Production and characterisation of monoclonal antibodies against a very small hapten, 3-methylindole

Monoclonal antibodies were produced against a very small (131.2 Da) hapten, 3-methylindole. Nine derivatives of 3-methylindole were synthesised with spacers ending in a carboxyl group, and coupled to immunogenic carriers and europium chelate labels. Almost all the antigens elicited an antihapten response, but the majority of the mAbs produced strongly recognised the spacer group and did not bind free 3-methylindole. However, specific antibodies were obtained with five immunogens. Specificity could be directed against the pyrrole ring by locating the bridging group to the aromatic moiety of the indole ring system. Any modification in the position 3 of the indole ring strongly hindered mAb binding to the compound, and the cross-reactivity of physiologically important compounds, such as tryptophan and tryptamine, was negligible for all of the mAbs. The developed hapten structures successfully focused antibody recognition to the important sub-determinants in the indole ring system. Similar constructs could also be useful in the development of antibodies against other indolic compounds.     

Three epitope-specific monoclonal antibodies against the hapten penicillin

Three monoclonal antibodies (Pen 4, Pen 7 and Pen 9) were raised against the benzylpenicilloyl group and used to investigate the antigenicity of this group. The binding of Pen 4 to carrier-bound penicillin derivatives was shown in an ELISA to be dependent on the structure of the side chain in the derivative. Hence Pen 4 recognizes this side chain. From the difference in binding to carrier-bound penicillin derivatives in a competitive enzyme immunoassay it was concluded that Pen 7 mainly recognizes the new antigenic determinant which emerges from the binding of the penicillin derivative to a carrier. The binding of Pen 9 to carrier-bound penicillin derivatives was not influenced by the nature of the side chain. Neither was the bound or free nature of the derivative of influence on the binding. Therefore it is concluded that Pen 9 mainly recognizes the thiazolidine ring of penicillin. This study thus shows that in the benzylpenicilloyl group at least three epitopes can be recognized.     

Induction of antibodies against a peptide hapten does not require covalent linkage between the hapten and a class II presentable T helper peptide.

Following immunization with a complex antigen, a B cell internalizes this antigen through an interaction between its surface immunoglobulins and an epitope of the antigen. Enzymatic processing of the antigen frees one or more short peptide determinants (TD) which bind to class II molecules of the B cell. If the complex TD-MHC II is recognized by the receptor of a T helper cell, T cell help is provided leading to the expansion of an antibody-producing B cell clone specific for the epitope. We present experimental evidence proving that the induction of anti-peptide hapten antibodies does not require covalent linkage between the peptide hapten and the peptide behaving as TD. Indeed, high anti-peptide hapten antibody titers are induced if an emulsion of TD and hapten are injected in the same immunization site. This result suggests a way to manipulate antibody production with useful applications to research and therapy.     

Molecular requirements for hapten binding to antibodies against glutamate and aspartate.

Molecular requirements for hapten recognition by antibodies raised in rabbits against glutaraldehyde conjugates of L-glutamate and L-aspartate were determined in enzyme immunoassays by measuring the displacement of binding of glutamate and aspartate, respectively, by a large number of selected haptens to two anti-glutamate and two anti-aspartate sera. The results indicate that N-terminal modifications of the amino acids, such as the presence of an N-acetyl or N-carbamyl group or the addition of a second amino acid to form dipeptides with C-terminal glutamate or aspartate, are tolerated to variable degrees, more so by the aspartate than the glutamate antisera. The antibodies possess point-to-point recognition sites for the two carboxyl groups present in both amino acids. Strong shape complementarity between the amino acids and their respective binding sites is suggested by the lack of recognition of the appropriate D stereoisomers by any of the antibodies. Changes in the distance between the two carboxyl groups, or modification, replacement or loss of either or both carboxyl groups, strongly reduce or eliminate binding. Based on these results, we suggest that other antibodies raised to similar conjugates of these amino acids are likely to share similar recognition characteristics. In addition, the results provide a rational background for the evaluation of antibody specificity and the interpretation of results in immunocytochemical studies using antisera to glutamate and aspartate.     

