Metalloantibodies

A metalloantibody has been constructed with a coordination site for metals in the antigen binding pocket. The Zn(II) binding site from carbonic anhydrase B was used as a model. Three histidine residues have been placed in the light chain complementarity determining regions of a single chain antibody molecule. In contrast to the native protein, the mutant displayed metal-dependent fluorescence-quenching behavior. This response was interpreted as evidence for metal binding in the three-histidine site with relative affinities in the order Cu(II) greater than Zn(II) greater than Cd(II). The presence of metal cofactors in immunoglobulins should facilitate antibody catalysis of redox and hydrolytic reactions.     

Accelerated electron transfer between metal complexes mediated by DNA

DNA-mediated long-range electron transfer from photoexcited 1,10-phenanthroline complexes of ruthenium, Ru(phen)2(3)+, to isostructural complexes of cobalt(III), rhodium(III), and chromium(III) bound along the helical strand. The efficiency of transfer depended upon binding mode and driving force. For a given donor-acceptor pair, surface-bound complexes showed greater rate enhancements than those that were intercalatively bound. Even in rigid glycerol at 253 K, the rates for donor-acceptor pairs bound to DNA remained enhanced. For the series of acceptors, the greatest enhancement in electron-transfer rate was found with chromium, the acceptor of intermediate driving force. The DNA polymer appears to provide an efficient intervening medium to couple donor and acceptor metal complexes for electron transfer.     

Structure, antigenic determinants of some clinically important insect allergens: chironomid hemoglobins

Determination of the molecular structure and properties of allergens that elicit severe immediate-type hypersensitivity diseases in humans and a knowledge of the structure of their antibody-binding sites should provide new insight into the pathogenetic mechanisms of allergic diseases. Monomeric and homodimeric hemoglobins (CTT I to X) have been identified as potent allergenic components of Chironomidae, a family of Diptera. Immunologic investigations of peptides of three of these hemoglobins (CTT IV, CTT VI, and CTT VIII) showed that human antibodies of the E and G classes recognize at least two different sites within each molecule. Individual hemoglobin peptides were aligned with homologous regions of chironomid hemoglobin CTT III, whose tertiary structure has been determined by x-ray analysis at a resolution of 1.4 angstroms. The antigenic site CTT IV(91 to 101) showed the following characteristics: (i) seven polar or hydroxylated amino acids, from a total of eleven, occupying predominantly superficial regions; (ii) the property of linkage to other molecules by hydrogen bonds or solvent clusters; and (iii) high thermal mobility factors. In contrast, peptide CTT IV(102 to 108), which does not bind human antibodies, contained no polar amino acids and had low thermal mobility factors. These results support the idea that the antigenicity of clinically relevant proteins is related to regions with a predominance of polar amino acids and with low energy barriers between different conformations, which allow high flexibility, including site-specific adaptation in antibody binding.     

Inhibition of self-binding antibodies (autobodies) by a VH-derived peptide

The self-binding properties of a dominant idiotypic antibody (T15) and a minor idiotypic antibody (M603), both specific for phosphorylcholine, were examined as models of self-binding antibodies (autobodies). Observed differences in the self-binding affinity of T15 and M603 relate to variable sequence differences in their respective heavy and light chains. A molecular recognition theory based on the translation of coding and noncoding DNA strands was used to identify complementary amino acid sequences responsible for self-binding. The second hypervariable region of the heavy chain domain, extending into the third framework region, was predicted as the primary self-binding locus. Among peptides synthesized with different variable heavy and light chain regions, a 24-residue peptide spanning the second hypervariable and third framework regions of the heavy chain of T15 was nearly as effective as phosphorycholine in inhibiting the self-binding complexes.     

Identification of an intracellular peptide segment involved in sodium channel inactivation

Antibodies directed against a conserved intracellular segment of the sodium channel alpha subunit slow the inactivation of sodium channels in rat muscle cells. Of four site-directed antibodies tested, only antibodies against the short intracellular segment between homologous transmembrane domains III and IV slowed inactivation, and their effects were blocked by the corresponding peptide antigen. No effects on the voltage dependence of sodium channel activation or of steady-state inactivation were observed, but the rate of onset of the antibody effect and the extent of slowing of inactivation were voltage-dependent. Antibody binding was more rapid at negative potentials, at which sodium channels are not inactivated; antibody-induced slowing of inactivation was greater during depolarizations to more positive membrane potentials. The peptide segment recognized by this antibody appears to participate directly in rapid sodium channel inactivation during large depolarizations and to undergo a conformational change that reduces its accessibility to antibodies as the channel inactivates.     

