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Die COVID-19-Pandemie machte deutlich, wie wichtig die Verfügbarkeit eines Schnelltests für ein funktionierendes Gesundheitssystem ist. Mit dieser einfachen Technologie kann die Unsicherheit in den unterschiedlichen und teils kontroversen statistischen Kennzahlen signifikant verringert werden. Immunoassays wie LFIA, FPIA und ELISA (Lateral Flow Immunoassay, Fluoreszenzpolarisationsimmunoassay und Enzymimmunoassay) werden in der Diagnostik und der Umweltanalyse eingesetzt. Die Verwendung von LFIA für ein Screening vor Ort bietet eine Alternative zu instrumentellen Methoden sowie komplizierteren Immunoassay-Formaten und liefert innerhalb von 10 bis 15 Minuten Ergebnisse. Verfahren mit Membranen als feste Träger erlauben parallele Assays in derselben Probe. In dieser Arbeit wurden Latex-Mikropartikel-basierte und Gold-Nanopartikel-basierte LFIAs zum Nachweis von Bisphenol A (BPA) entwickelt. Ein Ziel der Arbeit war die Entwicklung von Nitrocellulose-Membranen, Konjugatpads, Membranbehandlungen sowie einer geeigneten LFIA-Vorbehandlung zwecks Optimierung. Die Ergebnisse eines LFIA-Tests können sowohl mit bloßem Auge als auch instrumentell interpretiert werden. Die visuelle Nachweisgrenze (vLOD) wurde bei 10 μg/L gefunden, die berechnete instrumentelle Nachweisgrenze (cLOD) war 0,14 μg/L. Die Synthese der Haptenproteinkonjugate und deren weitere Bewertung in einem ELISA-Aufbau führte zu einem hochempfindlichen Assay mit einer Nachweisgrenze von 0,05 μg/L. Zusätzlich wurde ein Mix-and-Read-FPIA zur Bestimmung von BPA entwickelt. Dafür wurden neue Tracer-Moleküle, welche Fluorophore mit Derivaten des Analyten verbinden, einschließlich eines C6-Spacers (Ahx) synthetisiert. Der Einfluss der Ahx-Tracer-Brückenlänge auf die Assay-Empfindlichkeit wurde abgeschätzt, die Nachweisgrenze lag bei 1,0 μg/L mit einem Arbeitsbereich von 2 bis 155 μg/L. Die Methoden wurden für reale Proben gegen LC-MS/MS als Referenzmethode mit guter Übereinstimmung mit LFIA, FPIA und ELISA validiert. / The COVID-19 pandemic made it obvious how important the availability of a rapid test is for an efficient healthcare system. This simple technology can significantly reduce the uncertainty in the various and sometimes highly controversial statistical figures of a pandemic. Immunoassays, like LFIA, FPIA and ELISA (lateral flow immunoassay, fluorescence polarization immunoassay, enzyme immunoassay) are used in diagnostics and environmental analysis. The use of LFIA for on-site screening is an alternative to instrumental methods and to more sophisticated immunoassay formats. Results from LFIA are obtained within 10 to 15 min. Methods that use membranes as solid supports allow parallel assays to be performed in the same sample. In this work, latex microparticles-based and gold nanoparticles-based LFIAs for a rapid detection of bisphenol A (BPA) were developed. The work focused on the search for suitable nitrocellulose membranes, conjugate pads, membrane treatments, as well as for a proper LFIA pretreatment to optimize the performance. The results of an LFIA test can be interpreted both by naked eye and instrumentally. The visual limit of detection (vLOD) was found to be 10 μg/L, the calculated instrumental limit of detection (cLOD) was 0.14 μg/L. The synthesis of the required hapten-protein conjugates and further evaluation in an ELISA setup resulted in a highly sensitive assay with a limit of detection of 0.05 μg/L. Additionally, a mix-and-read FPIA for determination of BPA was developed. Here, new tracer molecules that link fluorophores to derivatives of the analyte were synthesized, including a C6 spacer (Ahx). The influence of the Ahx tracer bridge length on the assay sensitivity was estimated. The limit of detection was 1.0 μg/L with a working range from 2 to 155 μg/L. The methods were validated for real samples against LC-MS/MS as reference method with good agreement with LFIA, FPIA, and ELISA.

