Development and characterization of a radioimmunoassay to measure human tissue kallikrein in biological fluids

1984 ◽  
Vol 101 (2) ◽  
pp. 173-179 ◽  
Author(s):  
A. F. Bagshaw ◽  
K. D. Bhoola ◽  
M. J. C. Lemon ◽  
J. T. Whicher
Keyword(s):  
Pancreatic Juice ◽  
Coefficient Of Variation ◽  
Biological Fluids ◽  
Human Saliva ◽  
Tissue Kallikrein ◽  
Antibody Technique ◽  
Standard Curve ◽  
Interassay Coefficient ◽  
Antigen Antibody

ABSTRACT A direct radioimmunoassay has been developed to measure tissue kallikrein in human biological fluids. These fluids include serum, plasma, urine, pancreatic juice and saliva. Purified kallikreins from human urine and human saliva were used to raise rabbit antibody and each was labelled with Na125I for use in the radioimmunoassay. A comparison of the different antigen-antibody systems was then made. Bound and free enzyme were separated by a double-antibody technique. The usable range of the standard curve was from 2·5 to 100 μg kallikrein/l. The intra-assay coefficient of variation was 4·7%, the interassay coefficient of variation 8·9% and the recoveries of purified kallikrein added to the samples were 99·3, 96·0, 110·8 and 81·2% for urine, saliva, serum and plasma respectively. Parallel dilution curves were obtained for serum and plasma, as well as for urine, saliva and pancreatic juice. However, plasma anticoagulated with EDTA or heparin gave consistently lower values than serum, when measured in the radioimmunoassay. From eight different subjects plasma (EDTA) values were on average 50% lower than those of serum. Experiments designed to determine the cause of this difference revealed that treatment of blood with some anticoagulants, in particular heparin and EDTA, resulted in a marked reduction in the amount of measurable tissue kallikrein. J. Endocr. (1984) 101, 173–179

A Critical Evaluation of Separation Methods in Radiommunoassays for Total Triiodothyronine and Thyroxine in Unextracted Human Serum

1974 ◽  
Vol 11 (1-6) ◽  
pp. 224-229 ◽  
Author(s):  
W. A. Ratcliffe ◽  
G. S. Challand ◽  
J. G. Ratcliffe
Keyword(s):  
Human Serum ◽  
Coefficient Of Variation ◽  
Critical Evaluation ◽  
Antibody Technique ◽  
Wide Range ◽  
Total Triiodothyronine ◽  
Interassay Coefficient ◽  
Working Day ◽  
Separating Agents ◽  
Range Of Values

Methods for separating free and antibody-bound hormone in radioimmunoassays for total triiodothyronine (T-3) and thyroxine (T-4) in unextracted human serum are evaluated. For T-3 assay, a simplified second antibody technique has significant advantages over other methods and gives a mean interassay coefficient of variation of 7.2 % over a wide range of values. For T-4 assay, polyethylene glycol is the method of choice and has a mean interassay coefficient of variation of 4.7 %. By adding the separating agents initially, the assays are readily semi-automated and may be completed within a working day.

Quantitative Assessment of the BCR-ABL Transcript Using the Cepheid Xpert BCR-ABL Monitor Assay

2007 ◽  
Vol 131 (6) ◽  
pp. 947-950
Author(s):  
Scott D. Dufresne ◽  
Dorothy R. Belloni ◽  
Norman B. Levy ◽  
Gregory J. Tsongalis
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Coefficient Of Variation ◽  
Quantitative Assessment ◽  
Myelogenous Leukemia ◽  
Chain Reaction ◽  
Normal Peripheral Blood ◽  
Standard Curve ◽  
Polymerase Chain ◽  
Normal Controls

