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PubMed1994

A method for the quantitation of protamine in plasma.

A unique and simple colorimetric method for the quantitation of plasma protamine levels has been developed. The method is established on the competitive binding displacement mechanism between protamine and heparin-azure A dye complex, and the metachromatic color change of azure A dye in the presence of heparin. Because the method is based on the clinical specificity of protamine as the heparin antagonist, it is specific for protamine quantitation. Plasma protamine levels determined by this method are within 94% of accuracy when compared with their aqueous counterparts determined by the conventional Lowry protein assay. Since the method measures the protamine excess after heparin neutralization, it potentially could be employed during clinical heparin reversal with protamine to monitor protamine excess. In addition, the method may provide a useful means to identify the mechanism of the so-called "heparin rebound".

Azure Stains↗
PubMed1991

Modification of the fluorescent allergosorbent test as an inhibition assay for determination of cross-reactivity among aeroallergens.

The fluorescent allergosorbent test was adapted as an inhibition assay to determine cross-reactivity between aeroallergens. With this method, similar antigenic determinants were found between short ragweed and giant ragweed, cocklebur, lamb's-quarter, rough pigweed, marsh elder, and goldenrod. Cocklebur and giant ragweed were highly potent in their ability to competitively bind to short ragweed IgE. The other pollens demonstrated lower potency of cross-reacting antigens. The fluorescent allergosorbent test-inhibition assay appears to be a useful method to determine cross-reactivity among aeroallergens.

Binding, Competitive↗
PubMed1991

Reassessment of Fura-2 and the ratio method for determination of intracellular Ca2+ concentrations.

To determine intracellular Ca2+ concentrations more accurately, we examined Kd of Fura-2 for Ca2+ in conditions which were systemically changed. In a solution comprising of 150 mM KCI, 20 mM MOPS-KOH, pH 6.94, 60-100 microM EGTA and 1 microM Fura-2, Kd at 20 degrees C was 0.266 +/- 0.016 microM (mean +/- SEM) (21 determinations). The ionic strength (I) of the solution strongly affected Kd: the relation of -log Kd versus 2 square root of I/(1 + square root of I) - 0.4.I was 3.6 times as steep as that of EGTA. Kd was moderately changed by pH higher than 7.1, while it was very slightly changed by pH between 6.7 and 7.1. Kd was minimally affected by temperature. The apparent Kd values for Ca2+ in the presence of various concentrations of Mg2+ gave an estimate of the Kd for Mg2+ of about 100 mM, which is about 10 times as great as the estimated value by Grynkiewicz et al. [1]. This estimation assumes competitive binding between Ca2+ and Mg2+ for Fura-2. However, the possibility that Mg2+ may bind Fura-2 in a more complicated way is also suggested. Co-existing proteins in the solution dose-dependently increased an apparent Kd, independent of the type of proteins used, up to a limiting value of about 1.0 microM. With the ratio method, the Ca2+ concentration which gives (Rmin + Rmax)/2 is Kd.beta. The range of Ca2+ concentrations on which R values show steep dependence is determined not only by Kd but also by beta. This means that the excitation spectra and pair of excitation wavelengths selected as well as Kd are critical factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗
PubMed1982

Effect of sequential glycolysis of the lectin of Euonymus europaeus on activation of the classical complement pathway in normal human serum.

The lectin of Euonymus europaeus at concentrations of 5-21 micrograms/ml causes activation of the classical complement (C) pathway (C1, C4, C2) when added to normal human serum at 37 degrees C. At higher concentrations, C3 is also consumed. The effect is dependent on a 'natural antibody' in serum of the IgM class which reacts with an epitope of the lectin. With physicochemical methods, the carbohydrate of the lectin was shown to be involved in the activation of C, but not involved in the agglutination of group B erythrocytes. Removal of N-acetyl-D-glucosamine from the lectin with an exoglycosidase greatly reduced the activation of C in serum, but it did not affect erythroagglutination. Using competitive binding studies with various sugars, it was confirmed that N-acetyl-D-glucosamine is the dominant specificity of a determinant for activation of the classical C pathway in serum.

Binding, Competitive↗
PubMed1982

Antigenic sites on mouse 2.5S nerve growth factor.

