BACKGROUND
fungal infections have recently emerged as a growing threat to human health, especially in persons whose immune systems are compromised  <cit> .
nowadays, the most common treatment for fungal infections is based on the use of amphotericin b, 5-flucytosine and fluconazole.
however, these drugs have repeatedly failed against infections caused by candida and cryptococcus species  <cit> .
a series of antigenic peptides from fungal pathogens have been identified that can generate immune responses, and which may assist in developing an antifungal vaccine  <cit> .
therefore, development of novel molecules and alternative therapeutic strategies for the battle against fungal infections is becoming a topical and widely recognized need.
to combat against fungal diseases, we are trying to produce a single, but general, antifungal vaccine by using an improved and optimized phage-display panning method that elicits immune responses in immunocompromised individuals who are at risk of invasive fungal infections by opportunistic fungal pathogens, as well as other multiple fungal infections.
one of the most widely used library methods is based on the use of a filamentous phage, a virus that lives on escherichia coli.
using a phage-display system, various antibody fragments can be displayed on the surface of filamentous phages containing the antibody gene as a phagemid  <cit> .
however, antibody fragments, including single chain fragment variable  molecules, have been developed for potential clinical applications  <cit> .
production of recombinant antibodies using phage-display technology has many advantages over conventional polyclonal and monoclonal antibody production  <cit> .
phage-display technology and antibody engineering have been used to isolate scfvs that can interact with a wide variety of antigens  <cit> .
an effective and reliable method is needed to produce antibodies with high antigenic specificity and affinity.
in this study, we investigated the de novo selection of dna that encodes scfvs specific to the immunogen, hm- <dig> killer toxin -neutralizing monoclonal antibodies  from splenocytes of a hyper-immunized mouse.
conventional methods of phage-display panning immobilize purified antigen to plates or other solid supports to which libraries are applied, and then use extensive washings in detergent-supplemented buffers to select specific phage antibodies  <cit> .
these methods sometimes are not so effective due to tight antigen-antibody binding and attachment to the solid support of the tubes or plates.
to optimize the method of phage-display panning and also to recover high-affinity anti-idiotypic phage antibodies from immobilized antigens, different elution conditions have been used  <cit> .
each antigen-antibody interaction requires careful fine-tuning of elution conditions to produce antigen-specific scfv antibodies  <cit> .
we are trying to establish an improved, optimized subtractive panning method to select antigen-specific antibodies more efficiently.
killer toxins are protein molecules that disrupt cell functions in a number of ways, some by making ion channels in cell membranes and others by interacting with membrane channels or receptors or both  <cit> .
among yeast killer toxins, hm- <dig> is highly stable against heat treatment and a wide range of ph values  and also exhibits the wide spectrum of anti-microbial activity  <cit> .
these unique properties of this toxin have made it attractive for therapeutic applications, but not for clinical use because of their instability in the host physiological conditions as well as their antigenicity.
to overcome the intrinsic toxicity and chemical instability of hm- <dig>  moreover taking advantage of its immunogenicity, nmab-kt has been produced.
to produce a new anti-fungal drug, previously we used nmab-kt as an immunogen in mice to get an antigen-specific recombinant anti-idiotypic scfv antibody, sharing structural or functional or both similarities with the active site of hm- <dig>  <cit> .
in this study, selection of phage-displayed scfv was based on specific antigen nmab-kt coated on to immunotubes and that was chosen to explore the potential of the cdna library, constructed from nmab-kt, to isolate antigen-specific antibody fragments.
the strategy applied for the modified subtractive panning, tested within five cycles, was based on the premise that competitive binding of hm- <dig> to coated nmab-kt can improve selection efficiency by an easy and quick dissociation of scfv-displayed phages from the immobilized surface.
the ultimate goal of this study was to produce recombinant antibodies and to characterize their properties by immunoassay parameters, dna analysis, product stability and functionality.
RESULTS
selection of clones with improved characteristics
solid phase panning as described by griffiths et al.
<cit>  was used to select nmab-kt-specific phage scfv.
selection of phages with specificity for nmab-kt was based on increasing stringency of selection at each round of panning .
enrichment was determined to check the number of phages recaptured after each round of panning by counting the colony forming units  of the infected e. coli tg <dig>  the phage-display library consisted of  <dig>  ×  <dig> clones  with a final diversity of  <dig>  ×  <dig> separate clones after fifth round of modified subtractive panning.
figure  <dig> showed the phage recovery after each round of regular panning and modified subtractive panning.
transformed e. coli tg <dig> cells in which the amber stop codon between the e-tag dna sequence and g <dig> gene is read through, allowing the production of the scfv-e tag-g <dig> fusion protein.
in the presence of helper phage m13ko <dig>  scfv fusion products were displayed on the recombinant phage tips, allowing affinity selection.
in the regular panning method, small differences were in the stringency of selection between different rounds of panning, but in modified subtractive panning after two or three rounds of selection, the antigen-binding clones showed a clear enrichment .
in modified subtractive panning, the stringency of panning was significantly increased after the fourth and fifth rounds of panning.
during different selection rounds of modified subtractive panning, bound scfv-containing phages were eluted easily and quickly from the immobilized nmab-kt by using an elution buffer supplemented with hm- <dig> at concentration  <dig> μg/ml.
the phage enrichment for regular panning was not as impressive as for modified subtractive panning.
