Immunoadsorption of IgG onto second antibody covalently attached to Amino-Dylark beads for radioimmunoassays

1990 ◽  
Vol 36 (3) ◽  
pp. 497-500 ◽  
Author(s):  
S E Kakabakos ◽  
G P Evangelatos ◽  
D S Ithakissios
Keyword(s):  
Solid Phase ◽  
High Titer ◽  
Significant Modification ◽  
Assay Performance ◽  
Rabbit Igg ◽  
Assay Tube ◽  
Specific Igg ◽  
Coating Procedure ◽  
Glycol Solution ◽  
Immobilization Method

Abstract We present a solid-phase immobilization method for radioligand assays, using an immunoadsorption coating procedure of anti-triiodothyronine rabbit IgG (anti-T3 IgG) onto second antibody (sheep anti-rabbit IgG) covalently bound to Amino-Dylark beads. The second antibody was in excess, compared with the first antibody, thus eliminating reproducibility problems between immunoadsorptions. Beads coated with second antibody can be used to immobilize a variety of antigen-specific first antibodies. The amount of anti-T3 antibody required for solid-phase T3 radioimmunoassay (RIA) was only 10% more, per assay tube, than that utilized in liquid-phase T3 RIA, in which polyethylene glycol solution was the separation reagent; characteristics of assay performance were comparable. The immobilization procedure requires high-titer antisera or antigen-specific IgG and seems advantageous because of the decrease in antibody requirements without significant modification of antibody functionality.

A solid-phase radioimmunoassay for human corticosteroid binding globulin

1985 ◽  
Vol 104 (2) ◽  
pp. 259-267 ◽  
Author(s):  
P. A. Robinson ◽  
M. S. Langley ◽  
G. L. Hammond
Keyword(s):  
Solid Phase ◽  
Binding Capacity ◽  
High Serum ◽  
Protein L ◽  
Parallel Displacement ◽  
Standard Curve ◽  
Corticosteroid Binding Globulin ◽  
Assay Tube ◽  
The Mean

ABSTRACT A radioimmunoassay (RIA) for human corticosteroid binding globulin (CBG) has been developed using 125I-labelled CBG and a monospecific solid-phase CBG-antiserum (CBG-Ab-cellulose). In an RIA of serum CBG concentrations, pure CBG standards (1–100 ng protein) or samples (1 : 200) were incubated (16 h at 20 °C) with 125I-labelled CBG and CBG-Ab-cellulose. After addition of 2 ml 0·9% NaCl, the tubes were centrifuged, supernatants were aspirated and the 125I-labelled CBG bound to the CBG-Ab-cellulose pellet was counted. The specificity of the RIA was confirmed by parallel displacement curves for serial dilutions of male, female and pregnancy sera, as well as pure CBG standards. The mean ± s.d. recovery (99±8%) of pure CBG (1·6–25·0 ng) added to a diluted serum sample verified the accuracy of the method, and a good correlation (r = 0·97; n = 43) existed between serum CBG cortisol binding capacity (nmol/l) measurements and CBG concentrations (mg protein/l) measured by RIA. Intra- and interassay precisions (C.V.) at low to high serum CBG concentrations were <5% and <9% respectively. The mean ± s.d. serum CBG concentrations (mg protein/l) measured by the RIA were: 21·8±4·6 in boys (n = 12), 20·0±4·2 in girls (n = 9), 20·7±2·7 in men (n = 6), 20·5±2·9 in women (n = 6) and 47·1 ±10·5 in pregnant women (n = 5). The sensitivity of the standard curve used in the routine RIA of serum CBG was 1·0 ng CBG/assay tube, but this could be increased to 0·2 ng/assay tube by reducing the amount of CBG-Ab-cellulose used. The RIA is suitable for both clinical and research purposes, and will aid the identification of abnormal forms of CBG and facilitate studies of the regulation of CBG production in vitro. J. Endocr. (1985) 104, 259–267

A solid-phase enzymeimmunoassay for the determination of progesterone in bovine, porcine and ovine plasma

