BACKGROUND
because of their unique ability to recognize target antigens with extremely high fidelity, antibodies remain an essential tool in fundamental biomedical research and in clinical diagnostic testing.
in addition, they represent one of the most promising novel therapeutic options, in particular in the field of cancer treatment.
worldwide, more than a thousand clinical trials have been completed and almost as many are currently underway using monoclonal antibodies as pharmaceuticals http://www.clinicaltrials.gov.
traditionally, monoclonal antibodies have been produced from hybridoma cell lines in a laborious process involving animal experimentation and screening large numbers of clones.
more recently, recombinant monoclonal antibodies, which add an additional level of flexibility, have also become a viable treatment option for various human diseases  <cit> .
several alternatives to the classic in vivo selection of antibodies have been developed and refined .
one of the most prominent technique is antibody phage display, which allows in vitro selection of single-chain variable fragment  antibodies from complex phage or phagemid libraries  <cit> .
in this approach, which has been utilized successfully in many laboratories, animal work is obviated entirely .
it has been shown that scfv can even be manufactured for clinical use  <cit> .
in our own research laboratory, highly functional scfv antibodies that recognize a broad range of targets, including evolutionarily conserved proteins have been selected and characterized  <cit> .
regardless of the type of production or ultimate application, the effective use of antibodies depends on a detailed understanding of the underlying antibody-antigen interaction, i.e.
knowledge about a given antibody's epitope.
this holds true especially for clinical applications where such information would be of utmost importance to accurately predict wanted and unwanted  biological effects.
however, target characterization of both traditional and recombinant antibodies has been challenging.
the gold standard of such epitope mapping - analysis of the crystal structure of a given antigen-antibody complex - is labor intensive and sometimes nearly impossible due to technical difficulties.
thus, often cumbersome strategies have to be developed, like systematic target protein mutagenesis or sub-fragment analysis.
examples of successful characterization of antibody targets through scanning mutagenesis and other techniques have been described elsewhere  <cit> .
more recently, combinatorial peptide phage display libraries have also been used  <cit> .
in the future, computer based or in silico combinatorial methods for epitope mapping, like mapitope  <cit>  might prove useful.
finally, epitope mapping has also become a powerful strategy in the process of developing new vaccines  <cit> .
here we describe an alternative approach to characterize the interaction between antibodies and their respective targets.
time- and resource-demanding crystallography or mutagenesis experiments are circumvented.
instead the yeast two-hybrid  technology is employed.
in most two-hybrid applications in the recombinant antibody field, scfv are selected from a prey library  <cit> , here, the system is inverted: previously characterized scfv are used as bait and screened against a comprehensive cdna library.
this new approach yielded novel, detailed data about the targets of several recombinant antibodies, including the need for tertiary antigen structure for binding of conformational sensors, mapping of precise epitopes and unveiling of secondary targets.
RESULTS
antibody targets are characterized using the two-hybrid system with the scfv antibody as bait, a validated scoring system to remove non-specific binders, and determination of selected interacting domains
investigators studying and selecting recombinant antibodies have mostly used the y <dig> h technology to select antibody populations that fold correctly under the reducing conditions of the cytosol  <cit> .
in such experiments, the epitope of a given antigen is defined and used as a bait to sample a diverse scfv prey library.
here, we reversed the system, using as baits known scfv antibodies that had been previously selected in phage display screens, and, as preys, proteins expressed from high-complexity libraries of randomly primed cdna .
individual scfv antibodies were exposed to a cdna library with full coverage, derived from either human placenta or from drosophila.
more than  <dig> million potential interactions were tested during each screen.
prey fragments of positive clones were identified using sequence analysis and comparison with genbank databases using the blast algorithm  <cit> .
next, a predicted biological score  was computed for each clone as described before  <cit> , enabling classification according to interaction reliability .
this permitted ranking of the clones into categories from "a" to "d" in decreasing probability of having a specific interaction with the scfv-bait.
two additional categories were designated: "e", interacting clones, which were likely retained due to highly connected prey domains; and "f", clones that were experimentally proven y <dig> h artifacts.
