A syngeneic monoclonal anti-idiotypic antibody with internal image properties restricted to a single aldosterone-binding system

Immunization with a murine anti-aldosterone mAb (AAC) resulted in the isolation of a syngeneic monoclonal anti-idiotypic antibody, LH9G4. LH9G4 bound to Fab fragments of AAC and was affinity-purified on an AAC column. LH9G4 inhibited the binding of aldosterone to AAC in a dose-dependent manner with an apparent dissociation constant of 0.5 nM as determined by competitive inhibition assays in ELISA and RIA. LH9G4 and aldosterone have similar relative affinities for AAC. Kinetic studies and Scatchard plot analysis support a reversible and reciprocal competitive inhibition mechanism between LH9G4 and aldosterone for the paratope of AAC. The possibility of a steric hindrance mechanism was eliminated. No cross-reactivity was seen with six other murine anti-aldosterone mAb, with a rabbit polyclonal antibody or with aldosterone receptor. The anti-idiotypic antibody, defined as a "restricted" internal image of aldosterone, is apparently directed at a private idiotope present in the paratope of AAC but not in binding sites of other aldosterone-binding proteins. Biophysical considerations involving characteristics of nonbonded attractive forces can explain these findings. An advantage of the one-step auto-anti-idiotypic procedure for the generation of Ab2-beta or internal image antibodies is discussed.     

Dynorphin-A (1-8) is contained within perikarya, nerve fibres and nerve terminals of rat duodenum

By the use of well-characterized antibodies against porcine dynorphin-A(1-8), an endogenous opioid peptide, and the use of a modified immunofluorescence microscopic technique, dynorphin-A(1-8) stained perikarya, nerve fibres, and nerve terminals were visualized in the rat duodenum. Dynorphin-A(1-8) immunoreactive perikarya were revealed with certainty only in the myenteric plexus, while dynorphinergic nerve fibres could bee seen in the myenteric plexus and circular muscle layer, but not in the longitudinal muscle layer and submucous plexus. Dynorphin-A(1-8) immunofluorescent nerve endings were in close contacts with submucosal blood vessels, probably arterioles, and Brunner's gland cells. These findings suggest that the opioid peptide dynorphin-A(1-8) might be synthetized within myenteric plexus perikarya of the rat duodenum and that it might modulate the peristaltic activity, intestinal blood pressure, and production of mucopeptides synthetized within Brunner's gland cells.     

Altered secretion of pregnancy-associated glycoproteins during gestation in bovine somatic clones

Somatic nuclear transfer (NT) in cattle is often accompanied by severe placental anomalies, hypertrophy, and hydrallantois, which induce a high rate of pregnancy losses throughout gestation. These placental deficits are associated with an abnormal increase of the maternal plasma levels of pregnancy-associated glycoprotein (PAG), produced by the trophoblastic binucleate cells (BNC) of the placenta. The objective of this study was to analyze the origin of the abnormally elevated PAG concentrations in the peripheral circulation of NT recipients during pathological pregnancies. Concentrations of PAG were measured both in maternal blood, in chorionic and cotyledonary tissular extracts from control recipients (after artificial insemination, AI, or in vitro fertilization, IVF) and clone recipients on Day 32, Day 62, and during the third trimester of gestation. Three different radioimmunoassay (RIA) systems were used. One homologous RIA for PSP60, similar to bovine PAG-1 (PAG_67kDa), and two heterologous RIA with PAG_67kDa as standard and tracer, and antisera anti-caprine PAG (AS#706 and AS#708). Circulating and tissular concentrations of bovine placental lactogen (bPL), a glycoprotein also produced by BNC, were determined by RIA at the same stages. The number of BNC in the placental tissues was determined by cell counting after immunostaining with anti PSP60 antibody on tissue sections from control and NT pregnancies. Maternal plasma PAG concentrations were not different among groups on Day 32, but they were significantly higher in NT than in control pregnancies on Day 62 with all three RIA and during the third trimester with two RIA (RIA-PSP60 and RIA with AS#708). Circulating bPL concentrations were undetectable on Days 32 and 62 and were not different in the third trimester between NT and control pregnancies. Tissular amounts of PAG on total proteins were not different between the two groups at all stages studied. No difference was determined in the percentage of PSP60-positive BNC in placental tissues between controls and NT on Day 62 and during the third trimester of pregnancy. Western blots of tissular extracts from placenta showed no major molecular weight changes of PAG in NT pregnancies compared to controls. No differences in maternal circulation concentrations or tissular content of bPL were observed between control and NT pregnancies. In conclusion, the specific increase of PAG in maternal plasma concentrations during abnormal NT pregnancies do not result from a higher proportion of BNC, or an increased protein expression of PAG and could be due to changes in the composition of terminal glycosylation which result into a clearance decrease of PAG from the circulation.     

