Comparative properties of monoclonal antibodies comprising a high-affinity anti-fluorescyl idiotype family

The T-dependent BALB/c murine immune response to fluorescein (F1) is characterized by structural heterogeneity at the protein level exemplified in part by a significantly wide range of affinities (Ka), and apparent lack of dominant idiotypes. In order to generate an idiotype family, xenogenic anti-idiotype (anti-ID) antibodies raised against anti-F1 monoclonal antibody (MCA) 4-4 (Ka = 1.7 X 10(10) M-1) were used in a solid-phase radioimmunoassay (SPRIA) to screen 68 anti-F1 hybridomas generated from multiple cell fusions for idiotypically related immunoglobulins. Four affinity-purified MCAs (designated 9-40, 10-25, 5-14 and 5-27) bearing 4-4 idiotypic determinants (ID 4-4) exhibited discrete isoelectric focusing spectrotypes (pI range = 6.8-7.7), significantly different fluorescence quenching values (38-95%) of bound ligand, binding affinities ranging from 3.3 X 10(7) to 5.3 X 10(8) M-1, similar active site inaccessibility to iodide, and closely related fine-specificity patterns for fluorescyl analogues. Idiotypic relatedness of each MCA to prototype 4-4 was quantitated by SPRIA, the results demonstrating that: each 125I-labeled MCA bound significantly to solid-phase anti-ID 4-4, and the concns of heterologous MCAs 9-40, 10-25 and 5-14 required for 50% inhibition of 125I-4-4/anti-ID 4-4 binding were comparable to homologous Ig protein. The finding that ID 4-4 bearing anti-F1 MCAs exhibit various binding properties and affinities is consistent with variable-region somatic diversification in anti-F1 affinity maturation.     

Idiotypic analysis of monoclonal anti-fluorescyl antibodies: Localization and characterization of idiotypic determinants

Nine monoclonal IgG anti-fluorescyl antibodies, which exhibit diverse affinities for fluorescein (Fl) (K_a values ranging from 5 × 10⁶ to 10¹⁰ M⁻) were analyzed idiotypically. Each of the BALB/c hybridoma proteins (γ, κ) exhibited unique idiotypic determinants although two clones (6-10-6 and 20-19-1) were partially (15–20%) cross-reactive. Of two other clones (4-6-9 and 4-6-10) derived from the same cell line, 4-6-9 contained γl heavy (H) chains and 4-6-10 contained both γl and γ2b H-chains. In addition, 4-6-9 shared idiotypic determinants with 4-6-10 although the latter also displayed unique idiotypic specificities. Collectively, the nine clones demonstrated structural diversity analogous to previous studies which denned binding mechanism diversity. The location of determinants recognized by antiidiotype reagents directed against each of the monoclonal antibodies was examined by binding inhibition with free Fl and fluorescein-BSA (Fl-BSA). All clones contained determinants both within the active site (Fl-inhibitable) and in close proximity to it (Fl-BSA-inhibitable), although the relative proportions of these determinants varied among the clones. Inhibitor concns required for 50% inhibition varied independently of ligand binding affinity, and therefore were more likely influenced by the heterogeneous nature and affinity of the anti-idiotype reagents toward the individual determinants. Idiotypic analysis of H- and light (L) chains derived from five monoclonal antibodies of diverse affinities was performed. Fl binding and expression of idiotypic determinants by all clones required both H- and L-chains. Restoration of the idiotye by reassociated H- and L-chains was found to be highly restricted to homologous H- and L-chain pairs, as heterologous combinations did not result in the expression of either parental idiotype. The latter was true whether the heterologous pairs were derived from clones of the same isotype or the heterologous combination associated to form an intact molecule with greater affinity than the parental H- and L-chain combination. Heterologous recombinants from the two clones (6-10-6 and 20-19-1) exhibiting partial idiotypic cross-reactivity were able to restore a fraction (˜25%) of their idiotypic determinants. Results demonstrated the extensive conformational requirements of ligand binding and idiotype expression and indicated that a high degree of specificity in the V_H- and V_L-chain interaction must exist for the expression of these idiotypes.     

