Monoclonal antibodies for the structural analysis of the Na/H antiporter NhaA from Escherichia coli

Since their advent some 25 years ago, monoclonal antibodies have developed into powerful tools for structural and functional analysis of their cognate antigens. Together with the respective antigen binding fragments, antibodies offer exclusive capacities in detection, characterization, purification and functional assays for every given ligand.Antibody-fragment mediated crystallization represents a major advance in determining the three-dimensional structure of membrane-bound protein complexes. In this review, we focus on the methods used to generate monoclonal antibodies against the NhaA antiporter from Escherichia coli as a paradigm of secondary transporters. We describe examples on how antibodies are helpful in understanding structure and function relationships for this important class of integral membrane proteins.The generated conformation-specific antibody fragments are highly valuable reagents for co-crystallization attempts and structure determination of the antiporter.     

Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies

Antigen specific llama VHH antibody fragments were compared to antigen specific mouse monoclonal antibodies with respect to specificity, affinity and stability. The llama VHH antibody fragments and the mouse monoclonal antibodies investigated were shown to be highly specific for the protein antigen hCG or the hapten antigen RR-6. The affinity of the interaction between monovalent llama VHH antibody fragments and their antigen is close to the nanomolar range, similar to the bivalent mouse monoclonal antibodies studied. Llama VHH antibody fragments are similar to mouse monoclonal antibodies with respect to antigen binding in the presence of ammonium thiocyanate and ethanol. The results show that relative to antigen specific mouse monoclonal antibodies, antigen specific llama VHH fragments are extremely temperature stable. Two out of six llama VHHs are able to bind antigen specifically at temperatures as high as 90 degrees C, whereas four out of four mouse monoclonal antibodies are not functional at this temperature. Together with the finding that llama VHH fragments can be produced at high yield in Saccharomyces cerevisiae, these findings indicate that in the near future antigen specific llama VHH fragments can be used in for antibodies unexpected products and processes.     

The antigenic structure of the acetylcholine receptor from Torpedo californica

The immunological structure of the acetylcholine receptor (AChR) from the electric organ of Torpedo californica was studied using a large number of monoclonal antibodies which were initially selected for their abilities to bind to intact AChRs. The monoclonal antibodies were tested for their ability to bind to denatured AChR subunits labeled with 125I. Antibodies derived from rats immunized with individual denatured subunits or a mixture of subunits of Torpedo AChR reacted well in the assay. A much smaller proportion of antibodies derived from rats immunized with native Torpedo AChR or native AChR from Electrophorus electricus electric organ, bovine muscle, or human muscle reacted with denatured subunits of Torpedo AChR. Many monoclonal antibodies reacted with more than one subunit, but they always reacted best with the subunit used for immunization. Those monoclonal antibodies that bound to intact subunits were mapped more precisely by their ability to bind characteristic fragments of each subunit generated by proteolysis with Staphylococcal V8 protease. These fragments were analyzed by SDS polyacrylamide gel electrophoresis, and monoclonal antibodies that precipitated the same fragment pattern were placed in groups. By this method, we define a minimum of 28 determinants on Torpedo AChR.     

Characterization of monoclonal antibodies against ascorbate peroxidase isoenzymes: purification and epitope-mapping using immunoaffinity column chromatography

We have developed three monoclonal antibodies against spinach chloroplastic (chl-mAb3 and chl-mAb6) and cytosolic (cyt-mAb1) ascorbate peroxidase (APX) isoenzymes to analyze the cross-reactivity and the structure of the epitopes for each monoclonal antibody. All three antibodies reacted specifically with their respective isoenzymes, but none cross-reacted with the others. Immunoreactive fragments in proteolytic recombinant APX isoenzymes were detected by means of the absorption on the corresponding immunoaffinity column. The cyt-mAb1 reacted with a peptide fragment containing the distal His region obtained by the lysyl endopeptidase digestion. The chl-mAb6 was capable of binding to the fragment, D-I-K-E-K-R, which is consistent with an inherent region of chloroplastic isoenzymes. No fragments reacting to the chl-mAb3 could be found in this study, suggesting that the chl-mAb3 recognizes a conformationally constituted epitope of the chloroplastic APX molecule, which may be destroyed by the enzymatic cleavage. We concluded that the peptides identified as epitopes are characteristic evidence of monoclonal antibodies.     