for regular panning, the number of recaptured phages increased approximately 2-fold with low binding affinity in every round, but in modified subtractive panning, the number of recaptured phages increased  <dig> -  <dig> fold in the first three rounds of panning but increased 10-fold in the fourth and fifth rounds with higher binding strength to the antigen .
thus, the overall enrichment for modified subtractive panning was approximately 10-fold higher than for regular panning after the final fifth round of panning .
to verify the panning performance, we identified antigen-specific positive clones binding to the immobilized nmab-kt antigen.
the binding properties of  <dig> randomly selected clones binding to nmab-kt for both types of panning were measured by using microtiter plate enzyme-linked immunosorbent assay .
the supernatant containing soluble scfv antibodies was added to each well of a microtiter plate that had been pre-coated with nmab-kt, and detection was done by using horseradish peroxidase -conjugated anti-e-tag antiboy.
for regular panning, 24%  tested clones of the infected e. coli tg <dig> contained the anti-idiotypic scfv gene of hm- <dig> killer toxin, and so soluble scfv showed positive elisa signals after the fifth round of panning .
interestingly, for modified subtractive panning, 80%  tested clones showed positive elisa signal, also after the fifth round of panning with higher binding strength to nmab-kt .
positive binding affinity was defined as an absorbance for immobilized nmab-kt that was 2-fold above the background or negative controls.
negative controls consisted of microtiter plates coated with blocking buffer  or washing buffer .
* randomly selected clones were grown overnight and supernatants were tested for binding to the immobilized antigen by elisa.
to examine the integrity of the library after the final rounds of modified subtractive panning,  <dig> individual colonies in e. coli tg <dig> were randomly isolated and the presence of the scfv dna insert in these clones was confirmed by using polymerase chain reaction  amplification; these amplified products were analyzed on 4% agarose gels .
among these  <dig> colonies,  <dig> were positive.
only five restriction patterns were identified by the agarose gel electrophoresis; these patterns were confirmed by sequence data analysis.
among these five restriction patterns, four patterns contained inserts of the expected size  fragments and the other pattern contained a low molecular weight dna  fragment .
sequence analysis of scfv clones
at least five unique genes  were identified by nucleotide sequencing after the fifth round of modified subtractive panning.
table  <dig> showed the percentages of positive clones based on identical dna sequences.
among all positive clones, k <dig>  and k <dig>  were identified as the highest populated group of clones after successful screening.
among the other positive clones, k <dig> was 10%  and k <dig> and k <dig> were each only 2%.
figure  <dig> showed amino acid sequences deduced from the obtained nucleotide data.
all five sequences had different amino acid substitutions that were expected and mostly were in complementarity determining regions   <cit> .
the light chains of all clones were slightly different from each other, except scfv k <dig>  for scfv k <dig>  a light chain variable domain was partially synthesized together with an e-tag sequence.
in the cdr area, the amino acid sequences shared relatively low sequence homology.
apparently, the degree of homology was observed higher in the variable region of the light chain  domain compared with the variable region of the heavy chain  domain.
k <dig> and k <dig> differed by only one amino acid at position  <dig> in the vh domain.
production and characterization of nmab-kt-specific scfv
all  <dig> individual positive clones after e. coli tg <dig> infection were reinfected into e. coli hb <dig>  in the nonsuppressor strain of e. coli hb <dig>  the amber stop codon between e-tag and g <dig> fusion protein was recognized as a stop codon, and a soluble scfv-e tag fusion protein was produced as a consequence.
the expression of the scfv-e-tag fusion protein in the resultant e. coli hb <dig> clones was induced by isopropyl β-d-thiogalactopyranoside , and soluble scfv antibodies were secreted into the culture supernatants.
after the final fifth round of modified subtractive panning, 80% clones  were positive with a strong binding affinity to nmab-kt .
however, in regular panning after the fifth round of panning only 24%  were positive clones with low binding affinity .
the nmab-kt-binding pattern of soluble scfv antibodies from these  <dig> positive clones of e. coli hb <dig> was almost similar to that of the parent phage e. coli tg <dig> scfv antibodies, which was confirmed by using elisa .
after screening, clones scfv k <dig> - k <dig> were identified, and soluble scfvs were purified from the periplasmic extract of e. coli hb <dig> on an anti-e-tag affinity column.
sds-page and western blotting to check scfv expression
the culture supernatant without anti-e-tag column purification, an approximately  <dig> kda ) and two types of small  scfv-e tag fusion protein were expressed from the reinfected e. coli hb <dig> .
there expressions indicated that all  <dig> positive clones originated from our original cdna library constructed by pcantab  <dig> e vector using the e-tag sequence.
four clones of purified soluble scfv  showed two strong protein bands  by using sodium dodecyl sulphate-polyacrylamide gel electrophoresis  and by western blot analysis with hrp-conjugated anti-e-tag antibody .
both proteins contained e-tag sequences, so western blotting using hrp-conjugated anti-e-tag antibody detected bands.
for the short sequenced scfv k <dig> clone, a  <dig> kda e-tagged protein band showed before purification , but after anti-e-tag column purification a protein band was not detected by using sds-page and western blotting .
functional characterization and antifungal activity of recombinant scfvs
in vitro antifungal activity of recombinant scfv antibodies was examined for non-pathogenic and pathogenic strains of fungal species saccharomyces cerevisiae and candida albicans, respectively.
