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

scholarly journals Circulating antibody to transcobalamin II causing retention of vitamin B12 in the blood

Blood ◽  
1977 ◽  
Vol 49 (6) ◽  
pp. 987-1000 ◽  
Author(s):  
R Carmel ◽  
B Tatsis ◽  
L Baril
Keyword(s):  
Vitamin B12 ◽  
Binding Capacity ◽  
Megaloblastic Anemia ◽  
Serum Vitamin ◽  
Vitamin B12 Deficiency ◽  
High Serum ◽  
The Status ◽  
B12 Deficiency ◽  
Long Acting

A patient with recurrent pulmonary abscess, weight loss, and alcoholism was found to have extremely high serum vitamin B12 and unsaturated vitamin B12-binding capacity (UBBC) levels. While transcobalamin (TC) II was also increased, most of his UBBC was due to an abnormal binding protein which carried greater than 80% of the endogenous vitamin B12 and was not found in his saliva, granulocytes, or urine. This protein was shown to be a complex of TC II and a circulating immunoglobulin (IgGkappa and IgGlambda). Each IgG molecule appeared to bind two TC II molecules. The reacting site did not interfere with the ability of TC II to bind vitamin B12, but did interfere with its ability to transfer the vitamin to cells in vitro. The site was not identical to that reacting with anti-human TC II antibody produced in rabbits. Because of this abnormal complex, 57Co-vitamin B12 injected intravenously was cleared slowly by the patient. However, no metabolic evidence for vitamin B12 deficiency was demonstrable, although the patient initially had megaloblastic anemia apparently due to folate deficiency. The course of the vitamin B12-binding abnormalities was followed over 4 yr and appeared to fluctuate with the status of the patient's illness. The IgG-TC II complex resembled one induced in some patients with pernicious anemia by intensive treatment with long-acting vitamin B12 preparations. The mechanism of induction of the antibody formation in our patient is unknown.

The Effect of Human Serum on the Binding Activity of Radiolabelled Monoclonal Antibodies

1987 ◽  
Vol 73 (6) ◽  
pp. 547-554
Author(s):  
Silvia Camagni ◽  
Silvana Canevari ◽  
Marina Ripamonti ◽  
Delia Mezzanzanica ◽  
Rosaria Orlandi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Blood ◽  
Solid Phase ◽  
Binding Capacity ◽  
Binding Activity ◽  
Ovarian Carcinomas ◽  
Radiolabelled Monoclonal Antibodies ◽  
In Vitro Stability ◽  
Murine Monoclonal Antibodies

Three murine monoclonal antibodies (MoAbs), MBrl and MOv2 of IgM isotype and MOv8 of IgG isotype, with restricted reactivity for breast or ovarian carcinomas, were labelled with 125I in the perspective of obtaining specific and stable radioimmunopharmaceutical reagents. The radiolabeled MoAbs were analyzed for their « in vitro » stability in human blood. They were incubated at 37 °C for various lengths of time in human or, as a control, in murine blood and their binding capacity was evaluated by solid-phase RIA and compared with that obtained after incubation with buffer. In human blood, serum and plasma, but not with other components such as erythrocytes, leukocytes, HSA and IgG, the MoAbs revealed a loss of binding reactivity which was marked and constant for the IgM MoAbs, and only occasional for the IgG MoAb. In murine serum the decrease was not so rapid. The same change in the binding capacity was observed when the MoAbs were labelled with 3H or 35S, excluding the involvement of dehalogenating mechanisms. In the perspective of using MoAbs for intracavity therapy the effect of ascitic or pleural fluids on their binding activity was also evaluated. The inhibition of the binding reactivity was not as evident and was not related to the MoAb isotype.

BONE PHOSPHORUS METABOLISM IN VITRO IN THYROTOXICOSIS

1960 ◽  
Vol XXXIII (IV) ◽  
pp. 577-583 ◽  
Author(s):  
C. A. Hernberg
Keyword(s):  
Bone Formation ◽  
Binding Capacity ◽  
Phosphorus Uptake ◽  
Close Correlation ◽  
Phosphorus Metabolism ◽  
Relative Percentage ◽  
Bone Powder ◽  
The Mean ◽  
Normal Controls

ABSTRACT The capacity of bone to take up phosphorus from an incubation medium was used as an indicator of bone formation in 31 cases of thyrotoxicosis. Eleven patients with no thyrotoxicosis served as controls. Three cases of hypothyroidism and one of hyperparathyroidism were also studied. As compared with the phosphorus binding capacity of a standard bone powder the relative percentage of phosphorus uptake in the normal controls was 101.6 ±4.9. The values tended to decrease with increasing age. In the cases of hypothyroidism the mean relative percentage was 97. In the hyperparathyroid case it was 140. The cases of thyrotoxicosis showed values between 99 and 137, with a mean of 118, st. dev. 12.1. There was no close correlation with the severity of thyrotoxicosis or with the nature of the goitre.