Mapping the functional domains of the eukaryotic elongation factor 1 beta gamma

The functional domains of the eukaryotic elongation factor (EF) 1 beta gamma have been delineated with the use of limited proteolysis, protein microsequencing, gel electrophoresis under non-denaturing conditions and antibodies against EF-1 beta and EF-1 gamma. By means of limited proteolysis, it was possible to obtain large fragments of EF-1 beta. In contrast to amino-terminal fragments, those derived from the carboxy-terminal part of EF-1 beta were still active in enhancing the guanine nucleotide exchange of GDP bound to EF-1 alpha. With the same technique of limited proteolysis, it was possible to isolate a trypsin-resistant core from EF-1 beta gamma containing polypeptide chain fragments derived from both subunits. A polyvalent antiserum against EF-1 beta and two monoclonal antibodies against EF-1 gamma were used to identify the protein fragments in this core. The monoclonal antibodies were shown to recognize different epitopes, one localized on the amino-terminal and another on the carboxy-terminal half of EF-1 gamma. The antiserum against EF-1 beta and one of the monoclonal antibodies (mAb 36E5), which recognized the amino-terminal half of EF-1 gamma, reacted with this trypsin-resistant core. We conclude that the amino-terminal halves of both EF-1 beta and EF-1 gamma are firmly attached to each other, and that the carboxy-terminal part of EF-1 beta interacts with EF-1 alpha.     

Epitope mapping of the human immunodeficiency virus type 1 gp120 with monoclonal antibodies.

A soluble form of recombinant gp120 of human immunodeficiency virus type 1 was used as an immunogen for production of murine monoclonal antibodies. These monoclonal antibodies were characterized for their ability to block the interaction between gp120 and the acquired immunodeficiency syndrome virus receptor, CD4. Three of the monoclonal antibodies were found to inhibit this interaction, whereas the other antibodies were found to be ineffective at blocking binding. The gp120 epitopes which are recognized by these monoclonal antibodies were mapped by using a combination of Western blot (immunoblot) analysis of gp120 proteolytic fragments, immunoaffinity purification of fragments of gp120, and antibody screening of a random gp120 gene fragment expression library produced in the lambda gt11 expression system. Two monoclonal antibodies which blocked gp120-CD4 interaction were found to map to adjacent sites in the carboxy-terminal region of the glycoprotein, suggesting that this area is important in the interaction between gp120 and CD4. One nonblocking antibody was found to map to a position that was C terminal to this CD4 blocking region. Interestingly, the other nonblocking monoclonal antibodies were found to map either to a highly conserved region in the central part of the gp120 polypeptide or to a highly conserved region near the N terminus of the glycoprotein. N-terminal deletion mutants of the soluble envelope glycoprotein which lack these highly conserved domains but maintain the C-terminal CD4 interaction sites were unable to bind tightly to the CD4 receptor. These results suggest that although the N-terminal and central conserved domains of intact gp120 do not appear to be directly required for CD4 binding, they may contain information that allows other parts of the molecule to form the appropriate structure for CD4 interaction.     

Rationalization of gene regulation by a eukaryotic transcription factor: calculation of regulatory region occupancy from predicted binding affinities