Multispecificity of a recombinant anti‐ras monoclonal antibody

Recombinant monoclonal antibodies (Ab's) have widespread application as research tools, diagnostic reagents and as biotherapeutics. Whilst studying the cellular molecular switch protein m‐ras, a recombinant monoclonal antibody to m‐ras was generated for use as a research tool. Antibody genes from a single rabbit B cell secreting IgG to an m‐ras specific peptide sequence were expressed in mammalian cells, and monoclonal rabbit IgG binding was characterized by ELISA and peptide array blotting. Although the monoclonal Ab was selected for specificity to m‐ras peptide, it also bound to both recombinant full‐length m‐ras and h‐ras proteins. The cross‐reactive binding of the monoclonal Ab to h‐ras was defined by peptide array blot revealing that the Ab showed preference for peptide sequences containing multiple positively charged amino acid residues. These data reinforce the concept of antibody multispecificity through multiple interactions of the Ab paratope with diverse polypeptides. They also emphasize the importance of immunogen and Ab selection processes when generating recombinant monoclonal Ab's.     

中脑导水管周围灰质内注射抗阿片肽血清对神经降压素增强电针镇痛的影响

本工作以钾离子透入引起大鼠甩尾反应作为痛反应指标,观察纳洛酮（ＮＸ）和抗阿片肽血清对大鼠中脑导水管周围灰质（ＰＡＧ）内微量注射神经降压素（ＮＴ）增强电针镇痛作用的影响。结果显示：（１）ＰＡＧ内微量注射ＮＴ后,大鼠电针镇痛效应明显提高;（２）ＰＡＧ内注射大剂量ＮＸ,可以显著减弱ＮＴ增强电针镇痛效应;（３）ＰＡＧ内微量注射抗甲脑啡肽血清和抗β内啡肽血清后,ＮＴ增强电针镇痛的作用明显降低;而ＰＡＧ内微量注射抗强啡肽Ａ１１３血清,对于ＮＴ增强电针镇痛的作用并无明显影响。提示：ＰＡＧ内ＮＴ在针刺镇痛过程中发挥重要作用,ＮＴ增强电针镇痛的作用与甲脑啡肽和β内啡肽有较密切的关系。     

中脑导水管周围灰质内注射抗阿片肽血清对神经降压素增强电针镇痛的 …

本工作以钾离子透入引起大鼠甩尾反应作为痛反应指标,观察纳洛酮（ＮＸ）和抗阿片肽血清对大鼠中脑导水管周围灰质（ＰＡＧ）内微量注射神经降压素（ＮＴ）增强电针镇痛作用的影响。     

Detection of postvaccination mumps virus antibody by neutralization test, enzyme-linked immunosorbent assay and sensitive hemagglutination inhibition test

