Improved measurement of free alpha subunit of glycoprotein hormones by assay with use of a monoclonal antibody.

1988 ◽  
Vol 34 (10) ◽  
pp. 2022-2025 ◽  
Author(s):  
R W Whitcomb ◽  
J S Sangha ◽  
A L Schneyer ◽  
W F Crowley
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Cross Reactivity ◽  
Assay System ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Glycoprotein Hormones ◽  
Sensitive Assay ◽  
Precise Information ◽  
Sensitive Method

Abstract Any exploration of the physiology of secretion of free alpha subunit of glycoprotein hormones in humans requires a sensitive assay system that shows low cross reactivity with the intact hormones (lutropin, follitropin, thyrotropin, and choriogonadotropin). We established and validated such an assay system with a monoclonal antibody for detection of free alpha subunit in serum with better sensitivity (detection limit, 30 ng/L) and less cross reactivity (0.67% with intact human lutropin) than with available polyclonal antibody methods. Thus, the specific epitope recognized by this antibody apparently is exposed only on the free or uncombined form of alpha subunit and is concealed after non-covalent combination with any beta subunit. Using Western blot analysis, we show that most of the cross reactivity is probably the result of contamination of the human lutropin standard (obtained from the National Hormone and Pituitary Program, NIH) with small amounts of free alpha subunit. With such a specific and sensitive method, it should be possible to obtain more-precise information regarding the secretion of free alpha subunit.

Validation of highly specific and sensitive radioimmunoassays for lutropin, follitropin, and free alpha subunit in unextracted urine

1990 ◽  
Vol 36 (2) ◽  
pp. 340-344 ◽  
Author(s):  
H Landy ◽  
A L Schneyer ◽  
R W Whitcomb ◽  
W F Crowley
Keyword(s):  
Monoclonal Antibody ◽  
Urinary Excretion ◽  
Blood Volume ◽  
High Sensitivity ◽  
High Specificity ◽  
Urine Samples ◽  
Alpha Subunit ◽  
Glycoprotein Hormones ◽  
Urinary Ph ◽  
Freeze Thaw

Abstract Measurement of the urinary excretion of lutropin (LH) and follitropin (FSH) and their common free alpha subunit (FAS) assists in monitoring the maturation of the hypothalamic-pituitary-gonadal axis and in understanding the physiology of the pituitary glycoprotein hormones. Here we describe sensitive, specific polyclonal radioimmunoassays for LH and FSH and a monoclonal radioimmunoassay for FAS for use with urine--assays unperturbed by alterations in urinary pH or osmolarity within the broad physiological range encountered in urine. Concordance between LH, FSH, and FAS concentrations in extracted and unextracted urine samples was high. Linearity and parallelism with the standard curves was observed with addition of 25 to 200 microL of unextracted urine. No effect on glycoprotein concentration was seen after as many as 10 freeze-thaw cycles. The need for extraction was further obviated by the high sensitivity of each assay, reflected by minimum detectable doses well below the concentrations encountered in patients' samples. Thus we have measured gonadotropins in unextracted urine as precisely as in extracted urine. We also have demonstrated an equally versatile assay for urinary alpha subunit, using a monoclonal antibody of high specificity for this monomer in its free, uncombined form. These radioimmunoassays complement assays of gonadotropins and free alpha subunit in serum and will allow longitudinal investigations otherwise limited by the constraints of the patient's blood volume.

An accelerated enzyme immunoassay for human choriogonadotropin in urine, involving reflow of specimen through capillary tubes.

1987 ◽  
Vol 33 (4) ◽  
pp. 498-501 ◽  
Author(s):  
H M Chandler ◽  
S A Fuller ◽  
C H Nakagawa ◽  
P A Nagainis ◽  
J G Hurrell
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Enzyme Immunoassay ◽  
Test Specimen ◽  
Capillary Tube ◽  
Test Procedure ◽  
Beta Subunit ◽  
Sensitive Assay ◽  
Substrate Solution ◽  
Human Choriogonadotropin

Abstract A very rapid and sensitive assay for human choriogonadotropin (hCG) has been developed involving two beta-subunit-specific monoclonal antibodies. In the assay the test specimen is passed backward and forward (reflow) through a monoclonal-antibody-coated capillary tube for 1 min, then incubated for 1 min with a second monoclonal antibody conjugated to urease (EC 3.5.1.5). After addition of a urease substrate solution, 10 int. units of hCG per liter can be detected visually within 5 min, which compares very favorably with other currently available hCG assay procedures. Advantages of the reflow/capillary tube assay system and optimization of the test procedure are discussed.

