ABSTRACT
B and T lymphocyte attenuator (BTLA) is an immunoglobulin superfamily member surface protein expressed on B and T cells. Its ligand, herpesvirus entry mediator (HVEM), is believed to act as a monomeric agonist that signals via the CRD1 of HVEM to inhibit lymphocyte activation: HVEM is also the receptor for lymphotoxin-α and LIGHT, which both bind in the CRD2 and CRD3 domains of the HVEM molecule, and for CD160 which competes with BTLA. We have shown that recombinant HVEM and a panel of different monoclonal antibodies specifically bind murine BTLA on both B and T cells and that some antibodies inhibit anti-CD3ε-induced T cell proliferation in vitro, but only when constrained appropriately with a putatively cross-linking reagent. The antibodies had no significant effect on in vitro T cell proliferation in a mixed lymphocyte reaction (MLR) assay nor on in vitro DO11.10 antigen-induced T cell proliferation. None of these antibodies, nor HVEM-Fc, had any significant effect on in vitro B cell proliferation induced by anti-immunoglobulin M antibodies (±anti-CD40) or lipopolysaccharide. We further elucidated the requirements for inhibition of in vitro T cell proliferation using a beads-based system to demonstrate that the antibodies that inhibited T cell proliferation in vitro were required to be presented to the T cell in a cis, and not trans, format relative to the anti-CD3ε stimulus. We also found that antibodies that inhibited T cell proliferation in vitro had no significant effect on the antibody captured interleukin-2 associated with the in vivo activation of DO11.10 T cells transferred to syngeneic recipient BALB/c mice. These data suggest that there may be specific structural requirements for the BTLA molecule to exert its effect on lymphocyte activation and proliferation.
Subject(s)
Antibodies, Monoclonal/immunology , B-Lymphocytes/immunology , Lymphocyte Activation , Receptors, Immunologic/antagonists & inhibitors , Receptors, Immunologic/immunology , T-Lymphocytes/immunology , Animals , CD3 Complex/immunology , CD40 Antigens/immunology , Cross-Linking Reagents/chemistry , Immunoglobulin M/immunology , Interleukin-2/blood , Interleukin-2/immunology , Lymphotoxin-alpha/immunology , Mice , Mice, Inbred BALB C , Receptors, Tumor Necrosis Factor, Member 14/immunologyABSTRACT
PROBLEM: Human placental alkaline phosphatase (PLAP) is a unique placental antigen bound to the syncytiotrophoblast, which may be able to elicit a specific immune response in pregnancy. METHOD OF STUDY: Antibody to PLAP was purified from placental extracts by: a) acid elution of membrane vesicles; b) purifying complexes of PLAP with human antibody on monoclonal antibodies to PLAP followed by denaturation of the enzyme; and c) by denaturation of PLAP in placental extracts and purification of antibody to PLAP on PLAP columns. RESULTS AND CONCLUSIONS: Specific antibody to PLAP is present in placental extracts, and is mostly bound to placental membrane preparations. PLAP is therefore immunogenic in pregnancy and could serve as a useful monitor of pregnancy-specific immunological responses. Since a similar enzyme appears in some cancers, it is possible that the immunization against PLAP in pregnancy will help to protect against the development of ovarian and endometrial cancer (the 'fetal antigen' hypothesis).
Subject(s)
Antibodies/isolation & purification , Isoenzymes/immunology , Placenta/immunology , Alkaline Phosphatase , Antibodies, Monoclonal/immunology , Chromatography, Affinity/methods , Female , GPI-Linked Proteins , Humans , Hydrochloric Acid/pharmacology , Imidazoles/pharmacology , Immunoglobulin G/immunology , Immunoglobulin G/isolation & purification , Immunoglobulin M/isolation & purification , Isoenzymes/drug effects , Precipitin Tests/methods , Pregnancy , Protein Denaturation , Sodium Chloride/pharmacology , Tissue ExtractsABSTRACT
Detection of the products of the PCR reaction using nonisotopically labeled DNA molecules containing biotin, fluorescein, or digoxigenin has become a popular method for identification of specific products of polymerase chain reaction (PCR) (1,3). These labeled molecules are prepared either by PCR synthesis in the presence of labeled deoxyuridine triphosphate (1,3) or by hybridization of labeled probes to the unlabeled PCR product (1,2). Detection is then in a format very similar to enzyme-linked immunosorbent assays (ELISA) using similarly labeled antigens and antibodies, i.e., by capture on the receptor for one ligand (streptavidin or antibody) and using the other ligand for detection.