Selection and expression of recombinant single domain antibodies from a hyper-immunized library against the hapten azoxystrobin

Three V(H)Hs against the model hapten, azoxystrobin (MW 403), were isolated from a hyper-immunized phage-displayed V(H)H library. This library was constructed by isolating the V(H)H-coding genes from the lymphocytes collected from a Llama glama that was immunized with azoxystrobin conjugated to bovine serum albumin (BSA). Six rounds of panning were performed against azoxystrobin conjugated to either ovalbumin (OVA) or rabbit serum albumin (RSA) to enrich clones containing V(H)Hs specific to the hapten. After screening 95 clones, three V(H)Hs (A27, A72, and A85) with different amino acid sequences were identified, expressed in soluble format in Escherichia coli HB2151, and purified using nickel-immobilized metal affinity chromatography. Competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) showed that A27 and A85 were specific to azoxystrobin while A72 was not. The IC(50) values of A27 and A85 V(H)Hs were 7.2 and 2.0μM, respectively. To our knowledge A85 is one of the highest affinity V(H)Hs that has yet been isolated against a hydrophobic hapten such as azoxystrobin.     

Improved functional immobilization of llama single-domain antibody fragments to polystyrene surfaces using small peptides

We studied the effect of different fusion domains on the functional immobilization of three llama single-domain antibody fragments (VHHs) after passive adsorption to polystyrene in enzyme-linked immunosorbent assays (ELISA). Three VHHs produced without any fusion domain were efficiently adsorbed to polystyrene, which, however, resulted in inefficient antigen binding. Functional VHH immobilization was improved by VHH fusion to a consecutive myc-His6-tag and was even more improved by fusion to the llama antibody long hinge region containing an additional His6-tag (LHc-His6). The partial dimerization of VHH-LHc-His6 fusion proteins through LHc-mediated disulfide-bond formation was not essential for their improved functional immobilization. VHH fusions to specific polystyrene binding peptides, hydrophobins, or other, unrelated VHH domains were less suitable for increasing functional VHH immobilization because of reduced microbial expression levels. Thus, VHH-LHc-His6 fusion proteins are most suited for functional passive adsorption in ELISA.     

Production and utilisation of antibodies directed against oestrone sulphate using specific hapten synthesis.

Oestrone-3-sulphate (E13S) is an important metabolite of oestrone. Studies in cattle had previously shown that it is synthesised in the gravid uterus by the foetus, but not by the corpus luteum. Progesterone measurement in milk by radioimmunoassay (RIA) is routinely carried out in some laboratories as a pregnancy test for cattle. The major drawback of progesterone measurement in milk, by RIA, as a pregnancy test was the failure to detect the lack of conceptus in those cows where early embryonic death had occurred but the corpus luteum still persisted thereby giving false positive results. We have developed a direct RIA for E13S by raising antibodies to an immunogen prepared from a specific hapten synthesised by an unambiguous chemical synthesis. The sensitivity of the RIA in milk was found to be 0.368 nmol/l. The levels of E13S in non-pregnant cows are undetectable but during a viable pregnancy, the levels are elevated to greater than 0.40 nmol/l by day 100. There was no cross-reactions in the assay with any free oestrogens. The measurement of this metabolite of oestrone promises to provide an accurate marker for the detection of a viable conceptus in cattle.     

Noncompetitive immunoenzymometric assays for a hapten molecule employing anti-idiotype antibodies

Noncompetitive immunoenzymometric assays for a hapten molecule employing anti idiotype antibodies (Ids) are introduced to enable sensitive determination of endogenous steroids in clinical specimens. Ids recognize an epitope in the variable region (idiotope) of anti-hapten antibody (primary antibody); alpha-type Id (Id-alpha) recognizes an idiotope in the framework region, while beta-type Id (Id-beta) recognizes an idiotope on the paratope. The target hapten was captured by an excess amount of primary antibody, and the unoccupied paratope was blocked with Id-beta. The hapten-primary antibody complex was selectively detected with biotin-labeled Id-alpha that does not bind to the antibody blocked by Id-beta because of steric hindrance arising by Id-beta. This method was applied to the determination of ursodeoxycholic acid 7-N-acetylglucosamindes, which are bile acid conjugates expected to be diagnostic markers for primary biliary cirrhosis. A similar assay procedure for 11-deoxycortisol (11-DC), a diagnostic index for pituitary-adrenal function, was also established where the hapten was captured with a biotin-labeled primary antibody and then the complex was captured by immobilized Id-alpha. These methods were approximately 10 times more sensitive than conventional competitive assays employing the same primary antibody. To achieve higher sensitivity, antibody-engineering techniques have been introduced to prepare fusion protein of the single-chain Fv fragment (scFv) specific for 11-DC and alkaline phosphatase (ALP). Employing fusion protein in combination with Id-alpha and Id-beta, recognizing the idiotope on scFv, a highly sensitive single-antibody immunoenzymometric assay for 11-DC was developed. The detection limit of this novel immunometric assay was approximately 500-times lower than a competitive radioimmunoassay based on the corresponding anti-11-DC antibody.     

