroner's decision may be influenced by: intimation of intent, psychiatric history and method of death. Sex of the deceased, history of attempted suicide, social setting and history of alcohol problems did not appear significantly associated with coroner's verdict.