first, in vitro susceptibility of s. cerevisiae to scfv antibodies was examined by cell suspension assay as described in the materials and methods .
among all scfv antibodies, scfv k <dig> was the most effective antifungal agent.
figure 7b showed the elution profile and biological activity of purified scfv k <dig> against the growth of s. cerevisiae a <dig>  next, to check the candidacidal activity of scfv k <dig> antibodies against c. albicans atcc  <dig>  cell growth was also measured by cell suspension assay .
table  <dig> showed the ic <dig> values of scfv k <dig> against s. cerevisiae a <dig> and c. albicans atcc  <dig> cells growth in vitro.
extensive studies by using our assay system showed that  <dig>  × 10- <dig> m and  <dig>  × 10- <dig> m were sufficient to produce 50% inhibition of growth of s. cerevisiae a <dig> and c. albicans atcc  <dig>  respectively.
*ic <dig> values for recombinant scfv k <dig> against cell growth of candida and saccharomyces species were measured by using a cell suspension assay as described in the methods.
kinetic parameters of selected clone scfv k1
the binding specificities and kinetic parameters of purified scfv k <dig>  hm- <dig> and hyi killer toxin  were determined by using surface plasmon resonance  analysis.
biacore sensograms showed the degree of interaction between immobilized nmab-kt and scfv k <dig>  hm- <dig> or hyi .
kinetic data from spr analysis showed that scfv k <dig> had a strong binding affinity for nmab-kt, like hm- <dig>  but hyi had no binding affinity for nmab-kt.
hm- <dig> analyte was used for the positive control and hyi analyte was used for the negative control experiment  <cit> .
table  <dig> showed the calculated values of kon, koff, ka and kd.
the kd values of scfv k <dig> and hm- <dig> were  <dig>  × 10- <dig> m and  <dig>  × 10- <dig> m, respectively.
therefore, we can conclude that scfv k <dig> had strong reactivity with nmab-kt, like hm- <dig>  but not with hyi.
ligand nmab-kt was immobilized on the surface of a cm <dig> sensor chip, and scfv k <dig>  hm- <dig> peptide or hyi peptide, were then injected over this immobilized surface in the presence of hbs-ep buffer.
the affinity constant  and dissociation constant  were obtained from the ratio of the association  and dissociation  rate constants derived from global fits of response against time data to a 1: <dig> langmuir binding model.
ka = kon/koff; kd = koff/kon; nd, signals not detected.
hm- <dig> analyte was used for the positive control and hyi analyte was used for the negative control experiment  <cit> .
competitive binding of scfv k <dig> antibody with hm- <dig> to nmab-kt
to examine whether scfv k <dig> antibody compete with hm- <dig> to bind with nmab-kt, a competitive binding elisa was performed .
when the scfv k <dig> antibody concentration was increased, the binding of scfv k <dig> antibody to nmab-kt was also increased, but the hm- <dig> binding to nmab-kt was simultaneously decreased.
these results suggest that scfv k <dig> antibody clone was homologous in competition with hm- <dig> for binding to nmab-kt.
discussion
anti-idiotypic antibodies representing the internal image of some antigenic determinants have been proposed as surrogate vaccines  <cit> , and antibodies conjugated with toxins have been proposed for immunotherapy of cancer  <cit> .
in some cases, amino acid sequence homology exists between the protein antigen and the anti-idiotypic antibody variable region  <cit> .
antigenic mimicry is usually more functional than biochemical, and the functional mimicry of ligands of biological receptors by anti-idiotypic antibodies has been extensively studied  <cit> .
to select a useful and specific scfv antibody for a target antigen, a high quality primary phage library is needed  <cit> .
importantly, the library must have a large functional size to match diverse antibody sequences to facilitate the selection of a variety of high affinity scfvs, and these scfv genes must be well expressed to allow the panning process.
previously, we established a large mouse scfv phage display library , and we attempted to isolate antigen-specific scfvs by using nmab-kt as a model antigen  <cit> .
in this study, after proper selection of the library, phage-displaying anti-idiotypic scfv antibodies represented the internal image of the original antigen hm- <dig> and thus might be effective as a vaccine, avoiding immunization with a pathogen.
for traditional panning procedures, elution conditions are not same to recover a wide variety of specific phages from the immobilized antigens.
sometimes bound phages are not easily dissociated from the immobilized antigen because of tight antigen-antibody interaction and attachment with the solid support of the tubes or plates.
several methods have been reported to improve these problems to recover phage-displayed scfvs with the highest affinity, and some of these methods are applicable to the selection of anti-idiotypic antibodies  <cit> .
several elution methods that include the common acidic or basic conditions have been used to increase the stringency of selection  <cit> .
more acidic or basic conditions sometimes may disrupt the phage particles, and hence scfvs may lose or change its functional characteristics.
so, we were proceeding an alternative way that will be more susceptible to get a high affinity binders to the specific antigen.
therefore, during elution of the bound phages in modified subtractive panning, we selected neutral phosphate-buffered saline  solution  supplemented with hm- <dig> that shared structural or functional similarities with the active site of phage-displayed scfv antibodies.
as a result, elution with hm- <dig> facilitates and favors the panning process through competition of binding to immobilized nmab-kt .
