scholarly journals Solid-phase adsorption method for removing undesired antibodies from polyclonal antiserum.

1989 ◽  
Vol 37 (6) ◽  
pp. 903-908 ◽  
Author(s):  
K Köves ◽  
A Arimura
Keyword(s):  
Solid Phase ◽  
Polyclonal Antiserum ◽  
Adsorption Method ◽  
Purification Method ◽  
Simple Method ◽  
Control Serum ◽  
Antibody Complex ◽  
Lh Cells ◽  
Pap Method ◽  
Antigen Antibody

We have developed a novel and simple method, requiring only a small amount of antigen, for removal of undesired antibodies from antiserum. The method was established using a well-characterized antiserum against rat luteinizing hormone (anti-rLH). Wells of polystyrene tissue culture plates were coated with rat LH (rLH). Anti-rLH diluted 1:3000 was added to rLH-coated wells and shaken to remove LH antibodies. Control anti-rLH was treated in a similar manner in non-rLH-coated wells. Both antisera were tested by immunocytochemistry on rat pituitaries. Antiserum from rLH-coated wells stained no cells, whereas the control serum stained cells that were morphologically typical of LH cells. The effectiveness of this antibody removal was also confirmed in a modified ELISA. In another experiment, anti-rLH and anti-hTSH beta sera were mixed. The final dilution of both antisera was 1:10,000. Anti-rLH was removed by the purification method described. Completeness of antibody removal was confirmed by a double-immunohistochemical staining of rat pituitary in which sections were first stained by the PAP method and then stained with an immunofluorescence procedure after elution of the first antigen-antibody complex. The mixed antiserum incubated in rLH-coated wells did not stain LH cells. There was no co-localization between the LH immunopositivity demonstrated by an anti-rLH serum using immunofluorescence and cells immunostained with the purified antiserum using the PAP method. As indicated in ELISA, the titer of the TSH beta antiserum was not decreased compared to that of the untreated, mixed control antiserum, and the LH antibodies were eliminated by the treatment. This new purification method has four distinct advantages: (a) antiserum is not treated chemically; (b) it requires only a small amount of antigen compared with the amount required for affinity chromatography; (c) neither the undesired antigen-antibody complex(es) nor an excess amount of antigen is present in the purified antiserum; and (d) removal of undesired antibodies can be monitored by ELISA.

Application of Radioimmunoassay for Detection of Staphylococcal Enterotoxins in Foods

1980 ◽  
Vol 43 (1) ◽  
pp. 68-72 ◽  
Author(s):  
MERLIN S. BERGDOLL ◽  
RAOUL REISER
Keyword(s):  
Solid Phase ◽  
Protein A ◽  
Staphylococcal Enterotoxins ◽  
A Cells ◽  
Low Concentrations ◽  
Antibody Complex ◽  
Chloramine T ◽  
Antigen Antibody

The staphylococcal enterotoxins can be iodinated with chloramine-T, lactoperoxidase or gaseous iodine. Low concentrations of enterotoxin, chloramine-T and 125I are recommended to avoid possible damage to the enterotoxin. The antigen-antibody complex can be separated from the unreacted enterotoxin by antibodies adsorbed onto tubes or bromoacetyl-cellulose or by precipitation of the complex with a second antibody or protein A cells. As little as 1 ng of enterotoxin per gram of food can be detected in food extracts with the solid-phase tube method or by precipitation of the antigen-antibody complex with protein A cells.

Polyclonal antibodies with predetermined specificity against bovine αs1-casein: application to the detection of bovine milk in ovine milk and cheese

1993 ◽  
Vol 60 (3) ◽  
pp. 413-420 ◽  
Author(s):  
Marie-Paule Rolland ◽  
Lotfi Bitri ◽  
Pierre Besançon
Keyword(s):  
Solid Phase ◽  
Polyclonal Antibodies ◽  
Bovine Milk ◽  
Milk Proteins ◽  
Peptide Fragments ◽  
Short Peptide ◽  
Antibody Complex ◽  
Predetermined Specificity ◽  
Antigen Antibody ◽  
Monospecific Antibodies