Heparin-Associated Thrombocytopenia: The Antibody Is Not Heparin Specific

1992 ◽  
Vol 67 (05) ◽  
pp. 545-549 ◽  
Author(s):  
A Greinacher ◽  
I Michels ◽  
C Mueller-Eckhardt
Keyword(s):  
Platelet Activation ◽  
Dextran Sulfate ◽  
Dermatan Sulfate ◽  
Solid Phase ◽  
Binding Capacity ◽  
Low Grade ◽  
Type Ii ◽  
Pentosan Polysulfate ◽  
Heparin Administration

SummaryIn this study the hypothesis was assessed whether heparin-associated thrombocytopenia (HAT) may be caused by an antibody dependent on polysulfated oligosaccharide epitopes, present not only on heparin but also on different polysulfated substances such as dextran sulfate and pentosan polysulfate. We found that the major factor for eliciting platelet activation with sera of HAT type II patients is neither the structure nor the AT III binding capacity of an oligosaccharide, but rather its grade of sulfation. This was shown by in vitro crossreactivity studies with 40 sera of HAT type II patients using unfractionated heparins, LMW heparins (Fragmin, Fraxiparin), enoxaparin, LMW heparinoid (Org 10172 and its subfractions), de-N-sulfated heparin, dermatan sulfate, dextran sulfate, pentosan polysulfate and dextran. Platelet activation was measured by the heparin induced platelet activation (HIPA) assay and the serotonin release assay (SRA). The platelet activating factor was isolated with the IgG fraction, but did not bind to heparin and dextran sulfate fixed to a solid phase. By isoimmune fixation electrophoresis a monoclonal gammopathy was ruled out in the three sera assessed. The in vivo effect of different LMW heparins and the heparinoid Org 10172 was observed in 10 patients with HAT type II. In a prospective study, a compatible heparin-like anticoagulant was selected for 10 HAT patients for whom further parenteral anticoagulation was required. The only substance that showed no crossreactivity in vitro was the LMW heparinoid Org 10172, which differs from heparin and LMW heparins by its low-grade sulfation. Upon treatment with the heparinoid, all 10 patients had a good clinical outcome, even if they had previously developed thromboembolic complications under LMW heparin administration. As Org 10172 contains a small amount of a LMW heparin-like substance (3%) this heparinoid should not be used in HAT patients without prior in vitro testing. We conclude that heparin-associated thrombocytopenia is not caused by a heparin-specific antibody and that a major factor contributing to the pathomechanism is the high grade of sulfation present in a variety of polysulfated oligosaccharides.

scholarly journals Plasma and platelet HLA in normal individuals: quantitation by competitive enzyme-linked immunoassay

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 282-286 ◽  
Author(s):  
KJ Kao
Keyword(s):  
Solid Phase ◽  
Hla Antigens ◽  
Mean Value ◽  
Platelet Surface ◽  
Standard Curve ◽  
Quantitative Correlation ◽  
Normal Individuals ◽  
The Mean ◽  
Solid Phase Assay ◽  
Mean Concentration

Abstract Recent studies on platelet HLA indicate that greater than 50% of platelet HLA antigens are adsorbed on the platelet surface and may be derived from plasma. It has been speculated that platelet HLA may be directly proportional to plasma HLA concentration. To determine the quantitative correlation between plasma and platelet HLA, a precise competitive enzyme-linked immunoassay (ELISA) for measurements of soluble and cellular HLA antigens was developed by using purified HLA antigens and W6/32 anti-HLA monoclonal antibody. The useful range of the standard curve for the assay was 0.01 to 5.0 micrograms/mL. The intraassay and interassay variations were 7% and 14%, respectively. The plasma HLA concentrations measured in 61 healthy adults ranged from 0.25 to 4.1 micrograms/mL, and the mean plasma HLA concentration was 1.47 +/- 0.87 micrograms/mL (+/- SD). Platelet HLA concentrations determined in the same 61 persons ranged from 4.7 to 17.33 fg/platelet, and the mean concentration was 9.3 +/- 2.9 fg/platelet (+/- SD). Chloroquine-elutable platelet HLA concentrations were also determined in 42 of the 61 persons, with the mean value of 5.7 +/- 2.1 fg/platelet (+/- SD). The plasma HLA concentration of each individual was then correlated with the same person's total or chloroquine-elutable platelet HLA concentration. Linear regression analyses of the results revealed no significant correlation between platelet and plasma HLA concentrations. Thus, it is unlikely that chloroquine-elutable HLAs are derived from plasma. The developed solid-phase assay for HLA will be useful for further study of the quantitative significance of plasma HLA antigens in allosensitization of transfused individuals.