A group of 251 children aged 2-3 years given live attenuated mumps virus vaccine PAVIVAC of Czechoslovak production were tested for antiparotitis antibody levels in pre- and postvaccination sera by neutralization test (NT), enzyme-linked immunosorbent assay (ELISA) and sensitive hemagglutination inhibition test, enhanced by heterologous antibody to human immunoglobulin G (E-HIT). The prevaccination findings were as follows: positive ELISA IgG titres, neutralization antibodies and hemagglutination inhibition antibodies were present in, respectively, 35%, 25.9% and 27.9% of the sera. Postvaccination seroconversions were evaluated in 159 susceptible vaccinees whose prevaccination sera had been negative by all three tests. The lowest seroconversion was detected by NT (74.2%), seroconversions by ELISA and E-HIT were appreciably higher (82.4% and 86.8%, respectively). The seven children showing a seroconversion by E-HIT but not by ELISA had a 4 fold increase of anti-mumps ELISA IgG antibodies as well, but the rise of antibody titres was at a level falling in the range below the positivity criterion for ELISA. The statistically evaluated detection rate for antibodies was significantly higher (significance test "t") by ELISA as compared with neutralization test. However, antibody levels (geometric mean titres) were 8-10 times lower in postvaccination sera than in convalescent sera of 30 children with mumps in all three tests.     

Extraction of neurotensin-like immunoreactivities from porcine ileal mucosa

The extractability of neurotensin (NT) from porcine ileal mucosa was studied by comparison of eight extraction procedures. Tissue content of neurotensin-like immunoreactivity was quantitated and characterized by sequence-specific radioimmunoassays and gel filtration chromatography. Homogenization prior to boiling in extraction solvent produced higher levels of the intact peptide than the reverse procedure. N-terminal immunoreactivity was not influenced by the sequence of these steps. Tissue levels of intact NT were highest after extraction with 2.0 M acetic acid (mean 79.1 pmol/g, N = 6) and lowest with distilled water (mean 6.5 pmol/g, N = 6). The opposite was the case with levels of N-terminal immunoreactivity (mean 55.2 pmol/g and 105.7 pmol/g respectively, N = 6). Recovery experiments with addition of synthetic NT 1-13 and the N-terminal fragment NT 1-8 indicated that these differences could be explained by differences in recovery of intact NT and N-terminal immunoreactive components in tissue. Gel chromatography confirmed that in acetic acid almost only the intact peptide was extracted from ileal mucosa, and showed that after extraction in water or phosphate buffer several N-terminal components were present. The results suggest that a molecular heterogeneity may be present in ileal tissue. If this concept is supported by further studies differential extraction procedures may be needed in the future.     

Properties of a monoclonal antibody directed to the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase

A synthetic peptide representing the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase (K-R-R-W-K-K-N-F-I-A-V-S-A-A-N-R-F-K-K-I-S-S-S-G-A-L) was used as an antigen to produce a monoclonal antibody. The antibody (designated MAb RSkCBP1, of the IgM class) reacted with similar affinity (KD approximately 20 nM) by competitive enzyme-linked immunoassay (ELISA) with the antigen peptide and intact rabbit skeletal muscle myosin light chain kinase. MAb RSkCBP1 inhibited rabbit skeletal muscle myosin light chain kinase activity competitively with respect to calmodulin (Ki = 20 nM). The antibody also inhibited myosin light chain kinase activity in extracts of skeletal muscle from several mammalian species (rabbit, sheep, and bovine) and an avian species (chicken). The concentration of MAb RSKCBP1 required for 50% inhibition of enzyme activity was similar for the mammalian species (80 nM) but was significantly higher for the avian species (1.2 microM). A competitive ELISA protocol was used to analyze weak cross-reactivity to other calmodulin-binding peptides and proteins. This assay demonstrated no cross-reactivity with the venom peptides melittin or mastoparan; smooth muscle myosin light chain kinases from hog carotid, bovine trachea, or chicken gizzard; bovine brain calmodulin-dependent calcineurin; or rabbit skeletal muscle troponin I. These data support the contention that the synthetic peptide used as the antigen represents the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase and that the calmodulin-binding domains of different calmodulin-regulated proteins may have distinct primary and/or higher order structures.     

Neurotensin may function as a regulatory peptide in small cell lung cancer.