Supramolecular chemistry: receptors, catalysts, and carriers

Supramolecular chemistry is the study of the structures and functions of the supermolecules that result from binding substrates to molecular receptors. Macropolycyclic receptors and coreceptors have been designed that form cryptate inclusion complexes and display molecular recognition towards spherical, tetrahedral, and linear substrates of various kinds (metal cations, inorganic anions, and organic or biological cations or anions). Anion binding has led to the development of anion coordination chemistry. Metalloreceptors simultaneously bind organic molecules and metal ions; speleands combine polar and nonpolar binding subunits. Receptors bearing reactive functional groups may act as molecular reagents or catalysts, performing a chemical transformation on the bound substrates (by such reactions as hydrogen transfer, ester cleavage, and protoadenosinetriphosphatase and protokinase activities). Receptors fitted with lipophilic groups can operate as molecular carriers, translocating bound species through a membrane; this transport can be coupled to chemical potentials (proton and redox gradients).     

中草药与金霉素对生长猪的影响及其机理初探

75～80日龄体重相近仔猪150头,随机均分3组,每组5个重复,每个重复10只样本,研究中草药饲料添加剂与金霉素影响生长猪生产性能的免疫内分泌机制。以中草药添加剂（1％）和盒霉素（25mg／kg）分别添加饲喂组Ⅰ,Ⅱ猪群,组Ⅲ为对照,各组仔猪注射猪瘟疫苗2头份／头。分别于开始用药当天（d0）、35d（d35）时逐一称重,并各组前腔静脉采血10头,统计各组猪群生产性能,测定血清猪瘟抗体水平和胰岛素样生长因子-Ⅰ（IGF—Ⅰ）、胰岛素（Insulin）和三碘甲腺原氨酸（T3）和甲状腺素（T4）等与生长有关的内分泌激素。结果显示中草药组仔猪在净增重、日增重和增重率及料肉比方面都显著优于金霉素组和对照组猪群,而后2组间差异不显著。试验结束时,中药组猪群血清猪瘟抗体水平、IGF—Ⅰ、Insulin和T4水平显著高于金霉素组和对照组仔猪。表明该中草药添加刑可通过增强仔猪免疫功能．并显著提高仔猪血清IGF—Ⅰ和胰岛素的水平、减缓T4的下降,从而提高仔猪的生产性能,表现出1种整体调节效应。     

FIXATION OF COMPLEMENT TO FRAGMENTS OF ANTIBODY

A specific precipitate of ovalbumin and its rabbit-serum antibody, after fixing human serum complement, was digested with papain. The digest was analyzed immunochemically for complexes of antigen, antibody fragments, and components of complement. The results indicated that complement is not bound to Porter fragment III, but very likely is bound to fragments I and II.     

Human CD4 binds immunoglobulins

T cell glycoprotein CD4 binds to class II major histocompatibility molecules and to the human immunodeficiency virus (HIV) envelope protein gp120. Recombinant CD4 (rCD4) bound to polyclonal immunoglobulin (Ig) and 39 of 50 (78%) human myeloma proteins. This binding depended on the Fab and not the Fc portion of Ig and was independent of the light chain. Soluble rCD4, HIV gp120, and sulfated dextrans inhibited the CD4-Ig interaction. With the use of a panel of synthetic peptides, the region critical for binding to Ig was localized to amino acids 21 to 38 of the first extracellular domain of CD4. CD4-bound antibody (Ab) complexed with antigen approximately 100 times better than Ab alone. This activity may contribute to the Ab-mediated enhancement of cellular HIV interaction that appears to depend on a trimolecular complex of HIV, antibodies to gp120, and CD4.     

Angiotensin II- and angiotensin 3-induced aldosterone release vivo in the rat

The potential role of angiotensin II and its heptapeptide metabolite, des-aspartyl-angiotensin II, was studied in the conscious unanesthetized rat. Aldosterone release was induced by both peptides at physiologic doses (0.72 nanogram per minute). [I-Sarcosyl-8-alanyl]-angiotensin II (P-113 inhibited angiotensin II more effectively than des-aspartyl-angiotensin II (101 percent as compared to 82 percent). These results indicate that angiotensin controls aldosterone release in the rat and that des-aspartyl-angiotensin II (that is, angiotensin III) may be important in this sequence.     