SummaryComparing the primary sequences of bovine and ovine milk proteins, some short peptide fragments are cow-specific, in particular the 141–148 fragment of bovine αs1-casein, which is deleted in its ovine counterpart. The 140–149 peptide was chemically synthesized on a solid phase matrix and directly used as an immunogen to produce polyclonal monospecific antibodies in rabbits. These antibodies recognized this fragment both on the peptidyl resin and in the native protein. They appeared to be monospecific, since no antigen–antibody complex was formed with homologous ovine or caprine proteins. Subsequently, a competitive enzyme-linked immuno-sorbent assay was successfully developed for the detection of defined amounts of cows' milk in sheep's milk from 0·125 to 64% (v/v) and in cheese from 0·5 to 25% (v/v) that was not influenced by heat treatment of milk or the degree of ripening of cheese.

scholarly journals THE UNLABELED ANTIBODY ENZYME METHOD OF IMMUNOHISTOCHEMISTRY PREPARATION AND PROPERTIES OF SOLUBLE ANTIGEN-ANTIBODY COMPLEX (HORSERADISH PEROXIDASE-ANTIHORSERADISH PEROXIDASE) AND ITS USE IN IDENTIFICATION OF SPIROCHETES

1970 ◽  
Vol 18 (5) ◽  
pp. 315-333 ◽  
Author(s):  
LUDWIG A. STERNBERGER ◽  
PAUL H. HARDY ◽  
JOHN J. CUCULIS ◽  
HOWARD G. MEYER
Keyword(s):  
Horseradish Peroxidase ◽  
Rabbit Antiserum ◽  
Antibody Fragment ◽  
Soluble Antigen ◽  
Simple Method ◽  
Antibody Complex ◽  
High Yields ◽  
Antigen Antibody ◽  
Unlabeled Antibody ◽  
Specific Rabbit

Antigen was identified histochemically without the use of labeled antibodies by the sequential application of (a) specific rabbit antiserum, (b) sheep antiserum to rabbit immunoglobulin G, (c) specifically purified, soluble horseradish peroxidase-anti-horseradish peroxidase complex (PAP), (d) 3,3'-diaminobenzidine and hydrogen peroxide and (e) osmium tetroxide. A simple method for preparation of high yields of PAP consisted of precipitation of antibody from specific rabbit antiserum with horseradish peroxidase (PO) at equivalence, solubilization of the washed precipitate with excess PO at pH 2.3, 1°C, followed by immediate neutralization and separation of PAP from PO by half-saturation with ammonium sulfate. The ratio of PO to anti-PO in PAP was 3:2 irrespective of the source of antiserum. PAP was heterogeneous on electrophoresis, homogeneous on sedimentation, diffusion and electron microscopy and consisted of pentagons with diameters of 205 Å. s20, w, 11.98 x 10–13; d20, w, 2.48 x 10–7; molecular weight by sedimentation velocity, 429,000, and equilibrium, 413,000. Sensitivity and specificity of immunohistochemical staining of spirochetes was about 100- to 1000-fold that of immunofluorescence. The unexpected ratio of PO to anti-PO is presumed to be due to stabilization by the pentagonal shape in which three corners are suspected to be PO and two antibody fragment Fc.

Radioimmunoassay of T-2 Toxin in Corn and Wheat

1981 ◽  
Vol 64 (1) ◽  
pp. 156-161 ◽  
Author(s):  
Sungsoo Lee ◽  
Fun S Chu
Keyword(s):  
Sodium Phosphate ◽  
Methanolic Extract ◽  
Precipitation Method ◽  
Ammonium Sulfate Precipitation ◽  
Reverse Phase ◽  
Simple Method ◽  
Sodium Phosphate Buffer ◽  
Antibody Complex ◽  
Antigen Antibody

Abstract A radioimmunoassay (RIA) for the determination of T-2 toxin in corn and wheat was developed. The toxin was first extracted with methanol, and the methanolic extract was washed with hexane and passed through a Sep-Pak C18 reverse phase cartridge. The extract was then evaporated to remove the organic solvent, diluted with 0.1M sodium phosphate buffer (pH 7.2), and subjected to RIA using an ammonium sulfate precipitation method to separate the free T-2 toxin from the antigen-antibody complex. The recovery of T-2 toxin after the extraction and cleanup steps was 78-85% for corn and 90-95% for wheat. Sep-Pak cleanup removed a considerable amount of interfering substances from the sample, thus enabling the detection of 1 and 2.5 ppb of T-2 toxin in the wheat and corn samples, respectively. The recovery by RIA methods of T-2 toxin added to the corn and wheat samples was between 84 and 103% in the ranges of 1-20 ppb (for wheat) and 2.5-20 ppb (for corn). The new RIA proved to be a sensitive, accurate, reproducible, and relatively simple method for determining T-2 toxin.