this competition of binding accelerated the quick dissociation of scfv-containing phages from the immobilized antigen nmab-kt with increased binding strength.
the binding strength and specificity were analyzed by using elisa with scfv antibodies against nmab-kt.
figures 3a and 3b compared the bindings between antigen nmab-kt and scfv antibodies obtained after using regular and modified subtractive pannings.
the elisa signals with soluble scfv antibodies from modified subtractive panning were much stronger than those from regular panning, indicating that the binding strength improved for elution with hm- <dig> in the selection procedure.
for further confirmation of the binding specificity of phage-displayed scfvs to antigen nmab-kt after each round of panning, the number of recaptured phages was quantified.
figure  <dig> clearly demonstrated that after each round of panning the recaptured phages for modified subtractive panning were markedly higher than those for regular panning.
although the regular panning experiments were successful, they failed to enrich the phage clones sufficiently may be because of less dissociation of tightly bound scfv-containing phages from the solid support of the tubes or plates during selection.
to ascertain the scfv repertoire and the quality of the immunized antibody library, dna segments encoding the scfv genes from  <dig> randomly selected positive clones  were identified after the fifth round of modified subtractive panning .
however, after the fifth round of regular phage display panning only 24% clones  were positive but with low binding affinity .
the  <dig> positive clones that had higher binding affinity were amplified by using the polymerase chain reaction , and the amplified products were analyzed on agarose gels and their dna fingerprint patterns were compared .
the patterns of these clones were found to vary, indicating an excellent diversity in the immunized mouse scfv library.
nucleotide sequencing showed that at least five unique scfv genes were identified by using modified subtractive panning, i.e., clones scfv k1- scfv k <dig> were analyzed to deduce the amino acid sequences .
dna sequencing also confirmed that the highest population of the positive clones  was unique  with a higher binding strength to the antigen nmab-kt.
also, the second highest populated group scfv k <dig>  had short dna sequences in the vl domain compared with the other groups.
dna fingerprints of k <dig> and k <dig> -  <dig> were apparently identical , although their amino acid sequences differed.
therefore, we produced at least five different anti-idiotypic scfv antibody clones of hm- <dig>
the scfvs, carrying the e-tag sequence and fused to the c-terminus, were purified by using an anti-e-tag affinity column and were analyzed on sds-page followed by western blotting performed against hrp/anti-e tag conjugate.
silver-stained gel analysis of all purified scfvs showed a sharp, unique band  of the expected mw of  <dig> kda together with an unexpected band of lower mw ; these bands were detected by western blotting with the hrp/anti-e-tag conjugate .
the low mw  <dig> kda bands had the e-tag sequence detected by western blotting, indicating that they might be proteolytic cleavage derivatives of the original  <dig> kda scfvs.
interestingly, scfv k <dig> expressed a  <dig> kda smaller protein that also contained an e-tag sequence detected by western blotting before column purification of the sample .
this  <dig> kda scfv k <dig> had a partial vl region of the antibody, i.e., a residue of  <dig> amino acids instead of  <dig> amino acids that was confirmed from the dna sequence data .
this  <dig> kda scfv k <dig> represented prematurely terminated translation products because of mutation.
purification of scfv k <dig> by using an anti-e-tag column was not possible to confirm by sds-page and western blotting .
therefore, the e-tag containing short scfv k <dig>  passed through the purification column without binding to the resin of the column that might be because of conformational changes due to partial lack of a light chain  domain and might be because of cross-linking with other parts of the scfv antibody and therefore not having the open arm for binding with the antie-tag sequence of the purification column.
scfv k <dig> that had the highest population  of positive clones showed strong cytocidal activity against s. cerevisiae and candida species compared with other scfvs .
so, for further experiments we selected scfv k <dig> to characterize the functional activity and specificity.
in this study, the antifungal activity was evaluated by cell suspension assay as described in the materials and methods.
the scfv k <dig> antibody showed strong antifungal activity against s. cerevisiae and c. albicans in vitro with ic <dig> values  <dig>  × 10- <dig> m for s. cerevisiae a <dig> and  <dig>  × 10- <dig> m for c. albicans atcc  <dig>  the anti-idiotypic scfv k <dig> had high affinities as judged by spr.
biacore analysis indicated kd =  <dig>  × 10- <dig> m for scfv k <dig>  which was comparable to the hm- <dig> killer toxin .
therefore, we concluded that nmab-kt showed strong reactivity toward scfv k <dig>  like hm- <dig>  but not toward hyi.
furthermore, the binding of scfv k <dig> antibody to nmab-kt was competitive with that of hm- <dig> , i.e., equimolar concentration of hm- <dig> inhibited the binding of scfv k <dig> antibody to the immobilized nmab-kt antigen.
based on several factors, such as expression level, elisa using nmab-kt, biacore  and fungal killing activity, scfv k <dig> was considered the most desirable scfv.
so, scfv k <dig> is a candidate for future studies directed at killing of pathogenic fungal species using an scfv-drug conjugate or by development into a whole igg format for complement-mediated and antibody-dependent cytotoxic pathways.