Linkage of low and high affinity anti-fluorescein idiotype families

Data presented in this study describes the isolation and characterization of two anti-fluorescein (Fl) hybridoma proteins 3–24 and 12–40, both IgG₁, with a K_a = 2.8 and 3.4 × 10⁶ M⁻¹, respectively, at 37°C. These clones inhibited (6.8 ± 2.8 − 20.8 ± 0.6% at l μg/well) the idiotype-anti-idiotype interactions (IAII) of anti-Fl clones 3–13 and 3–17, which define a previously described low affinity idiotype family. Antibodies 3–24 and 12–40 also inhibited (45.0 ± 3.0 and 61.3 ± 5.6%, respectively, at 1 μg/well) an IAII denfied by a high affinity (K_a = 5.2 ± 1.5 × 10⁹ M⁻¹ at 37°C) anti-Fl clone, 4-4. Hybridoma proteins 3–13 and 3–17 possess similar affinities for Fl (K_a = 3.8 ± 5.1 and 5.9 ± 4.0 × 10⁴ M⁻¹) and are known to be idiotypically unrelated to clone 4-4. While 3–24 and 12–40 appeared very similar, non-identity of their active sites was established by heterologous idiotypic inhibitions, fine specificity of binding and spectral measurements (Q_max and λ_max) of bound Fl. All IAII (3–13, 3–17, 9–40 and 4-4) were inhibited>80% by the presence of 10⁻⁴M F1 or F1-BSA, In addition, four intermediate affinity (6.0 × 10⁶ K_a 5.3 × 10⁸ M⁻¹) anti-FI clones, comprising a second previously described idiotype family (designated the 9–40 family) were further analyzed. Inhibition of the 9–40 IAII by all heterologous proteins in the 9–40 family (except clone 5–27), and clones 3–24, 12–40 and 4-4 ranged from 87.7 ± 1.3 to 95.4 ± 1.0% at 1μg/well. Titration of the 9–40 IAII inhibition by antibodies 9–40, 3–24, 12–40 or 4-4 generated essentially superimposable profiles. In reciprocal inhibition experiments, using the 4-4 IAII, clones 3–24, 12–40, 9–40 and 4-4 gave distinct idiotypic titration patterns. Thus, members of the 9–40 family, 3–24 and 12–40 were more closely related to intermediate affinity clone 9–40 than high affinity clone 4-4. Finally all members of the 9–40 family also significantly inhibited both the 3–13 and 3–17 IAII (11.8 ± 3.1 − 32.9 ± 6.1 at 1 μg/well) and gave distinct idiotypic inhibition profiles. Clones 3–24 and 12–40, characterized in this report, and the 9–40 family provide linkage between idiotypically distinct anti-Fl hybridoma proteins differing in affinity by> 20,000-fold. This linkage provides a greater span in affinity, than in all previously reported idiotypic families, within restricted or unrestricted systems.     

Novel anti-digoxin monoclonal antibodies with different binding specificities for digoxin metabolites and other glycosides

Spleen cells of BALB/c mice immunized with a digoxin-bovine serum albumin conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Seven monoclonal antibodies (MAb) selected by indirect ELISA were produced, purified and characterized. All the MAb were IgG1 isotypes. The apparent equilibrium association constants (Ka) of four of the MAb, determined by Scatchard analysis of the RIA data, ranged from 1 x 10(9) M-1 to 5.9 x 10(9) M-1. The estimated Ka values of the three other MAb were found to be between 4.8 x 10(7) M-1 and 5.9 x 10(8) M-1. Using digoxin and eighteen structurally-related compounds, the seven MAb could be divided into five groups based on their binding specificities assessed by an inhibition immunoenzymatic test. The MAb in Groups I and II, in particular, showed very different specificity profiles: the two MAb in Group I had low cross-reactivity with cardioinactive digoxin metabolites, whereas the high affinity MAb in Group II recognized all the digoxin metabolites tested. The MAb in Group I might be useful in a digoxin immunoassay and the Group II MAb in therapeutic reversal of digoxin intoxication.     