Antibodies specific for insulin and human immunoglobulin G (HlgG) were attached to controlled pore glass (CPG) particles which had been silanized with a diol-bearing silane. Up to 20 mg of antibody protein could be attached covalently to 1 gram of CPG. Such immobilized antibodies, or immunosorbents, would bind specific antigens, but not unrelated proteins, when used in a high pressure liquid chromatographic configuration. This technique was given the name "high performance immunoaffinity chromatography" (HPIC). The HPIC properties of these immunosorbents were evaluated by an equilibrium theory and were found to be comparable to batch values. An immunosorbent for HIgG antigen showed an HPIC association constant of 10⁷·⁶; the batch equilibrium constant for the same immunosorbent was 10⁷·⁸. Two different anti-insulin immunosorbents retained the intrinsic affinity (10⁶ and 10⁹) of the antibody used to make them. The total active antibody concentrations of these immunosorbents were evaluated by HPIC and batch methods with good agreement between the two. The immobilization reaction was seen to result typically in the loss of 90% of the original antibody activity. HPIC was shown to be applicable to the rapid analysis of antigens at levels as low as ng/mL. This was found to be possible in part because of the rapid forward kinetics which were assessed by HPIC. A forward rate constant of 3 X 10⁷ L·mol⁻¹·sec⁻¹ for the binding of insulin by a specific HPIC column could be determined. The possibility of HPIC fluorescence immunoassays was investigated using a highly sensitive fluorescence detector. An Eimac collimated xenon arc lamp provided sufficient power to detect picomolar levels of fluorescamine labeled insulin and other compounds. The limitations of HPIC in performing picomolar immunoassays were thus shown to be immunochemical rather than instrumental. The ability of immunoaffinity purifications to overcome these limitations was demonstrated.

M.Tech. (Biomedical Technology) / Please read abstract in the full-text document

Ziel: Im Mittelpunkt dieser prospektiven Studie steht die Neubestimmung eines geschlechtspezifischen Referenzbereiches für Calcitonin-Konzentrationen, sowohl basal als auch nach Stimulation mit Pentagastrin bzw. Calcium unter Verwendung eines vollautomatischen Assays (Analyseautomat IMMULITE®2000). Aufgrund des gewählten Studiendesigns ist es möglich, die Wertigkeit des etablierten Pentagastrin-Stimulationstests im Vergleich zu einem alternativen Calcium-Stimulationstest zu beurteilen. Methodik: Insgesamt wurden 50 schilddrüsengesunde, nichtrauchende Versuchspersonen (davon 25 weiblich) im Alter von 20 bis 60 Jahren (Mittelwert: 33 Jahre) in die Studie eingeschlossen. Im Vorfeld wurde bei jedem Probanden mittels sonographischer und labortechnischer Untersuchungen (fT3, FT4, TSH, TPO-Antikörper, TG-Antikörper) das Vorliegen krankhafter Veränderungen der Schilddrüse ausgeschlossen. Um einen intraindividuellen Vergleich der intravenösen Stimulationsverfahren zu ermöglichen, erfolgten die Stimulationsversuche unter gleichen Bedingungen (Nahrungskarenz >4h) in einem zeitlichen Abstand von mehreren Wochen. Die Anzahl der Probanden, die an beiden Versuchen teilnahmen, lag bei 42 (davon 18 Frauen). Die Durchführung des Pentagastrin-Tests erfolgte nach dem in unserer Klinik etablierten Protokoll: 0,5 μg Pentagastrin/ kg Körpergewicht Injektion innerhalb von 10 sec.. Die Dosierung des Stimulans Calcium richtete sich nach Angaben der Literatur. Die Stimulation mit Calcium wurde mit Calciumgluconatlösung durchgeführt (2,5 mg Calcium/kg Körpergewicht, mit einer Injektionsgeschwindigkeit von etwa 10ml/min). Vor der Stimulation wurde jeweils der basale Calcitoninspiegel bestimmt. Weitere Blutabnahmen erfolgten direkt im Anschluss an die Injektion sowie 2, 5 und 15 Minuten nach Injektionsende. Sämtliche Calcitoninkonzentrationen wurden mit Hilfe eines Festphasen, Enzym-markierten, Sandwich, immunometrischen Chemilunineszenz Assay (IMMULITE®2000 Calcitonin) bestimmt. Ergebnisse Bei der Betrachtung der 95. Perzentile des basalen Calcitoninspiegels zeigte sich kein deutlicher geschlechtspezifischer Unterschied (95. Perzentile: Männer: 5,0 pg/ml vs. Frauen: 5,7 pg/ml; Mittelwert: Männer: 2,6±1,3 pg/ml vs. Frauen 1,6±1,3 pg/ml). Bei den Stimulationsverfahren hingegen lagen die Calcitoninkonzentrationen in der Gruppe der Männer im Vergleich zur Gruppe der Frauen jeweils signifikant höher (Pentagastrin-Test: p=0,001; Calcium-Test: p=0,004; Mann-Whitney Test). In beiden Testverfahren wurde der Calcitonin Peak nach 2 bis 5 Minuten erreicht. Bei der