CONCLUSIONS
in conclusion, we successfully obtained recombinant scfv antibodies with high specificity against nmab-kt directly from the phage-displayed scfv library of a hyper-immunized mouse.
filamentous phage-displaying scfvs were selected against nmab-kt and were enriched through five rounds of modified subtractive panning.
the scfv antibodies generated after modified subtractive panning appear to have superior binding properties than for regular panning.
we showed a simple modification of the elution condition in the phage-display panning protocol, and so make a large difference in determining success.
the deduced amino acid sequences of cdrs might also help us to create other scfv fragments with even higher affinity and specificity for nmab-kt and fungal cells by further mutagenesis study.

 fungal infections have recently emerged as a growing threat to human health, especially in persons whose immune systems are compromised. nowadays, the most common treatment for fungal infections is based on the use of amphotericin b, 5-flucytosine and fluconazole. however, these drugs have repeatedly failed against infections caused by candida and cryptococcus species. a series of antigenic peptides from fungal pathogens have been identified that can generate immune responses, and which may assist in developing an antifungal vaccine. therefore, development of novel molecules and alternative therapeutic strategies for the battle against fungal infections is becoming a topical and widely recognized need. to combat against fungal diseases, we are trying to produce a single, but general, antifungal vaccine by using an improved and optimized phage-display panning method that elicits immune responses in immunocompromised individuals who are at risk of invasive fungal infections by opportunistic fungal pathogens, as well as other multiple fungal infections. one of the most widely used library methods is based on the use of a filamentous phage, a virus that lives on escherichia coli. using a phage-display system, various antibody fragments can be displayed on the surface of filamentous phages containing the antibody gene as a phagemid. however, antibody fragments, including single chain fragment variable molecules, have been developed for potential clinical applications. production of recombinant antibodies using phage-display technology has many advantages over conventional polyclonal and monoclonal antibody production. phage-display technology and antibody engineering have been used to isolate scfvs that can interact with a wide variety of antigens. an effective and reliable method is needed to produce antibodies with high antigenic specificity and affinity. in this study, we investigated the de novo selection of dna that encodes scfvs specific to the immunogen, hm- killer toxin -neutralizing monoclonal antibodies from splenocytes of a hyper-immunized mouse. conventional methods of phage-display panning immobilize purified antigen to plates or other solid supports to which libraries are applied, and then use extensive washings in detergent-supplemented buffers to select specific phage antibodies. these methods sometimes are not so effective due to tight antigen-antibody binding and attachment to the solid support of the tubes or plates. to optimize the method of phage-display panning and also to recover high-affinity anti-idiotypic phage antibodies from immobilized antigens, different elution conditions have been used. each antigen-antibody interaction requires careful fine-tuning of elution conditions to produce antigen-specific scfv antibodies. we are trying to establish an improved, optimized subtractive panning method to select antigen-specific antibodies more efficiently. killer toxins are protein molecules that disrupt cell functions in a number of ways, some by making ion channels in cell membranes and others by interacting with membrane channels or receptors or both. among yeast killer toxins, hm- is highly stable against heat treatment and a wide range of ph values and also exhibits the wide spectrum of anti-microbial activity. these unique properties of this toxin have made it attractive for therapeutic applications, but not for clinical use because of their instability in the host physiological conditions as well as their antigenicity. to overcome the intrinsic toxicity and chemical instability of hm- moreover taking advantage of its immunogenicity, nmab-kt has been produced. to produce a new anti-fungal drug, previously we used nmab-kt as an immunogen in mice to get an antigen-specific recombinant anti-idiotypic scfv antibody, sharing structural or functional or both similarities with the active site of hm-. in this study, selection of phage-displayed scfv was based on specific antigen nmab-kt coated on to immunotubes and that was chosen to explore the potential of the cdna library, constructed from nmab-kt, to isolate antigen-specific antibody fragments. the strategy applied for the modified subtractive panning, tested within five cycles, was based on the premise that competitive binding of hm- to coated nmab-kt can improve selection efficiency by an easy and quick dissociation of scfv-displayed phages from the immobilized surface. the ultimate goal of this study was to produce recombinant antibodies and to characterize their properties by immunoassay parameters, dna analysis, product stability and functionality.  selection of clones with improved characteristics solid phase panning as described by griffiths et al. was used to select nmab-kt-specific phage scfv. selection of phages with specificity for nmab-kt was based on increasing stringency of selection at each round of panning. enrichment was determined to check the number of phages recaptured after each round of panning by counting the colony forming units of the infected e. coli tg the phage-display library consisted of × clones with a final diversity of × separate clones after fifth round of modified subtractive panning. figure showed the phage recovery after each round of regular panning and modified subtractive panning. transformed e. coli tg cells in which the amber stop codon between the e-tag dna sequence and g gene is read through, allowing the production of the scfv-e tag-g fusion protein. in the presence of helper phage m13ko scfv fusion products were displayed on the recombinant phage tips, allowing affinity selection. in the regular panning method, small differences were in the stringency of selection between different rounds of panning, but in modified subtractive panning after two or three rounds of selection, the antigen-binding clones showed a clear enrichment. in modified subtractive panning, the stringency of panning was significantly increased after the fourth and fifth rounds of panning. during