1995 ◽  
Vol 75 (4) ◽  
pp. 525-529 ◽  
Author(s):  
R. P. Del Vecchio ◽  
W. D. Sutherland ◽  
M. L. Connor
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Petroleum Ether ◽  
Bovine Serum ◽  
Solid Phase ◽  
Plasma Samples ◽  
Assay Sensitivity ◽  
Coefficients Of Variation ◽  
Rabbit Igg

The purpose of this project was to develop a valid quantitative enzymeimmunoassay (EIA) for progesterone in blood plasma of cattle, pigs and sheep. Rabbit anti-progesterone, mouse monoclonal anti-rabbit IgG, authentic progesterone, and acetylcholine esterase bound covalently to progesterone were the principal reagents used to develop the EIA. Ninety-six well microliter plates were coated with mouse monoclonal anti-rabbit IgG and saturated with bovine serum albumin before use. Rabbit anti-progesterone was diluted to a working dilution of 1:2.0 × 106. Standard curves were linear and ranged from 1.56 to 400 pg of progesterone per well which allowed for the measurement of 0.03125 to 8.0 ng mL−1. Assay sensitivity averaged 1.56 pg well−1. Progesterone was extracted from plasma samples with petroleum ether. Plasma samples (n = 3 or 4 from each species) with unknown amounts of progesterone that were extracted and serially diluted with EIA buffer did not deviate from parallelism with progesterone standard curves in buffer. The correlation between EIA and radioimmunoassay (RIA) measurements of progesterone in the same plasma samples was high (P < 0.0001) for all three species (r = 0.96 for bovine; r = 0.96 for porcine; r = 0.94 for ovine). The regression of EIA data on RIA data produced the following equations:[Formula: see text]The intra- and inter-assay coefficients of variation were 5.4 and 10.6% for bovine, 5.8 and 11.0% for porcine and, 6.1 and 12.3% for ovine, respectively. These data show that this EIA is a valid and reliable memod for quantitating progesterone in extracts of bovine, porcine and ovine plasma. Key words: Enzymeimmunoassay, progesterone, plasma, bovine, porcine, ovine

Improved radioimmunoassay for vitamin D and its use in assessing vitamin D status.

1985 ◽  
Vol 31 (11) ◽  
pp. 1815-1819 ◽  
Author(s):  
B W Hollis ◽  
J L Napoli
Keyword(s):  
Vitamin D ◽  
High Titer ◽  
Rabbit Serum ◽  
Vitamin D Status ◽  
Ultraviolet Detection ◽  
Vitamin D Analog ◽  
Assay Tube ◽  
Chromatographic Procedure ◽  
Liquid Chromatographic ◽  
25 Hydroxycholecalciferol

Abstract We describe a faster, more-sensitive radioimmunoassay for vitamin D in plasma. Antibodies were generated in rabbits immunized with a new vitamin D analog, the 23,24,25,26,27-pentanor-C(22)-carboxylic acid of vitamin D, coupled directly with bovine serum albumin. After several months, Rivanol-treated sera from the rabbits contained high-titer antibodies, as determined by their abilities to bind 25-hydroxy-[3H]cholecalciferol. The antibody, used at a 1:15 000 final dilution, cross reacted equally with all cholecalciferol and ergocalciferol metabolites tested except 1,25-dihydroxycalciferol metabolites and the parent calciferols. 25-Hydroxycalciferol and similar forms were efficiently extracted from plasma or serum with acetonitrile. We separated bound from free 25-hydroxy-[3H]cholecalciferol by using a second antibody: goat antiserum to rabbit serum. The detection limit of the assay was 3.0 pg of 25-hydroxycholecalciferol and its equivalents per tube; thus only 1 microL of plasma is needed per assay tube. Results compared well with those from a liquid-chromatographic procedure involving specific ultraviolet detection of 25-hydroxycalciferol in plasma.