Equilibrium binding characteristics of monoclonal antibodies recognizing melanoma cell surface antigens

The equilibrium binding characteristics of a panel of six monoclonal antibodies (MAb) recognizing melanoma cell surface antigens (125 kdal cell surface melanoma associated glycoprotein antigen, 125kD-MAA; high molecular weight melanoma associated antigen, HMW-MAA; and a non-protein melanoma associated antigen, NP-MAA) were investigated using the cell lines SK-MEL-2, SK-MEL-5, and M21. The MAbs displayed equilibrium association constant (K) values ranging from 10(7) M-1 to 10(10) M-1 and maximum MAb binding values (Qmax) from 2 x 10(4) to 2 x 10(6) MAb molecules bound per cell. High trypsin concentrations were shown to have deleterious effects on Qmax values obtained for antibodies recognizing the 125kD-MAA, and even low trypsin concentrations affected Qmax values obtained for MAbs recognizing the HMW-MAA (although a complete linear recovery of HMW-MAA antigen was observed in 20-25 hours). Significant changes in Qmax were also noted for different cell passages. Except for MAb 43.2, little variation in K was observed when different cell lines were used. Linear Scatchard plots were obtained for all MAbs except 43.2 in which case concave down behavior was observed suggesting the existence of positive cooperativity between the binding sites of this MAb.     

Human IgG1 and its Fc fragment bind with different affinities to the Fc receptors on the human U937, HL-60 and ML-1 cell lines

Measurements were made of the binding of human monomeric 125I-IgG1 and 125I-Fc to U937 cells at room temp. Analyses of the binding data showed that these cells possessed a single class of receptor (FcR) for Fc or IgG and, although both ligands were found to bind to the same number of sites per cell, Fc was found to bind with about twice the affinity of IgG. At 20 degrees C estimates of the forward rate constants and the dissociation rate constants for IgG and Fe were 1.13 and 3.65 X 10(7) M-1 min-1 and 0.33 and 0.57 X 10(-2) min-1 respectively. Independent determinations of the association constants (Ka) under the same experimental conditions gave values of 0.98 X 10(9) M-1 for IgG and 3.1 X 10(9) M-1 for Fc. Thus the Fc fragment of IgG appears to bind to U937 FcR at 3-4 times the rate of IgG and to dissociate at about twice the rate, resulting in higher values of Ka for the Fc-FcR than for the IgG-FcR interaction. Also, in competitive-binding experiments and in EA rosette inhibition assay the Fc fragment was consistently found to be more efficient in FcR binding than IgG. Similar results were obtained using HL-60 and ML-1 cells which possess FcR like those on U937 cells and with IgG1 and Fc prepared from other myelomas. IgG and Fe which had undergone mild reduction and alkylation bound to the same number of FcR per U937 cell as the non-reduced ligands but the affinity of binding was diminished to a similar degree with both ligands, suggesting that the major effect of cleavage of the interchain disulfide bonds on cytophilic binding is due to alteration of the native quaternary relationships of the C gamma 2 and C gamma 3 domains.     

Production of stable anti-digoxin Fv in Escherichia coli.

We have created a bacterial expression-export system and have used it to express (14 mg l-1) the variable region fragment (Fv) of an anti-digoxin antibody (26-10) in Escherichia coli. The expression-export plasmid contains a T7 promoter and the E. coli signal sequences ompA [Movva et al., J. biol. Chem. 255, 27-29 (1980)] and phoA [Inouye et al., J. Bacteriol. 149, 434-439 (1982)] fused to heavy chain (VH) and light chain (VL) variable region sequences to generate an artificial cistron. The 26-10 Fv protein made using this system was soluble, unlike many other expression systems which produce insoluble proteins in the form of inclusion bodies. The 26-10 VH and VL proteins were cleaved at their mature N-termini and exported into the bacterial periplasm where they could be easily extracted and affinity purified on ouabain-Sepharose. 26-10 Fv bound to digoxin with similar affinity and specificity as the whole 26-10 antibody (Ka for Fv, 1.3 x 10(9) M-1, Ka for IgG, 7 x 10(9) M-1). 26-10 Fv appears to be remarkably stable in comparison with other Fv fragments. The half-life for chain dissociation of 26-10 Fv was 48 hr compared to the reported 1.5 hr half-life of McPC603 Fv. We present the proton NMR spectra of the 26-10 Fv as preliminary evidence that this expression-export system can be used to facilitate the analysis of the solution structure of 26-10 Fv by NMR.     