The antigen-binding fragments (Fab) of antibodies are powerful tools in clinical therapy, molecular diagnostics and basic research. However, their principal applications require pure recombinant molecules in large amounts, which are challenging to obtain. Severe limitations in yield, folding and functionality are commonly encountered in bacterial production of Fab fragments. Secretion into the oxidizing periplasm generally results in low yield, whereas expression in the reducing cytoplasmic environment produces unfolded or non-functional protein. We hypothesized that an impaired reducing environment of the cytoplasm would permit correctly folded, functional cytoplasmic expression of Fabs with high yield. We used the Escherichia coli strain FA113, which has no activity of both thioredoxin and glutathione reductase, and thus has an oxidizing cytoplasmic environment. With the newly constructed vector pFAB1 we tested the cytoplasmic expression of two Fab fragments, which recognize the integral membrane protein NhaA, a bacterial Na(+)/H(+) antiporter. These antibodies differ in terms of DNA sequence and stability. Both antibody fragments were produced to very high yields (10-30 mg/l from bacterial cultures at an A(600 nm)=1.2-1.3). This is a factor 50-250 times higher than any other reported over-expression strategy for Fab fragments and currently represents the highest production rate ever been reported for antibody Fab fragments in bacteria grown to similar cell densities. The fragments are fully functional and can be efficiently purified by His-tag chromatography. Expression of active Fab fragments in the bacterial cytoplasm unlocks the possibility of using antibody specific targeting in an intracellular environment. Such a capacity opens new perspectives for investigating metabolic and regulatory pathways in vivo and also provides a powerful selection system for functional genomics.     

Type VI collagen. Studies on its localization, structure, and biosynthetic form with monoclonal antibodies

Monoclonal antibodies were prepared by immunization with whole tissue and were selected for their reactivity with extracellular matrices in tissue immunofluorescence. Two such antibodies were used to isolate the corresponding antigen from pepsin extracts of human placental tissue by immunochromatography. In each case, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the isolated material was composed of four polypeptides of Mr between 57,000 and 85,000 that were disulfide-bonded into a high molecular weight aggregate. Amino acid analyses showed that the isolated material was partly collagenous. The material was shown to be antigenically related to previously isolated peptic fragments of type VI collagen and it shared their unique structure as revealed by electron microscopy. Based on these findings, it was concluded that the isolated material was a form of type VI collagen. In immunofluorescence, the monoclonal antibodies localized type VI collagen throughout the connective tissue and in the extracellular matrix of cultured fibroblasts. Polypeptides presumably comprising the intact form of this collagen were isolated from cultures of metabolically radiolabeled fibroblast cell cultures using the two monoclonal antibodies. The isolated material consisted of two polypeptides of Mr 240,000 and 140,000 that were extensively disulfide cross-linked. Four additional monoclonal antibodies bound the same radioactive polypeptides from fibroblast cultures, but only one of them reacted with the fragments isolated from pepsin-digested placenta. Since all six antibodies were originally selected based on tissue immunofluorescence, and therefore react with the tissue form of the protein, the tissue form appears to be more similar to the polypeptides detected in fibroblast cultures than to the pepsin-resistant fragments. Since these monoclonal antibodies apparently recognize different parts of the molecule, they will be useful for further study of the structure and function of the intact form of type VI collagen.     

Functional characterization of two anti-estradiol antibodies as deduced from modelling and site-directed mutagenesis experiments

Monoclonal antibodies are now widely used to measure the concentration of steroid hormones in human serum samples. The great development of molecular engineering techniques over the past 10 years has made possible the improvement of specificity and/or sensitivity of selected antibodies. We have obtained two monoclonal antibodies, 17E12E5 and 10G6D6, using estradiol-6-ethyl methoxy carbonyl (EMC)-bovine serum albumin (BSA) as immunogen. To tentatively improve their affinities for natural estradiol, we have initiated their structural and functional studies. For this purpose, we have cloned and sequenced the genes encoding the variable fragments of each antibody. Single chain variable fragments (scFv) were produced into the periplasmic space of E. coli using the pLIP6 expression vector. Mapping of the functional structures of both antibodies was obtained by combination of modelling and mutational analyses together with cross-reaction studies. The two binding pockets are described and models of estradiol complexed to 17E12E5 and 10G6D6 are proposed.     

Identification of an immunodominant linear neutralization domain on the S2 portion of the murine coronavirus spike glycoprotein and evidence that it forms part of complex tridimensional structure.