Specific enzyme immunoassay for human chorionic gonadotrophin

1981 ◽  
Vol 97 (4) ◽  
pp. 562-568 ◽  
Author(s):  
Tatsuhiro Sekiya ◽  
Yoshihito Furuhashi ◽  
Setsuko Goto ◽  
Shigeaki Kaseki ◽  
Yutaka Tomoda ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Serum Samples ◽  
Coefficients Of Variation ◽  
Highly Sensitive ◽  
Assay Tube ◽  
Sandwich Type ◽  
Serial Samples

Abstract. A sandwich-type enzyme immunoassay system specific for human chorionic gonadotrophin (hCG) was prepared with the antibody Fab'-β-D-galactosidase complex and the antibody F(ab')2-immobilized silicone rubber solid phase by using a purified antibody to β subunit of hCG (hCGβ). The assay system cross-reacted less than 4% with human luteinizing hormone (hLH) and human follicular stimulating hormone (hFSH), and proved to be highly sensitive with hCG measurable at levels as low as 0.3 mIU per assay tube. Using 50 μl of serum sample, 6–600 mIU/ml of hCG in serum could be determined specifically with the same degree of precision as in radioimmunoassay but without sample interference with the assay. The coefficients of variation within-run and between-run were 8.6–8.9%, and 4.9– 10.7%, respectively. Values obtained with the enzyme immunoassay correlated well with those of radioimmunoassay ([unk] = 0.98, slope = 0.94, y-intercept = 10.2 mIU/ml for 75 serum samples). Results of the immunoassay of hCG levels in serial samples of serum from healthy women and patients with choriocarcinoma show that this method is useful in the clinical diagnosis of trophoblastic disease.

scholarly journals Stereocontrolled 1-S-glycosylation and comparative binding studies of photoprobe-thiosaccharide conjugates with their O-linked analogs

2013 ◽  
Vol 85 (9) ◽  
pp. 1789-1801 ◽  
Author(s):  
Lingquan Deng ◽  
Xin Wang ◽  
Suji Uppalapati ◽  
Oscar Norberg ◽  
Hai Dong ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Solid Phase ◽  
Solid Support ◽  
Binding Studies ◽  
Highly Efficient ◽  
Comparative Binding ◽  
Kinetic Effects ◽  
Immobilization Method ◽  
Carbohydrate Immobilization

The use of thioglycosides and other glycan derivatives with anomeric sulfur linkages is gaining increasing interest, both in synthesis and in various biological contexts. Herein, we demonstrate the occurrence and circumvention of anomerization during 1-S-glycosylation reactions, and present highly efficient and stereocontrolled syntheses of a series of photoprobe-thiosaccharide conjugates. Mutarotation of glycosyl thiols proved to be the origin of the anomeric mixtures formed, and kinetic effects could be used to circumvent anomerization. The synthesized carbohydrate conjugates were then evaluated by both solution- and solid-phase-based techniques. Both binding results showed that the S-linked glycosides interact with their cognate lectins comparably to the corresponding O-analogs in the present cases, thus demonstrating the reliability of the solid-support platform built upon our photo-initiated carbohydrate immobilization method for probing protein bindings, and showing the potential of combining these two means for studying carbohydrate&ndash;protein interactions.

Solid-phase radioimmunoassay with protein-A-bearing Staphylococcus aureus cells used to assay a protein (ferritin) and a hapten (digoxin).

1980 ◽  
Vol 26 (1) ◽  
pp. 37-40
Author(s):  
J Gauldie ◽  
H K Tang ◽  
A Corsini ◽  
W H Walker
Keyword(s):  
Staphylococcus Aureus ◽  
Matrix Effects ◽  
Solid Phase ◽  
Protein A ◽  
General Applicability ◽  
Rabbit Igg ◽  
Human Sera ◽  
Solid Phase Radioimmunoassay ◽  
Formalin Fixed ◽  
Economical Alternative

Abstract We compared use of protein-A-containing Staphylococcus aureus bacteria with conventional ammonium sulfate precipitation and second-antibody methods of separating bound and free antigen in the radioimmunoassay of a hapten (digoxin) and protein (ferritin) in human sera. In each case, values obtained with the heat-killed, formalin-fixed bacteria correlated well with those found by established methods. No matrix effects were detected in either hapten or protein measurements. Because of the affinity of S. aureus for rabbit IgG, rabbit antisera could be used with a small number of bacteria to detect antigen in the presence of 50-fold excess human IgG. The availability of S. aureus and ease of handling make this reagent a rapid, economical alternative of general applicability in radioimmunoassay.