日本血吸虫合成表位肽疫苗对小鼠的保护性免疫

为观察日本血吸虫合成表位多肽疫苗诱导的保护性免疫,将用日本血吸虫抗原免疫血清筛选噬菌体随机12肽库得到的、具有较好免疫保护性的4个模拟抗原表位短肽进行人工合成,用酶联免疫吸附实验(ELISA)检测它们的抗原性.将合成多肽分别与钥孔戚血蓝蛋白(KLH)连接后免疫昆明小鼠,第3次免疫后收集血清,检测其针对各个多肽和可溶性成虫抗原(AWA)的IgG抗体滴度,以及补体介导的小鼠体外杀日本血吸虫童虫作用.结果显示,4个合成多肽均能被相应的抗体识别,具有良好的抗原性;能诱导产生特异性IgG抗体.这些抗体在补体参与下能在体外有效毒杀血吸虫童虫.与正常小鼠血清比较,4个合成多肽免疫血清的杀童虫率分别为31.7%,41.3%,21.1%和17.3%,均具有显著性;与KLH免疫血清相比时,杀童虫率分别为23.7%,34.4%,11.8%(P=0.077,无显著性)和7.5%(P=0.102,无显著性).这些结果表明,这4个合成表位多肽具有良好的抗原性和免疫原性,与KLH连接后能诱导明显的抗体反应和显著的细胞毒作用.     

Autologous peptides constitutively occupy the antigen binding site on Ia

Low molecular weight material associated with affinity-purified class II major histocompatibility complex (MHC) molecules of mouse (Ia) had the expected properties of peptides bound to the antigen binding site of Ia. Thus, the low molecular weight material derived from the I-Ad isotype was efficient in inhibiting the binding of 125I-labeled I-Ad-specific peptide to I-Ad, but did not significantly inhibit the binding of an I-Ed-specific peptide to I-Ed; the reciprocal isotype-specific inhibition was demonstrated with low molecular weight material derived from I-Ed. The inhibitory material was predominantly peptide in nature, as shown by its susceptibility to protease digestion. It was heterogeneous as measured by gel filtration (mean molecular weight approximately 3000), and when characterized by high-performance liquid chromatography, it eluted over a wide concentration of solvent. Such self peptide-MHC complexes may have broad significance in the biology of T cell responses, including generation of the T cell repertoire, the specificity of mixed lymphocyte responses, and the immune surveillance of self and nonself antigens in peripheral lymphoid tissues.     

Inhibition of antibodies to nuclear antigen and to DNA in New Zealand mice infected with lactate dehydrogenase virus

New Zealand mice developed antibodies to nuclear antigen leading to immune-complex nephritis. Humoral antibody was directed primarily against denatured DNA, although antibody to native DNA was also found. Persistent infection with lactate dehydrogenase virus significantly lowered antibodies both to nuclear antigen and to DNA in these mice. In addition, female (NZB x W)F(1) mice infected with lactate dehydrogenase virus were protected from the usual nephritic death occurring after the trapping of complexes of nuclear antigen and its antibody and of DNA and its antibody in the glomerular filter.     

Antibody-mediated bacteriorhodopsin orientation for molecular device architectures

A rational method for constructing highly oriented films of purple membrane (PM) has been established by using two kinds of bispecific antibodies with different antigen-binding sites, one binding to a specific side of bacteriorhodopsin and the other to a phospholipid hapten. A hapten monolayer deposited on a metal electrode was treated with a bispecific antibody solution and incubated with a PM suspension to produce a highly oriented PM film, as confirmed by electron microscopy in which an immunogold labeling technique was used. This antibody-mediated PM monolayer was then used in the construction of a light-sensing photoelectric device. A comparison of the two incorporated PM monolayers showed that highly efficient photocurrents were produced by the PM monolayer whose unidirectionally oriented cytoplasmic surface faces the electrode.     