Numerous studies have demonstrated that the spike glycoprotein of coronaviruses bears major determinants of pathogenesis. To elucidate the antigenic structure of the protein, a panel of monoclonal antibodies was studied by competitive ELISA, and their reactivities were assayed against fragments of the murine coronavirus murine hepatitis virus strain A59 S gene expressed in prokaryotic vectors. An immunodominant linear domain was localized within the predicted stalk, S2, of the peplomer. It is recognized by several neutralizing antibodies. Other domains were also identified near the proteolytic cleavage site, in the predicted globular head, S1, and in another part of the stalk. Furthermore, competition results suggest that the immunodominant functional domain forms part of a complex three-dimensional structure. Surprisingly, some antibodies which have no antiviral biological activities were shown to bind the immunodominant neutralization domain.     

Multivalency: the hallmark of antibodies used for optimization of tumor targeting by design

High-precision tumor targeting with conventional therapeutics is based on the concept of the ideal drug as a "magic bullet"; this became possible after techniques were developed for production of monoclonal antibodies (mAbs). Innovative DNA technologies have revolutionized this area and enhanced clinical efficiency of mAbs. The experience of applying small-size recombinant antibodies (monovalent binding fragments and their derivatives) to cancer targeting showed that even high-affinity monovalent interactions provide fast blood clearance but only modest retention time on the target antigen. Conversion of recombinant antibodies into multivalent format increases their functional affinity, decreases dissociation rates for cell-surface and optimizes biodistribution. In addition, it allows the creation of bispecific antibody molecules that can target two different antigens simultaneously and do not exist in nature. Different multimerization strategies used now in antibody engineering make it possible to optimize biodistribution and tumor targeting of recombinant antibody constructs for cancer diagnostics and therapy.     

Topological and epitope mapping of the cellular retinaldehyde-binding protein from retina.

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinol or 11-cis-retinaldehyde as endogenous ligands and may function as a substrate carrier protein that modulates interaction of these retinoids with visual cycle enzymes. As a first approach to identifying functional domains and protein recognition sites in CRALBP, a low resolution topological and epitope map has been developed using monoclonal and polyclonal antibodies and limited proteolysis. Fifteen peptides of 8-31 residues spanning 99% of the 316-residue bovine CRALBP were synthesized and used to prepare 13 anti-peptide polyclonal antibodies. Using a competitive ELISA procedure, peptide epitopes were classified as either accessible or inaccessible in the native protein based on the extent of their recognition by these site-specific antibodies. Use of the synthetic peptides to map the epitopes of a polyclonal antibody to intact CRALBP confirmed that the amino terminus and carboxyl terminus are immunodominate regions and hence likely to be exposed, at least in part. Limited tryptic proteolysis of native CRALBP produced three major fragments which were shown by microsequence and Western analysis to be derived from sequential loss of short peptides from the amino terminus. None of these major fragments reacted with four monoclonal antibodies (mAbs) to intact CRALBP although each mAb immunoprecipitated native CRALBP. These results and the lack of mAb recognition of any of the synthetic peptides indicates that the amino terminus of the protein is exposed and contains part of an assembly epitope recognized by the mAbs. Overall this study indicates that residues 1-30, 100-124, and 257-285 contain highly exposed segments in the native protein and therefore constitute potential interaction domains for CRALBP and visual cycle enzymes. Residues 30-99 and 176-229 are inaccessible in the native structure and may be involved with retinoid binding. These results provide a basis for a systematic higher resolution mutagenesis study directed toward correlating CRALBP structural domains with function.     

Tenascin-C is expressed by human glioma in vivo and shows a strong association with tumor blood vessels

The extracellular matrix (ECM) protein tenascin-C (TN-C) is upregulated within glioma tissues and cultured glioma cell lines. TN-C possesses a multi-modular structure and a variety of functional properties have been reported for its domains. We describe five novel monoclonal antibodies identifying different domains of TN-C. The epitopes for these antibodies were investigated by using recombinantly expressed fibronectin type III domains of TN-C. The biological effects of TN-C fragments on glioma cell proliferation and adhesion were analyzed. The expression pattern of TN-C in human glioma tissue sections and in glioma cell lines was studied with the novel library of monoclonal antibodies. The immunocytochemical analyses of the established human glioma cell lines U-251-MG, U-373-MG and U-87-MG revealed distinct staining patterns for each antibody. Robust expression of TN-C was found within the tumor mass of surgery specimens from glioblastoma. In many cases, the expression of this ECM molecule was clearly associated with blood vessels, particularly with microvessels. Three of the new antibodies highlighted individual TN-C-expressing single cells in glioma tissues. The effect of TN-C domains on glioma cells was examined by a BrdU-proliferation assay and an adhesion assay. Short fragments of constitutively expressed TN-C-domains did not exert significant effects on the proliferation of glioma cells, whereas the intact molecule increased cell division rates. In contrast, the long fragment TNfnALL containing all of the FNIII domains of TN-C decreased proliferation. Additionally, we found strong differences between the adhesion-influencing properties of the recombinant fragments on glioma cells.     