Preparation and Identification of Monoclonal Antibody against BPA

2012 ◽  
Vol 550-553 ◽  
pp. 1438-1442
Author(s):  
Lei Zhang ◽  
Su Qing Zhao ◽  
Hong Huang
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Linear Regression ◽  
Regression Line ◽  
High Specificity ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hydroxybenzoic Acid ◽  
Coating Antigen ◽  
Line Equation

Firstly, BPA structure was modified, then coupling BPA with BSA or OVA to prepare immunogen and coating antigen. Five Balb/C mice were immunized with BPA-BSA. Finally an antibody was prepared and the indirect competitive enzyme-linked immunosorbent assay was founded. Results:(1) The monoclonal antibody belongs to IgG1 subtype and К light chain.(2) The antibody titer is 1：256000, the most suitable concentration of coating antigen is 2μg/mL, and the optimal dilution of antibody and HRP are 1:16000 and 1:10000 respectively. (3)The linear regression line equation is y = 0.1139x + 0.1046, correlation coefficient is R2=0.97, the detection limit is 0.911ng/mL and IC50 is 2.454×103ng/mL. (4)The monoclonal antibody has high specificity for the cross reactivity with phenol, hydroquinone, and tert-butyl hydroquinone being lower than 0.01%, except ortho-hydroxybenzoic acid 2.1%. (5)The recovery range is 93%～116% and 89%～112% when adding BPA into black samples.（6）When the method was used in real materials to detect BPA residual, the results were proximate to the dates by HPLC.

Measurement of the free alpha subunit of human glycoprotein hormones by a monoclonal antibody-based immunoradiometric assay, and further exploration of antigenic sites on the choriogonadotropin molecule.

1987 ◽  
Vol 33 (7) ◽  
pp. 1147-1151 ◽  
Author(s):  
R J Norman ◽  
R Haneef ◽  
R H Buck ◽  
S M Joubert
Keyword(s):  
Solid Phase ◽  
Polyclonal Antibodies ◽  
Antigenic Site ◽  
Reference Interval ◽  
Cross Reaction ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Immunoradiometric Assay ◽  
Glycoprotein Hormones ◽  
Antigenic Sites

Abstract Three monoclonal antibodies were raised against the free alpha subunit of choriogonadotropin (hCG); each recognized a different antigenic site on the molecule. One (antibody 42) preferentially bound to the alpha subunit when it was coupled to the beta subunit as dimeric choriogonadotropin (hCG), thyrotropin (TSH), lutropin (LH), or follitropin (FSH). Antibody 71 showed some cross-reaction with intact FSH; antibody 75 was more specific for the alpha subunit. All were of low affinity (10(-7) to 10(-8) mol/L), but when combined in immunoradiometric assays (IRMAS) they proved to be as sensitive as current radioimmunoassays involving polyclonal antibodies. Advantages of the combination of antibody 75 bound to the solid phase and antibody 71 as the radiolabeled antibody were: detection limit of at least 0.1 micrograms/L; linear dilution of serum and urine; insignificant cross-reaction with intact hCG, allowing direct assay in pregnancy fluids; and a coefficient of variation less than 3% over the reference interval for nonpregnant women. There was 4% cross-reaction with intact FSH, suggesting that the epitopes recognized by nos. 71 and 75 are more exposed in FSH and that perhaps there is less folding in this molecule than in intact hCG.

scholarly journals Immunoassays for the Rapid Detection of Gentamicin and Micronomicin in Swine Muscle

2010 ◽  
Vol 93 (1) ◽  
pp. 335-342 ◽  
Author(s):  
Yiqiang Chen ◽  
Xiangmei Li ◽  
Lidong He ◽  
Shuheng Tang ◽  
Xilong Xiao
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Rapid Detection ◽  
Cross Reactivity ◽  
Hplc Method ◽  
Rapid Screening ◽  
Test Elisa ◽  
Inhibition Concentration ◽  
Strip Test ◽  
Phosphate Buffered Saline