different selection rounds of modified subtractive panning, bound scfv-containing phages were eluted easily and quickly from the immobilized nmab-kt by using an elution buffer supplemented with hm- at concentration μg/ml. the phage enrichment for regular panning was not as impressive as for modified subtractive panning. for regular panning, the number of recaptured phages increased approximately 2-fold with low binding affinity in every round, but in modified subtractive panning, the number of recaptured phages increased - fold in the first three rounds of panning but increased 10-fold in the fourth and fifth rounds with higher binding strength to the antigen. thus, the overall enrichment for modified subtractive panning was approximately 10-fold higher than for regular panning after the final fifth round of panning. to verify the panning performance, we identified antigen-specific positive clones binding to the immobilized nmab-kt antigen. the binding properties of randomly selected clones binding to nmab-kt for both types of panning were measured by using microtiter plate enzyme-linked immunosorbent assay. the supernatant containing soluble scfv antibodies was added to each well of a microtiter plate that had been pre-coated with nmab-kt, and detection was done by using horseradish peroxidase -conjugated anti-e-tag antiboy. for regular panning, 24% tested clones of the infected e. coli tg contained the anti-idiotypic scfv gene of hm- killer toxin, and so soluble scfv showed positive elisa signals after the fifth round of panning. interestingly, for modified subtractive panning, 80% tested clones showed positive elisa signal, also after the fifth round of panning with higher binding strength to nmab-kt. positive binding affinity was defined as an absorbance for immobilized nmab-kt that was 2-fold above the background or negative controls. negative controls consisted of microtiter plates coated with blocking buffer or washing buffer. * randomly selected clones were grown overnight and supernatants were tested for binding to the immobilized antigen by elisa. to examine the integrity of the library after the final rounds of modified subtractive panning, individual colonies in e. coli tg were randomly isolated and the presence of the scfv dna insert in these clones was confirmed by using polymerase chain reaction amplification; these amplified products were analyzed on 4% agarose gels. among these colonies, were positive. only five restriction patterns were identified by the agarose gel electrophoresis; these patterns were confirmed by sequence data analysis. among these five restriction patterns, four patterns contained inserts of the expected size fragments and the other pattern contained a low molecular weight dna fragment. sequence analysis of scfv clones at least five unique genes were identified by nucleotide sequencing after the fifth round of modified subtractive panning. table showed the percentages of positive clones based on identical dna sequences. among all positive clones, k and k were identified as the highest populated group of clones after successful screening. among the other positive clones, k was 10% and k and k were each only 2%. figure showed amino acid sequences deduced from the obtained nucleotide data. all five sequences had different amino acid substitutions that were expected and mostly were in complementarity determining regions. the light chains of all clones were slightly different from each other, except scfv k for scfv k a light chain variable domain was partially synthesized together with an e-tag sequence. in the cdr area, the amino acid sequences shared relatively low sequence homology. apparently, the degree of homology was observed higher in the variable region of the light chain domain compared with the variable region of the heavy chain domain. k and k differed by only one amino acid at position in the vh domain. production and characterization of nmab-kt-specific scfv all individual positive clones after e. coli tg infection were reinfected into e. coli hb in the nonsuppressor strain of e. coli hb the amber stop codon between e-tag and g fusion protein was recognized as a stop codon, and a soluble scfv-e tag fusion protein was produced as a consequence. the expression of the scfv-e-tag fusion protein in the resultant e. coli hb clones was induced by isopropyl β-d-thiogalactopyranoside, and soluble scfv antibodies were secreted into the culture supernatants. after the final fifth round of modified subtractive panning, 80% clones were positive with a strong binding affinity to nmab-kt. however, in regular panning after the fifth round of panning only 24% were positive clones with low binding affinity. the nmab-kt-binding pattern of soluble scfv antibodies from these positive clones of e. coli hb was almost similar to that of the parent phage e. coli tg scfv antibodies, which was confirmed by using elisa. after screening, clones scfv k - k were identified, and soluble scfvs were purified from the periplasmic extract of e. coli hb on an anti-e-tag affinity column. sds-page and western blotting to check scfv expression the culture supernatant without anti-e-tag column purification, an approximately kda ) and two types of small scfv-e tag fusion protein were expressed from the reinfected e. coli hb. there expressions indicated that all positive clones originated from our original cdna library constructed by pcantab e vector using the e-tag sequence. four clones of purified soluble scfv showed two strong protein bands by using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by western blot analysis with hrp-conjugated anti-e-tag antibody. both proteins contained e-tag sequences, so western blotting using hrp-conjugated anti-e-tag antibody detected bands. for the short sequenced scfv k clone, a kda e-tagged protein band showed before purification, but after anti-e-tag column purification a protein band was not detected by using sds-page and western blotting. functional characterization and antifungal activity of recombinant scfvs in vitro antifungal activity of recombinant scfv antibodies was examined for non-pathogenic and pathogenic strains of fungal species saccharomyces cerevisiae and candida albicans, respectively. first, in vitro susceptibility of s. cerevisiae to scfv antibodies was examined by cell suspension assay as described in the materials and methods. among all scfv antibodies, scfv k was the most effective antifungal agent. figure 7b showed the elution profile and biological activity of purified scfv k against the growth of s. cerevisiae a next, to check the candidacidal activity of scfv k antibodies against c. albicans atcc cell growth was also measured by cell suspension assay. table showed the ic values of scfv