In-Vitro Adsorption Studies of Isoniazid

1991 ◽  
Vol 10 (2) ◽  
pp. 133-135 ◽  
Author(s):  
O.E. Orisakwe ◽  
A. Akintonwa
Keyword(s):  
Activated Carbon ◽  
Carbon Black ◽  
Adsorption Capacity ◽  
Adsorption Isotherms ◽  
Activated Charcoal ◽  
Binding Capacity ◽  
In Vitro Experiments ◽  
Adsorption Studies ◽  
The Mean

In-vitro experiments were performed to investigate the extent of adsorption of isoniazid to activated charcoal and locally produced activated carbon black (N220) and to explore the effect of varying pH on this adsorption. The results of the study indicated that activated charcoal and activated carbon black adsorbed isoniazid effectively. Adsorption was dependent upon the quantity of charcoal used. With charcoal quantity at 0.5 g, adsorption was virtually completed within 60 min. The mean or composite adsorption capacity of activated charcoal and activated carbon black (?g ml-1 of charcoal) were 325 and 278, respectively. The result of adsorption isotherms indicated no change in binding capacity of the drug from solutions of different pH.

Determination of serum ferritin by a one-step immunoenzymoassay, and comparison of four liver-ferritin standards.

1985 ◽  
Vol 31 (4) ◽  
pp. 640-642 ◽  
Author(s):  
M C Revenant ◽  
J Goudable ◽  
A Beaudonnet ◽  
A Mailliavin ◽  
J Pichot ◽  
...  
Keyword(s):  
New England ◽  
Solid Phase ◽  
Rabbit Antibody ◽  
Standard Curve ◽  
Sandwich Type ◽  
Human Ferritin ◽  
One Step ◽  
The Mean ◽  
Liver Ferritin

Abstract We describe a one step "sandwich"-type immunoenzymoassay for ferritin in human serum. The solid-phase consists of glutaraldehyde-treated polypropylene tubes coated with rabbit antibody to human ferritin. Liver ferritin is the standard. Peroxidase-conjugated antiserum to ferritin and a sensitive chromogen, o-phenylenediamine, are used. The assay requires 90 min. The standard curve is linear up to 400 micrograms of ferritin per liter of serum. Within- and between-run CVs are less than 6% for low, high, and medium concentrations and are about 13.0% at the decision level for iron deficiency. Results by a two-step "sandwich" procedure (New England Immunology Associates kit) correlated well, r = 0.98. We assessed four liver ferritin standards from different manufacturers with the described method. The mean absorbance for the 40 micrograms/L ferritin standard was 1.5 for that from Diagnostics Biochem and National Institute for Biological Standards and Controls, 1.0 for that from Dako, and 0.4 for that from Sigma. Consequently, to standardize results, all liver ferritin standards should be calibrated vs the National Institute for Biological Standards and Controls reference standard.

scholarly journals Plasma and platelet HLA in normal individuals: quantitation by competitive enzyme-linked immunoassay

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 282-286
Author(s):  
KJ Kao
Keyword(s):  
Solid Phase ◽  
Hla Antigens ◽  
Mean Value ◽  
Platelet Surface ◽  
Standard Curve ◽  
Quantitative Correlation ◽  
Normal Individuals ◽  
The Mean ◽  
Solid Phase Assay ◽  
Mean Concentration