ABSTRACT
The measurement of chicken and human antibodies to Clostridium botulinum neurotoxins A, B, and E was accomplished by affinity isolation of complexes containing these antibodies. By this approach, a mixture of toxin with the test antibody, fluoresceinated antibody, and enzyme (Russell's viper venom factor X activator)-labeled antibody is allowed to form a complex in solution phase. This complex is then bound to a matrix containing antifluorescein antibody. All components not bound to the matrix are washed off, and the complex is isolated intact by elution with fluorescein, which competes with the complex for binding to the antifluorescein matrix. The eluted complex is then bound to a matrix which specifically binds the test antibody (anti-chicken immunoglobulin Y [IgY] or anti-human IgG), and the bound complex is measured by using the enzyme label. Using this approach, we were able to measure as little as 1 ng of specific antibody per ml from affinity-isolated, monospecific chicken antibody preparations and to measure antibody specifically from IgY fractions of monospecific chicken antibody preparations. Human antibodies from subjects immunized with pentavalent toxoid preparations were detectable at dilutions as great as 24,300-fold, and undiluted serum from most control subjects showed no measurable antibody. Antibody was also measured in 65 subjects who were receiving preparations of neurotoxin A (BOTOX) for the treatment of spastic disorders. Eighteen of them had toxin-specific antibody reactive with toxin B, and two of them had toxin-specific antibody reactive with toxin A. The two patients having antibody to toxin A were refractory to treatment with this toxin. This approach of isolation of hapten-labeled immune complexes under nondenaturing conditions with hapten is broadly applicable to the specific measurement of antibodies present at very low concentrations in serum.
Subject(s)
Antibodies, Bacterial/analysis , Bacteriological Techniques , Botulinum Toxins/immunology , Immunoassay/methods , Animals , Antibodies, Bacterial/blood , Antibodies, Monoclonal , Antigen-Antibody Complex/isolation & purification , Bacteriological Techniques/statistics & numerical data , Botulinum Toxins, Type A/administration & dosage , Botulinum Toxins, Type A/immunology , Chickens , Evaluation Studies as Topic , Fluorescein , Fluoresceins , Haptens , Humans , Immunization , Immunoassay/statistics & numerical data , Sensitivity and SpecificityABSTRACT
The role of tissue factor (TF) as an initiator of the thrombotic complications secondary to atherosclerosis has been acknowledged, and in situ expression of TF activity by monocyte-derived macrophages and lesion-associated macrophage foam cells has been documented. Macrophages express TF activity upon exposure in vitro to either oxidized low density lipoprotein LDL (Ox-LDL) or endotoxin (lipopolysaccharide). This activity has been associated with membrane vesicles that apparently are shed after procoagulant expression. The present study based upon the correlative use of an enzyme-linked coagulant assay and three-dimensional multi-antigen, immunogold electron microscopy, reports the ultrastructural localization of TF antigen and spatially correlates TF with OX-LDL binding and the presence of nascent fibrin polymers on the plasma membrane of cultured macrophages. Pigeon monocyte/macrophages, after a 4-hour induction with lipopolysaccharide (2 micrograms/ml) or minimally oxidized LDL (50 micrograms/ml; thiobarbituric acid reducing substance, 5 to 8 nmol/mg protein) were incubated for 40 minutes in a Tris-buffered medium containing factors VII, V, X, II, and I before either assaying for coagulant activity or processing for gold-colloid cytochemistry. TF activity, as measured by enzyme-linked coagulant assay peaked 6 hours after agonist exposure with lipopolysaccharide and Ox-LDL giving, respectively, 115- and 60-fold stimulation as compared with control. This activity corresponded to the elaboration of membrane ruffles and microvilli on the cell surfaces. Through correlative immunogold cytochemistry (15-nm-diameter colloid) and gold-ligand cytochemistry (30-nm-diameter colloid), TF antigen (83%) and Ox-LDL (78%) were primarily associated with the membrane ruffles and microvilli. Multi-antigen immunogold cytochemistry when used in conjunction with ligand-gold cytochemistry documented co-localization of Ox-LDL (22-nm gold), TF antigen (15-nm gold) and a delicate three-dimensional network of short fibrin fibers that were decorated in a linear fashion with the immunogold probes (30-nm gold). These results provide evidence that TF antigen is located at selected regions on the cell surfaces. Furthermore, these same regions provide binding sites for agonist uptake and organization sites for fibrin polymerization. Hypothetically, the localized membrane regions could be shed from the cell surface as a means for regulating coagulation potential.