COMPARATIVE STUDY OF RADIOIMMUNOASSAYS FOR FRAGMENT E-NEOANTIGEN IN PLASMA AND E-ANTIGEN IN SERUM

1987 ◽  
Author(s):  
J P Chen ◽  
M H Goldman ◽  
M stegner
Keyword(s):  
Conformational Changes ◽  
Solid Phase ◽  
Degradation Products ◽  
Normal Serum ◽  
Specific Marker ◽  
Vein Thrombosis ◽  
Antibody Complex ◽  
Normal Males ◽  
Antigen Antibody ◽  
Deep Vein

Fragment E-neoantigen (Eneo) is a specific marker of structual and conformational changes associated with the plasmic degradation of fibrinogen and fibrin. The Eneo radioimmunoassay (RIA) is capable of determining normal and pathological plasma levels of fibrin(ogen) degradation products (FDP). However, the detection of the minute quantity of E-related FDP present in the blood of a normal individual requires subjecting the Eneo RIA to a delayed (18 hr) addition of labeled ligand. This step increases the already lengthy period of incubation (48 hr) for the primary antigen-antibody complex to 66 hr. An additional incubation period (24 hr) is required to precipitate the primary complex with goat anti-rabbit IgG.In an attempt to simplify and speed up the diagnostic blood test for deep vein thrombosis (DVT), the serum fragment E-antigen (E:Ag) RIA has been compared with the Eneo RIA in plasma. Four patients with DVT were followed sequentially with respect to the pre- and post-operative change in response to vascular surgery. Both the Eneo RIA and E:Ag RIAs showed a gradation in plasma (132 - 463 ng/ml) and serum (171 - 1400 ng/ml) E-related FDP levels respectively. Significantly, the sequential measurements of plasma Eneo and serum E:Ag immunoreactivities for these patients appear to fluctuate in parallel with each other. Normal females gave higher plasma Eneo (15.2 ± 4.3 ng/ml) as well as serum E:Ag (42.7 ± 17.8 ng/ml) values than normal males (Eneo = 10.2 ± 5.5 ng/ml, E:Ag = 38.8 ± 19.5 ng/ml). Normal serum E:Ag levels represent the increase in Fg-E concentration, 3-4 times that of normal plasma Eneo levels.Considerably higher plasma Eneo (2.2 - 7.1 pg/ml of Fg-E equivalent) and serum E:Ag levels were observed in DVT patients under thrombolytic therapy with streptokinase. Western immuno-blotting of patients1 plasmas and sera revealed that the Fg-E antigenic sites of both pathological plasmas and sera showed a similar pattern with a major immunoreactive FDP at Mr-360,000.The serum E:Ag assay as currently practiced takes 24 hr to complete, but the time of the assay can be shortened considerably by adapting it to a solid-phase RIA.

Visualization of Antibody Transport in Rat Visceral Yolk-Sac Placenta

1982 ◽  
Vol 40 ◽  
pp. 246-247
Author(s):  
William P. Jollie
Keyword(s):  
Phosphate Buffer ◽  
Yolk Sac ◽  
Female Rats ◽  
Thin Sections ◽  
Visceral Yolk Sac ◽  
Antibody Complex ◽  
Cytochemical Reaction ◽  
Hind Foot ◽  
Antigen Antibody ◽  
Yolk Sac Placenta