Cyanogen bromide and tryptic peptides of mouse 2.5S Nerve Growth Factor (NGF) have been used to inhibit competitively binding of rabbit antiserum anti-NGF to native NGF in a solid-phase radioimmunoassay. The results showed that the larger of the two peptides obtained by cyanogen bromide cleavage (amino-acid residues 10-118) was indistinguishable from NGF in this respect, whereas the smaller N-terminal peptide (residues 1-9) was virtually non-inhibitory. Ten peptides were purified from a tryptic digest of the larger cyanogen bromide fragment. One of them, peptide G7 (residues 58-59, 60-69, 104-114, linked by disulfide bridges), was capable of almost full inhibition of binding, though only when used at a concentration about 100 times higher than that necessary to get the same effect with NGF. One other peptide, G10-H1 (residues 70-74), showed some inhibition at relatively high concentrations, but the majority of peptides, including peptide DE-5 which has been shown to be active in the NGF bioassay, were essentially non-inhibitory. A significant co-operative effect was seen when peptides G7 and G10-H1 were used in conjunction. No such effects were observed with any other combination tested.

Animals↗
PubMed1984

Delineation of serologically distinct monomorphic determinants of human MHC class II antigens: evidence of heterogeneity in their topographical distribution.

Three monoclonal antibodies (mAbs), FMC4, FMC14 and FMC15, which react with invariant sites of major histocompatibility complex (MHC) class II (Ia-like) molecules were studied in various serological assays. Sequential immunodepletion experiments show that all three epitopes are present on the same class II molecules. However, a minor subset may exist which does not express epitope 15. In competitive binding assays, using several different B-lymphoblastoid cell lines (B-LCLs), e.g. BRISTOL-8 8392, B-85, RAJI, 8866, CESS-B and LD-B, FMC4 did not block the binding of FMC14, or FMC15, and vice versa. In contrast, mutual inhibition was observed between FMC14 and FMC15. Furthermore, pairwise combinations of saturating amounts of FMC4 + FMC14 and FMC4 + FMC15 gave additive binding whilst FMC14 + FMC15 did not. These results demonstrate that epitopes 4 and 14/15 are spatially distinct; 14 and 15 on the other hand appear to be spatially related. However, contrary to this partial and reciprocal inhibition was consistently observed between FMC4 and FMC14 on two other LCLs, namely DAUDI and BALM-2. Furthermore, on certain cells, FMC14 and FMC15 show markedly disparate binding. Taken together, these observations indicate that the juxtaposition of certain epitopes on class II antigens can vary according to the cell type. This demonstrates a hitherto unreported heterogeneity of antigenic determinants and of their topographical distribution on the class II molecule.

Animals↗
PubMed1988

Monoclonal antibodies to bovine serum albumin: affinity and specificity determinations.

A panel of 12 monoclonal antibodies (MAb) to bovine serum albumin (BSA) was developed and characterized as to their physiochemical and immunological properties. Affinity constants of the MAb varied over a wide range from 10(5) to 10(8) M-1. MAb were assembled into several groups of non- or minimally interacting antibodies by analysis of competitive binding experiments, and BSA domain and subdomain specificities of the MAb were assigned by analysis of results of MAb binding to purified BSA fragments. Further fine specificity delineation was accomplished by examination of cross-reactivity patterns to several mammalian albumins. The data suggest that some of the low affinity MAb recognize sites on different portions of the BSA molecule, indicating that similar epitopes exist on different domains of the BSA molecule.

Animals↗
PubMed1991

Epitope specificity of three anti-pertussis toxin monoclonal antibodies with dissimilar effects in assays of toxin neutralizing activity.

The epitope specificity of two monoclonal antibodies against the S1 subunit (A4, A12) and one MAb against the S3 subunit (B9) of pertussis toxin, all protective in the mouse aerosol model of B. pertussis infection, but with different effects in assays of toxin-neutralizing activity, was examined in competitive binding enzyme immunoassays using biotinylated anti-pertussis toxin monoclonal antibodies or biotinylated goat anti-pertussis toxin polyclonal antibody after preincubation with unlabelled antibody. Biotinylated A4 was blocked by A4, A12, and B9; A12 was blocked by A4, A12, and B9. In contrast, biotinylated B9 was blocked by B9 and A4, but not by A12. All three monoclonal antibodies successfully blocked the anti-pertussis toxin polyclonal antibody; a mixture of the three anti-pertussis toxin monoclonal antibodies was more effective than any monoclonal antibody alone P less than or equal to 0.01). These data suggest that these three anti-pertussis toxin monoclonal antibodies recognize separate, but closely linked epitopes on pertussis toxin, and that epitopes on the S1 subunit and B-oligomer may induce protective immunity.