Subject(s)
Antigens/metabolism , Fibrin/physiology , Lipoproteins, LDL/pharmacology , Macrophages/metabolism , Thromboplastin/metabolism , Animals , Cell Membrane/metabolism , Cells, Cultured , Columbidae/blood , Lipoproteins, LDL/metabolism , Macrophages/cytology , Microscopy, Electron , Microscopy, Electron, Scanning , Monocytes/cytology , Oxidation-Reduction , Thromboplastin/immunology , Tissue DistributionABSTRACT
We describe a microtiter-plate-based, colorimetric assay for DNA, the enzyme-linked DNA-enzyme-linked coagulation assay (EDNA-ELCA). The EDNA-ELCA uses amplification of the common pathway of coagulation for the ultrasensitive detection of DNA which is tagged by incorporation of functional groups such as biotin and fluorescein. The EDNA-ELCA enables detection of attomole amounts of DNA (< 1 pg per microtiter well), with a sensitivity 200-1000 times higher than other colorimetric techniques. The assay has been applied as an adjunct to PCR for quantitative determination of methicillin-resistant Staphylococcus aureus DNA at levels corresponding to 1-10(5) organisms. The EDNA-ELCA can also be used to assay DNA by hybridization; < 50 amol of an unlabeled DNA template is detected by hybridization to biotin- and fluorescein-labeled probes.
Subject(s)
DNA Primers/chemical synthesis , DNA/chemical synthesis , Polymerase Chain Reaction/methods , Animals , Antibodies , Base Sequence , Cloning, Molecular , Colorimetry/methods , DNA/chemistry , Fibrinogen , Fluorescein-5-isothiocyanate , Goats/immunology , Indicators and Reagents , Microchemistry/methods , Molecular Sequence Data , Sensitivity and SpecificityABSTRACT
The solution-phase complex assay for toxins A, B, and E from Clostridium botulinum (Elcatech, Inc., Winston-Salem, N.C.) was modified to measure antibody. The addition of unlabeled polyclonal antibodies to a mixture consisting of toxin with chicken antibody and RVV-XA-labeled horse antibody reduces the sensitivity of detection of neurotoxin. This reduction in sensitivity can be used as a measure of the specific antibody titer.