Recent studies on platelet HLA indicate that greater than 50% of platelet HLA antigens are adsorbed on the platelet surface and may be derived from plasma. It has been speculated that platelet HLA may be directly proportional to plasma HLA concentration. To determine the quantitative correlation between plasma and platelet HLA, a precise competitive enzyme-linked immunoassay (ELISA) for measurements of soluble and cellular HLA antigens was developed by using purified HLA antigens and W6/32 anti-HLA monoclonal antibody. The useful range of the standard curve for the assay was 0.01 to 5.0 micrograms/mL. The intraassay and interassay variations were 7% and 14%, respectively. The plasma HLA concentrations measured in 61 healthy adults ranged from 0.25 to 4.1 micrograms/mL, and the mean plasma HLA concentration was 1.47 +/- 0.87 micrograms/mL (+/- SD). Platelet HLA concentrations determined in the same 61 persons ranged from 4.7 to 17.33 fg/platelet, and the mean concentration was 9.3 +/- 2.9 fg/platelet (+/- SD). Chloroquine-elutable platelet HLA concentrations were also determined in 42 of the 61 persons, with the mean value of 5.7 +/- 2.1 fg/platelet (+/- SD). The plasma HLA concentration of each individual was then correlated with the same person's total or chloroquine-elutable platelet HLA concentration. Linear regression analyses of the results revealed no significant correlation between platelet and plasma HLA concentrations. Thus, it is unlikely that chloroquine-elutable HLAs are derived from plasma. The developed solid-phase assay for HLA will be useful for further study of the quantitative significance of plasma HLA antigens in allosensitization of transfused individuals.

scholarly journals Elevated Serum Iron, Low Unbound Transferrin and Candidiasis in Acute Leukemia

Blood ◽  
1969 ◽  
Vol 34 (4) ◽  
pp. 441-451 ◽  
Author(s):  
LEONA CAROLINE ◽  
FRED ROSNER ◽  
PHILIP J. KOZINN
Keyword(s):  
Acute Leukemia ◽  
Serum Iron ◽  
Binding Capacity ◽  
Lymphoblastic Leukemia ◽  
Elevated Serum ◽  
High Serum ◽  
Systemic Candidiasis ◽  
Candidal Infection ◽  
Iron Binding Capacity

Abstract Patients with acute leukemia have been shown to have elevated serum iron levels and markedly diminished or absent unbound iron binding capacity (21 of 34 patients with myeloblastic and 2 of 6 patients with lymphoblastic leukemia). Four patients (2 myeloblastic and 2 lymphoblastic) developed systemic candidiasis with elevated Candida agglutination titers and positive precipitin tests against a somatic antigen of Candida albicans. It is postulated that high serum iron and/or low unbound transferrin predispose patients with acute leukemia to candidal infection by enhancing growth of this fungus. In vitro studies support this hypothesis.

Enzyme-linked immunoabsorbant assay of apolipoprotein AII in plasma, with use of a monoclonal antibody.

1986 ◽  
Vol 32 (6) ◽  
pp. 967-971 ◽  
Author(s):  
E A Stein ◽  
L DiPersio ◽  
A J Pesce ◽  
M Kashyap ◽  
J T Kao ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Solid Phase ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Cross Reactivity ◽  
Lipoprotein Subfractions ◽  
Parallel Displacement ◽  
Standard Curve ◽  
Human Apolipoprotein ◽  
Plasma High Density

Abstract We produced a monoclonal antibody (C2-22) to human apolipoprotein (Apo) AII and describe its use in an enzyme-linked immunoabsorbant assay (ELISA) for Apo AII in human plasma and lipoprotein subfractions. No cross reactivity of the antibody with Apo CI, CII, CIII, E, or ablumin was detected. Apo AI and low- and very-low-density lipoprotein cross reacted by 0.25%, less than 0.2%, and less than 0.3%, respectively. Whole plasma high-density lipoprotein (HDL) and HDL subfractions (HDL2 and HDL3) produced parallel displacement curves. This quantitative ELISA is based on competition between solid-phase-bound Apo AII and free Apo AII. Bound C2-22 is detected by alkaline-phosphatase-labeled second antibody. The standard curve for the assay is linear for plasma diluted 500-fold originally containing 140 to 1140 mg of Apo AII per liter. Delipidation of plasma samples exposed no additional antigenic sites. Within- and between-run CVs were respectively 8.4% and 8.7% at 327 mg/L of Apo AII, and 6.8% and 7.4% at 587 mg/L. Results correlated well with those by a polyvalent-antisera-based RIA procedure: r = 0.916, p less than 0.01, RIA = 0.896 ELISA -19.1 mg/L.

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