Analysis of the Temporal Compressibility of Breast Tumour Marker Assays: Development of a “Near Patient” Assay

1995 ◽  
Vol 10 (4) ◽  
pp. 200-205 ◽  
Author(s):  
A. Murray ◽  
J. F. R. Robertson ◽  
M. R. Price
Keyword(s):  
Breast Cancer ◽  
Monoclonal Antibodies ◽  
Liquid Phase ◽  
Solid Phase ◽  
Breast Tumour ◽  
Tumour Marker ◽  
Binding Kinetics ◽  
Immunoradiometric Assay ◽  
Assay Performance ◽  
Kinetics Of

The aim of this study was to investigate whether immunoassays for circulating MUC1 antigen in breast cancer could be compressed in time so that serum level results would be made available during the time of the patient's visit to clinic. Two assays were used: - The EMCA (Euro DPC) is a liquid phase immunoassay and the ELSA CA15-3 (CIS) is a double determinant solid phase immunoradiometric assay. The effects of shortened incubation times were investigated by assaying standards and unknown samples and comparing the results with those using the standard kit protocols. The binding kinetics of the monoclonal antibodies employed in the assays were analysed separately. We conclude that the EMCA assay can be shortened to 35 min and we have attributed this to the fast binding kinetics inherent in a liquid phase assay. This shortened assay may produce the basis for a useful “near patient” assay. By comparison, the solid phase ELSA CA15-3 assay cannot be compressed without loss in assay performance.

Solid-phase radioimmunoassay with protein-A-bearing Staphylococcus aureus cells used to assay a protein (ferritin) and a hapten (digoxin).

1980 ◽  
Vol 26 (1) ◽  
pp. 37-40 ◽  
Author(s):  
J Gauldie ◽  
H K Tang ◽  
A Corsini ◽  
W H Walker
Keyword(s):  
Staphylococcus Aureus ◽  
Matrix Effects ◽  
Solid Phase ◽  
Protein A ◽  
General Applicability ◽  
Rabbit Igg ◽  
Human Sera ◽  
Solid Phase Radioimmunoassay ◽  
Formalin Fixed ◽  
Economical Alternative

Abstract We compared use of protein-A-containing Staphylococcus aureus bacteria with conventional ammonium sulfate precipitation and second-antibody methods of separating bound and free antigen in the radioimmunoassay of a hapten (digoxin) and protein (ferritin) in human sera. In each case, values obtained with the heat-killed, formalin-fixed bacteria correlated well with those found by established methods. No matrix effects were detected in either hapten or protein measurements. Because of the affinity of S. aureus for rabbit IgG, rabbit antisera could be used with a small number of bacteria to detect antigen in the presence of 50-fold excess human IgG. The availability of S. aureus and ease of handling make this reagent a rapid, economical alternative of general applicability in radioimmunoassay.

scholarly journals Cytomegalovirus-specific lymphocyte proliferation and in vitro cytomegalovirus IgG synthesis for diagnosis of cytomegalovirus infections after bone marrow transplantation

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 108-112 ◽  
Author(s):  
P Ljungman ◽  
B Lonnqvist ◽  
G Gahrton ◽  
O Ringden ◽  
B Wahren
Keyword(s):  
Bone Marrow ◽  
Lymphocyte Proliferation ◽  
Solid Phase ◽  
Marrow Transplant ◽  
Cmv Infection ◽  
Igg Synthesis ◽  
Specific Igg ◽  
Lymphocyte Responses ◽  
Cytomegalovirus Infections

Abstract With new techniques 19 bone marrow transplant (BMT) recipients were monitored for lymphocyte proliferation and specific IgG production in vitro by cytomegalovirus (CMV) antigen in solid phase. Twelve patients got a reactivated CMV infection as defined by virus isolation or serum IgG conversion. Lymphocyte proliferation and in vitro IgG production responses were significantly stronger in these 12 patients than in seven without ongoing CMV infection (P = .02). CMV infection was indicated by the lymphocyte responses at a mean of 45 days after BMT as against a mean of 79 days that passed before CMV growth in culture was detected (P less than .05). Lymphocyte proliferation and in vitro IgG production may thus be used as tools for diagnosis and for monitoring of CMV infections in BMT recipients.

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