ELISA-based determination of immunological binding constants

An expression for the time-dependent concn of antibody in a hemispherical antigen-coated well is derived by taking the Laplace transformation of the diffusion equation. From this expression, and from antibody adsorption kinetics measured using ELISA, it is possible to evaluate the rate constant of bimolecular association, k1, the rate constant of first order antibody-antigen dissociation, k2, and their ratio, the binding equilibrium constant or affinity, Ka. For the interaction of an anti-arsanilate monoclonal antibody with arsanilate-coupled albumin, analysis yields k1 = 8.8 X 10(3) M-1 sec-1, k2 = 2.5 X 10(-4) sec-1 and Ka = 3.5 X 10(7) M-1, for mean values over 10 experiments. These results are discussed in reference to the conventionally-obtained values for binding constants, including the affinity of the anti-arsanilate monoclonal for the hapten (p-azobenzenearsonate)-N-acetyl-L-tyrosine, determined by equilibrium dialysis.     

The binding of acetaminophen, lidocaine, and valproic acid to human milk

The binding of acetaminophen, lidocaine, and valproic acid to pooled normal mature human milk was studied in vitro by using equilibrium dialysis. Scatchard analysis revealed high-affinity, low-capacity binding for acetaminophen (Ka [affinity constant of association], 1.47 x 10(4) L/mol; Bo [concentration of binding sites], 9.01 x 10(-4) mol/L) and some minimal, nonspecific binding. Binding ranged up to 85%. For lidocaine, low-affinity, high-capacity binding was noted (Ka, 1.42 x 10(2) L/mol; Bo, 1.69 x 10(-2) mol/L). Binding ranged up to 72%. For valproic acid, only minimal, nonspecific binding was noted at low drug concentrations with binding ranging up to 64%. The binding of these drugs to milk might enhance their excretion and subsequent ingestion by infants who are breast-fed. In addition, the low pH of the milk (6.24) may cause "ion trapping" of acetaminophen (pKa, 9.5) and lidocaine (pKa, 7.9).     

Binding of antiarrhythmic drugs to human placenta in vitro.

Lidocaine, procainamide and quinidine binding to human placenta was investigated in vitro. Pooled whole human placental homogenate supplemented with either non-radiolabelled lidocaine, procainamide or quinidine over the concentration range 50-5,000 x 10(-7) mol/L was submitted to equilibrium dialysis against phosphate buffer, pH 7.4, 0.1 mol/L. Post-dialysis drug concentrations were measured by enzyme immunoassay. Data were analyzed by the method of Scatchard. No binding to placenta was noted for either lidocaine or procainamide. In contrast, up to 22 percent of quinidine was bound. Two binders were defined as follows: #1 (Ka, 7.37 x 10(5) L/mol; Bo, 1.55 x 10(-7) mol/L) and #2 (Ka, 7.11 x 10(4) L/mol; Bo, 4.05 x 10(-6) mol/L). The concentrations of quinidine binding sites in moles per gram of placenta were 1.55 x 10(-9) and 4.05 x 10(-8), respectively. These data suggest that quinidine may be accumulated in human placenta.     

Characterization of the interaction between recombinant human interferon-gamma and its receptor on human polymorphonuclear leukocytes

The interaction of human recombinant interferon-gamma (rIFN-gamma) with human polymorphonuclear cells (PMN) was investigated. Bolton-Hunter radioiodinated rIFN-gamma bound to PMN in a specific and saturable manner. Eleven hundred binding sites were observed with a Ka of 0.56 x 10(10) M-1. Binding to PMN was rapid with a K1 of 9 x 10(5) M-1 sec-1 at 4 degrees C. At 37 degrees C binding was complete within 6 min. About 50% of bound ligand was internalized within 30 min at 37 degrees C. The receptor demonstrated moderate lability at 37 degrees C in culture. After 1 h at 37 degrees C, PMN lost 80% of their 125I-rIFN-gamma binding sites. This loss was reversed in part by the presence of interleukin-1 in the culture, but not tumor necrosis factor. These studies provide a framework for further investigation into the signalling process of rIFN-gamma on PMN.     