Abstract A monoclonal antibody (mAb)-based ELISA and strip test for gentamicin (GEN) and its analogue micronomicin (MIN), are reported in this study. The conjugate gentamicin-glutaraldehyde-bovine serum albumin (GEN-GDA-BSA) was used as an immunogen. The produced anti-GEN mAB exhibited high cross-reactivity with micronomicin (MIN; 131.2) and slight or negligible cross-reactivity with other aminoglycosides. Based on this mAB, an ELISA and a strip test for GEN and MIN were developed and evaluated. The ELISA showed a 50 inhibition concentration (IC50) of 0.75 ng/mL for GEN and 0.58 ng/mL for MIN. For GEN, the average recoveries at 25200 µg/kg ranged from 73 to 91, with intraday CVs of 916 and interday CVs of 815. For MIN, the average recoveries ranged from 108 to 131, with intraday CVs of 1016 and interday CVs of 815. In contrast, the strip test for GEN or MIN had a detection limit of 5 ng/mL in phosphate-buffered saline and 50 µg/kg in muscle (n = 24), and the results could be judged within 10 min. The detection results of incurred samples analyzed by the strip test, ELISA, and HPLC indicated that the two immunoassays correlated well with the HPLC method and could be used as convenient tools for the rapid screening of GEN and MIN residues in swine muscle.

Enzyme immunoassay of the glycoprotein tropic hormones--choriogonadotropin, lutropin, thyrotropin--with solid-phase monoclonal antibody for the alpha-subunit and enzyme-coupled monoclonal antibody specific for the beta-subunit.

1982 ◽  
Vol 28 (9) ◽  
pp. 1862-1866 ◽  
Author(s):  
H G Wada ◽  
R J Danisch ◽  
S R Baxter ◽  
M M Federici ◽  
R C Fraser ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Room Temperature ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Enzyme Conjugate ◽  
Capture Antibody ◽  
Assay Precision

Abstract Monoclonal antibody technology has made it possible to produce homogeneous populations of antibodies to discrete determinants on an antigen surface. We have produced monoclonal antibodies to the alpha-subunit and beta-subunits of the glycoprotein hormones choriogonadotropin, thyrotropin, and lutropin, and developed two-site simultaneous enzyme-linked immunospecific assays for these hormones. The anti-alpha-subunit monoclonal antibody was used as the solid-phase (coated tube) capture antibody for all three hormones; the anti-beta-subunit monoclonal antibodies were coupled to horseradish peroxidase (EC 1.11.1.7). Cross reactions between the closely related choriogonadotropin and lutropin were apparently greater in this method than in RIA, with use of the same antibodies. Ka of the antibodies did not appear to be as critical to sensitivity of the sandwich assay as it was for RIA. The lower limit of detection was 0.2 microgram/L after a 2-h incubation with serum sample at room temperature and a 30-min incubation with enzyme substrate at room temperature after washing away excess enzyme conjugate. Within-assay precision (CV) was very good, less than 6%.

scholarly journals The beta subunit of the interleukin-2 receptor mediates interleukin-2 induction of anti-CD3 redirected cytotoxic capability in large granular lymphocytes

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 825-828 ◽  
Author(s):  
OR Colamonici ◽  
R Quinones ◽  
A Rosolen ◽  
JB Trepel ◽  
E Sausville ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Sodium Dodecyl ◽  
Interleukin 2 ◽  
Electrophoretic Analysis ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Marker Enzyme ◽  
Large Granular Lymphocytes ◽  
Granular Lymphocytes ◽  
Stimulation Of

Abstract The interleukin 2 (IL 2) receptor was studied in three cases of large granular lymphocyte (LGL) lymphocytosis. All cases were nonreactive with anti-Tac monoclonal antibody (MoAb; recognizing the p55 alpha subunit of the IL 2 receptor). Sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoretic analysis (PAGE) of cells to which radio-labeled rIL 2 had been chemically crosslinked revealed uniform expression of the p70/75 beta subunit of the IL 2 receptor in the absence of the alpha subunit. Stimulation of this receptor with 2 nmol/L rIL 2 for five days led to acquisition of anti-CD3 redirected cytotoxicity. This was accompanied by a fivefold to tenfold elevation in the activity of intracellular N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl esterase, an LGL granule marker enzyme. These effects of IL 2 did not require induction of the Tac peptide.

Highly specific enzyme immunoassay for the beta-subunit of human thyrotropin with use of monoclonal antibodies.

