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Biomedical subjects

L J Kricka

Publications and source records attributed to L J Kricka.

At least 55 records · Page 3Linked to original sources
PubMed1994

Selected strategies for improving sensitivity and reliability of immunoassays.

Selected recent advances in immunoassay are reviewed. Development has continued on new labels (beta-lactamase, pyrophosphatase, luciferases, photoproteins, pyridopyridazines, europium cryptates, metal carbonyl complexes, porphines, phosphors) and label-detection methods (e.g., chemiluminescence assays, thermometric assays, NADP(+)- and FADP-based coupled assays). Various methods have been explored to increase assay sensitivity, including label amplification via catalyzed reporter deposition (peroxidase label) and immuno-polymerase chain reaction (DNA label). The focus of new immunoassay strategies has been on improved reliability (bispecific antibodies), assay simplification (piezoelectric and surface plasmon resonance immunosensors, phase-modulation fluorescence spectroscopy, polymerized bilayer assemblies), and simultaneous multianalyte testing (e.g., quadruple labeling with lanthanides, one-step test devices for drugs of abuse).

Enzymes↗
PubMed1994

Chemiluminescent reporter gene assays: sensitive detection of the GUS and SEAP gene products.

Chemiluminescent assays are described for the secreted alkaline phosphatase (SEAP) and beta-glucuronidase (GUS) reporter gene products. These assays provide simple, sensitive, non-isotopic alternatives to existing detection methods and are performed in microplate or tube luminometers or in a scintillation counter. The SEAP reporter gene product is secreted from mammalian cells and is thus easily detected in a sample of culture medium. Sensitive detection of secreted placental alkaline phosphatase is performed with CSPD chemiluminescent alkaline phosphatase substrate, and approximately 3 fg of enzyme can be detected. GUS has become the major reporter gene used for the analysis of plant gene expression. Sensitive chemiluminescent detection of GUS activity can be performed with an assay system we have developed using Glucuron, a beta-glucuronidase substrate. This chemiluminescent assay detects 60 fg of GUS and is linear over six orders of magnitude of enzyme concentration. CSPD and Glucuron substrates have been incorporated into two new chemiluminescent reporter gene assay kits for SEAP and GUS.

Alkaline Phosphatase↗
PubMed1994

PCR in a silicon microstructure.

Devices for performing polymerase chain reactions (PCR) have been developed for use with photolithographed silicon. Microchambers capable of holding between 5.0 and 10 microL of PCR reagents were constructed by etching specific areas of rectangular silicon chips (17 x 15 mm), which were then capped with Pyrex glass. These silicon devices (PCRChips), which were etched to depths of 40-80 microns, permitted free flow of fluids in the microchannels and microchambers. Access to the microchambers was through holes in the silicon. Thermal cycling of the PCR reagents was achieved by placing the disposable PCRChip in a small holder containing a computer-controlled Peltier heater-cooler. Successful amplification was demonstrated by electrophoresis of products in agarose gel containing ethidium bromide, and the migration of the product was compared with that obtained in a commercially available thermal cycler. The thermal characteristics of the silicon, coupled with the high surface area to volume ratio in the new devices, are particularly advantageous features for amplification by PCR.

Bacteriophage lambda↗
PubMed1994

Manipulation and flow of biological fluids in straight channels micromachined in silicon.

Analysis of minute sample volumes is a major analytical challenge that requires an understanding of fluid flow in microstructures. Accordingly, flow dynamics of biological fluids and cell suspensions in straight glass-capped silicon microchannels (40 to 150 microns wide, 20 and 40 microns deep) were studied. We demonstrated that these microstructures are appropriate components for microfluidic analytical devices. Different fluids were easily manipulated in the microchannels, and measurements of flow rate as a function of pressure for whole human blood, serum, plasma, and cell suspensions revealed non-Newtonian behavior. By means of micromachined filters (5 microns) located in channels, blood cells and microparticles were effectively separated from nanoliter-sized samples, clearly indicating the future role of microstructures for a variety of analytical purposes.

Albumins↗
PubMed1993

Measurement of intracellular calcium using bioluminescent aequorin expressed in human cells.

Changes in intracellular free calcium concentration ([Ca2+]i) are involved in many important physiological responses. Detecting changes in [Ca2+]i is crucial to understanding the physiologic roles of intracellular free calcium. We have characterized changes of [Ca2+]i in human cells transfected with apoaequorin cDNA. When reconstituted in vivo by incubating transfected cells with coelenterazine, aequorin emits light upon binding free calcium and acts as a bioluminescent indicator for calcium. We have used this system to determine the concentration response relationship of serotonin for its receptor. Cells cotransfected with serotonin receptor cDNA and apoaequorin cDNA emitted light upon treatment with serotonin. The light emission responses were saturable and serotonin concentration-dependent, and they were inhibited by serotonin antagonists. Human 293 cells that stably express apoaequorin have been created. This system should facilitate the investigation of [Ca2+]i involvement in physiological and pathophysiological responses.

Aequorin↗
PubMed1993

Ultrasensitive immunoassay techniques.

Ultrasensitive detection of clinically important substances using assays based on ligand:binder interactions has revolutionized laboratory medicine. Various strategies have been perfected to push the analytical sensitivity of ligand:binder assays (e.g., immunoassay, blotting, nucleic acid hybridization assay) into the attomole and zeptomole region (10(-18)-10(-21) mol). These include the use of labels with amplifying properties (e.g., enzymes), multiple labeling, and cascade reactions. In addition a wide range of ultrasensitive luminescent detection reactions have been developed for conventional enzyme labels based on chemiluminescent, bioluminescent, and time-resolved fluorescent reactions.

Affinity Labels↗
PubMed1993

Applications of a microfabricated device for evaluating sperm function.

Mesoscale structures (microns dimensions, nL-pL volumes) have been designed and fabricated in silicon for use in various analytical tasks. We studied sperm motility and performed sperm selection in channels (80 microns wide x 20 microns deep), branching structures (40 microns wide x 20 microns deep, eight bifurcations), and channels containing barriers (7 microns feature size). Sperm-cervical mucus and sperm-hyaluronic acid interactions were assessed by using appropriate microchannel-chamber structures filled with either cervical mucus or hyaluronic acid. Simultaneous assessment of the potency of different spermicides (e.g., nonoxynol-9, C13G) and spermicide concentrations was achieved with structures comprising chambers containing spermicide connected via channels to a central chamber into which semen was introduced. Semen was also tested for the presence of sperm-specific antibodies by using microchannels filled with human anti-IgG antibody-coated microbeads.

Humans↗
PubMed1992

Improved chemiluminescent western blotting procedure.

A chemiluminescent Western blotting procedure and its application in assays for human transferrin and human immunodeficiency virus-I antibodies are described. The procedure is based on a chemiluminescent substrate, adamantyl 1,2-dioxetane aryl phosphate and alkaline phosphatase-labeled detection antibodies. Different membranes (polyvinylidene fluoride, nitrocellulose, nylon) and a proprietary membrane treatment agent (Nitro-Block) have been studied. This sensitive blotting procedure utilizing AMPPD, a polyclonal rabbit anti-transferrin:goat anti-rabbit IgG-alkaline phosphatase detection complex, and a PVDF membrane blocked with Nitro-Block permits the detection of 125 pg (1.6 fmol) of human transferrin. A novel 1,2-dioxetane substrate, CSPD, has also been evaluated.

Adamantane↗