Abstract Context.—Chronic myelogenous leukemia (CML) and the assessment of the BCR-ABL transcript has become a new paradigm. Novel tyrosine kinase inhibitors as mainstream therapeutic options for the CML patient warrant routine quantification of the BCR-ABL transcript. The Xpert BCR-ABL Monitor assay is a nested reverse transcriptase polymerase chain reaction that greatly reduces technical time by using a single cartridge to isolate RNA and run a quantitative reverse transcriptase polymerase chain reaction. Objective.—To evaluate the Xpert BCR-ABL Monitor assay for quantitative assessment of the BCR-ABL transcript in CML patients. Design.—A standard curve of K-562 cells diluted in normal peripheral blood was used to test the sensitivity, linearity, and percent coefficient of variation of the assay. Specimen stability was tested by running standard curves immediately and after 24 hours or 96 hours of storage at 4°C. Specimens from normal controls, patients known to have CML, or patients suspected of having CML were also tested. Results.—The sensitivity of the assay was sufficient to detect 1 K-562 cell in 105 normal cells. The R2 of the standard curve was 0.98 and the percent coefficient of variation for each data point was 15% to 24%. Eleven of 14 patients with known CML on imatinib treatment tested positive for the BCR-ABL transcript, whereas 10 normal controls tested negative. Conclusions.—The Xpert BCR-ABL Monitor assay is a rapid, sensitive method for monitoring the presence of the BCR-ABL transcript in CML patients. The single-use cartridge minimizes hands-on technical time, minimizes the potential for contamination, and allows quantitative BCR-ABL testing to be performed in a random access fashion.

scholarly journals Characterization of Mouse Tissue Kallikrein 5

2006 ◽  
Vol 23 (11) ◽  
pp. 963-968 ◽  
Author(s):  
Sanath Rajapakse ◽  
Katsueki Ogiwara ◽  
Noriko Yamano ◽  
Atsushi Kimura ◽  
Kensaku Hirata ◽  
...  
Keyword(s):  
Mouse Tissue ◽  
Tissue Kallikrein

scholarly journals Quantitation of Friend spleen focus-forming virus by a nine-day 59Fe assay

1976 ◽  
Vol 4 (6) ◽  
pp. 486-491
Author(s):  
J H Menna ◽  
W D Hankins ◽  
S B Krantz
Keyword(s):  
Coefficient Of Variation ◽  
Intraperitoneal Injection ◽  
Dose Range ◽  
Intravenous Injections ◽  
Counting Procedure ◽  
Virus Dose ◽  
Ad Libitum

A previously described 3-day 59Fe assay for quantitation of Friend spleen focus-forming virus has been modified to produce a 200-fold more sensitive 9-day 59Fe assay. A characterization of this assay is reported here. Male BALB/c mice received intravenous injections of appropriately diluted Friend polycythemia virus (FVP); control mice received virus diluent. All mice were allowed food and water ad libitum for 6 days, and on day 6 after virus injection were fasted by removal of food but not water. On day 3 of the fast (the 9th day after virus injection) each mouse received an intraperitoneal injection of 1 muCi of 59Fe. Six hours later the mice were sacrificed and the splenic radioactivity was determined. The percent splenic incorporation of 59Fe was directly related to the logarithm of spleen focus-forming units (SFFU) of FVP injected in a range of approximately 25 to 1,000 SFFU. Using a standard FVP preparation in a dose range of 25 to 1,000 SFFU, it was possible to determine the SFFU titers of unknown samples by extrapolation of the percent splenic 59Fe incorporation to the logarithm of SFFU. SFFU titers obtained by the 9-day 59Fe assay were similar to those obtained by the enumerative-response assay. Advantages of the 9-day 59Fe assay over the enumerative-response assay include a 50-fold greater virus dose range, an easier and a more objective counting procedure, and a reduced coefficient of variation.

scholarly journals Quantification of a novel dense granule protein (granulophysin) in platelets of patients with dense granule storage pool deficiency

Blood ◽  
1992 ◽  
Vol 80 (5) ◽  
pp. 1231-1237 ◽  
Author(s):  
A Shalev ◽  
G Michaud ◽  
SJ Israels ◽  
A McNicol ◽  
S Singhroy ◽  
...  
Keyword(s):  
Membrane Vesicles ◽  
Dense Granule ◽  
Enzyme Linked Immunosorbent Assay ◽  
Granule Formation ◽  
Standard Curve ◽  
Storage Pool ◽  
Granule Membrane ◽  
Hermansky Pudlak Syndrome ◽  
Novel Protein

An antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) was developed for a novel protein granulophysin, a constituent of the platelet dense granule (DG) membrane and used to characterize patients with dense granule storage pool deficiency (delta-SPD). The assay uses two monoclonal antibodies against the protein, one of which is conjugated to peroxidase. Purified DGs, an enriched source of the protein, were used for the standard curve. Granulophysin levels were only low in forms of delta-SPD associated with albinism. Granulophysin levels in platelet homogenates of 30 patients with the Hermansky-Pudlak syndrome form of delta-SPD were 1/4 to 1/5 of levels in controls or obligate heterozygotes. Two patients with the Chediak-Higashi form of delta-SPD syndrome also had markedly reduced levels of granulophysin. Patients with other forms of delta-SPD had normal levels of granulophysin. Two sisters with delta-SPD in one family had normal granulophysin present in empty dense granule membrane vesicles. Three members of another family with delta-SPD had low DG counts but normal granulophysin levels, indicating that in this group the level of granulophysin was maintained despite the reduction in granule formation. Thus, granulophysin quantitation facilitates characterization of delta-SPD patients and may provide clues to the nature of defective granules in delta-SPD subtypes.