Animals↗
PubMed1996

Heteroligation of a mouse monoclonal IgE antibody (La2) with small molecules, analysed by computer-aided automated docking.

A mouse monoclonal anti-TNP IgE antibody (IgE-La2) was screened by a competitive-binding ELISA with a random pool of over 2000 small molecules, mostly drugs, drug derivatives and metabolites. Thirteen of these (naproxene, beta-carboxy-chi-naphthol, oxolinic acid, hymecromone, 8-aminoquinoline, beta-naphthylamine, chi-nitrilo-cinnamic acid, 1,5-diaminonaphthaline, prolonium iodide, diaspirin, 3,4,5-trimethoxy-cinnamic acid, cycrimine, hemimellitic acid) were found to bind as strongly, or stronger, to the antibody as the immunizing hapten. We have used a Monte Carlo search technique for simulated docking of the DNP and non-DNP ligands to a model of the Fv region of IgE(La2). The validity of structural predictions made by the AutoDock program were tested on IgG(ANO2), the three-dimensional structure of which had been obtained previously by X-ray crystallography and 2D-NMR. The rms differences between the experimentally determined and auto-docked complexes in the energetically most favored binding modes were 0.31-0.44 A. Evaluation of structures of IgE(La2)-ligand complexes [including 2,4-dinitrophenol (DNP), 16 DNP amino acids, and the 13 non-DNP ligands listed above] obtained by computer-aided automated docking, suggested the existence of two subsites within an approximately 12 x 18 A2 groove extending between the H and L CDRs. Some of the ligands (DNP-Glu, 8-aminoquinoline, prolonium-I, beta-naphthylamine) were found to bind exclusively to subsite 1, others (DNP-Ala, chi-nitrilo-cinnamic acid, hemimellitic acid, beta-carboxy-chi-naphthol) to subsite 2. The majority of DNP amino acids and other ligands (oxolinic acid, 3,4,5-trimethoxy-cinnamic acid, diaspirin, [R]-cycrimine) were found to occupy an overlapping area including subsites 1 and 2, while some of the compounds (DNP-Asn, DNP-Pro, hymecromone, 1,5-naphthylenediamine) were predicted to interact with either of these subsites with comparable probabilities. When all of the docked La2-ligand complexes were taken into account, five tyrosine residues (H33, L32, L91, L92, L96) were found to provide the majority (53.4%) of all observed contact points. Thus, a multitude of interactions with aromatic residues, and a combinatorial type of interaction within the binding region, seem to be the major factors to explain the mechanism of heteroligation by IgE(La2).

Amino Acid Sequence↗
PubMed1992

Determination of endothelin by an immobilized receptor assay utilizing a 96-well format.

Bovine cerebellar membranes immobilized on 96-well microtiter plates provide receptors for 125I-labeled endothelin-1 as the basis for a competitive binding assay. Adsorption of the membranes to a surface does not significantly alter the ligand-receptor interaction and reduces non-specific binding to 3-7% of total binding compared to 10-20% for a filtration technique. Considerable savings in reagents are realized since assays can be performed in 100 microliter volumes with only 10-20 micrograms of membrane protein. The 96-well format allows the rapid quantitation of large numbers of samples, and the assay is especially attractive in that it utilizes readily available reagents and equipment without the need for specific antibodies. The endothelin-receptor-based assay may be used to measure conversion of big endothelin-1 to endothelin-1 in aqueous assays. Since the presence of serum does not affect this method, tissue culture medium may be directly analyzed for endothelin production by cultured cells. All three isoforms of endothelin are detected, and the specificity of the receptor is retained since fragments and precursor forms of endothelin are not recognized. In cases where multiple endothelin isoforms may be present or where specificity of binding is in question, this assay may be used in conjunction with high pressure liquid chromatography to distinguish active peptides.

Animals↗
PubMed1994

Characterization of monoclonal antibodies to ovine tumor necrosis factor-alpha and development of a sensitive immunoassay.