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

Abstract. A panel of 10 mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFA) has been isolated using porcine GFA as antigen. Although all antibodies recognize GFA purified from porcine spinal cord in the western blot technique, they can be subdivided into at least three groups on the basis of their reactivity against defined fragments of the molecule. Immunofluorescence staining patterns with the monoclonal antibodies performed on tissues and cell lines resemble those reported with conventional polyclonal antibodies directed against GFA. In particular astrocytes and Bergmann glia are strongly stained. In addition mouse monoclonal antibodies specific for either the 200 kd, or the 160 kd, or the 68 kd neurofilament triplet protein have been isolated and characterized. These antibodies are specific for neuronal cells and support conclusions made with similar antigen affinity-purified polyclonal antibodies. The combined set of monoclonal antibodies seems a valuable tool to characterize the different cell types of the nervous system.     

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

A panel of 10 mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFA) has been isolated using porcine GFA as antigen. Although all antibodies recognize GFA purified from porcine spinal cord in the western blot technique, they can be subdivided into at least three groups on the basis of their reactivity against defined fragments of the molecule. Immunofluorescence staining patterns with the monoclonal antibodies performed on tissues and cell lines resemble those reported with conventional polyclonal antibodies directed against GFA. In particular astrocytes and Bergmann glia are strongly stained. In addition mouse monoclonal antibodies specific for either the 200 kd, or the 160 kd, or the 68 kd neurofilament triplet protein have been isolated and characterized. These antibodies are specific for neuronal cells and support conclusions made with similar antigen affinity-purified polyclonal antibodies. The combined set of monoclonal antibodies seems a valuable tool to characterize the different cell types of the nervous system.     

Purification of Monoclonal Antibodies Using High Performance Liquid Chromatography (HPLC)

Recent increases in the ability to detect low levels of immunofluorescence have shown the need for highly purified primary immunoreagents. There are now reports of purification of monoclonal antibodies using HPLC with reverse phase columns. In this study we have utilized standard size exclusion HPLC to purify both biotinylated and non-biotinylated monoclonal antibodies from hybridoma culture supernatants. Results indicated that both biotinylated and non-biotinylated monoclonal antibodies retained their antigen binding capacity after purification, and were not different in this capacity from commercially available, affinity purified reagents. These findings indicate that size exclusion HPLC may be used in the purification of biologically active monoclonal antibodies, and suggest that this technique may be used in the large scale production of antibodies and their fragments, in antibody purification from ascites fluid, and in antisera quality control.     

Purification of monoclonal antibodies using high performance liquid chromatography (HPLC)

Recent increases in the ability to detect low levels of immunofluorescence have shown the need for highly purified primary immunoreagents. There are now reports of purification of monoclonal antibodies using HPLC with reverse phase columns. In this study we have utilized standard size exclusion HPLC to purify both biotinylated and non-biotinylated monoclonal antibodies from hybridoma culture supernatants. Results indicated that both biotinylated and non-biotinylated monoclonal antibodies retained their antigen binding capacity after purification, and were not different in this capacity from commercially available, affinity purified reagents. These findings indicate that size exclusion HPLC may be used in the purification of biologically active monoclonal antibodies, and suggest that this technique may be used in the large scale production of antibodies and their fragments, in antibody purification from ascites fluid, and in antisera quality control.     