Gegenüberstellung des Pentagastrin-Tests und des Calcium-Tests bewirkte letzterer den größeren Calcitoninanstieg (Männer: p<0,001, Frauen: p<0,001). Im Einzelnen lag der Wert der 95. Perzentile – zum Zeitpunkt der 2-Minuten-Messung - für Männer im Pentagastrin-Test bei 37,8 pg/ml (Frauen: 26,2 pg/ml) und im Calcium Test bei 95,4 pg/ml (Frauen: 90,2 pg/ml). Die Daten zeigten keinen Anhalt für einen Einfluss von Alter oder Gewicht. Schlussfolgerung Die mit Hilfe eines verbreiteten Analyseautomaten ermittelten geschlechtsspezifischen Referenzbereiche für Calcitonin liegen unterhalb der bisherigen für andere Messverfahren erarbeiteten Angaben. Bei einem schilddrüsengesunden Kollektiv bewirkte die Stimulation mit Calcium im Vergleich zu Pentagastrin einen stärkeren Calcitoninanstieg. / Background: Calcitonin (hCT) - produced by the C-cells of the thyroid gland - plays an essential part in diagnosis and follow-up of medullary thyroid cancer. To increase specificity of this tumor marker, several stimulation tests have been developed e.g. pentagastrin-stimulation test. Since pentagastrin is no longer available in the United States of America, it seems important to evaluate whether calcium stimulation is equivalent to pentagastrin stimulation for this purpose. Our aim was to investigate healthy adults in order to determine the normal range of stimulated serum hCT levels (applying the two-site chemiluminescent immunometric assay IMMULITE®2000 Calcitonin) and to compare intravenous calcitonin stimulation in an intraindividual study set-up using either pentagastrin or calcium as agent. Methods: Having obtained approval from the local Ethics Committee we included 50 healthy, non-smoking volunteers aged 22 - 57 years (25 women) showing no evidence of thyroid abnormality in a preceding screening. 42 subjects – after having given written informed consent – participated in both intravenous stimulation tests, which were performed on separate days using either Pentagastrin (0.5 μg/kg bodyweight over 10 seconds) or calcium gluconate 10% (calcium 2.5 mg/kg bodyweight at a rate of 10ml/min). Tested subjects were committed to fasting before stimulation; drawing of blood samples (at baseline, immediately after application and after 2, 5 and 15 min.). We used a solid phase, enzyme-labeled, two-site chemiluminescent immunometric assay (IMMULITE 2000 Calcitonin) to measure serum hCT. Results: Baseline values did not differ significantly between males and females (mean: 2.6±1.3 vs. 1.6±1.3 pg/ml; 95th percentile 5.0 vs. 5.7 pg/ml). Calcium yielded a greater rise in hCT than did pentagastrin (men: p<0.001; women: p<0.001). Referring to the value of the 95th percentile: after Pentagastrin stimulation maximal hCT-peak of 37.8 pg/ml in men (26.2 pg/ml in women); after calcium stimulation maximal hCT-peak of 95.4 pg/ml in men (90.2pg/ml in women). Conclusions: We established a reference range for basal and stimulated hCT for healthy adults using an automated chemiluminescent assay, which are lower than reported for other methods. Our results emphasize that adequate reference values need to be validated individually for the assay used as well as for the method of stimulation. see also: Journal of Clinical Endocrinology & Metabolism (Aug 2009, 94 (8): 2970-4) Potency and Tolerance of Calcitonin Stimulation with High-Dose Calcium versus Pentagastrin in Normal Adults. Patricia Doyle, Christian Düren, Kai Nerlich, Frederik A. Verburg, Inge Grelle, Hanne Jahn, Martin Fassnacht, Uwe Mäder, Christoph Reiners, and Markus Luster

Microcystin-LR (MCLR) is hepatotoxic to animals and humans with disruption of liver structure causing cytoskeletal damage, necrosis and pooling of blood in the liver, leading to large increase in liver weight. It is also a strong liver tumor promoter and protein phosphatase inhibitor. Microcysin-LR binds protein phosphatases 1 and 2A, and influences regulation of cellular protein phosphorylation. In the present study, a colloidal gold based immunoassay test strip was developed for Microcystin-LR detection. The detection limit was found to be 1 ng/mL. 5 nm colloidal gold test strips exhibits more efficient for detection, compared with 20 nm colloidal gold test strips. The interaction between Microcystin-LR antibody (immunoglobulin G) and colloidal gold nanoparticles was investigated by various analytical methods, including Ultraviolet/Visible (UV/VIS), Fourier Transform Infrared (FTIR) and Fluorescence spectroscopy as well as transmission electron microscopy (TEM). / by Jiesi Xu. / Thesis (M.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