Neurotensin (NT) has been postulated to act as a modulatory agent in the central nervous system. Besides its presence in mammalian brain, NT is produced by small cell carcinoma of the lung (SCLC) and cell lines derived from these tumors. Receptors have also been characterized in some SCLC cell lines leading to the suggestion that NT could regulate the growth of SCLC in an autocrine fashion similar to bombesin/GRP. Previously, we had reported that a 10 nM dose of NT and NT(8-13), but not NT(1-8), elevated cytosolic Ca2+, indicating that SCLC NT receptors may use Ca2+ as a second messenger. Using intact SCLC cells we report that time-course incubations with NT lead to the formation of the amino-terminal fragment NT(1-8) and small amounts of the C-terminal fragment NT(9-13). These fragments are formed by metalloendopeptidase 3.4.24.15 cleaving enzyme at the Arg8-Arg9 bond of NT. Significant levels of soluble 3.4.24.15 (10-17 nmoles/mg Pr-/min) are present in SCLC cell lines. Using the in vitro clonogenic assay we tested the effect of 0.5, 5.0 and 10.0 nM doses of NT, NT(1-8) and NT(8-13) on SCLC clonal growth. NT and the C-terminal fragment NT(8-13) stimulated colony formation whereas the N-terminal fragment did not. In summary, NT may function as a regulatory peptide in SCLC through the formation of peptide fragments.     

Human immunodeficiency virus type 1-neutralizing monoclonal antibody 2F5 is multispecific for sequences flanking the DKW core epitope

Human monoclonal antibody 2F5 is one of a few human antibodies that neutralize a broad range of HIV-1 primary isolates. The 2F5 epitope on gp41 includes the sequence ELDKWA, with the core residues, DKW, being critical for antibody binding. HIV-neutralizing antibodies have never been elicited by immunization with peptides bearing ELDKWA, suggesting that important part(s) of the 2F5 paratope remain unidentified. The use of longer peptides extending beyond ELDKWA has resulted in increased epitope antigenicity, but neutralizing antibodies have not been generated. We sought to develop peptides that bind to 2F5, and that function as specific probes of the 2F5 paratope. Thus, we used 2F5 to screen a set of phage-displayed, random peptide libraries. Tight-binding clones from the random peptide libraries displayed sequence variability in the regions flanking the DKW motif. To further reveal flanking regions involved in 2F5 binding, two semi-defined libraries were constructed having 12 variegated residues either N-terminal or C-terminal to the DKW core (X(12)-AADKW and AADKW-X(12), respectively). Three clones isolated from the AADKW-X(12) library had similar high affinities, despite a lack of sequence homology among them, or with gp41. The contribution of each residue of these clones to 2F5 binding was evaluated by Ala substitution and amino acid deletion studies, and revealed that each clone bound 2F5 by a different mechanism. These results suggest that the 2F5 paratope is formed by at least two functionally distinct regions: one that displays specificity for the DKW core epitope, and another that is multispecific for sequences C-terminal to the core epitope. The implications of this second, multispecific region of the 2F5 paratope for its unique biological function are discussed.     

Peptide-specific antibodies as probes of the orientation of the glucose transporter in the human erythrocyte membrane

Antibodies were raised in rabbits against synthetic peptides corresponding to the N-terminal (residues 1-15) and the C-terminal (residues 477-492) regions of the human erythrocyte glucose transporter. The antisera recognized the intact transporter in enzyme-linked immunosorbent assays (ELISA) and Western blots. In addition, the anti-C-terminal peptide antibodies were demonstrated, by competitive ELISA and by immunoadsorption experiments, to bind to the native transporter. Competitive ELISA, using intact erythrocytes, unsealed erythrocyte membranes, or membrane vesicles of known sidedness as competing antigen, showed that these antibodies bound only to the cytoplasmic surface of the membrane, indicating that the C terminus of the protein is exposed to the cytoplasm. On Western blots, the anti-N-terminal peptide antiserum labeled the glycosylated tryptic fragment of the transporter, of apparent Mr = 23,000-42,000, showing that this originates from the N-terminal half of the protein. The anti-C-terminal peptide antiserum labeled higher Mr precursors of the Mr = 18,000 tryptic fragment, although not the fragment itself, indicating that the latter, with its associated cytochalasin B binding site, is derived from the C-terminal half of the protein. Antiserum against the intact transporter recognized the C-terminal peptide on ELISA, and the Mr = 18,000 fragment but not the glycosylated tryptic fragment on Western blots.     