Monoclonal antibodies (mAbs) and a polyclonal rabbit antiserum were raised against recombinant ovine tumor necrosis factor-alpha (rovTNF alpha). Ten mAbs specific for rovTNF alpha were isolated and designated TNF1-10. All mAbs were of the IgG1 isotype and reacted with rovTNF alpha in Western blot analysis. Eight of the ten mAbs, TNF1, TNF3-7 and TNF9 and 10, completely blocked the activity of rovTNF alpha and macrophage derived native ovTNF alpha, as measured by their ability to inhibit TNF alpha-mediated lysis of WEHI-164 or L929 cells. In addition, TNF3, -7, -9 and -10 blocked the cytolytic activity of recombinant human TNF alpha (rhuTNF alpha). However, when tested for the ability to inhibit TNF alpha induced thymocyte proliferation, only mAbs TNF1, -3, -5, -7, -9 and -10 could completely block activity. Competitive binding analysis using unlabelled and horseradish peroxidase (HRPO) labelled mAbs indicated that the mAbs could be divided into five groups based on their reactivity with rovTNF alpha. The mAbs were used to develop a sensitive sandwich immunoassay for the detection of ovTNF alpha. All combinations of mAbs and the polyclonal antiserum were tested to determine which pair of antibodies gave the most sensitive assay. The combination of TNF5 as the capture antibody and the polyclonal antiserum gave the most sensitive result, detecting less than 0.24 ng rovTNF alpha ml-1. A similar sensitivity was obtained when TNF4 was used as the capture antibody and TNF10 HRPO labelled mAb as the second antibody. The immunoassay was more sensitive than the WEHI-164 bioassay which had a detection limit of 1 ng ml-1 for rovTNF alpha. This immunoassay also detected glycosylated ovTNF alpha in the supernatant of COS-7 cells which had been transfected with an ovTNF alpha cDNA.

Animals↗
PubMed1994

CD11/CD18 panel report for swine CD workshop.

Five monoclonal antibodies (mAbs), PNK-I (W #037), H20A (W #077), MUC76A (W #078), MUC93A (W #079) and MHM23 (W #136) of CD11/18 panels reacted with 80-96% of porcine PBMC and PMN. Epitope mapping studies by competitive binding of mAb by flow cytometric analysis based on PNK-I as CD18 epitope defining antibody resulted H20A and MHM23 mAbs bind to the same shared epitope as PNK-I, but MUC76A and MUC93A mAbs were distinct from PNK-I. Thus, PNK-I, H20A and MHM23 were designated as CD18a mAbs.

Animals↗
PubMed1994

Expression and antigenicity of Plasmodium falciparum major merozoite surface protein (MSP1(19)) variants secreted from Saccharomyces cerevisiae.

Four antigenic variants of the 19-kDa carboxy terminal fragment of Plasmodium falciparum merozoite surface protein, MSP1 (MSP1(19)), were expressed in Saccharomyces cerevisiae as a histidine-tagged, secreted polypeptides (rMSP1(19)s). Structural analysis of the rMSP1(19)s indicated that a single amino acid change (E to Q) in the first EGF-like domain of the yeast-secreted rMSP1(19) proteins caused a significant change in their disulfide bond-dependent conformation. The antigenicity of the rMSP1(19)s were qualitatively and quantitatively analyzed by direct and competitive binding ELISAs. The data indicate that conserved and variant B cell determinants of MSP1(19), as well as epitopes that are known targets of protective antibodies, were recreated authentically in the rMSP1(19)s. Secretion of histidine-tagged rMSP1(19)s using the expression system described may be an efficient and effective means of producing a properly folded immunogen for a human vaccine against the blood stages of P. falciparum.

Amino Acid Sequence↗
PubMed1993

Identification of IGF-1 receptors in primitive vertebrates.

This is the first report of the existence of insulin-like growth factor (IGF-1) receptors in three representatives of lower vertebrates: the osteichtyes, chondrichtyes and cyclostomi. Competitive binding studies and affinity labelling of brain membranes from Cottus scorpius (sea scorpion), Raja clavata (ray) and Myxine glutinosa (atlantic hagfish) identified a mammalian type 1 or IGF-1 receptor by its binding specificity and the molecular size of its alpha-subunit. IGF-1 and IGF-2 are almost equally potent in displacing receptor-bound 125I-IGF-1 or 125I-IGF-2, and the proteins labeled with both tracers have a molecular size of 100,000-120,000 under reducing conditions. There was no evidence for the presence of a mammalian type 2 or IGF-2/mannose 6-phosphate receptor in brains of Cottus, Raja or Myxine. In all three species the binding of 125I-IGF-1 and 125I-IGF-2 was significantly higher in brain compared with liver and gastrointestinal tract, and the IGF-1 receptor could only be identified with certainty in Raja liver. It is concluded that the brain of three lower vertebrates express mammalian IGF-1 receptors, whereas IGF-2-mannose 6-phosphate receptors could not be detected.