Location and chemical synthesis of a binding site for HIV-1 on the CD4 protein

The human immunodeficiency virus type 1 (HIV-1) uses the CD4 protein as a receptor for infection of susceptible cells. A candidate structure for the HIV-1 binding site on the CD4 protein was identified by epitope mapping with a family of eight functionally distinct CD4-specific monoclonal antibodies in conjunction with a panel of large CD4-derived synthetic peptides. All of the seven epitopes that were located reside within two immunoglobulin-like disulfide loops situated between residues 1 and 168 of the CD4 protein. The CD4-specific monoclonal antibody OKT4A, a potent inhibitor of HIV-1 binding, recognized a site between residues 32 and 47 on the CD4 protein. By analogy to other members of the immunoglobulin superfamily of proteins, this particular region has been predicted to exist as a protruding loop. A synthetic analog of this loop (residues 25 to 58) showed a concentration-dependent inhibition of HIV-1-induced cell fusion. It is proposed that a loop extending from residues 37 to 53 of the CD4 protein is a binding site for the AIDS virus.     

新型鸭呼肠孤病毒卵黄抗体被动免疫持续期试验

为了确定鸭新型鸭呼肠孤病毒卵黄抗体被动免疫持续时间,试验取3批哈药集团生物疫苗有限公司试制的新型鸭呼肠孤病毒卵黄抗体,按1.0 mL·只^-1颈部皮下接种1日龄易感雏鸭,分别在注射抗体后1、3、5、7 d攻毒(抗体试验组),分析攻毒保护情况,剖检试验雏鸭,观察病理变化,同时设攻毒对照组(不接种卵黄抗体但攻毒)和健康对照组(不接种卵黄抗体也不攻毒)。结果显示:雏鸭接种卵黄抗体后1~5 d,卵黄抗体对雏鸭的攻毒保护率均在90%以上;注射后第7天的攻毒保护率为53.3%;攻毒对照组雏鸭均90%~100%发病。剖检发病雏鸭,可见肝脏和脾脏均呈典型的坏死及出血性病理变化;抗体试验组存活雏鸭剖检无病理变化;健康对照组雏鸭均100%健活,剖检无病理变化。说明注射卵黄抗体5 d内能对机体产生良好的保护作用,而7 d后保护作用大幅减弱,确定新型鸭呼肠孤病毒卵黄抗体的被动免疫持续期为5 d。     

天然鼠源噬菌体抗体库的构建及抗羊抑制素基因工程单抗的筛选

 【目的】构建单链抗体（ScFv）噬菌体表面展示文库,从中筛选抗羊抑制素单抗并进行表达,建立制备羊抑制素单抗的新方法。【方法】用未经免疫的多种小鼠脾细胞作为基因来源,采用噬菌体抗体库技术,构建天然噬菌体表面抗体文库。用羊抑制素对其进行3轮吸附-洗脱-富集,筛选抗羊抑制素单抗,用ELISA检测其抗原结合活性。【结果】构建了天然鼠源噬菌体抗体库,为筛选和制备各种单链抗体提供了一个平台。用羊抑制素对其进行筛选,制备抗羊抑制素单抗,经ELISA检测,55/96克隆具有与羊抑制素结合活性,阳性率为57%。【结论】制备的羊抑制素可溶性ScFv及其表面展示噬菌体抗体有很好的抗原结合活性,为羊抑制素单抗的制备提供了一种新的方法。     

M13噬菌体肽库扩增及兔抗血清制备

为制备M13噬菌体多克隆抗体,将噬菌体十二肽原始文库进行大量扩增,作为免疫原制备兔抗M13的多克隆抗体血清并进行间接ELISA鉴定。结果表明,该多抗效价达1 1 048 576,说明此多抗可与扩增噬菌体文库发生很好的抗原抗体反应。将制备的噬菌体M13多克隆抗体与商业化M13抗体水平比较,制备多抗与商业化M13抗体效果相当。本研究成功制备兔抗M13噬菌体多克隆抗体,为深入研究噬菌体展示技术提供了材料。     

Immunogenicity of synthetic peptides from circumsporozoite protein of Plasmodium falciparum

In a study of recombinant proteins that might be useful in developing a vaccine against malaria, synthetic peptides from the circumsporozoite (CS) protein of Plasmodium falciparum were found to be immunogenic for mice and rabbits. Antibody to peptides from the repeating region of the CS protein recognized native CS protein and blocked sporozoite invasion of human hepatoma cells in vitro. Antibodies to peptides from regions I and II had no biologic activity, although antibody to region I recognized processed CS protein by Western blot analysis. These data support the feasibility of developing a vaccine against the sporozoite stage of the malaria parasite by using synthetic peptides of the repeating region of the CS protein conjugated to a carrier protein.