Subject(s)
Antibodies, Bacterial/analysis , Botulinum Toxins/immunology , Clostridium botulinum/immunology , Enzyme-Linked Immunosorbent Assay/methods , Animals , Antibody Specificity , Binding, Competitive , Biological Assay , Chickens , Enzyme-Linked Immunosorbent Assay/statistics & numerical data , Evaluation Studies as Topic , Horses , Humans , Mice , Neutralization Tests , Sensitivity and SpecificityABSTRACT
The measurement of toxins A, B, and E from Clostridium botulinum was accomplished by use of a modified sandwich enzyme-linked immunosorbent assay (ELISA) employing labeled horse antibody and either chicken antibody or biotinylated horse antibody. The complexes formed in solution phase were captured onto solid phases coated with rabbit anti-chicken immunoglobulin G (chicken antibody) or avidin (biotinylated antibody). The assay was brought to the sensitivity of the mouse bioassay (5 to 10 pg/ml, or 0.03 to 0.07 pM) by employing as labeling enzyme the factor X activator of Russell's viper venom (RVV-XA) and a sensitive coagulation-based assay amplification system known as enzyme-linked coagulation assay. Complex formation was found to be a slower reaction than binding to the capture plate, and so the assay used a preincubation step to produce the solution-phase complexes before they were bound to the solid phase. Keeping the concentrations of Russell's viper venom factor X activator antibody and capture antibody constant for diluted samples and diluting complexes into buffer without keeping labeled antibody concentrations constant were equivalent in allowing the detection of low neurotoxin concentrations. This ELISA-enzyme-linked coagulation assay procedure is a convenient alternative to the mouse bioassay, which shows complete resolution of the neurotoxins in addition to the requisite sensitivity.
Subject(s)
Blood Coagulation Tests/methods , Botulinum Toxins/analysis , Clostridium botulinum/chemistry , Enzyme-Linked Immunosorbent Assay/methods , Neurotoxins/analysis , Antibodies, Bacterial , Avidin , Biotin , Immunoglobulin G , Sensitivity and Specificity , Species SpecificityABSTRACT
Monocyte-derived macrophages, focal to initiation and progression of atherosclerosis, have been implicated in thrombotic complication of this disease. In the present study we demonstrated tissue factor based procoagulant activity in cultured macrophages from the White Carneau pigeon following endotoxin (1-2 micrograms/ml) stimulation. This macrophage procoagulant activity paralleled activity obtained with pigeon brain homogenate. We used Enzyme-Linked Coagulation Assay (ELCA), an ultrasensitive microtiter plate assay, to measure procoagulant activity in these cells. Through the use of clotting factors purified from pigeon plasma, procoagulant activity could be detected with as few as 1-3 cells. Tissue factor antigen, detected through the use of immunogold labelling in conjunction with a polyclonal antibody which was highly specific to human tissue factor, was distributed uniformly over the plasma membrane of the endotoxin-stimulated cells. These studies suggest that this procoagulant activity might play an important role in the pathobiology of atherosclerosis in White Carneau pigeons by initiating fibrin polymerization and thus leading to thrombotic complications of the disease.
Subject(s)
Columbidae/blood , Macrophages/chemistry , Monocytes/cytology , Thromboplastin/analysis , Animals , Blood Coagulation Tests , Cells, Cultured , Endotoxins/pharmacology , Factor V/isolation & purification , Factor VII/isolation & purification , Factor X/isolation & purification , Humans , Immunohistochemistry , Monocytes/drug effects , Prothrombin/isolation & purification , Sensitivity and Specificity , Species SpecificityABSTRACT
RVV-X, the factor X activator from Russell's viper venom, has been isolated using affinity chromatography on agarose columns of a monoclonal antibody specific for this enzyme. Upon testing acid, alkaline, and high concentrations of MgCl2 for elution, it was found that use of high concentrations of MgCl2 was most effective in elution of RVV-X. It was nondenaturing and yielded 90% recovery of homogeneous enzyme without measurable contamination by other proteins of the venom.
