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Results for “antibody characterization”

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At least 19 recordsLinked to original sources
PubMed1979

Heterogeneity of anti-mitochondrial antibodies: characterization and separation of the antigen associated with the pseudolupus erythematosus syndrome.

It could be shown that anti-mitochondria antibodies (AMA) found in drug-induced pseudolupus erythematosus syndrome (PLE) had a different specificity from those found previously in primary biliary cirrhosis (PBC). The PLE antigen could be easily separated from the PBC antigen either by isopycnic sucrose density gradient centrifugation of cytoplasmic extracts (supernatant 40) or purified sonicated rat liver or kidney mitochondria. The PLE antigen was firmly membrane-bound and, in contrast to the PBC antigen, not solubilized by treatment with various salts or enzymes. The ATP-ase complex, the probable target antigen of PBC-specific antibodies, did not react with sera from patients with the PLE syndrome. There is evidence that the PLE-associated antibodies (M3) occur exclusively in patients who have been sensitized to derivatives of pyrazolone or their metabolites, indicating that PLE antibodies may be specific markers for this type of drug allergy.

Antibody Specificity↗
PubMed1975

The antibody-enzyme analogy. Characterization of antibodies to phosphopyridoxyltyrosine derivatives.

Stable analogs of the crucial Schiff base intermediate of enzymatic and nonenzymatic pyridoxal phosphate catalysis have been used as haptens for induction of specific antibodies. N-(5-phosphopyridoxyl)-3'-amino-L-tyrosine and its conformationally distinct cyclized derivative resemble the Schiff base formed upon mixing tyrosine with pyridoxal phosphate. These compounds were covalently coupled to a protein carrier via the 3'-amino group so as to confer a prescribed orientation, with the coenzyme region farthest removed from the carrier. A third antigen, with the phosphopyridoxyl group alone as the hapten, was prepared by linkage of pyridoxal phosphate directly to free amino groups on the carrier protein. Antibodies elicited for each determinant were purified by means of appropriate affinity columns. Antibody heterogeneity was observed in that different species could be separated from a given serum by sequential elution from the affinity columns with 1 M sodium phosphate buffers of pH 7.6, 5.2, 2.6 and 1.5. In assays of quantitative precipitation, inhibition of precipitation, equilibrium dialysis, and fluorescence quenching, antibodies to the phosphopyridoxyltyrosine haptens showed specificity for the phosphorylated form of the coenzyme and binding activity for both the coenzyme and tyrosine portions of the hapten. Antibodies to the phosphopyridoxyl groups alone did not display a similar reactivity toward the tyrosine portion of the complex haptens. The cyclic and noncyclic conformations of the hapten were serologically distinct, as antibody to each reacted preferentially with the homologous form.

Animals↗
PubMed1976

The use of the radioimmunoassay in the characterization of antibodies to basement membrane collagen.

This study describes the use of the radioimmunoassay for the characterization of antibodies to basement membrane (type IV) collagen from bovine anterior lens capsule. The immunogen was extracted from calf anterior lens capsules by limited pepsin digestion and injected into rabbits. The antisera were characterized using gel diffusion, haemagglutination and the radioimmunoassay in which 125I-labelled types I, II, III, and IV bovine collagen were employed. In the direct radioimmunoassay there was no reaction with either native or denatured types I, II or III bovine collagen, whereas there were high titres towards both native and denatured type IV bovine collagen. Radioimmune inhibition studies using unlabelled types I, II, III and IV bovine collagen, collagenase digested and repepsinized type IV collagen showed that there was marked inhibition by either native, denatured or repepsinized type IV collagen, and slight inhibition by native type I collagen; native type II and type III, denatured types I, II and III, and collagenase digested type IV collagen had no inhibitory effects.

Amino Acids↗
PubMed1978

Speckled (particulate) epidermal nuclear IgG deposition in normal skin. Correlation of clinical features and laboratory findings in 46 patients with a subset of connective tissue disease characterized by antibody to extractable nuclear antigen.

Clinical and laboratory findings were correlated from 46 patients with IgG localization in epidermal nuclei in a speckled (particulate) pattern on direct immunofluorescence of normal skin. Cutaneous manifestations included lupus erythematosus (LE), swollen hands or sclerodactyly, alopecia, vasculitis, and dyspigmentation. Systemic manifestations included arthritis or arthralgia, Raynaud's phenomenon, serositis, vascular headaches, mild renal disease, myositis, and sicca syndrome. High titer (mean = 1:142, 800) serum antibody to extractable nuclear antigen (ENA) was found in 81%. Eighty-six percent had antibody to an RNase-sensitive antigenic component of ENA (ribonucleoprotein or RNP); 14% had antibody to an RNase-resistant ENA termed Sm. Deposition of IgG in a speckled pattern in epidermal nuclei is an immunopathologic marker for a subset of connective tissue disease characterized by antibody to ENA. Those with Sm specificity had systemic LE (SLE); Those with RNP specificity had Raynaud's phenomenon usually associated with overlapping features of SLE, scleroderma, and/or dermatomyositis.

Adolescent↗
PubMed1978

Quantitation and characterization of antibody binding to tubulin.