in a third step, overlapping prey fragments originating from the same gene were designated as clusters and their translated amino acid sequences were aligned and superimposed onto the open reading frame .
overlapping regions shared by all fragments were designated as "selected interacting domain" , as described before  <cit> .
for the conformation-specific antibodies aa <dig> and rof <dig>  only large fragments are obtained, suggesting a non-linear antibody binding-site
in the first screen, the scfv aa <dig> was used as bait.
aa <dig> is a well-described conformation-specific antibody that exclusively binds the activated  form of the small gtpase rab <dig>  <cit> .
from the human cdna prey library only three specific binders were retained by y <dig> h .
two  encoded for rab <dig> .
the screen was repeated using a drosophila cdna library.
eight specific clones were retained , four  representing drosophila rab <dig>  .
the result of the drosophila screen suggested that aa <dig>  which had been selected against human rab6a, can also recognize the fly rab6a homologue.
in our experience, polyclonal anti-rab <dig> antibodies  have failed to label drab <dig> in immunofluorescence .
in contrast, and in agreement with the y <dig> h data, aa <dig> labeling of drab <dig> on golgi membranes was seen by immunofluorescence .
furthermore, analogous to what had been shown in mammalian cells  <cit> , aa <dig> was also functional as an intrabody and labeled golgi stacks in living drosophila s <dig> cells .
our y <dig> h approach thus revealed that in addition to mammalian rab <dig>  aa <dig> also detects the drosophila homologue.
this is likely due to the fact that the three-dimensional structure of gtp-bound rab <dig> remained highly conserved throughout evolution.
in both y <dig> h screens using aa <dig> as bait, only prey fragments spanning most of the orf were retained .
apparently, the entire core region of rab <dig> had to be expressed to allow binding of this conformation-sensitive antibody.
this further suggested that this method of target determination could validate non-linear epitopes.
to confirm this, we next characterized the target of another recently obtained scfv, which was also observed to be conformation specific .
this antibody, called rof <dig>  detects the small gtpase rab1a and/or rab1b only after their activation through gtp-loading.
when the screen was performed using rof <dig> as bait, a total of  <dig> specifically interacting clones were retained from the mammalian cdna library.
eighty percent  encoded for rab1a or rab1b, once again all encompassing the full core region of the orf .
in the two y <dig> h screens using conformational sensors as bait  and comprehensive prey libraries, only targets expressing a sizable portion of the protein  were retained.
likely, fragments had to be large enough to allow folding into proper tertiary structure and gtp loading.
the first few amino-terminal residues and the last  <dig> or so amino-acid long carboxy-terminal hypervariable tail are known to be dispensable for correct three-dimensional assembly of small gtpases.
in fact, the crystal structure of rab6b was solved by expressing a recombinant protein that contained only the core region of the protein   <cit> .
we believe that the residues, which make up the epitopes for the binding of aa <dig> and rof <dig>  respectively, are non-adjacent in the primary amino acid sequence and only come together after correct folding of the polypeptide into tertiary structure and subsequent activation through gtp binding.
lack of reactivity of both antibodies on immunoblotting, and results from the y <dig> h screens strongly supports this hypothesis.
in summary, our approach showed that for the conformation-specific sensors aa <dig> and rof <dig> correct three-dimensional antigen structures are needed to present the antibody binding sites.
it furthermore revealed that the aa <dig> epitope is conserved in evolution since drab <dig> is recognized by the scfv.
for the anti-giantin antibody ta <dig>  a small binding site within the large coiled-coil protein giantin is determined
next, we characterized the target of ta <dig>  this scfv is a recombinant antibody directed against a very large  golgi-associated matrix protein called giantin.
ta <dig> was originally selected via phage display using intact, purified, golgi stacks as target  <cit> .
the mammalian cdna prey library was screened using ta <dig> as bait.
sixty interacting clones were retained .
only one cluster, containing seven clones, obtained a high pbs ranking; they all fell within the central portion of the giantin orf.
alignment of the seven clones defined a ~ <dig> kda-large sid  located in the center portion of giantin.