利用噬菌体展示技术筛选胰岛素模拟肽

为了寻找能够模拟胰岛素生物活性的小肽,以胰岛素多克隆抗体为靶标,筛选噬菌体展示随机C7C环肽库.3轮筛选后,通过ELISA方法挑取与靶分子特异性结合的15个阳性克隆,测序获得两条序列,分析所得序列并合成相应短肽.通过细胞生物学活性检测,小肽CPTSQANSC（ZJ1）能够竞争性的抑制胰岛素与其受体的结合,并对正常小鼠和四氧嘧啶诱导的糖尿病小鼠,都有明显的降血糖作用.上述结果表明,小肽CPTSQANSC具有胰岛素样生物学活性.而小肽CVQPSHSSC（ZJ2）表现出胰岛素拮抗活性,能引起正常小鼠血糖升高.这表明筛选到了能够模拟胰岛素表位的短肽CPTSQANSC,可能为治疗胰岛素依赖性糖尿病提供了新线索.     

Endocrine control of amphibian sexual behavior: Evidence for a neurohormone-androgen interaction

The incidence of sexual behavior increased after an injection of arginine-8 vasotocin or arginine-8 vasopressin into intact male newts (Taricha granulosa). Administration of arginine vasopressin to males that were castrated 35 days earlier enhanced sexual behaviors in only those males implanted with androgen.     

Identification, synthesis and properties of a consensus peptide recognized by a monoclonal antibody directed to various type I cytokine receptors

Previous works demonstrated that the monoclonal antibody (MAb) called R7B4 is directed to an epitope shared by receptors for lactogenic and somatogenic hormones as well as interleukins 2 and 6 (IL-2 and IL-6). The MAb inhibited the biological effects of those hormones and cytokines by impairing their binding to receptors. It is known that the receptors for growth hormones (GH), prolactins (PRL), IL-2, and IL-6 pertain to the type I cytokine receptor family, sharing the common motif WSXWS or the homologous F(Y)GEFS. Thus, a set of 34 decapeptides corresponding to diverse receptors containing those sequences were synthesized by the PEPSCAN method and their reactions with MAb R7B4 were measured by ELISA. The MAb significantly recognized 21 peptides, allowing us to establish the consensus sequence HGYWSEWSPE as a portion of the R7B4 epitope. The consensus peptide was synthesized and purified by conventional methods, and its capacity to bind to MAb R7B4 paratope confirmed. Moreover, polyclonal Ab to the peptide elicited in mice were able to inhibit the hGH binding to lactogenic, somatogenic and human specific liver receptors. This fact suggests that the consensus peptide could be used as an immunogen to produce anti-hGH receptor Ab behaving as hormone or cytokine antagonists in certain pathological conditions.     

中脑导水管周围灰质内神经降压素在电针镇痛中的作用

本工作以钾离子透入法引起大鼠甩尾反应的电流强度为痛反应指标,测定动物痛阈,观察到大鼠中脑导水管周围灰质（ＰＡＧ）内注入神经降压素（ＮＴ）后,大鼠痛阈和电针镇痛效应明显升高;注入抗神经降压素血清后,痛阈和电针镇痛效应明显降低。注入纳洛酮后,可明显减弱ＮＴ镇痛和电针镇痛的效应。提示,ＰＡＧ内ＮＴ参与电针镇痛的病理生理过程,且至少部分效应是通过内源性阿片肽系统中介的     

Minimum requirements for immunogenic and antigenic activities of homologs of a synthetic peptide of influenza virus hemagglutinin.