有机膦半抗原诱导的抗神经性毒剂单克隆抗体的制备及鉴定

目的 :制备新化合物 1 羟基 1 对胺基苯基甲磷酸单频哪基醇酯 (P4 )的单克隆抗体 (mAb)。方法 :以P4为半抗原 ,与血蓝蛋白 (LPH)化学偶联制备人工抗原 (P4 LPH) ,并以其免疫BALB/c小鼠 ,制备抗P4的mAb。用竞争抑制酶免疫分析法 ,测定mAb对梭曼等 7种配体的结合活性 ,结果以mAb与P4 BSA的结合被抑制 5 0 %时的配体浓度 (IC50 ) ,表示mAb的交叉反应性。结果 :建立了 9株对P4 BSA阳性反应的mAb ,它们分属 3种Ig亚类 ;6株 (2B2、3A10、3D5、4F1、6C9和 6H4 )为IgG1,1株 (3B9)为IgG2b ,2株 (1B3和 6H5 )为IgM。 9株mAb中 ,8株具有与半抗原P4的结合的特异性。4株 (1B3、2B2、3D5和 4F1)具有梭曼结合活性 ,其IC50 值分别为 10 -3 、10 -3 、10 -5和 10 -6mol/L ;1株 (4F1)具有与对氧磷结合的特异性 ,其IC50 值为 10 -5mol/L。 4株腹水的mAb鼠滴度高 ,分别达 10 -6(1B3和 2B2 )和 10 -8(3D5和 4F1)。结论 :用新的半抗原P4成功地制得抗梭曼和抗对氧磷的mAb ,可用于梭曼抗体酶、梭曼的免疫抗毒和免疫检测研究     

Generation of genetic engineering monoclonal antibodies against prion protein

Two strains of Fab monoclonal antibodies (mAbs) against prion protein, designated as IV-66 and IV-78, were selected from the phage display libraries. The gene sequences encoding the light κ chain and heavy Fd chain of IV-78 were inserted into a baculovirus expression cassette vector for mouse IgG expression. Western blot, Dot-ELISA and immunoprecipitation confirmed that these Fab and IgG mAbs reacted well with the recombinant hamster and human PrP proteins expressed in prokaryotic and in mammalian cells and PrP^Sc from scrapie-infected hamsters. It demonstrates that mAbs against prion protein are successfully generated by phage-display technique.     

人源抗狂犬病毒基因工程单链抗体的研究

目的 研制人源抗狂犬病毒基因工程抗体.方法 从具有高滴度狂犬病毒抗体的疫苗接种者采集外周血淋巴细胞,通过pHAL14载体和高效噬菌体系统构建人源抗狂犬病毒单链基因工程抗体库,经纯化的狂犬病毒aG株富集筛选,通过ELISA和IFA鉴定阳性抗体克隆并进行序列测定.利用IgG表达载体VH/VK双质粒系统瞬时转染293T细胞实现IgG抗体的分泌型表达,通过亲和力测定和中和试验鉴定IgG抗体功能.结果 成功地获得了6株抗狂犬病毒糖蛋白的人源单克隆抗体,非竞争ELISA显示6株抗体具有较高的亲和力,体外中和试验证实这6株人源单抗对狂犬病毒CVS株无中和活性,对aG株具有较强的中和活性.结论 从免疫库中获得了6株人源单克隆抗体,为狂犬病毒的鉴别诊断和治疗奠定了基础.     

Generation of a panel of antibodies against proteins encoded on human chromosome 21

Background  

Down syndrome (DS) is caused by trisomy of all or part of chromosome 21. To further understanding of DS we are working with a mouse model, the Tc1 mouse, which carries most of human chromosome 21 in addition to the normal mouse chromosome complement. This mouse is a model for human DS and recapitulates many of the features of the human syndrome such as specific heart defects, and cerebellar neuronal loss. The Tc1 mouse is mosaic for the human chromosome such that not all cells in the model carry it. Thus to help our investigations we aimed to develop a method to identify cells that carry human chromosome 21 in the Tc1 mouse. To this end, we have generated a panel of antibodies raised against proteins encoded by genes on human chromosome 21 that are known to be expressed in the adult brain of Tc1 mice     

TNT detection using llama antibodies and a two-step competitive fluid array immunoassay