binding of ta <dig> within this region of  <dig> amino acids was confirmed via overexpression experiments .
ta <dig> is a blotting antibody and thus, in contrast to aa <dig> and rof <dig>  should detect the denatured protein at a small linear binding site.
taking advantage of the flexibility of yeast genetics, we therefore attempted to narrow the binding domain of ta <dig> further using a method based on gap repair.
primers were designed along the shortest of the seven prey fragments and pcr fragments were generated .
the y <dig> h screen was then repeated, this time using the resulting small pcr products as prey .
in this manner, the ta <dig> binding region could be narrowed further to a single coiled coil domain measuring 9kda .
binding of ta <dig> to this region was confirmed in overexpression experiments using both immunofluorescence and western blotting .
finally, carrying out additional biochemical and overexpression experiments we narrowed the antibody-binding region even further .
in summary, the y <dig> h screen enabled rapid and accurate target characterization for ta <dig>  a precise region of  <dig> amino acids, representing less than  <dig> % of the entire protein, was determined as its binding site.
for the non-muscle myosin iia-targeting antibody sf <dig>  a precise epitope within a long coiled-coil protein is determined and cross reactivity with human and other, non-human, homologues is revealed
sf <dig> was selected alongside ta <dig> in a phage display screen using purified rat liver golgi stacks as the antigen.
sf9's target, non-muscle myosin iia, was identified using immunoblotting followed by mass spectrometry analysis  <cit> .
from the y <dig> h screen with sf <dig> as bait a total of  <dig> specifically interacting clones were retained .
more than half  encoded for non-muscle myosin iia and their alignment allowed identification of a very small sid of only  <dig> amino acids .
an additional  <dig> clones represented one of two very closely related myosins: non-muscle myosin iib and smooth-muscle myosin .
their alignments yielded similarly precise sid .
all three sid overlapped with one another inside a conserved stretch of the tail region of the myosin protein family, further narrowing the putative binding domain for sf <dig> .
binding of the antibody to the 29-amino-acid long common sid was confirmed in a series of overexpression experiments.
gfp-tagged non-muscle myosin iia constructs, which either included or lacked the sid , were overexpressed in mammalian cells.
in addition to the endogenous myosin pool, only the overexpressed full-length but not the truncated protein was recognized in immunofluorescence by sf <dig> .
immunoblot analysis revealed that a recombinant protein containing the 29-amino-acid long sid was detected while no binding was seen with the construct lacking this short region .
together, this confirmed our y <dig> h results.
non-muscle myosins are evolutionarily conserved proteins.
in addition, sf <dig> was found to bind to a region of the protein that shares near-identical amino acid sequences between human homologues .
alignment of the sid with non-muscle myosins of other species revealed a high degree of evolutionary conservation in this region even with the drosophila counterpart .
we thus speculated that sf <dig> might also detect the insect form of myosin.
indeed, when using total insect cell lysates a single ca.
200-kda-large band was detected by sf <dig>  this signal completely disappeared after specific anti-myosin rnai-mediated gene silencing .
in summary, our y2h-approach allowed precise characterization of the sf <dig> target, defining a very small  epitope within a ~200kda protein.
in addition, it revealed cross-reactivity with closely related proteins of the same and across species.
results of screens using antibodies that do not work as intrabodies
our novel approach yielded important additional information about the targets of several distinct monoclonal antibodies.
however, some variability in the strength and reliability of the data could be noted when comparing individual screens.
for instance, when screening using yet another scfv, named f2c , results were less conclusive.
while a total of  <dig> interacting clones were obtained, only  <dig> encoded for α-tubulin .
their alignment allowed narrowing of the epitope-containing region to the c-terminal portion of the protein.
however, many of the remaining  <dig> clones were also classified as possible binders using the pbs scoring method.
nonetheless, it is unlikely that they represent secondary targets for f2c given a highly specific signal of f2c in both immunofluorescence and western blotting  <cit> .
another scfv originating from the above mentioned golgi-stack screen, called te <dig>  <cit> , was tested in our approach.
