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.

scholarly journals Ultrastructural immunohistochemical localization of anti-invasion factor (AIF) in bovine cartilage matrix.

1982 ◽  
Vol 30 (6) ◽  
pp. 524-531 ◽  
Author(s):  
F H Wezeman ◽  
G V Childs
Keyword(s):  
Staining Technique ◽  
Ultrastructural Localization ◽  
Immunohistochemical Localization ◽  
Cartilage Matrix ◽  
Fixed Tissue ◽  
Antibody Complex ◽  
Protein Components ◽  
Antigen Antibody ◽  
Unlabeled Antibody ◽  
Bovine Cartilage

Rabbit antibodies prepared against bovine cartilage anti-invasion factor (AIF) were tested for their affinity toward antigenic sites in glutaraldehyde-fixed bovine hyaline cartilage matrix. Ultrastructural localization of the antigen-antibody complex was accomplished by the unlabeled antibody peroxidase-antiperoxidase staining technique. Unextracted and salt-extracted (1 M NaCl or 3 M GuHCl) cartilage slices were incubated with anti-AIF antibodies at a working dilution of 1:20,000. Staining occurred in unextracted matrix distributed throughout the tissue, but with regional variation in the lacunar matrix. Significantly less stain was noted in extracted tissues. The results suggest that at least certain protein components in AIF are morphologically associated with matrix complexes in aldehyde-fixed tissue.

STUDIES ON A THYROID STIMULATING FACTOR IN URINARY CHORIONIC GONADOTROPHIN PREPARATIONS

1967 ◽  
Vol 55 (4) ◽  
pp. 587-599 ◽  
Author(s):  
A. Burger ◽  
R. Kunz
Keyword(s):  
Biological Response ◽  
Chorionic Gonadotrophin ◽  
Rabbit Serum ◽  
Soluble Antigen ◽  
Globulin Fraction ◽  
Human Pituitary ◽  
Antibody Complex ◽  
Antigen Antibody ◽  
Stimulating Factor ◽  
Immune Rabbit

ABSTRACT Thyroid stimulating activity was demonstrated in human urinary chorionic gonadotrophin preparations (HCG) by the method of McKenzie (1958). The biological response of the material was similar to that of bovine pituitary thyrotrophin (TSH), but no neutralization of the activity occurred with antibodies against human pituitary TSH and, even more interesting, with antibodies against human urinary chorionic gonadotrophin preparations. The latter antibodies perfectly neutralized human pituitary TSH. The Gamma-G-globulins precipitated from a mixture of HCG and anti-HCG contained biological activity whereas no activity was recovered in the Gamma-G-globulin fraction when HCG was mixed with non immune rabbit serum. This may indicate the presence of a soluble antigen-antibody complex.

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.

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.

scholarly journals The conglutination phenomenon. IV. An experimental investigation of the factors determining the adsorption of complement by an antigne-antiserum mixture

1950 ◽  
Vol 48 (1) ◽  
pp. 73-95 ◽  
Author(s):  
A. M. Blomfield ◽  
R. R. A. Coombs ◽  
N. H. Hole
Keyword(s):  
General Pattern ◽  
Complex Problem ◽  
Specific Factor ◽  
Soluble Antigen ◽  
Particulate Antigen ◽  
Two Factors ◽  
Initial Intervention ◽  
Antibody Complex ◽  
The One ◽  
Antigen Antibody

1. Salmonella antisera from a number of animals, including man, have been examined by conglutinating complement-absorption and haemolytic complement-fixation tests for the presence of anitbodies. Both a soluble antigen and a particulate antigen of washed suspended orgainsms have been used. The behavior of the antisera was comparable with that previously recorded with the mallein antimallein system, in so far as the reactions obtained with the different complements followed the same general pattern.2. Two factors were found to explain the failure of certain antisera, in conjunction with the antigen, to adsorb certain complements. On the one hand, a few antibodies themselves appeared incapable of fixing a particular complement. On the other hand, although some antisera failed to fix specific complements, their antibodies, isolated from the serum, were able to fix the complements concerned. In these cases it appeared that the heated serum contained a non-specific factor that prevented the adsorption of the complements.3. The existence of such non-specific factors in certain heat-inactivated sera is illustrated in Figs. 11–14. An examination of the mechanism involved does not appear to implicate the procomplementary effect of sera, although this effect in itself is a complex problem requiring further elucidation. The few experiments carried out indicate that the factor in the inactivated serum does not come into play and inhibit the adsorption of complement by an antigen-antibody complex without the initial intervention of some component of complement. Only after this has occurred is the inhibitory factor able to block any further complement adsorption.4. We have discussed the implications of these experiments and their possible relationships to the complementoid phenomena described by previous workers.

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.

Unlabeled Antibody Immunocytochemistry Applied to Murine Mammary Tumor Cells

1975 ◽  
Vol 33 ◽  
pp. 390-391
Author(s):  
Wm. J. Arnold ◽  
J. Russo ◽  
H. D. Soule ◽  
M. A. Rich
Keyword(s):  
Horseradish Peroxidase ◽  
Mammary Tumor ◽  
Covalent Binding ◽  
Petri Dish ◽  
Gamma Chain ◽  
Mammary Tumor Virus ◽  
Mammary Tumor Cells ◽  
Murine Mammary Tumor ◽  
Tumor Virus ◽  
Unlabeled Antibody

Our studies of mammary tumor virus have included the application of the unlabeled antibody enzyme method of Sternberger to mammary tumor derived mouse cells in culture and observation with an electron microscope. The method avoids the extravagance of covalent binding of indicator molecules (horseradish peroxidase) with precious antibody locator molecules by relying instead upon specific antibody-antigen linkages. Our reagents included: Primary Antibody, rabbit anti-murine mammary tumor virus (MuMTV) which was antiserum 113 AV-2; Secondary Antibody, goat anti-rabbit IgG gamma chain (Cappel Laboratories); andthe Indicator, rabbit anti-horseradish peroxidase - horseradish peroxidase complex (PAP) (Cappel Labs.). Dilutions and washes were made in 0.05 M Tris 0.15 M saline buffered to pH 7.4. Cell monolayers, after light fixation in glutaraldehyde, were incubated in place by a protocol adapted from Sternberger and Graham and Karnovsky, then embedded by our usual method for monolayers. Reagents were confined to specific areas by neoprene 0-rings (Parker Seal Co.) reducing the amount of reagent needed to 50 microliters, 1/6th of that required to wet a 35 mm petri dish.

A Simple Method for Preparing Fibrinogen

1966 ◽  
Vol 16 (01/02) ◽  
pp. 198-206 ◽  
Author(s):  
W Straughn ◽  
R. H Wagner
Keyword(s):  
Barium Sulfate ◽  
Caproic Acid ◽  
Aminobutyric Acid ◽  
Gamma Aminobutyric Acid ◽  
Human Fibrinogen ◽  
Simple Method ◽  
High Yields ◽  
Marked Stability

SummaryA simple new procedure is reported for the isolation of canine, bovine, porcine, and human fibrinogen. Two molar β-alanine is used to precipitate fibrinogen from barium sulfate adsorbed plasma. The procedure is characterized by dependability and high yields. The material is 95% to 98% clottable protein but still contains impurities such as plasminogen and fibrin-stabilizing factor. Plasminogen may be removed by adsorption with charcoal. The fibrinogen preparations exhibit marked stability to freezing, lyophilization, and dialysis. Epsilon-amino-n-caproic acid and gamma-aminobutyric acid which were also studied have the property of precipitating proteins from plasma but lack the specificity for fibrinogen found with β-alanine.

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.

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