k against s. cerevisiae a and c. albicans atcc cells growth in vitro. extensive studies by using our assay system showed that × 10- m and × 10- m were sufficient to produce 50% inhibition of growth of s. cerevisiae a and c. albicans atcc respectively. *ic values for recombinant scfv k against cell growth of candida and saccharomyces species were measured by using a cell suspension assay as described in the methods. kinetic parameters of selected clone scfv k1 the binding specificities and kinetic parameters of purified scfv k hm- and hyi killer toxin were determined by using surface plasmon resonance analysis. biacore sensograms showed the degree of interaction between immobilized nmab-kt and scfv k hm- or hyi. kinetic data from spr analysis showed that scfv k had a strong binding affinity for nmab-kt, like hm- but hyi had no binding affinity for nmab-kt. hm- analyte was used for the positive control and hyi analyte was used for the negative control experiment. table showed the calculated values of kon, koff, ka and kd. the kd values of scfv k and hm- were × 10- m and × 10- m, respectively. therefore, we can conclude that scfv k had strong reactivity with nmab-kt, like hm- but not with hyi. ligand nmab-kt was immobilized on the surface of a cm sensor chip, and scfv k hm- peptide or hyi peptide, were then injected over this immobilized surface in the presence of hbs-ep buffer. the affinity constant and dissociation constant were obtained from the ratio of the association and dissociation rate constants derived from global fits of response against time data to a 1: langmuir binding model. ka = kon/koff; kd = koff/kon; nd, signals not detected. hm- analyte was used for the positive control and hyi analyte was used for the negative control experiment. competitive binding of scfv k antibody with hm- to nmab-kt to examine whether scfv k antibody compete with hm- to bind with nmab-kt, a competitive binding elisa was performed. when the scfv k antibody concentration was increased, the binding of scfv k antibody to nmab-kt was also increased, but the hm- binding to nmab-kt was simultaneously decreased. these results suggest that scfv k antibody clone was homologous in competition with hm- for binding to nmab-kt. discussion anti-idiotypic antibodies representing the internal image of some antigenic determinants have been proposed as surrogate vaccines, and antibodies conjugated with toxins have been proposed for immunotherapy of cancer. in some cases, amino acid sequence homology exists between the protein antigen and the anti-idiotypic antibody variable region. antigenic mimicry is usually more functional than biochemical, and the functional mimicry of ligands of biological receptors by anti-idiotypic antibodies has been extensively studied. to select a useful and specific scfv antibody for a target antigen, a high quality primary phage library is needed. importantly, the library must have a large functional size to match diverse antibody sequences to facilitate the selection of a variety of high affinity scfvs, and these scfv genes must be well expressed to allow the panning process. previously, we established a large mouse scfv phage display library, and we attempted to isolate antigen-specific scfvs by using nmab-kt as a model antigen. in this study, after proper selection of the library, phage-displaying anti-idiotypic scfv antibodies represented the internal image of the original antigen hm- and thus might be effective as a vaccine, avoiding immunization with a pathogen. for traditional panning procedures, elution conditions are not same to recover a wide variety of specific phages from the immobilized antigens. sometimes bound phages are not easily dissociated from the immobilized antigen because of tight antigen-antibody interaction and attachment with the solid support of the tubes or plates. several methods have been reported to improve these problems to recover phage-displayed scfvs with the highest affinity, and some of these methods are applicable to the selection of anti-idiotypic antibodies. several elution methods that include the common acidic or basic conditions have been used to increase the stringency of selection. more acidic or basic conditions sometimes may disrupt the phage particles, and hence scfvs may lose or change its functional characteristics. so, we were proceeding an alternative way that will be more susceptible to get a high affinity binders to the specific antigen. therefore, during elution of the bound phages in modified subtractive panning, we selected neutral phosphate-buffered saline solution supplemented with hm- that shared structural or functional similarities with the active site of phage-displayed scfv antibodies. as a result, elution with hm- facilitates and favors the panning process through competition of binding to immobilized nmab-kt. this competition of binding accelerated the quick dissociation of scfv-containing phages from the immobilized antigen nmab-kt with increased binding strength. the binding strength and specificity were analyzed by using elisa with scfv antibodies against nmab-kt. figures 3a and 3b compared the bindings between antigen nmab-kt and scfv antibodies obtained after using regular and modified subtractive pannings. the elisa signals with soluble scfv antibodies from modified subtractive panning were much stronger than those from regular panning, indicating that the binding strength improved for elution with hm- in the selection procedure. for further confirmation of the binding specificity of phage-displayed scfvs to antigen nmab-kt after each round of panning, the number of recaptured phages was quantified. figure clearly demonstrated that after each round of panning the recaptured phages for modified subtractive panning were markedly higher than those for regular panning. although the regular panning experiments were successful, they failed to enrich the phage clones sufficiently may be because of less dissociation of tightly bound scfv-containing phages from the solid support of the tubes or plates during selection. to ascertain the scfv repertoire and the quality of the immunized antibody library, dna segments encoding the scfv genes from randomly selected positive clones were identified after the fifth round of modified subtractive panning. however, after the fifth round of regular phage display panning only 24% clones were positive but with low binding affinity. the positive clones that had higher binding affinity were amplified by using the polymerase chain reaction, and the amplified products were analyzed on agarose gels and their dna fingerprint patterns were compared. the patterns of these clones were found to vary, indicating an excellent diversity in the immunized mouse scfv library. nucleotide