1988 ◽  
Vol 34 (1) ◽  
pp. 98-102 ◽  
Author(s):  
Y Iijima ◽  
Y Endo ◽  
N Hata ◽  
H Fujita ◽  
M Unoki ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Solid Phase ◽  
Pituitary Tumors ◽  
Cross Reactivity ◽  
Beta Subunit ◽  
Specific Enzyme ◽  
Human Choriogonadotropin ◽  
Pituitary Thyroid Axis ◽  
Thyroid Axis ◽  
Hypothyroid Patients

Abstract A highly specific enzyme-linked "sandwich" immunoassay is described for determining free human thyrotropin (hTSH) beta-subunit in serum by using a anti-hTSH beta-subunit monoclonal antibody conjugated with beta-D-galactosidase (EC 3.2.1.23) and a solid phase consisting of silicone rods coated with another monoclonal antibody. We could detect as little as 0.04 ng of beta-subunit per assay. The measurable range of hTSH beta-subunit concentrations in serum was 0.4 to 50 micrograms/L. The assay demonstrated little or no cross reactivity with intact hTSH, hTSH alpha-subunit, or human choriogonadotropin. The mean CVs were 12.2% within assay, 13.9% between assay. The hTSH beta-subunit was not detectable in sera from healthy subjects, patients with hyperthyroidism, or two patients with pituitary tumors producing TSH. It was measurable (at concentrations of 0.65 to 2.70 micrograms/L) in sera from eight of 23 hypothyroid patients. In five of the hypothyroid patients examined, the concentration of hTSH beta-subunit in serum increased after administration of thyroliberin. This method may be useful in elucidating the physiological and pathological significance of the hTSH beta-subunit and in examining the function of the hypothalamus-pituitary-thyroid axis.

Sensitive, Specific Radioimmunoassay for Quantifying Pergolide in Plasma

1992 ◽  
Vol 38 (10) ◽  
pp. 1975-1980 ◽  
Author(s):  
R R Bowsher ◽  
J M Apathy ◽  
J A Compton ◽  
R L Wolen ◽  
K H Carlson ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Parkinson Disease ◽  
Clinical Studies ◽  
Rhesus Monkeys ◽  
Plasma Concentrations ◽  
Cross Reactivity ◽  
Major Metabolite ◽  
Dopaminergic Activity ◽  
Specific Radioimmunoassay

Abstract Pergolide, a synthetic ergoline with potent dopaminergic activity, is used to treat Parkinson disease. The low plasma concentrations of pergolide achieved during therapy complicate the development of a method for its analysis. Because radioimmunoassay successfully measures other structurally related ergolines in physiological fluids, we undertook the development of a radioimmunoassay of pergolide. The detection limit of the radioimmunoassay is 21 ng/L with an optimal working range from 100 to 1000 ng/L. We maximized assay specificity by using a monoclonal antibody that displayed low cross-reactivity with pergolide sulfoxide, a major metabolite found in animals. The radioimmunoassay has performed acceptably for > 2 years during toxicology studies with rats and rhesus monkeys and in clinical studies involving patients with Parkinson disease. We consider the radioimmunoassay a valid method for quantifying therapeutic concentrations of pergolide in plasma.

scholarly journals Both recombinant African catfish LH and FSH are able to activate the African catfish FSH receptor

2003 ◽  
Vol 31 (1) ◽  
pp. 133-140 ◽  
Author(s):  
HF Vischer ◽  
JC Granneman ◽  
MH Linskens ◽  
RW Schulz ◽  
J Bogerd
Keyword(s):  
Culture System ◽  
Expression Patterns ◽  
Biologically Active ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
African Catfish ◽  
Fsh Receptor ◽  
Glycoprotein Hormones ◽  
Soil Amoeba ◽  
Testis Tissue

LH and FSH are heterodimeric glycoprotein hormones, composed of a common alpha-subunit non-covalently associated with a hormone-specific beta-subunit. Repeated efforts to isolate catfish FSH (cfFSH) have not been successful and only catfish LH (cfLH) has been purified from catfish pituitaries. Recently, however, we succeeded in cloning the cDNA encoding the putative cfFSHbeta; the cDNAs for the alpha- and beta-subunit of cfLH have been cloned before. Here we report the expression of biologically active cfLH and cfFSH in the soil amoeba, Dictyostelium discoideum. The biological activity of the recombinant hormones was analyzed using cell lines transiently expressing either the cfLH receptor or the cfFSH receptor. Moreover, a primary testis tIssue culture system served to study the steroidogenic potency of the recombinant hormones. Our results demonstrated that Dictyostelium produced biologically active, recombinant catfish gonadotropins, with recombinant cfLH being almost indistinguishable from its native counterpart, purified from pituitaries. Although recombinant cfFSH has significant effects in the bioassays used in this study, the specific function of native cfFSH in the control of reproduction and its expression patterns are not yet understood.

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