Radioimmunoassay of inhibin based on synthetic human inhibin alpha-chain peptide

1991 ◽  
Vol 37 (1) ◽  
pp. 40-46 ◽  
Author(s):  
M J Sinosich ◽  
S Sieg ◽  
A Zakher ◽  
N Ling ◽  
D M Saunders ◽  
...  
Keyword(s):  
Specific Activity ◽  
Biological Fluids ◽  
Mammalian Species ◽  
Protein A ◽  
Divalent Metal Cations ◽  
Polyethylene Glycol 200 ◽  
Separation Phase ◽  
Ovarian Inhibin ◽  
Placental Extract

Abstract Polyclonal rabbit antisera were produced against cyclic human inhibin [(Cys6, Tyr7) alpha-(6-30)NH2] peptide, covalently conjugated to bovine serum albumin. The tyrosine residue introduced at position 7 facilitated the oxidative incorporation of radiolabel (125I) to yield a tracer with specific activity of 73.9 Ci/g. These reagents were used to develop a homologous equilibrium radioimmunoassay for human inhibin, with polyethylene glycol, 200 g/L, serving as the separation phase. At a detection limit of 2 micrograms/L (n = 7), immunoactive inhibin was detectable in human pre-ovulatory follicular fluid (128 micrograms/L), seminal plasma (2374 micrograms/L), amniotic fluid (66 micrograms/L), and placental extract (347 micrograms/L). We also demonstrated inhibin immunoreactivity in biological fluids from other mammalian species: macaque, chimpanzee, porcine, and bovine, but not rodent (guinea pig). Although the antisera were raised against a nonbioactive inhibin peptide, immunoglobulins fractionated on Protein A-Sepharose neutralized the bioactivity of human ovarian inhibin. Further characterization of inhibin immuno- and bioactivity was undertaken with immobilized heparin, divalent metal cations, and dye ligands. Only heparin-Sepharose distinguished between immuno- and bioactive inhibin.

scholarly journals Characterization of a kininogenase from rat vascular tissue resembling tissue kallikrein.

1985 ◽  
Vol 56 (6) ◽  
pp. 816-821 ◽  
Author(s):  
H Nolly ◽  
A G Scicli ◽  
G Scicli ◽  
O A Carretero
Keyword(s):  
Vascular Tissue ◽  
Tissue Kallikrein

Apolipoprotein C-II deficiency associated with nonfunctional mutant forms of apolipoprotein C-II

1984 ◽  
Vol 62 (9) ◽  
pp. 847-852 ◽  
Author(s):  
Graham F. Maguire ◽  
J. Alick Little ◽  
Gary Kakis ◽  
W. Carl Breckenridge
Keyword(s):  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Normal Subjects ◽  
Two Dimensional ◽  
Antibody Technique ◽  
Molecular Weights ◽  
Apolipoprotein C ◽  
Antigen Antibody ◽  
Mutant Forms

Two previously unidentified apolipoproteins (apo) designated apo C-II-X and C-II-Y have been found in plasma of homozygotes and obligate heterozygotes of a family with apo C-II deficiency. Because the plasmas of homozygotes do not activate lipoprotein lipase, apo C-II-X and C-II-Y are apparently nonfunctional. These apolipoproteins have isoelectric focusing points of 5.15 and 5.54, respectively, compared with 4.88 and 4.74 for the known isomorphs, C-II-1 and C-II-2, respectively. They have approximately similar molecular weights to apo C-II-1 and C-II-2 on two-dimensional sodium dodecyl sulphate – glycerol – polyacrylamide slab gel electrophoresis. They do not form insoluble antigen–antibody complexes with antibodies to apo C-II in single antibody immunodiffusion or electroimmunoassay systems. However, using a double-antibody technique in which immunoblotting is coupled with polyacrylamide isoelectric focusing slab gel electrophoresis, apo C-II-1, C-II-2, C-II-X, and C-II-Y have similar reactivity with antibodies to apo C-II. In this family the presence of apo C-II-X and C-II-Y discriminates obligate heterozygotes from normal subjects.

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