Subject(s)
Chromatography, Affinity/methods , Endopeptidases/isolation & purification , Magnesium Chloride , Metalloendopeptidases , Viper Venoms/enzymology , Animals , Antibodies, Monoclonal/immunology , Antibody Affinity , Electrophoresis, Polyacrylamide Gel , Endopeptidases/immunology , Hydrogen-Ion Concentration , Immunosorbent Techniques , Mice , Mice, Inbred BALB C/immunology , Molecular WeightABSTRACT
A new immunoassay amplification method has been applied to the measurement of toxins A, B, and E from Clostridium botulinum. The technique is a modified enzyme-linked immunosorbent assay (ELISA) which relies on the detection of sandwich complexes on microtiter plates by a solid-phase coagulation assay known as ELCA, or enzyme-linked coagulation assay. In the method, a coagulation activating enzyme (RVV-XA) isolated from the venom of Russell's viper is conjugated to affinity-purified horse antibodies specific for toxin type A, B, or E. Plates are coated with affinity-purified antibodies, and standard captag (capture-tag) protocols using labeled antibody are employed to bind the toxin from solution. Complexes are detected by adding a modified plasma substrate which contains all the coagulation factors mixed with alkaline phosphatase-labeled fibrinogen and solid-phase fibrinogen; deposition of solid-phase, enzyme-labeled fibrin on the solid phase is then a reflection of formation of toxin-RVV-XA-antibody complexes on the solid phase. Because of the ability to detect RVV-XA by this coagulation assay at concentrations < 0.1 pg/ml, it was possible to measure C. botulinum toxins A, B, and E at mouse bioassay levels (< 10 pg/ml, or < 0.07 pM) for both purified neurotoxin and crude culture filtrates obtained from strains known to produce appropriate single toxins. ELISA-ELCA should be applicable to measurement of toxins in most of the materials (contaminated food, blood, and excreta) for which the comparably sensitive mouse bioassay is currently employed. This method has the potential of broad application to the measurement of low concentrations of any antigen for which appropriate immunochemical reagents are available, in a color test format.
Subject(s)
Botulinum Toxins/analysis , Enzyme-Linked Immunosorbent Assay/methods , Animals , Blood Coagulation , Cattle , Hydrogen-Ion Concentration , Mice , Sensitivity and SpecificityABSTRACT
Cyst and ascites fluids from patients with ovarian epithelial neoplasms contain immunoglobulins with antitumor activity. Autologous antibodies bound to the cellular membrane fragments obtained from human ovarian neoplastic effusions react with cell-surface antigens on different human ovarian cell lines, surgical specimens of human ovarian adenocarcinoma, and human ovarian tumors grown in athymic Balb/c mice. The antibodies do not react with tissue preparations from normal human ovaries, other nonovarian normal or neoplastic tissues, and nonovarian human cell lines. These studies indicate that these antibodies are capable of complement-mediated lysis of human ovarian tumor cell lines in vitro. Preliminary characterization of the autologous ovarian tumor-associated antigen(s) indicates that it may be composed of three large-molecular-weight proteins of 182,000, 164,000, and 122,000 d.
Subject(s)
Antigens, Neoplasm/analysis , Autoantibodies/analysis , Cytotoxicity, Immunologic , Ovarian Neoplasms/immunology , Adenocarcinoma/immunology , Adenocarcinoma, Papillary/immunology , Animals , Antigens, Surface/analysis , Cystadenocarcinoma/immunology , Cytotoxicity Tests, Immunologic , Female , Fluorescent Antibody Technique , Humans , Immunoglobulins/analysis , Mice , Mice, Inbred BALB C , Tumor Cells, CulturedABSTRACT
We present an assay for components of the fibrinolytic system based on hydrolysis of solid-phase associated enzyme-labeled fibrin. This Enzyme-linked Fibrinolytic Assay, (ELFA) permits the measurement of less than 1 IU/ml of t-PA in 50 microliters of plasma diluted to 1:80 within six hours, in a microtiter plate format, with a colorimetric endpoint. High levels (greater than 10 IU/ml) of tissue plasminogen activator can be measured in less than 30 minutes. The assay was approximately 100 times more sensitive than a clot lysis assay performed in microtiter plates and used less reagents. By comparison with the parabolic rate, coupled assay using chromogenic substrates and soluble fibrin, ELFA performed assays with equal sensitivity and in less time. The ELFA assay uses solid phase fibrin as the substrate, activator and indicator for the assay. For this reason, it has the advantage of the clot lysis assays in that it is more analogous to the physiological hydrolysis of fibrin but with the sensitivity and convenience of the parabolic rate, coupled assay.