A goat antiserum, raised to native hog brain tubulin, was characterized by conventional immunological techniques and by employing a method for precipitation of tubulin.anti-tubulin complexes with heat-inactivated Staphylococcus aureus. Antiserum dilution experiments indicated that antibodies to native tubulin were raised, and maximal binding was observed to microtubules fixed with 1 mM glutaraldehyde. Competition experiments, using iodinated fixed microtubules as tracer, demonstrated that equivalent binding occurred with microtubules at protein concentrations 100- to 1000-fold lower than those for monomeric tubulin. A rabbit antiserum raised to sodium dodecyl sulfate-treated axonemal tubulin was also characterized. The serum bound maximally (90%) to either sodium dodecyl sulfate-treated or native iodinated hog brain tubulin, and competition for antibody binding has been observed with tubulin from diverse sources (Tetrahymena pyriformis ciliary axonemes, Lytechinus pictus flagellar axonemes, and mouse neuroblastoma extracts). Using these two antisera, radioimmunoassays are being developed for quantitation of polymeric and total tubulin in cellular systems.

Animals↗
PubMed2024

Phage Immunoprecipitation and Sequencing-a Versatile Technique for Mapping the Antibody Reactome.

Characterizing the antibody reactome for circulating antibodies provide insight into pathogen exposure, allergies, and autoimmune diseases. This is important for biomarker discovery, clinical diagnosis, and prognosis of disease progression, as well as population-level insights into the immune system. The emerging technology phage display immunoprecipitation and sequencing (PhIP-seq) is a high-throughput method for identifying antigens/epitopes of the antibody reactome. In PhIP-seq, libraries with sequences of defined lengths and overlapping segments are bioinformatically designed using naturally occurring proteins and cloned into phage genomes to be displayed on the surface. These libraries are used in immunoprecipitation experiments of circulating antibodies. This can be done with parallel samples from multiple sources, and the DNA inserts from the bound phages are barcoded and subjected to next-generation sequencing for hit determination. PhIP-seq is a powerful technique for characterizing the antibody reactome that has undergone rapid advances in recent years. In this review, we comprehensively describe the history of PhIP-seq and discuss recent advances in library design and applications.

Humans↗
PubMed1976

Characterization of antibodies to the insulin receptor: a cause of insulin-resistant diabetes in man.

We have characterized the circulating inhibitor of insulin receptor binding found in several patients with a new syndrome of extreme insulin resistance. The inhibitor is an immunoglobulin by multiple criteria, including precipitation by 33% ammonium sulfate, migration on G-200 Sephadex gel filtration and DEAE chromatography, and immuno-precipitation with specific anti-human immuno-globulins. Although predominantly IgG, some activity is found in the IgM fraction of the immunoglobulins in one patient. The inhibitory immunoglobulins reacted with antisera to both kappa and lambda light chain determinants and are therefore polyclonal. In addition, activity is retained in the F(ab')2 fraction of pepsin-digested IgG. Evidence suggests that these antibodies are directed at determinants on or near the insulin receptor, and that they are responsible for the observed clinical insulin resistance.

Antibodies↗
PubMed1975

Production and characterization of antibodies to meperidine.

A mepridine-bovine serum albumin (Mep-BSA) conjugate with 15-20 moles of meperidine per mole of BSA was synthesized and characterized. Rabbits immunized with Mep-BSA produced antibodies that were assayed utilizing saturated ammonium sulfate to separate 3H-meperidine (3H-M) bound to antibody from free 3H-M. Antibody specificity was assessed by competitive inhibition studies. The nanomoles of inhibitor required to decrease the binding of 3H-M by 50% were: meperidine .046; meperidine acid, 3.2; alphaprodine, 7.8; dextromethorphan, 20; codeine, 50; and morphine 55. The sensitivity of the assay is approximately 30 ng/ml; sufficient for pharmacokinetic studies of the disposition of meperidine in man.

Animals↗
PubMedDate not supplied

Antibodies to radiographic contrast agents. Induction and characterization of rabbit antibody.

An allergic, immunologic mechanism for certain adverse reactions to radiocontrast agents has been postulated on the basis of clinical and theoretical considerations. One obstacle to the acceptance of this allergic theory has been the inability to demonstrate significant antibodies with specificity for these agents. We attempted the induction of antibodies in the rabbit using analogues of radiocontrast media. The synthesis of four different analogues of commonly used radiocontrast agents allowed for testing the haptenic potential of various portions of the contrast medium molecule. Each analogue was conjugated to a carrier protein, emulsified in a suitable adjuvant, and repeatedly injected into several rabbits. Optimal immunization conditions were selected to increase the probability of antibody induction. Specific antibody was produced in good titer to each of the four haptens and in all injected rabbits. Antibodies were characterized by means of precipitation assays and radioimmunoassay using contrast agents labeled with 125I. Antibody concentrations ranged from 0.23 to 2.7 mg/ml and varied with interval and frequency of booster injections. In addition to antibodies specific for the contrast media analogues, antibodies to the carrier protein were also detected. Antibodies were of the IgG class and in selected sera were 83-92% precipitable. The induction of antibodies to radiocontrast agents supports the allergic theory of contrast medium toxicity.