The spontaneous ability of normal human IgG to inhibit the Fc receptors of normal human monocytes is related to their binding capacity to lectins

The lectin-binding capacity of 96 normal human IgG, assessed by solid-phase radioimmunoassay, strikingly varied according to the lectin considered. Indeed, half of the IgG samples exhibited peanut agglutinin (PNA)- and pokeweed mitogen-specific binding capacities superior or equal to 4%, whereas less than 15% of IgG similarly bound to concanavalin A (Con A) and to phytohemagglutinin. The ability of those IgG to inhibit the Fc receptor (Fc-R) function of human monocytes, measured by a classical rosette assay, was inversely correlated to their binding ratios to PNA and Con A only. By affinity chromatography, three groups of IgG were separated: the IgG purified on agarose-PNA columns slightly reduced the Fc-R function (40-45% inhibition); the IgG purified on Sepharose-Con A columns exhibited the highest inhibitory properties (80-85% inhibition); the IgG that did not bind to PNA and Con A columns possessed intermediate inhibitory properties (65-70% inhibition). The different effect of IgG on Fc receptors was conserved when monocytes were first treated by trypsin and was unrelated to their specific binding to human monocytes, to their subclasses, and to their C1q- or protein A-binding capacities. Incubation of monocytes with D-galactose (10 mM) significantly improved their capacity to form IgG rosettes, whereas their incubation with D-mannose (10 mM) significantly reduced the Fc-R function. Scatchard plots of 125I-IgG1 myeloma protein binding to monocytes were linear under basal conditions, as well as after a prior incubation of the cells with D-galactose or D-mannose. Monocytes bound about 16,000 molecules of IgG1 per cell in each instance. In contrast, the mean association constant (Ka) for IgG1 binding was 2.59 +/- 0.50 X 10(8) M-1 under basal conditions, 4.4 +/- 0.75 X 10(8) M-1 after D-galactose incubation, and 1.35 +/- 0.50 X 10(8) M-1 after D-mannose incubation. These data suggest that the level of human monocyte Fc-R function blockade induced by human IgG depends mainly on the presence of "accessible" galactosyl or mannosyl residues in the Fc domain and that the modulation of the Fc-R function induced by these carbohydrates is due to a change in the affinity rather than in the number of single class of high-affinity binding sites.     

The use of monoclonal antibodies to define levels of cystatin C in normal human serum

We established four hybridoma cell lines which secreted monoclonal antibodies against human cystatin C. These monoclonal antibodies were of IgA(kappa), IgG2a(lambda), IgG1(kappa), and IgG1(lambda) isotypes, respectively. The association constants (Ka) of the three IgG monoclonal antibodies ranged from 3.6 x 10(9) M-1 to 7.3 x 10(10) M-1. An ELISA technique for the measurement of cystatin C was established by using one of these monoclonal antibodies. The assay procedure was highly specific, sensitive, and reproducible. The lower detectable limit of cystatin C by this procedure was 1.9 ng/ml. The level of cystatin C in normal human serum was 1.16 +/- 0.91 micrograms/ml (mean +/- S.D., n = 274).     

牛磺酸对离体大鼠脊髓缺氧／复氧损伤的保护作用

本文在孵育的离体大鼠脊髓组织切片上，观察牛磺酸对缺氧／复氧损伤的影响，缺氧／复氧损伤引起大鼠脊髓组织ＡＴＰ含量减少和ＬＤＨ漏出增多，ＮＯ生成和ＮＯＳ活性明显增加，Ｌ－精氨酸转运Ｖｍａｘ增加９７％（Ｐ〈０．０１），Ｋｍ值无明显变化，用１０和２０ｍｍｏｌ／Ｌ牛磺酸卵育与单纯缺氧／臭氧组比较，显著减轻了缺氧／复氧引起的组织ＡＴＰ减少和ＬＤＨ漏出，ＮＯ生成分别低３４％（Ｐ〈０．０１）和４０％（Ｐ〈０．０１     

