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L J Kricka

Publications and source records attributed to L J Kricka.

At least 73 records · Page 4Linked to original sources
PubMed1991

Chemiluminescent and bioluminescent techniques.

Light-emitting chemical reactions (chemiluminescence, CL) and biological reactions (bioluminescence, BL) have a diverse range of analytical applications but relatively few have been adopted by routine clinical laboratories. Advantages of CL and BL assays include sensitivity (attomole and sub-attomole detection limits), speed (signal generated in a few seconds and in some cases stable for several hours), nonhazardous reagents, and simple procedures. The most promising clinical applications are in immunoassay, protein blotting, and DNA probe assays. Chemiluminescent molecules exploited as labels include luminol, isoluminol, acridinium esters, thioesters and sulfonamides, and phenanthridinium esters. Separation and nonseparation assays have been devised, based on isoluminol and acridinium ester labels. The combination of the amplification properties of an enzyme and a CL or BL detection reaction provides a highly sensitive analytical system. Since 1983, CL and BL methods have been developed for many enzyme labels, e.g., alkaline phosphatase, glucose-6-phosphate dehydrogenase, horseradish peroxidase, Renilla luciferase, and xanthine oxidase. Currently, the most successful enzyme assays are the enhanced CL method for a peroxidase label involving a mixture of luminol, hydrogen peroxide, and an enhancer (e.g., p-iodophenol) and the direct CL method for alkaline phosphatase, with an adamantyl 1,2-dioxetane phenyl phosphate as substrate. Both systems are very sensitive (the detection limit for alkaline phosphatase when using the dioxetane reagent is 0.001 amol) and produce long-lived light emission (greater than 30 min), which is ideal for membrane applications in which light emission is detected with photographic film or a charge-coupled device camera.

Blotting, Western↗
PubMed1990

Interference by human anti-mouse antibody in two-site immunoassays.

Studies with goat and rabbit anti-mouse antibody, as models of human anti-mouse antibody (HAMA), have shown that several of the commonly used two -site immunoassays (e.g., for CA-125, carcinoembryonic antigen, choriogonadotropin, lutropin, hepatitis B surface antigen, thyrotropin) are susceptible to interference by this type of bridging heterophile antibody. In most cases the interference can be blocked by incubation with mouse IgG. We studied HAMA interference in an assay of hepatitis B surface antigen by using HAMA-positive sera from a transplant patient given OKT3 and from a cancer patient given CYT-103 (modified antibody B72.3). The HAMA interference attributable to the OKT3 could be blocked by incubation with mouse IgG at room temperature. In contrast, the interference caused by the B72.3-induced HAMA could be blocked by prolonged incubation with high concentrations of the B72.3 antibody at 4 degrees C. A limited survey of 50 hospital patients selected without conscious bias revealed two HAMA-positive patients, only one of whom was known to have been exposed to mouse immunoglobulin. HAMA interferences are currently a minor problem in routine laboratory medicine, but the increasing use of diagnostic and therapeutic products involving mouse-origin monoclonal antibodies will make the detection and elimination of HAMA interferences an important part of laboratory practice in the future.

Animals↗
PubMed1990

Rapid and sensitive detection of DNA in Southern blots with chemiluminescence.

A new chemiluminescent Southern blot procedure offers molecular biologists a safe, ultrasensitive and rapid alternative to conventional 32P-based systems. This new DNA detection system, SOUTHERN-LIGHT, has been developed by Tropix, Inc. The luminescent signal is produced from a direct chemiluminescent substrate, disodium 3-(4-methoxyspiro[1,2-dioxetane-3-2'-tricyclo-[3.3.1.1 .3,7]decan]-4-yl) phenyl phosphate (AMPPD), which decomposes upon dephosphorylation with alkaline phosphatase. SOUTHERN-LIGHT is an ultrasensitive, rapid detection kit for use with membrane-bound DNA. It is the first test kit to incorporate AMPPD. It requires no specialized equipment and results can be conveniently imaged on instant film or x-ray film within 5-60 min of exposure.

Adamantane↗
PubMed1989

Clinical applications of luminescent assays for enzymes and enzyme labels.

A wide range of luminescent assays for enzyme labels has been developed. The clinical application of luminescent assays for horseradish peroxidase, alkaline phosphatase, xanthine oxidase, beta-D galactosidase, and glucose 6-phosphate dehydrogenase is discussed. The enzyme luminescent methods provide improved sensitivity compared with colorimetric and fluorimetric assays. A new reagent, dioxetane phosphate, has provided a simple and extremely sensitive assay for alkaline phosphatase. The available instruments and new photographic detection techniques for luminescent assays are briefly discussed.

Alkaline Phosphatase↗
PubMed1989

Advantages of firefly luciferase as a reporter gene: application to the interleukin-2 gene promoter.

The chloramphenicol acetyltransferase (CAT) gene is widely used in recombinant constructs employed to study promoter and enhancer control of gene expression. However, CAT-based assays require a laborious, multi-step procedure for quantitation of promoter activity. We have applied the recently described firefly luciferase (LUC) reporter gene to the study of the interleukin-2 (IL2) promoter and have further defined the properties of this reporter gene system. We find that IL2-LUC constructs have multiple advantages over IL2-CAT constructs. The LUC assay is highly sensitive and requires 1/10 the cells used in the CAT system. A final quantitative measure of promoter activity can be obtained within 25 h following transfection with IL2-LUC, compared to 108-160 h with IL2-CAT. Light emission significantly (fourfold) above background is detectable 3 h after induction in a direct assay of extracts from transfected cells. We have described the variability of the assay, the minimum number of transfected cells required to detect light, the stability of luciferase in cell extracts, the effect of Triton X-100 on the assay, and a rapid cell lysis procedure. The luciferase system is a simple, rapid, and sensitive method for the study of promoter activity in transfected cells, particularly for weakly expressed genes such as IL2 which give low activity in the CAT assay.