while many interacting clones were retained, none could be confirmed in overexpression experiments and the te <dig> target remained elusive so far .
in sharp contrast to the just mentioned examples  our rof7- and sf9-screens not only confirmed the antibodies' targets but also gave detailed information about their respective epitopes: in each case almost 90% of clones aligned within its target .
in agreement with the more classical use of scfv in y <dig> h  <cit>  there seems to be a direct correlation between the ability of an antibody to fold correctly under the reducing conditions of the eukaryotic  cytosol and their use in y <dig> h. while sf <dig>  ta <dig>  aa <dig> and rof <dig> efficiently bind their respective targets when expressed as intrabodies, f2c is only faintly staining microtubules in living cells and te <dig> never yielded specific staining as an intrabody.
we thus hypothesize that the same relative efficiency for target recognition is present during the yeast two hybrid experiments.
to make full use of our method, it will therefore be important to obtain good intrabodies, for example via screening of dedicated libraries  <cit>  or through the use of single domain antibodies, like camelidae antibodies, that seem to be more resistant to the reducing conditions of the intracellular milieu  <cit> .
CONCLUSIONS
we present a novel approach, which allows in-depth characterization of antibody target using the y <dig> h technology.
in our approach the traditional orientation of prey and bait is reversed, complex cdna prey libraries are screened, and a robust scoring system to eliminate non-specific binders is employed.
this way, critical new information about antibodies' targets can be obtained.
this includes strong corroborative evidence for conformational specificity and the need for a non-linear epitope for conformational antibodies, precise mapping of small epitopes, and information about cross reactivity within and across species.
in summary, our y <dig> h approach represents a powerful method for the detailed characterization of scfv/antibody targets.
given the high quantity and quality of new data that can be obtained from a simple experiment, we believe that our approach might in the future serve as an invaluable tool for the detailed target characterization of many types of antibodies.

 because of their unique ability to recognize target antigens with extremely high fidelity, antibodies remain an essential tool in fundamental biomedical research and in clinical diagnostic testing. in addition, they represent one of the most promising novel therapeutic options, in particular in the field of cancer treatment. worldwide, more than a thousand clinical trials have been completed and almost as many are currently underway using monoclonal antibodies as pharmaceuticals http://www.clinicaltrials.gov. traditionally, monoclonal antibodies have been produced from hybridoma cell lines in a laborious process involving animal experimentation and screening large numbers of clones. more recently, recombinant monoclonal antibodies, which add an additional level of flexibility, have also become a viable treatment option for various human diseases. several alternatives to the classic in vivo selection of antibodies have been developed and refined. one of the most prominent technique is antibody phage display, which allows in vitro selection of single-chain variable fragment antibodies from complex phage or phagemid libraries. in this approach, which has been utilized successfully in many laboratories, animal work is obviated entirely. it has been shown that scfv can even be manufactured for clinical use. in our own research laboratory, highly functional scfv antibodies that recognize a broad range of targets, including evolutionarily conserved proteins have been selected and characterized. regardless of the type of production or ultimate application, the effective use of antibodies depends on a detailed understanding of the underlying antibody-antigen interaction, i.e. knowledge about a given antibody's epitope. this holds true especially for clinical applications where such information would be of utmost importance to accurately predict wanted and unwanted biological effects. however, target characterization of both traditional and recombinant antibodies has been challenging. the gold standard of such epitope mapping - analysis of the crystal structure of a given antigen-antibody complex - is labor intensive and sometimes nearly impossible due to technical difficulties. thus, often cumbersome strategies have to be developed, like systematic target protein mutagenesis or sub-fragment analysis. examples of successful characterization of antibody targets through scanning mutagenesis and other techniques have been described elsewhere. more recently, combinatorial peptide phage display libraries have also been used. in the future, computer based or in silico combinatorial methods for epitope mapping, like mapitope might prove useful. finally, epitope mapping has also become a powerful strategy in the process of developing new vaccines. here we describe an alternative approach to characterize the interaction between antibodies and their respective targets. time- and resource-demanding crystallography or mutagenesis experiments are circumvented. instead the yeast two-hybrid technology is employed. in most two-hybrid applications in the recombinant antibody field, scfv are selected from a prey library, here, the system is inverted: previously characterized scfv are used as bait and screened against a comprehensive cdna library. this new approach yielded novel, detailed data about the targets of several recombinant antibodies, including the need for tertiary antigen structure for binding of conformational sensors, mapping of precise epitopes and unveiling of secondary targets.  