An approach for characterization and purification of a human monoclonal hybridoma antibody

An IgG human-human monoclonal hybridoma antibody, AML-19, reactive with human myeloid cells of non-malignant and malignant origin has been produced by fusion of blood mononuclear cells from a patient with acute myeloid leukemia (AML) and the human B-lymphoma cell line RH-L4. The monoclonal antibody (MAb) AML-19 was purified from hybridoma supernatant by primarily anion-exchange chromatography, in order to separate the AML-19 MAb from contaminating immunoglobulin (Ig), e.g. bovine Ig and MAb derived from the parental fusion partner, and followed by immunoaffinity chromatography. This purification method gave the highest yield and purity of the AML-19 MAb. The isoelectric point (pI) of the MAb was estimated to be 5. Inhibition assays indicate an apparent dissociation constant (Kd) corresponding to 4 x 10(-9) M and an affinity constant (Ka) to 2.5 x 10(8)M-1 to K562 erythroleukemia cells. Scatchard plot demonstrated a linear slope as a manifestation of monoclonality and a low number of AML-19 specific epitopes, estimated to 1500 per cell.     

Secretory Component-Binding Properties of Normal Serum IgM

Our aim was to investigate why serum IgM is poorly transferred into secretions in normal subjects. Indeed, the low IgM level in secretions contrasts with the capacity of monoclonal IgM to bind to secretory component (SC), but it is not well established to what extent normal serum IgM can do so. The mean SC affinity was studied with a polyclonal IgM preparation from 250 normal subjects and with a representative pool of 100 different monoclonal IgM. The SC-binding percentages varied as a function of the IgM/SC molar ratio according to a common hyperbolic curve, with similar association constants: Ka = 4.19 +/- 2.61 x 10(7) M-1 (polyclonal pool) and Ka = 5.80 +/- 2.73 x 10(7) (monoclonal pool). It thus appears that the large difference in IgM concentrations between blood and secretions cannot be due to an SC-binding defect of serum IgM, but is probably explained by its low diffusion from blood to the extravascular compartment.     

The production of high affinity monoclonal antibodies to human chorionic gonadotropin and their application to immunoradiometric assay

Production of monoclonal antibodies against hCG has been studied using hCG as the antigen. This study reports the successful isolation of hybrid clones secreting monoclonal antibodies specific for hCG with an affinity constant higher than 10(10) M-1. Of 23 fusions, only 17 fusions have produced positive clones which secrete antibodies giving high levels of binding with 125I-labelled hCG in the supernatant. Finally, 6 different monoclonal antibodies have been isolated; 4 of them, specific for the beta-subunit, with a Ka approximately 1.1-4.0 X 10(11) M-1 and 2 others, specific for the alpha-subunit, presenting an affinity of 2.5 X 10(10) M-1. When the antibodies specific for the beta-subunit are used, specific and highly sensitive radioimmunoassays are obtained after only 3 hrs of incubation. Using iodinated monoclonal antibodies specific for the alpha-subunit and tubes coated with antibodies against the beta-subunit, we have developed sensitive immunoradiometric assays.     

Degradation of airway neuropeptides by human lung tryptase.

Several lines of evidence suggest a possible role for mast cell proteases in modulating the biologic effects of neuropeptides. To explore the potential of such interactions in human airway, we examined the activity of human tryptase, the major secretory protease of human lung mast cells, against several neuropeptides with proposed regulatory functions in human airway. Using highly purified tryptase obtained from extracts of human lung, we determined the sites and rats of hydrolysis of vasoactive intestinal peptide (VIP), peptide histidine-methionine (PHM), calcitonin gene-related peptide (CGRP), and the tachykinins substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). Tryptase hydrolyzes VIP rapidly at several sites (Arg12, Arg14, Lys20, and Lys21) with an overall kcat/Km of 1.5 x 10(5) M-1 s-1 and hydrolyzes PHM primarily at a single site (Lys20) with a kcat/Km of 1.9 x 10(4) M-1 s-1. Tryptase also rapidly hydrolyzes CGRP at two sites (Arg18 and Lys24) with a kcat/Km of 2.7 x 10(5) M-1 s-1. The tachykinins are not hydrolyzed by tryptase. These observations raise the possibility that tryptase-mediated degradation of the bronchodilators VIP and PHM combined with exaggerated mast cell release of tryptase may contribute to the increase in bronchial responsiveness and the decrease in immunoreactive VIP in airway nerves associated with asthma. The favorable rates of hydrolysis of CGRP suggest that tryptase may also terminate the effects of CGRP on bronchial and vascular smooth muscle tone and permeability.     