Regional specificities of monoclonal anti-human apolipoprotein B antibodies

The usefulness of monoclonal antibodies as probes of protein structure is directly related to knowledge of the structures and locations of the epitopes with which they interact. In this report we provide a detailed map of 13 epitopes on apoB-100 defined by our anti-apoB monoclonal antibodies based on current information on the amino acid sequence of apoB-100. To localize antibody specificities to smaller regions along the linear sequence of the apoB-100 molecule we used a) thrombin- and kallikrein-generated fragments of apoB-100; b) beta-galactosidase- apoB fusion proteins; c) heparin; and d) antibody versus antibody competition experiments. Most of the monoclonal antibodies elicited by immunization with LDL were directed towards epitopes within the first 1279 amino terminal (T4/K2 fragments) or last 1292 carboxyl terminal amino acid residues (T2/K4 fragments) of apoB-100. One epitope localized to the mid-portion of apoB-100 was elicited by immunization with VLDL (D7.2). Saturating amounts of heparin bound to LDL did not inhibit the binding of any of the monoclonal antibodies to their respective epitopes on apoB-100, indicating that none of the antibody determinants is situated close to any of the reported heparin binding sites on LDL apoB. We examined the expression of apoB epitopes on VLDL subfractions and LDL isolated from a normolipidemic donor. The apparent affinities with which the antibodies interacted with their respective epitopes on the VLDL subfractions and LDL uniformly increased as follows: LDL greater than VLDL3 greater than VLDL2 greater than VLDL1, suggesting that each of the major regions of apoB-100 is progressively more exposed as normal VLDL particles become smaller in size and epitopes are most exposed in LDL. Previous experiments utilizing hypertriglyceridemic VLDL subfractions yielded similar results, but the rank order of VLDL subfractions and LDL was not the same for all antibodies tested. Thus, differences in apoB epitope expression on VLDL particles of differing sizes is a general phenomenon, but the expression of apoB epitopes in hypertriglyceridemic VLDL appears to be more heterogeneous than is the case for VLDL from normolipidemic donors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Surface plasmon resonance detection of interactions between peptide fragments of N-telopeptide and its monoclonal antibodies.

As populations age, osteoporosis is becoming an important public health care problem. Urinary level of the cross-linked N-telopeptide of type I collagen has been reported to be a sensitive marker of bone resorption. Recently, we synthesized and characterized 10 overlapping peptides covering the N-telopeptide of alpha-2 type I collagen and reported their relative binding response to anti-type I collagen cross-linked N-telopeptide (NTX) antibodies determined by a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). In this study, we design an assay based on the surface plasmon resonance (SPR) technology to detect binding interaction of each peptide fragment of NTX with the anti-NTX monoclonal antibodies. Anti-NTX monoclonal antibodies were immobilized on the surface of sensor chip by amine-coupling procedure. Serial dilutions of each peptide were prepared and injected separately onto the antibodies-immobilized sensor chip. The real-time association and dissociation interactions of each peptide were detected and reported as sensorgrams. Binding response of each peptide to the monoclonal antibodies was determined, and the SPR results were compared with the ELISA results. We demonstrate that the trends of binding potency of peptide fragments detected by SPR are in good correlation to the results obtained by ELISA, indicating that our developed SPR-based method can be further applied to detect the NTX fragments in urine and to monitor the bone loss in humans. The potent peptide fragments identified by both assays are promising for further preparation of specific monoclonal antibodies in order to develop bioassays for bone loss in humans.     

Localization of antigenic determinants using monoclonal antibodies and alpha-NH2-terminal labeling of polypeptide chains

A method for localization of antigenic determinants in a polypeptide chain of unknown primary structure was proposed. A protein is modified at NH2-terminal and epsilon-NH2-groups of lysine residues with maleic anhydride and then is subjected to partial enzymatic cleavage. Newly formed NH2-terminal groups are tagged with radioiodinated Bolton--Hunter's reagent. The labeled fragments of the antigen are then demaleylated. Comparison of the two longest labeled fragments, only one of which still binds monoclonal antibody, makes it possible to define the location of the antigenic determinant along the polypeptide chain. The method was tested on the bovine tryptophanyl-tRNA synthetase using earlier prepared monoclonal antibodies against this enzyme.