antibody targets are characterized using the two-hybrid system with the scfv antibody as bait, a validated scoring system to remove non-specific binders, and determination of selected interacting domains investigators studying and selecting recombinant antibodies have mostly used the y h technology to select antibody populations that fold correctly under the reducing conditions of the cytosol. in such experiments, the epitope of a given antigen is defined and used as a bait to sample a diverse scfv prey library. here, we reversed the system, using as baits known scfv antibodies that had been previously selected in phage display screens, and, as preys, proteins expressed from high-complexity libraries of randomly primed cdna. individual scfv antibodies were exposed to a cdna library with full coverage, derived from either human placenta or from drosophila. more than million potential interactions were tested during each screen. prey fragments of positive clones were identified using sequence analysis and comparison with genbank databases using the blast algorithm. next, a predicted biological score was computed for each clone as described before, enabling classification according to interaction reliability. this permitted ranking of the clones into categories from "a" to "d" in decreasing probability of having a specific interaction with the scfv-bait. two additional categories were designated: "e", interacting clones, which were likely retained due to highly connected prey domains; and "f", clones that were experimentally proven y h artifacts. in a third step, overlapping prey fragments originating from the same gene were designated as clusters and their translated amino acid sequences were aligned and superimposed onto the open reading frame. overlapping regions shared by all fragments were designated as "selected interacting domain", as described before. for the conformation-specific antibodies aa and rof only large fragments are obtained, suggesting a non-linear antibody binding-site in the first screen, the scfv aa was used as bait. aa is a well-described conformation-specific antibody that exclusively binds the activated form of the small gtpase rab. from the human cdna prey library only three specific binders were retained by y h. two encoded for rab. the screen was repeated using a drosophila cdna library. eight specific clones were retained, four representing drosophila rab. the result of the drosophila screen suggested that aa which had been selected against human rab6a, can also recognize the fly rab6a homologue. in our experience, polyclonal anti-rab antibodies have failed to label drab in immunofluorescence. in contrast, and in agreement with the y h data, aa labeling of drab on golgi membranes was seen by immunofluorescence. furthermore, analogous to what had been shown in mammalian cells, aa was also functional as an intrabody and labeled golgi stacks in living drosophila s cells. our y h approach thus revealed that in addition to mammalian rab aa also detects the drosophila homologue. this is likely due to the fact that the three-dimensional structure of gtp-bound rab remained highly conserved throughout evolution. in both y h screens using aa as bait, only prey fragments spanning most of the orf were retained. apparently, the entire core region of rab had to be expressed to allow binding of this conformation-sensitive antibody. this further suggested that this method of target determination could validate non-linear epitopes. to confirm this, we next characterized the target of another recently obtained scfv, which was also observed to be conformation specific. this antibody, called rof detects the small gtpase rab1a and/or rab1b only after their activation through gtp-loading. when the screen was performed using rof as bait, a total of specifically interacting clones were retained from the mammalian cdna library. eighty percent encoded for rab1a or rab1b, once again all encompassing the full core region of the orf. in the two y h screens using conformational sensors as bait and comprehensive prey libraries, only targets expressing a sizable portion of the protein were retained. likely, fragments had to be large enough to allow folding into proper tertiary structure and gtp loading. the first few amino-terminal residues and the last or so amino-acid long carboxy-terminal hypervariable tail are known to be dispensable for correct three-dimensional assembly of small gtpases. in fact, the crystal structure of rab6b was solved by expressing a recombinant protein that contained only the core region of the protein. we believe that the residues, which make up the epitopes for the binding of aa and rof respectively, are non-adjacent in the primary amino acid sequence and only come together after correct folding of the polypeptide into tertiary structure and subsequent activation through gtp binding. lack of reactivity of both antibodies on immunoblotting, and results from the y h screens strongly supports this hypothesis. in summary, our