Llamas possess unique subclasses of antibodies that lack light chains, and thus are made by the pairing of two heavy chains. IgG was purified from two llamas which had been immunized with trinitrobenzene-keyhole limpet hemocyanin. Conventional IgG1 and heavy chain IgG2 and IgG3 subclasses were fractionated using affinity chromatography. The effectiveness of heavy chain antibodies for the detection of trinitrotoluene (TNT) using a competitive fluid array immunoassay was evaluated and compared to both the llama IgG1 as well as a murine monoclonal anti-TNT antibody. It was found that heavy chain antibody bound TNT with selectivity similar to conventional antibodies, yet the heavy chain antibodies possessed greater thermal stability. The titer of the heavy chain antibodies however was found to be 10-fold lower than the IgG1; thus analytical assays were best demonstrated using the llama IgG1 conventional antibody. The TNT competitive immunoassay on the Luminex fluid analyzer had a dynamic range from ∼ 100 ng/mL to 10 μg/mL. Utilizing the same two-step competitive assay format the dynamic range of the monoclonal antibody was found to have a broad range (1 ng/mL to 1 μg/mL). This method was demonstrated on TNT contaminated soil extracts using both the llama IgG1 and the mouse monoclonal validating the utility of method for analysis of field samples.     

Generation and characterization of monoclonal antibodies against Staphylococcus aureus thermonuclease

Monoclonal antibodies (MAbs) against Staphylococcus aureus thermonuclease (TN) were raised by immunizing BALB/c mice with a commercial TN preparation. Six monoclones were generated producing MAbs specific for S. aureus TN as tested in Western blots and ELISA. They all combined with a 17 kD and a 21 kD protein, respectively, both of which showed DNase activity. All MAbs were of IgG1 isotype with kappa light chain. Competition ELISA showed that five of the MAbs recognized a total of three different binding sites of TN, designated I, II and III, respectively. Only the anti-site II MAbs inhibited the DNase activity. A MAb-based sandwich ELISA showed a lower detection limit for TN of approximately 0.5 ng/ml protein. Only S. aureus strains (culture supernatants) showed positive ELISA (31 positive/31 tested), although other tested gram positive cocci produced thermostable nucleases. The MAbs have potentials as reagents for rapid and specific detection of S. aureus.     

Generation of rabbit antibodies against death ligands by cDNA immunization

Specificity problems, especially in immunoblot analysis, have been shown for several commercial antibodies raised against the death ligand Fas ligand (FasL) using conventional protein and/or peptide immunizations. In this work, we have optimized the development of rabbit antisera and isolated pAb against the death ligands FasL, Apo2 ligand/TRAIL and Apo3 ligand/TWEAK by cDNA intramuscular immunization. This alternative approach has generated specific pAb in all three cases, which are useful for immunoblot purposes. The present data suggest that for the production of antibodies against certain glycosylated membrane proteins, cDNA immunization could be the method of choice.     

Generation of polyclonal antibodies against recombinant human activin A.

A goat antiserum to purified recombinant human activin A (rhAct-A), a dimer formed by two beta A-subunits of inhibin, has been produced. The immunoreactivity of the antiserum has been evaluated in an antigen coated enzyme-linked immunosorbent assay, in a radioimmunoassay using iodinated rhAct-A, and by Western blot analysis. The antiserum demonstrated some cross reactivity to inhibin A, a structurally related heterodimer which contains an identical beta A-subunit coupled to a distinct, though similar, alpha subunit. A simple radioimmunoassay for rhAct-A in tissue culture supernatant has been developed with rhAct-A affinity column purified polyclonal antiserum. The assay is precise and sensitive with a range of 0.31-40 ng/ml. The cross reactivity of inhibin A in the RIA is about 4.3%. Despite its cross-reactivity this antiserum will facilitate studies of the physiology of activin A and inhibin A which includes a Western blot analysis where a molecular size distinction is accomplished.     

Generation and characterization of a panel of anti-phosphoprotein monoclonal antibodies directed against Mokola virus

The generation of a new panel of 21 monoclonal antibodies (MAbs) reactive with the P protein of Mokola virus (MOKV) is described. Through competitive ELISA and immunoblotting analyses, these MAbs were classified into several groups. Consistent with prior studies on lyssavirus P protein antigenic structure, many of the sites recognized by these Mabs appear to correspond to sites identified previously. Studies on the reactivity of these anti-MOKV P MAbs against a collection of lyssaviruses identified MAbs that were broadly cross-reactive to all genus members and others that bound selectively to members of different species. In particular the utility of this MAb panel for differentiation of African lyssaviruses was explored. Such a panel will be useful for further examination of the extent of functional complementation between lyssavirus P proteins.