H-2 expression by lymphoid cells of different mouse strains: quantitative interaction of H-2 with monoclonal antibodies and their Fab fragments.

Monoclonal antibodies (H100-30/3 and 11-4.1) to H-2k were used to study H-2 antigen expression and characteristics of the H-2 antigen-antibody interaction at the cell surface. Studies with radiolabelled F(ab')2 and Fab' fragments of 11-4.1 antibody confirmed that monoclonal IgG binding to cells is directly proportional to the number of H-2 sites and shows a high proportion of monovalent binding over a wide range of concentrations. Scatchard plots showed no difference in the binding affinity constant (Ka) of a given monoclonal antibody on lymphoblasts from various H-2k F1 and congenic strains, but only in the number of antigenic sites per cell. F1 (k x d) lymphoblasts show 1 x 10(5) H-2k sites/cell, about 50% of the expression in homozygotes. Dk expression in C3H.OH is 1.4 x 10(4) sites/cell. While normal cells appear to have a constant amount of H-2 (2-3 x 10(5) sites/cell), BW thymoma cells show unstable H-2 expression, having an average of five times fewer H-2 sites per cell when grown in vitro as compared to in vivo growth. Another BW cell surface marker, Thy-1.1, does not fluctuate in parallel with H-2. The 30/3 and 11-4.1 antibodies bind to topologically distinct sites on H-2Kk. The binding of these antibodies can be perturbed differentially: paraformaldehyde fixation of cells abolishes binding of 11-4.1 antibody but not of 30/3 antibody; increasing temperature increases the Ka of 30/3 antibody binding but decreases the Ka of 11-4.1 antibody binding to cells.     

Production and characterization of four anti-neuropeptide Y monoclonal antibodies.

Human neuropeptide Y (hNPY) is a potent vasoconstrictor peptide of 36 aminoacid residues. We isolated hybridomas secreting four monoclonal antibodies directed against various epitopes of neuropeptide Y and studied their cross-reactivity with peptide YY (PYY) and the pancreatic polypeptide (PP), two peptides sharing sequence homologies with hNPY (respectively 70% and 50%). The antibody NPY02 is an IgG1 with a Ka of 5.5 x 10(10) liters/mol. It binds to the 11-24 region of NPY (IC50 = 2 x 10(-7)M), does not recognize PP but cross-reacts weakly with PYY. Antibodies NPY03 and NPY05 are IgG2 with respective Ka's of 6.7 x 10(9) and 2.5 x 10(10) liters/mol. They interact with a C-terminal epitope on NPY (NPY 27-34, IC50 = 2 x 10(-9) M for NPY03 and NPY 32-36, IC50 = 1 x 10(-9) M for NPY05). These two antibodies cross-react with PYY whereas only NPY05 binds PP. NPY05 is unable to bind the free acid form of neuropeptide Y. The 32-36 COOH free subpeptide is recognized 50,000 less efficiently by NPY05 than its amidated form. Antibody NPY04 is an IgG3 with a Ka of 3.8 x 10(8) liters/mol. It recognizes a N-terminal epitope between aminoacids 1 and 12 (IC50 = 2.5 x 10(-6) M). NPY04 interacts weakly with PYY but not detectably with PP. These results obtained with 4 different monoclonal antibodies demonstrate the presence of at least four epitopes on hNPY, two of them being continuous. These antibodies will be used to study the interaction of NPY with its receptor and to develop sensitive and specific assays for determination of NPY concentrations in biological fluids.