A technique has been developed for visualizing antibody against horseradish peroxidase (HRP) in rat visceral yolk sac, the placental membrane across which passive immunity previously has been shown to be transferred from mother to young just prior to birth. Female rats were immunized by injecting both hind foot pads with 1 mg HRP emulsified in complete Freund's adjuvant. They were given a booster of 0.5mg HRP in 0.1 ml normal saline i.v. after one week, then bred and autopsied at selected stages of pregnancy, viz., 12, 1 7 and 22 days post coitum, receiving a second booster, injected as above, five days before autopsy. Yolk sacs were removed surgically and fixed immediately in 2% paraformaldehye, 1% glutaraldehye in 0.1 M phosphate buffer with 0.01% CaCl2 at pH 7.4, room temperature, for 3 hr, rinsed 3X in 0.1 M phosphate buffer plus 5% sucrose, then exposed to 1 mg HRP in 1 ml 0.1 M phosphate buffer at pH 7.4 for 1 hr. They were refixed in aldehydes, as above, for 1 5 min (to assure binding of antigen-antibody complex). Following buffer washes, the tissues were incubated in 3 mg diaminobenzidine tetrahydrochloride and 0.01% H2O2 in 0.05 M Tris-HCl buffer for 30 min. After brief buffer washes, they were postfixed in 2% OsO4. in phosphate buffer at pH 7.4, 4°C for 2 hr, dehydrated through a graded series of ethanols, and embedded in Durcupan. Thin sections were observed and photographed without contrast-enhancement with heavy metals. Cytochemical reaction product marked the site of HRP (i.e., antigen) which, in turn, was present only where it was bound with anti-HRP antibody.

INHIBITION OF LIPOLYTIC ACTIVITY OF SYNTHETIC β1–24 AND β1–39 CORTICOTROPHIN BY ANTI-ACTH ANTISERUM

1966 ◽  
Vol 51 (1) ◽  
pp. 88-94 ◽  
Author(s):  
A. Villanueva ◽  
S. J. H. Ashcroft ◽  
J. P. Felber
Keyword(s):  
Guinea Pig ◽  
Lipolytic Activity ◽  
Peptide Hormones ◽  
Fat Pad ◽  
Double Antibody ◽  
Epididymal Fat ◽  
Antibody Complex ◽  
Radio Immunoassay ◽  
Antigen Antibody

ABSTRACT The synthetic ACTH peptides β1–39 and β1–24 stimulated lipolysis as determined by the rat epididymal fat pad in vitro. The stimulating effect of these peptides was diminished by prior incubation of the peptides with antibodies produced by the guinea-pig against ACTH. The stimulating effect of these hormones was also diminished by the double antibody system used in the radio-immunoassay of ACTH and other peptide hormones, in which incubation with antiserum is followed by precipitation of the antigen-antibody complex by rabbit anti-guinea-pig-γ-globulin.

IDENTIFICATION OF LATS AS AN ANTIGEN-ANTIBODY COMPLEX

1973 ◽  
Vol 71 (4_Suppl) ◽  
pp. S11
Author(s):  
K. Schemmel ◽  
L. Weisbecker ◽  
H. Norden ◽  
V. Mokmol ◽  
V. Becker ◽  
...  
Keyword(s):  
Antibody Complex ◽  
Antigen Antibody

Solid-phase crystallization of ultra-thin amorphous Ge layers on insulators

2021 ◽  
Author(s):  
Ryo Oishi ◽  
Koji ASAKA ◽  
Bolotov Leonid ◽  
Noriyuki Uchida ◽  
Masashi Kurosawa ◽  
...  
Keyword(s):  
Grain Size ◽  
Grain Boundary ◽  
Barrier Height ◽  
Carrier Mobility ◽  
Solid Phase ◽  
Hole Mobility ◽  
Solid Phase Crystallization ◽  
Simple Method ◽  
20 Nm ◽  
Grain Boundary Barrier

Abstract A simple method to form ultra-thin (< 20 nm) semiconductor layers with a higher mobility on a 3D-structured insulating surface is required for next-generation nanoelectronics. We have investigated the solid-phase crystallization of amorphous Ge layers with thicknesses of 10−80 nm on insulators of SiO2 and Si3N4. We found that decreasing the Ge thickness reduces the grain size and increases the grain boundary barrier height, causing the carrier mobility degradation. We examined two methods, known effective to enhance the grain size in the thicker Ge (>100 nm). As a result, a relatively high Hall hole mobility (59 cm2/Vs) has been achieved with a 20-nm-thick polycrystalline Ge layer on Si3N4, which is the highest value among the previously reported works.

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