approach showed that for the conformation-specific sensors aa and rof correct three-dimensional antigen structures are needed to present the antibody binding sites. it furthermore revealed that the aa epitope is conserved in evolution since drab is recognized by the scfv. for the anti-giantin antibody ta a small binding site within the large coiled-coil protein giantin is determined next, we characterized the target of ta this scfv is a recombinant antibody directed against a very large golgi-associated matrix protein called giantin. ta was originally selected via phage display using intact, purified, golgi stacks as target. the mammalian cdna prey library was screened using ta as bait. sixty interacting clones were retained. only one cluster, containing seven clones, obtained a high pbs ranking; they all fell within the central portion of the giantin orf. alignment of the seven clones defined a ~ kda-large sid located in the center portion of giantin. binding of ta within this region of amino acids was confirmed via overexpression experiments. ta is a blotting antibody and thus, in contrast to aa and rof should detect the denatured protein at a small linear binding site. taking advantage of the flexibility of yeast genetics, we therefore attempted to narrow the binding domain of ta further using a method based on gap repair. primers were designed along the shortest of the seven prey fragments and pcr fragments were generated. the y h screen was then repeated, this time using the resulting small pcr products as prey. in this manner, the ta binding region could be narrowed further to a single coiled coil domain measuring 9kda. binding of ta to this region was confirmed in overexpression experiments using both immunofluorescence and western blotting. finally, carrying out additional biochemical and overexpression experiments we narrowed the antibody-binding region even further. in summary, the y h screen enabled rapid and accurate target characterization for ta a precise region of amino acids, representing less than % of the entire protein, was determined as its binding site. for the non-muscle myosin iia-targeting antibody sf a precise epitope within a long coiled-coil protein is determined and cross reactivity with human and other, non-human, homologues is revealed sf was selected alongside ta in a phage display screen using purified rat liver golgi stacks as the antigen. sf9's target, non-muscle myosin iia, was identified using immunoblotting followed by mass spectrometry analysis. from the y h screen with sf as bait a total of specifically interacting clones were retained. more than half encoded for non-muscle myosin iia and their alignment allowed identification of a very small sid of only amino acids. an additional clones represented one of two very closely related myosins: non-muscle myosin iib and smooth-muscle myosin. their alignments yielded similarly precise sid. all three sid overlapped with one another inside a conserved stretch of the tail region of the myosin protein family, further narrowing the putative binding domain for sf. binding of the antibody to the 29-amino-acid long common sid was confirmed in a series of overexpression experiments. gfp-tagged non-muscle myosin iia constructs, which either included or lacked the sid, were overexpressed in mammalian cells. in addition to the endogenous myosin pool, only the overexpressed full-length but not the truncated protein was recognized in immunofluorescence by sf. immunoblot analysis revealed that a recombinant protein containing the 29-amino-acid long sid was detected while no binding was seen with the construct lacking this short region. together, this confirmed our y h results. non-muscle myosins are evolutionarily conserved proteins. in addition, sf was found to bind to a region of the protein that shares near-identical amino acid sequences between human homologues. alignment of the sid with non-muscle myosins of other species revealed a high degree of evolutionary conservation in this region even with the drosophila counterpart. we thus speculated that sf might also detect the insect form of myosin. indeed, when using total insect cell lysates a single ca. 200-kda-large band was detected by sf this signal completely disappeared after specific anti-myosin rnai-mediated gene silencing. in summary, our y2h-approach allowed precise characterization of the sf target, defining a very small epitope within a ~200kda protein. in addition, it revealed cross-reactivity with closely related proteins of the same and across species. results of screens using antibodies that do not work as intrabodies our novel approach yielded important additional information about the targets of several distinct monoclonal antibodies. however, some variability in the strength and reliability of the data could be noted when comparing individual screens. for instance, when screening using yet another scfv, named f2c, results were less conclusive. while a total of interacting clones were obtained, only encoded for α-tubulin. their alignment allowed narrowing of the epitope-containing region to the c-terminal portion of the protein. however, many of the remaining clones were also classified as possible binders using the pbs scoring method. nonetheless, it is unlikely that they represent secondary targets for f2c given a highly specific signal of f2c in both immunofluorescence and western blotting. another scfv originating from the