Subject(s)
Fibrin/metabolism , Peroxidases , Tissue Plasminogen Activator/blood , Chromogenic Compounds , Humans , Reagent Kits, DiagnosticABSTRACT
We have purified the integrin GPIIb:IIIa from the membrane fraction of human blood platelets by lentil lectin affinity chromatography followed by gel filtration chromatography. With purified GPIIb:IIIa as an antigen, we have produced monoclonal antibody CS-1, which immunoblotting demonstrates to be specific for native GPIIIa; disulfide bond reduction of GPIIIa resulted in loss of immunoreactivity. Radiolabelled ligand binding studies revealed that CS-1 recognized approximately 55,000 sites per platelet and bound with a Kd in the nanomolar range, independent of the state of platelet activation. Binding of CS-1 or its Fab fragment to ADP- and thrombin-stimulated gel filtered platelets caused a 2-3 fold inhibition of binding the soluble ligands fibrinogen and fibrin protofibrils. CS-1 also inhibited aggregation of ADP- and thrombin-stimulated platelets by 2- and 4-fold, respectively. Since CS-1 inhibits fibrin(ogen) interactions with GPIIb:IIIa, we propose that the conformationally dependent epitope on GPIIIa recognized by CS-1 constitutes a region of the receptor which is involved in fibrin(ogen) binding.
Subject(s)
Antibodies, Monoclonal , Blood Platelets/metabolism , Fibrin/metabolism , Fibrinogen/metabolism , Platelet Membrane Glycoproteins/immunology , Adenosine Diphosphate/pharmacology , Amino Acids/analysis , Antibodies, Monoclonal/biosynthesis , Antibody Specificity/immunology , Blood Platelets/drug effects , Enzyme-Linked Immunosorbent Assay , Humans , Iodine Radioisotopes , Kinetics , Platelet Aggregation/drug effects , Platelet Aggregation/immunology , Platelet Membrane Glycoproteins/isolation & purification , Protein Binding , Protein Conformation , Spectrophotometry, Ultraviolet , Thrombin/pharmacologyABSTRACT
We have previously reported an ultrasensitive microtiter plate assay, enzyme-linked coagulation assay (ELCA), which can measure a factor X activator isolated from Russell's viper venom (RVV-XA) at concentrations less than 0.1 amol/sample. The high sensitivity of this assay is derived from enzyme amplification via the clotting cascade in combination with the utilization of enzyme-labeled solution-phase and unlabeled solid-phase fibrinogen. Modification of the ELCA assay to detect RVV-XA directly bound to nitrocellulose, ELCA blot, as described in this report, allowed the detection of blotted RVV-XA at amounts as low as 2 fg. The high sensitivity of the ELCA blot was utilized to develop an immunodetection system for Western blots, the ELCA immunoblot, and a biotin/avidin protein stain for blotted membranes, biotin/avidin ELCA blot. For the ELCA immunoblot, using RVV-XA-labeled antibodies we were able to detect blotted placental alkaline phosphatase at amounts two orders of magnitude lower than those when using peroxidase-labeled antibodies. Using an avidin-RVV-XA conjugate in the biotin/avidin ELCA blot, 1 ng of biotinylated fibrinogen and 100 pg of biotinylated placental alkaline phosphatase, which had been subjected to electrophoresis and transferred to a nitrocellulose membrane, were visualized. These data support the general utility of the ELCA system for assay amplification on solid-phase matrices and demonstrate considerable potential of this methodology in "blotting" applications.