Animals↗
PubMed1975

Endogenous immune complex nephropathy associated with malignancy I. Studies on the nature and immunopathogenic significance of glomerular bound antigen and antibody, isolation and characterization of tumor specific antigen and antibody and circulating immune complexes.

Three patients with clear cell renal carcinoma and one with another intrarenal malignancy were studied for the presence of glomerular localized immunoglobulins, complement components and tumor specific antigen and antibody by immunofluorescence. To determine the association and elucidate the pathogenic mechanisms involved in the relationship between tumors and glomerular deposits, antibody eluted from tumor tissue and renal glomeruli, cryoproteins, serum antibodies and rabbit antisera to tumor tissue were tested for specificity to antigen. The relationship between tumor antigens and the lipoprotein antigen localized in normal proximal tubular brush border (RTE) and the small bowel mucosa, was studied by immunofluorescence, absorption and blocking studies as well as complement fixation. Immunoglobulins and complement components were localized in the glomeruli and tumor membrane of all patients. Sera and glomerular fixed antibody from three patients with renal cell carcinoma localized to normal proximal tubular brush border and jejunal mucosa as well as to tumor membrane and the glomeruli and proximal tubules of all of these three patients. Anti RTE activity was also detected by complement fixation. Immunologic similarity between RTE and renal cell carcinoma antigen was confirmed by absorption studies. Furthermore, cryoprecipitable complexes of tumor antigen and specific antibody were isolated from the serum. The tumor antibody was immunologically similar to RTE. In the other case the rabbit anti-tumor antibody and the patient's serum fixed to the tumor membrane and kidney of the patient but did not show cross reactivity with the renal cell carcinoma or RTE. These studies suggest that the tumor antigen in renal cell carcinoma is similar to RTE and the glomerular deposits represent tumor antigen and antibody complexes. In addition these investigations support the hypothesis that tumor immune complexes are significant in the glomerular lesions, and that the origin of renal cell carcinoma is in the proximal tubule. The investigations also show that tumor antibodies are specific for tumors of the same morphological type but not for other tumors in the same tissue. Moreover, the renal glomerulus appears to be a chosen anatomic site for deposition of tumor antigens and antibodies and studies of the kidney may provide insight into the nature of tumor antigens and antibodies. Cryoprecipitation appears to be a valuable method in isolation of tumor complexes and characterization of tumor specific antigen and antibody.

Absorption↗
PubMed1976

Circular dichroism of hapten--antibody complexes: characterization of the combining sites of native and reformed MOPC-315 protein, its isolated subunits, and its Fv fragment.

Extrinsic Cotton effects generated by binding haptens to native and reformed MOPC-315 protein, its subunits, and its Fv fragment have been examined. The identity of the combining sites of native and reassociated proteins and Fv-315 was demonstrated by the identity of their circular dichroism (CD) difference spectra. The spectrum of TNP-aminocaproate complexed with L chains differed in maxima and minima and cross-over points and lacked the 495-nm CD peak of TNP-aminocaproate-MOPC-315 protein and Nalpha-TNP-tryptophan spectra. A negative 293-nm tryptophanyl CD band, present in spectra of MOPC-315 protins and Fv-315 but absent from spectra of L and H chains, was blue-shifted by haptens and may represent electronic interactions occurring within the MOPC-315 combining site between tryptophanyl and chromophoric residues of different subunits. This conclusion is supported by molecular models of the MOPC-315 combining site.

Animals↗
PubMed1979

Production and characterization of antibody against aflatoxin M1.

Antibody against aflatoxin M1 was obtained after immunization of rabbits with bovine serum albumin-afla M1 oxime conjugate. The antibody has greatest binding efficiency for afla M1, and was less efficient for afla B1. Cross-reaction of antibody with aflatoxin Q1, aflatoxicol, and aflatoxin B2a was weak. Aflatoxin B2, G1, and G2 and afla B1-guanine adducts showed almost no cross-reaction with the antibody. The sensitivity of the binding assay for aflatoxin M1 detection is in the range of 1-10 ng per assay. Detailed methods for the preparation of the conjugate, production of immune serum, and methods for antibody determination are described.

Aflatoxins↗
PubMed1977

Characterization of antibody to human phosphatidylcholine: cholesterol acyltransferase.

Purified preparations of phosphatidylcholine (lecithin): cholesterol acyltransferase (EC 2.3.1.43), were injected into goats to produce antisera reacting with this enzyme. The antisera and the gamma-globulin derived thereform were examined by the technics of immunodiffusion, immunoelectrophoresis and immunoinhibition of the enzyme. The antisera gave no precipitation lines with human high density lipoproteins (HDL) and human low density lipoproteins (LDL). A weak antibody titer towards human serum albumin was noted only after prolonged immunization. The enzymatically active band isolated from acrylamide gels gave a single arc in immunodiffusion and immunoelectrophoresis. The gamma-globulin derived from the antisera inhibited human phosphatidylcholine:cholesterol acyltransferase activity.

Acyltransferases↗