O presente estudo teve como objetivo utilizar a técnica de enzimaimunoensaio (EIE) com o Ac monoclonal CL425 na dosagem de metabólitos de progestinas fecais para caracterizar o perfil no início da atividade ovariana e durante o ciclo estral em onça pintada (Panthera onca) e durante a gestação e lactação em gato mourisco (Puma yagouaroundi). Foram estudadas três fêmeas de onça pintada em fases distintas (pré-puberes n=2 e adulta n=1) e três fêmeas de gato mourisco em duas fases distintas (gestantes n=2 e lactantes n=3). O protocolo empregado no EIE foi validado para a mensuração de progestinas em fezes de onça pintada e gato mourisco (r=0,98, p=0,0078; r=0,97, p=0,0130, respectivamente). Observou-se que os animais pré-puberes apresentaram início da produção de progesterona nos meses de setembro e novembro. As elevações das progestinas fecais na fêmea adulta de onça pintada não se sustentaram, indicando que não ocorreu ovulação espontânea nessa espécie. Houve diferença significativa entre as concentrações médias de progestinas fecais (p<0,001) dos animais pré-púberes e adultos no grupo das fêmeas de onça pintada. No grupo das fêmeas de gato mourisco, não foi possível diferenciar as concentrações de progestinas fecais durante a gestação e lactação.Obtivemos correlação entre as concentrações de progestinas fecais medida pelos métodos de radioimunoensaio (RIE) e enzimaimunoensaio (r=0,98, p<0,0001). / The present study had as objective to use the technique of enzyme immunoassay (EIA) with monoclonal antibody CL425 in the dosage of faecal progestin to characterize the profile during ovarian activity beginning and estral cycle in jaguar (Panthera onca) and gestation and lactation in jaguarondi (Puma yagouaroundi). Three female jaguars were studied in distinct phases (pre-pubertal n=2 and adult n=1) and three female jaguarondi were studied during two distinct phases (gestation n=2 and lactation n=3). The EIA used was validated for faecal progestin measurement in jaguar and jaguarondi (r=0,98, p=0,0078; r=0,97, p=0,0130, respectively). The beginning of progesterone production for pre-pubertal animals was in September and November. Elevations of progestin in the adult jaguar were not supported, showing that there was not spontaneous ovulation for this specie. There was significant difference between medium progestin concentrations (p<0,001) of pre-pubertal and adult jaguars. It was not possible to identify different progestin concentrations during gestation and lactation in female jaguarondi. The faecal progestin profiles measured by radioimmunoassay (RIA) and enzyme immunoassay (EIA) corresponded well and were positively correlated (r=0,98, p<0,0001).

Vários autores já enfatizaram a importância do monitoramento do ciclo estral em cadelas e citaram exemplos de como ele pode ser feito. O objetivo deste estudo foi avaliar a técnica de dosagem de progesterona salivar para monitorar o ciclo estral da espécie. Para composição do grupo experimental, foram utilizadas 13 cadelas. As amostras de sangue e saliva foram colhidas paralelamente em todos os animais, a partir dos primeiros sinais de proestro. As amostras salivares foram obtidas com o uso de dispositivo específico para coleta Salivette®, método que se mostrou eficaz, visto que foi possível obter volume suficiente para dosagem de progesterona na grande maioria das amostras. As concentrações de progesterona no soro foram determinadas pela técnica de RIE e na saliva por EIE. Embora haja uma relação linear crescente e positiva entre a progesterona sérica e salivar (r=0,704; p<0,0001), não é possível utilizar o parâmetro salivar para determinar o momento da ovulação. / Several authors have already emphasized the importance of monitoring estrous cycle in bitches and mentioned examples of how it can be done. The aim of this study was to evaluate the salivary progesterone quantification technique in order to monitor the estrous cycle in this species. To compound the experimental group, 13 bitches were used. Blood and saliva samples were collected simultaneously in all animals, starting about the first day of proestrus signs. Salivary samples were collected with a specific device: Salivette®. This method was effective, since it was possible to obtain enough volume in almost all samples to quantify progesterone. Serum progesterone was quantified by radioimmunoassay and salivary progesterone by enzyme immunoassay. Although there is an increasing, linear and positive correlation between salivary and serum progesterone (r=0,704; p<0,0001), it is not possible to use the salivary parameter to set the moment of ovulation.