Affinity Labels↗
PubMed1984

Interaction of rat prostate androgen receptors with polynucleotides, RNA, DNA and cloned DNA fragments.

Androgen receptors were partially purified from prostates of mature (non-castrated) rats by chromatography on 2',5'-ADP-Sepharose and labelled by exchange with 5 alpha-[3H]dihydrotestosterone. The partially purified receptor preparation was free of DNAase activity and sedimented at approx. 3 S. The specificity of the interaction of this androgen receptor with nucleotides was investigated in a competitive binding assay using inhibition of binding of the steroid receptor complex to ADP-Sepharose. Certain polyribonucleotides were strongly bound (e.g., poly(UG), poly(AU), poly(G) and poly(U] and competed more effectively for the receptor binding sites than prostate RNA. Restriction fragments of genomic clones from the genes which code for prostatic binding protein showed only moderate affinity for the 3 S receptor form. These data suggest that the 3 S form of the androgen receptor lacks the specific domain or conformation necessary for specific interaction with DNA, but retains a high affinity for certain forms of RNA. Some potent inhibitors of proteolysis (diisopropylfluorophosphate, leupeptin) did not have any effect on the form of the receptor isolated from mature intact animals. A possible function of the 3 S form in post-transcriptional processing is discussed.

Adenosine Diphosphate↗
PubMed1989

Human seminal ribonuclease. Immunological quantitation of cross-reactive enzymes in serum, urine and seminal plasma.

The distribution of secretory-type ribonuclease in human serum, urine and seminal plasma has been studied by immunological measurements. Inhibition of enzyme activity by antibodies against pure human seminal RNAase shows that a cross-reactive enzyme is predominant (90%) in seminal plasma and is a significant component (70-80%) in urine and serum. A competitive binding radioimmunoassay has been developed by using specific antibodies and 125I-labelled RNAase as radioligand. The procedure, very sensitive, reproducible and specific, has been used to determine seminal RNAase levels in seminal plasma samples from 48 healthy individuals (age range, 20-58 years). The mean concentration of the enzyme was found to be 6.6 micrograms/ml (S.D. +/- 1.9).

Adult↗
PubMed1987

Antigenic characterization of the internal proteins of Newcastle disease virus by monoclonal antibodies.

We have prepared and characterized monoclonal antibodies against the three internal structural proteins, M, P and NP, of Newcastle disease virus. At least two non-overlapping antigenic sites were delineated on the M protein, four on the P, and two on the NP by competitive binding assay. One of the two non-overlapping antigenic sites on the M protein was found to be a cluster of at least three distinct epitopes. Enhancement of antibody binding by the binding of a second antibody was observed with the M protein. The reactivity of these monoclonal antibodies with heterologous strains was studied by enzyme-linked immunosorbent assay. The results indicated that there are both highly conserved antigenic sites and those subject to remarkable change on both M and P proteins. On the other hand, NP appeared to be antigenically more stable.

Antibodies, Monoclonal↗
PubMed1988

Mapping of three antigenic sites on the haemagglutinin-neuraminidase protein of Newcastle disease virus.

Nine neutralizing monoclonal antibodies (MAbs), each of which react with the haemagglutinin-neuraminidase (HN) glycoprotein of the Beaudette C strain of Newcastle disease virus (NDV), have been used in competitive binding assays to delineate three non-overlapping antigenic sites A, B and C. Epitopes within these sites have been identified on the basis of cross-reactivity of MAb-resistant mutants against the panel of MAbs, determined by plaque assays and Western blotting. Site A contains three non-overlapping epitopes (A1, A2 and A3). A1 is the only linear epitope; all remaining epitopes are conformational. MAbs which react with epitopes A2 and A3 inhibit neuraminidase activity (NA) when assayed with neuraminlactose. Site B contains three partially overlapping epitopes (B1, B2 and B3) and site C is represented by a single epitope (C1). HN gene sequence analysis of MAb-resistant mutants showed that they each had only single amino acid substitutions which range from amino acid residues 347-460 for site A, 284-325 for site B, and at 481 for the C1 epitope. The apparent molecular mass of the HN glycoprotein of one mutant was increased from 72 to 75 kDa. This correlates well with the creation of an additional potential glycosylation site in this mutant from Asn-Ser-Pro(325) to Asn-Ser-Ser(325).

Amino Acid Sequence↗