Subject(s)
Endopeptidases/analysis , Immunoblotting/methods , Metalloendopeptidases , Alkaline Phosphatase/analysis , Avidin , Biotin , Blotting, Western/methods , Endopeptidases/immunology , Fibrinogen/analysis , Humans , Immunoenzyme Techniques , MicrochemistryABSTRACT
This solid-phase colorimetric microtiter-plate clotting assay is much more sensitive than standard clotting tests. In enzyme-linked coagulation assay (ELCA), enzyme-labeled fibrinogen and solid-phase fibrinogen are the substrate for thrombin generated in the clotting cascade. We used this assay to measure the factors of the extrinsic pathway by an extrinsic pathway-specific assay (EP-ELCA) and to determine the individual factors of the extrinsic pathway (VII, X, V, II) in plasmas of coumadin-treated and heparin-treated patients, with prothrombin time (PT) values used as a reference. In the ELCA method, samples and controls are incubated on the same plate, eliminating the requirement for pre-standardization of the substrate "plasma" before the factor assay is done. Concentrations of factors are determined by serially diluting sample and control plasmas to yield equivalent activity at given dilutions, a more direct approach for measuring specific factors than determining log concentrations vs log clotting time. Changes in the concentrations of clotting factors are seen before changes are apparent by PT. For coumadin-treated patients, all vitamin K-dependent factors were significantly (P less than or equal to 0.001) less than in normal controls, whereas factor V concentrations were normal, as expected. For patients treated with heparin, concentrations of factors X and VII were less than in normal controls (P less than 0.01) and results for EP-ELCA, II, and V assays were normal. This methodology can readily be automated.
Subject(s)
Blood Coagulation Factors/analysis , Blood Coagulation Tests , Fibrinogen , Peroxidase , Adult , Aged , Aged, 80 and over , Disseminated Intravascular Coagulation/blood , Disseminated Intravascular Coagulation/drug therapy , Factor V/analysis , Factor VII/analysis , Factor X/analysis , Heparin/therapeutic use , Humans , Male , Prothrombin/analysis , Prothrombin Time , Thrombosis/drug therapy , Vitamin K/pharmacology , Warfarin/poisoning , Warfarin/therapeutic useABSTRACT
We have applied the enzyme-linked coagulation assay (ELCA) system to the development of an amplified immunoassay using the clotting cascade to enhance sensitivity of detection of immune complexes. The factor X-activating enzyme of Russell's viper venom was detectable using ELCA in amounts as low as 0.25 fg per assay. Monoclonal antibodies to beta-hCG, placental alkaline phosphatase (PLAP), and the P-24 antigen of HTLV-III were labeled with this enzyme or peroxidase and used for "sandwich" immunoassays using another monoclonal antibody (beta-hCG, PLAP) or polyclonal patient IgG (P-24 antigen) bound to a polylysine-glutaraldehyde-coated plate as a "capture" reagent. After the immunobinding step, the plate was washed and substrate consisting of a mixture of factors X, V, and II in buffer containing calcium and lipid was incubated for various lengths of time. The mixture was transferred to another plate coated with fibrinogen and containing peroxidase-fibrinogen in EDTA solution to measure the amount of thrombin generated. Using this protocol, we were able to measure the presence of 2-10 pg/ml of beta-hCG and PLAP (5-30 amol per sample). All three model antigens were detectable at concentrations 2-3 orders of magnitude less using RVV-XA-labeled antibodies and ELCA than they were using peroxidase-labeled antibodies. The assay has considerable potential as a general immunoassay amplification system, yielding a "color test" for antigens of interest with a detection limit not readily attainable using other chromogenic methodologies.