above mentioned golgi-stack screen, called te, was tested in our approach. while many interacting clones were retained, none could be confirmed in overexpression experiments and the te target remained elusive so far. in sharp contrast to the just mentioned examples our rof7- and sf9-screens not only confirmed the antibodies' targets but also gave detailed information about their respective epitopes: in each case almost 90% of clones aligned within its target. in agreement with the more classical use of scfv in y h there seems to be a direct correlation between the ability of an antibody to fold correctly under the reducing conditions of the eukaryotic cytosol and their use in y h. while sf ta aa and rof efficiently bind their respective targets when expressed as intrabodies, f2c is only faintly staining microtubules in living cells and te never yielded specific staining as an intrabody. we thus hypothesize that the same relative efficiency for target recognition is present during the yeast two hybrid experiments. to make full use of our method, it will therefore be important to obtain good intrabodies, for example via screening of dedicated libraries or through the use of single domain antibodies, like camelidae antibodies, that seem to be more resistant to the reducing conditions of the intracellular milieu.  we present a novel approach, which allows in-depth characterization of antibody target using the y h technology. in our approach the traditional orientation of prey and bait is reversed, complex cdna prey libraries are screened, and a robust scoring system to eliminate non-specific binders is employed. this way, critical new information about antibodies' targets can be obtained. this includes strong corroborative evidence for conformational specificity and the need for a non-linear epitope for conformational antibodies, precise mapping of small epitopes, and information about cross reactivity within and across species. in summary, our y h approach represents a powerful method for the detailed characterization of scfv/antibody targets. given the high quantity and quality of new data that can be obtained from a simple experiment, we believe that our approach might in the future serve as an invaluable tool for the detailed target characterization of many types of antibodies. 
this approach may thus prove to be an elegant and rapid method for the target characterization of newly obtained scfv antibodies. regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope is of great importance. our method, utilizing the yeast two-hybrid technology and scfv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. recombinant antibodies, including single chain variable fragments, are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. it allows precise domain mapping for linear epitopes, confirmation of non-linear epitopes for conformational sensors, and detection of secondary binding partners. for two other scfv, ta and sf which recognize the large proteins giantin and non-muscle myosin iia, respectively, precise antibody-binding regions within the target were defined. due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. this strongly suggested interaction with a non-linear epitope. it may be considered prior to any research application and particularly before any use of such recombinant antibodies in clinical medicine. interactions between antibodies and their respective targets were characterized for several scfv. finally, for some antibodies, secondary targets within and across species could be revealed.
 due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. recombinant antibodies, including single chain variable fragments, are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope is of great importance. however, such data is frequently difficult to obtain.  we describe an approach that allows detailed characterization of a given antibody's target using the yeast two-hybrid system. several recombinant scfv were used as bait and screened against highly complex cdna libraries. systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. multiple alignment of the obtained cdna fragments provided a selected interacting domain, efficiently narrowing the epitope-containing region. interactions between antibodies and their respective targets were characterized for several scfv. for aa and rof two conformation-specific sensors that exclusively bind the activated forms of the small gtpases rab and rab respectively, only fragments expressing the entire target protein's core region were retained. this strongly suggested interaction with a non-linear epitope. for two other scfv, ta and sf which recognize the large proteins giantin and non-muscle myosin iia, respectively, precise antibody-binding regions within the target were defined. finally, for some antibodies, secondary targets within and across species could be revealed.  our method, utilizing the yeast two-hybrid technology and scfv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. it allows precise domain mapping for linear epitopes, confirmation of non-linear epitopes for conformational sensors, and detection of secondary binding partners. this approach may thus prove to be an elegant and rapid method for the target characterization of newly obtained scfv antibodies. it may be considered prior to any research application and particularly before any use of such recombinant antibodies in clinical medicine. 