Synthetic peptides of increasing length and corresponding in sequence to the C-terminal end of the HA1 molecule of influenza virus were constructed and examined for their immunogenic and antigenic properties. Peptides containing at least the four C-terminal amino acids, when coupled to keyhole limpet hemocyanin, were capable of eliciting antibody in BALB/c mice that bound to the 24-residue parent peptide H3 HA1 (305 to 328). In the absence of a carrier, the C-terminal decapeptide was the shortest peptide capable of eliciting antibody. The specificity of this antibody was indistinguishable from that of a monoclonal antibody to the parent peptide which recognizes an epitope encompassed by the C-terminal seven residues. All peptides containing at least the C-terminal four residues were able to inhibit completely the binding of this monoclonal antibody to the parent peptide. Taken together, these results indicate that (i) the tetrapeptide is capable of eliciting specific antibody when coupled to a carrier, (ii) this tetrapeptide possesses all of the antigenic information necessary to occupy the paratope of a monoclonal antibody elicited by the longer parent peptide, and (iii) the decapeptide contains all of the information necessary to elicit a specific immune response and therefore carries an epitope recognized by T cells as well as one recognized by B cells.     

传染性法氏囊病病毒五个抗原表位短肽的鉴定与序列分析

以5株传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)单克隆抗体HNF1、HNF7、B34、2B1和2G8作为筛选分子,对噬菌体展示12肽库进行3轮"吸附-洗脱-扩增"淘洗,从每株单克隆抗体筛选到的噬菌斑中随机挑取12个单克隆蓝色噬菌斑,合计60个,用间接ELISA检测,A值大于1.00;用竞争抑制ELISA分析,单克隆抗体和IBDV抗原均能竞争抑制筛选12肽与固相包被单克隆抗体的反应,抑制率大于40%,表明在该12肽内含有IBDV抗原表位。选取35个单克隆噬菌斑,测定噬菌体gIII部分基因的核苷酸序列,确定了这5个含有不同IBDV抗原表位12肽的核苷酸和氨基酸序列。进一步将其与GenBank中IBDV基因组编码蛋白的氨基酸序列进行比较,发现2B1筛选肽有4个连续氨基酸残基Leu-Ala-Ser-Pro与IBDV基因组A片段编码多聚蛋白的第536-599氨基酸残基一致,推测2B1为线性表位;而HNF1、HNF7、B34和2G8筛选肽均没找到有3个以上连续氨基酸残基与IBDV蛋白序列相同之处,推测可能是构象依赖性表位。     

Variable Region Identical Immunoglobulins Differing in Isotype Express Different Paratopes

The finding that the antibody (Ab) constant (C) region can influence fine specificity suggests that isotype switching contributes to the generation of Ab diversity and idiotype restriction. Despite the centrality of this observation for diverse immunological effects such as vaccine responses, isotype-restricted antibody responses, and the origin of primary and secondary responses, the molecular mechanism(s) responsible for this phenomenon are not understood. In this study, we have taken a novel approach to the problem by probing the paratope with ¹⁵N label peptide mimetics followed by NMR spectroscopy and fluorescence emission spectroscopy. Specifically, we have explored the hypothesis that the C region imposes conformational constraints on the variable (V) region to affect paratope structure in a V region identical IgG₁, IgG_2a, IgG_2b, and IgG₃ mAbs. The results reveal isotype-related differences in fluorescence emission spectroscopy and temperature-related differences in binding and cleavage of a peptide mimetic. We conclude that the C region can modify the V region structure to alter the Ab paratope, thus providing an explanation for how isotype can affect Ab specificity.     

Neurotensin-like immunoreactivity in the pituitary and hypothalamus of bony fishes

Using an antiserum raised against synthetic neurotensin (NT), the distribution of immunoreactivity in the pituitary and hypothalamus has been examined by immunocytochemistry at light and electron microscope level in a number of species of bony fishes. In most species immunoreactive perikarya were found in the preoptic region of the hypothalamus, with fibres throughout the tuberal hypothalamus and neurohypophysis (neural lobe and median eminence). In the neurohypophysis of teleosts NT-like immunoreactivity was seen in a dense band of fibres bordering the ACTH cells of the rostral pars distalis: absorption controls showed that this was due to the presence of an NT(8-13)-like or xenopsin-like sequence, which, according to electron microscopic observations, was contained in small dense cored vesicles. The antiserum also stained the pituitary ACTH cells of some species, apparently due to cross-reaction with the 17-19 sequence of ACTH. These results suggest that an NT-like peptide may have a role in control of the adenohypophysis in fishes.