Subject(s)
Antibodies, Monoclonal , Serine Endopeptidases/analysis , Viper Venoms/immunology , Alkaline Phosphatase/analysis , Chorionic Gonadotropin/analysis , Enzyme-Linked Immunosorbent Assay , Factor Xa , HumansABSTRACT
The solid-phase clotting assay utilizing fibrinogen coated on the wells of a microtiter plate and peroxidase-fibrinogen in solution as a substrate for thrombin (enzyme-linked coagulation assay, ELCA) has been modified for use as an immunoassay. Direct inhibition of factors II, VII, and X by polyclonal (rabbit) antibodies and of factor X by monoclonal antibodies has been demonstrated at high dilution of these antibodies and detection of the specific factors using ELCA. Using plates coated with a second antibody (goat anti-mouse IgG) as well as fibrinogen, monoclonal antibodies to factors X and VII were measured by binding the active factor to the plate and detection of the bound factor using ELCA. The assay was very sensitive, permitting the detection of as little as 0.2 ng/ml (30 pg/assay) of monoclonal antibody, or less than 0.4 ng/ml (60 pg/assay) of factor Xa. When plates were coated with monoclonal antibody to factor X and fibrinogen, the assay permitted the identification of distinct epitope specificities for two monoclonal antibodies to factor X by distinct competition of the monoclonal antibodies added in the solution phase for binding of factor Xa to the plate. This assay could be applied generally for immunoassay of clotting factors, and could have application in general as an immunoassay amplification system.
Subject(s)
Factor VII/analysis , Factor X/analysis , Prothrombin/analysis , Animals , Antibodies, Monoclonal/immunology , Binding Sites, Antibody , Binding, Competitive , Enzyme-Linked Immunosorbent Assay , Epitopes , Factor VII/immunology , Factor X/immunology , Humans , Immunoassay , Immunoglobulin G , Prothrombin/immunology , RabbitsABSTRACT
Cyst and ascitic fluids from patients with ovarian epithelial neoplasms were studied to determine whether they contained immunoglobulins with antitumor activity. The results demonstrated the presence of autologous antibodies bound to the cellular membrane fragments obtained from human ovarian neoplastic effusions. Membrane fragments were prepared from more than 60 samples of human ovarian effusions, and the amounts of membrane-bound IgG and IgA were determined. Six fluids obtained from patients with malignant ovarian neoplasms were selected for large-scale preparation of IgG, on the basis of the quantity of fluid available (greater than 200 ml) and amount of membrane-bound IgG (greater than 400 ng/ml) determined by enzyme-linked immunoassay. The antibodies were strongly reactive with cell-surface antigens on 4 different human ovarian cell lines, 4 surgical specimens of human ovarian adenocarcinoma, and 2 human ovarian tumors grown in athymic BALB/c mice, as demonstrated by indirect immunofluorescence. The antibodies did not react, or reacted only weakly, with tissue preparations from 4 normal human ovaries, other nonovarian normal or neoplastic tissues, and nonovarian human cell lines. These studies indicate that patients with ovarian cancer have the capability to recognize and form antibodies against autologous ovarian tumor-associated antigens.
Subject(s)
Antibodies, Neoplasm/isolation & purification , Antibody Specificity , Ovarian Neoplasms/immunology , Animals , Antibodies, Neoplasm/analysis , Antibodies, Neoplasm/immunology , Antigens, Neoplasm/analysis , Ascitic Fluid/immunology , Female , Fluorescent Antibody Technique , Humans , Immunoglobulin A/analysis , Immunoglobulin G/analysis , Immunoglobulin G/immunology , Mice , Organ SpecificityABSTRACT
We have developed a solid-phase clotting assay which uses peroxidase-fibrinogen in solution and fibrinogen bound to microtiter plates as a substrate for the thrombin generated from the clotting cascade. We have developed this assay for measurement of the extrinsic pathway factors thromboplastin (tissue factor, factor III), VII and VIIa, X, and II. Using long incubation times (40-90 min), thromboplastin could be measured in extracts of human brain at very low concentrations. Specificity for thromboplastin was demonstrated by showing a requirement for factors II, V, X, and VII but not for VIII, IX, XI, or XII; both substrate plasmas monodeficient in single factors and mixtures of the pure factors were used in demonstrating this specificity. The assay was modified to measure factors II, VII, VIIa, and X using appropriate deficient plasmas. The limit of detection was 2-3 orders of magnitude lower than a one-stage clotting test for all factors assayed. This assay has the advantages of convenience, specificity comparable to standard clotting tests, and high sensitivity.