O presente estudo teve como objetivo utilizar a técnica de enzimaimunoensaio (EIE) com o Ac monoclonal CL425 na dosagem de metabólitos de progestinas fecais para caracterizar o perfil no início da atividade ovariana e durante o ciclo estral em onça pintada (Panthera onca) e durante a gestação e lactação em gato mourisco (Puma yagouaroundi). Foram estudadas três fêmeas de onça pintada em fases distintas (pré-puberes n=2 e adulta n=1) e três fêmeas de gato mourisco em duas fases distintas (gestantes n=2 e lactantes n=3). O protocolo empregado no EIE foi validado para a mensuração de progestinas em fezes de onça pintada e gato mourisco (r=0,98, p=0,0078; r=0,97, p=0,0130, respectivamente). Observou-se que os animais pré-puberes apresentaram início da produção de progesterona nos meses de setembro e novembro. As elevações das progestinas fecais na fêmea adulta de onça pintada não se sustentaram, indicando que não ocorreu ovulação espontânea nessa espécie. Houve diferença significativa entre as concentrações médias de progestinas fecais (p<0,001) dos animais pré-púberes e adultos no grupo das fêmeas de onça pintada. No grupo das fêmeas de gato mourisco, não foi possível diferenciar as concentrações de progestinas fecais durante a gestação e lactação.Obtivemos correlação entre as concentrações de progestinas fecais medida pelos métodos de radioimunoensaio (RIE) e enzimaimunoensaio (r=0,98, p<0,0001). / The present study had as objective to use the technique of enzyme immunoassay (EIA) with monoclonal antibody CL425 in the dosage of faecal progestin to characterize the profile during ovarian activity beginning and estral cycle in jaguar (Panthera onca) and gestation and lactation in jaguarondi (Puma yagouaroundi). Three female jaguars were studied in distinct phases (pre-pubertal n=2 and adult n=1) and three female jaguarondi were studied during two distinct phases (gestation n=2 and lactation n=3). The EIA used was validated for faecal progestin measurement in jaguar and jaguarondi (r=0,98, p=0,0078; r=0,97, p=0,0130, respectively). The beginning of progesterone production for pre-pubertal animals was in September and November. Elevations of progestin in the adult jaguar were not supported, showing that there was not spontaneous ovulation for this specie. There was significant difference between medium progestin concentrations (p<0,001) of pre-pubertal and adult jaguars. It was not possible to identify different progestin concentrations during gestation and lactation in female jaguarondi. The faecal progestin profiles measured by radioimmunoassay (RIA) and enzyme immunoassay (EIA) corresponded well and were positively correlated (r=0,98, p<0,0001).

In this paper, a parametric study of the airbrush deposition technique was investigated for the deposition biomolecular thin films. The airbrush parameters under investigation were intake valve opening, carrier gas pressure, distance between the airbrush and substrate, concentration of solution, vapor pressure of solvent, and hydrophobic/hydrophilic substrate surface. This study was assessed through the characterization of dried droplet residues of Bovine Serum Albumin (BSA) and of complete films of BSA by means of scanning electron microscopy (SEM) and atomic force microscopy (AFM). It was determined that droplet size was mainly affected by carrier gas pressure and vapor pressure. The parameters intake valve opening, distance between the airbrush and substrate, and concentration of solution control the rate of spray, or solution flux, onto the substrate. Solution flux was determined to have the greatest impact on film roughness. This allowed for flexibility in the airbrush deposition technique to produce films with various substrate wetting rates. Low flux films were produced when the droplets dried on the substrate surface before the next droplet arrived. High flux films were generated when droplets on the surface arrive before subsequent droplets are given time to dry. Finally, as an extension of the results of these experiments, a practical application of the airbrush deposition technique was conducted using appropriate deposition parameters. An E. coli wave guide biosensor was produced on a glass substrate. A sandwich immunoassay was used to confirm E. coli capture.