Animals↗
PubMed1989

An enhanced chemiluminescent enzyme immunoassay for follicle stimulating hormone.

An enhanced chemiluminescent enzyme immunoassay for serum follicle stimulating hormone is described which involves sequential reaction of anti-follicle stimulating hormone antibody immobilised to the inside surface of an opaque microtitre plate with sample, monoclonal anti-alpha thyroid stimulating hormone antibody, and an anti-mouse IgG - horseradish peroxidase conjungate. Bound peroxidase activity was measured using a p-hydroxycinnamic acid enhanced chemiluminescent luminol-hydrogen peroxide reaction. The assay was sensitive (detection limit 0.01 mU/well) precise (intra-assay precision 2.5-8.1%, inter-assay precision 6.7-11.9%) and results obtained with this assay and a competitive radioimmunoassay were in good agreement (correlation coefficient 0.98).

Chorionic Gonadotropin↗
PubMed1989

Chemiluminescent enzyme immunoassay of alpha-fetoprotein based on an adamantyl dioxetane phenyl phosphate substrate.

We have evaluated a new chemiluminescent substrate for the alkaline phosphatase (EC 3.1.3.1) label used in a Hybritech Tandem-E immunoassay of alpha-fetoprotein (AFP). The new substrate, adamantyl 1,2-dioxetane phenyl phosphate (AMPPD), emits light at 477 nm when acted upon by the enzyme. Detection limits for AFP with this method were 33 ng/L (mean of 20 replicates of the zero standard + 2 SD) and 470 ng/L (twice background). Between-batch CVs ranged from 4.31% to 9.60% for AFP in the range 29.1-132.0 micrograms/L. Comparison of results for 49 specimens assayed with use of the chemiluminescent kit and a colorimetric version of the AFP assay gave statistical values as follows: slope = 0.88, intercept = 4.19, and r = 0.94.

Adamantane↗
PubMed1989

Chemiluminescent assay of alkaline phosphatase applied in an ultrasensitive enzyme immunoassay of thyrotropin.

We compared a chemiluminescent assay and a colorimetric endpoint assay for measuring an alkaline phosphatase (EC 3.1.3.1) label in an enzyme immunoassay of thyrotropin (TSH). The substrate in the chemiluminescent assay is a derivative of adamantyl 1,2-dioxetane phosphate. On dephosphorylation, catalyzed by alkaline phosphatase, the 1,2-dioxetane decomposes further and emits a glow of light (lambda max 470 nm). We modified the Hybritech Tandem-E TSH High Sensitivity assay for chemiluminescent detection of bound alkaline phosphatase label by using this substrate (with 20-, 40-, and 60-min incubations). Detection limits (mean +2 SD of zero standard) were 6.0, 5.2, and 4.5 micro-int. units/L for these incubation periods, respectively, vs 20 micro-int. units/L for the conventional colorimetric version of the assay. Comparison of results for 44 clinical specimens assayed by the chemiluminescent (20-min incubation, y) and colorimetric (60-min endpoint, x) TSH immunoassays gave statistical values of: slope = 1.17, intercept = -0.22, and r = 0.98. Hemoglobin, but not bilirubin, lipids, or protein, interfered; but these interferents were removed by the washing steps in the enzyme immunoassay.

Alkaline Phosphatase↗
PubMed1988

Clinical and biochemical applications of luciferases and luciferins.

Recent advances in the analytical applications of bacterial and firefly luciferases and firefly luciferin are reviewed. Luciferases have been used in soluble and immobilized/co-immobilized forms in assays for a variety of enzymes, substrates, and cofactors. The firefly luciferase reaction forms the basis of rapid microbiological tests which have found application in susceptibility testing, detection of bacteriuria, activated sludge analysis, and food testing. Rapid microbiological assays are also possible using bacteriophages containing the lux genes from Virbrio harveyi. Both the firefly and the bacterial luciferase reaction have been applied in immunoassay and DNA probe assays and the firefly luciferin phosphate substrate for alkaline phosphatase labels has proven particularly successful.

Adenosine Triphosphate↗
PubMed1988

An enhanced chemiluminescence enzyme immunoassay for serum oestradiol.

Oestradiol in serum was determined with a simple enhanced chemiluminescent enzyme immunoassay. The assay is based on oestradiol labelled with horseradish peroxidase and the IgG fraction of an oestradiol antiserum coated on a black polystyrene microtitre plate. The enzyme activity of bound label was determined using a p-hydroxycinnamic acid-enhanced chemiluminescent reaction. The assay was sensitive (1.8 fmol/well), precise (intra- and inter-assay CV 4-10% and 8-12%, respectively for sample concentration in the range 122-1330 pmol/L) and showed good agreement with conventional radioimmunoassays (r = 0.99).

Buffers↗
PubMed1987

Improvement in the antibody binding characteristics of microtitre wells by pretreatment with anti-IgG Fc immunoglobulin.

A preliminary coating of the surface of polystyrene microtitre wells with anti-IgG Fc immunoglobulin improves the binding capacity of the wells for antibody and leads to a marked improvement in the reproducibility of immunoassays carried out in the wells. In an enhanced chemiluminescent enzyme immunoassay for oestradiol the binding capacity for oestradiol of wells precoated with anti-IgG Fc immunoglobulin and then coated with anti-oestradiol immunoglobulin was more than twice that of wells coated directly with anti-oestradiol immunoglobulin. Intra-assay precision in the precoated wells ranged from 1.3 to 4.0% compared to 2.9 to 6.7% (0-3200 fmol oestradiol) in wells which had not been precoated.

Antibodies, Anti-Idiotypic↗