sequencing showed that at least five unique scfv genes were identified by using modified subtractive panning, i.e., clones scfv k1- scfv k were analyzed to deduce the amino acid sequences. dna sequencing also confirmed that the highest population of the positive clones was unique with a higher binding strength to the antigen nmab-kt. also, the second highest populated group scfv k had short dna sequences in the vl domain compared with the other groups. dna fingerprints of k and k - were apparently identical, although their amino acid sequences differed. therefore, we produced at least five different anti-idiotypic scfv antibody clones of hm- the scfvs, carrying the e-tag sequence and fused to the c-terminus, were purified by using an anti-e-tag affinity column and were analyzed on sds-page followed by western blotting performed against hrp/anti-e tag conjugate. silver-stained gel analysis of all purified scfvs showed a sharp, unique band of the expected mw of kda together with an unexpected band of lower mw; these bands were detected by western blotting with the hrp/anti-e-tag conjugate. the low mw kda bands had the e-tag sequence detected by western blotting, indicating that they might be proteolytic cleavage derivatives of the original kda scfvs. interestingly, scfv k expressed a kda smaller protein that also contained an e-tag sequence detected by western blotting before column purification of the sample. this kda scfv k had a partial vl region of the antibody, i.e., a residue of amino acids instead of amino acids that was confirmed from the dna sequence data. this kda scfv k represented prematurely terminated translation products because of mutation. purification of scfv k by using an anti-e-tag column was not possible to confirm by sds-page and western blotting. therefore, the e-tag containing short scfv k passed through the purification column without binding to the resin of the column that might be because of conformational changes due to partial lack of a light chain domain and might be because of cross-linking with other parts of the scfv antibody and therefore not having the open arm for binding with the antie-tag sequence of the purification column. scfv k that had the highest population of positive clones showed strong cytocidal activity against s. cerevisiae and candida species compared with other scfvs. so, for further experiments we selected scfv k to characterize the functional activity and specificity. in this study, the antifungal activity was evaluated by cell suspension assay as described in the materials and methods. the scfv k antibody showed strong antifungal activity against s. cerevisiae and c. albicans in vitro with ic values × 10- m for s. cerevisiae a and × 10- m for c. albicans atcc the anti-idiotypic scfv k had high affinities as judged by spr. biacore analysis indicated kd = × 10- m for scfv k which was comparable to the hm- killer toxin. therefore, we concluded that nmab-kt showed strong reactivity toward scfv k like hm- but not toward hyi. furthermore, the binding of scfv k antibody to nmab-kt was competitive with that of hm-, i.e., equimolar concentration of hm- inhibited the binding of scfv k antibody to the immobilized nmab-kt antigen. based on several factors, such as expression level, elisa using nmab-kt, biacore and fungal killing activity, scfv k was considered the most desirable scfv. so, scfv k is a candidate for future studies directed at killing of pathogenic fungal species using an scfv-drug conjugate or by development into a whole igg format for complement-mediated and antibody-dependent cytotoxic pathways.  in conclusion, we successfully obtained recombinant scfv antibodies with high specificity against nmab-kt directly from the phage-displayed scfv library of a hyper-immunized mouse. filamentous phage-displaying scfvs were selected against nmab-kt and were enriched through five rounds of modified subtractive panning. the scfv antibodies generated after modified subtractive panning appear to have superior binding properties than for regular panning. we showed a simple modification of the elution condition in the phage-display panning protocol, and so make a large difference in determining success. the deduced amino acid sequences of cdrs might also help us to create other scfv fragments with even higher affinity and specificity for nmab-kt and fungal cells by further mutagenesis study. 
to produce more selective and specific anti-idiotypic scfv-antibodies from a cdna library, constructed from hm- killer toxin -neutralizing monoclonal antibodies, the method was modified by using an elution buffer supplemented with hm- that shares structural and functional similarities with the active site of the scfv antibody. the major populations of these clones were genotypically same and had strong cytocidal activity against saccharomyces and candida species. the scfv k had strong reactivity toward nmab-kt, like hm- as judged by spr analysis. regular panning methods are sometimes complicated by inefficient detachment of the captured phages from the antigen-coated solid supports, which prompted us to modify. competitive binding of hm- to nmab-kt allowed easy and quick dissociation of scfv-displayed phages from immobilized nmab-kt to select specific anti-idiotypic scfv antibodies of hm- after modified panning, 80% clones showed several times higher binding affinity to nmab-kt than regular panning. a simple modification of the elution condition in the phage-display panning protocol makes a large difference in determining success. the scfv antibodies generated after modified subtractive panning appear to have superior binding properties and cytocidal activity than regular panning.
 phage-display panning is an integral part of biomedical research. regular panning methods are sometimes complicated by inefficient detachment of the captured phages from the antigen-coated solid supports, which prompted us to modify. here, we produce an efficient antigen-specific single chain fragment variable antibody by using a target-related molecule that favored selection ofrecombinant antibodies.  to produce more selective and specific anti-idiotypic scfv-antibodies from a cdna library, constructed from hm- killer toxin -neutralizing monoclonal antibodies, the method was modified by using an elution buffer supplemented with hm- that shares structural and functional similarities with the active site of the scfv antibody. competitive binding of hm- to nmab-kt allowed easy and quick dissociation of scfv-displayed phages from immobilized nmab-kt to select specific anti-idiotypic scfv antibodies of hm- after modified panning, 80% clones showed several times higher binding affinity to nmab-kt than regular panning. the major populations of these clones were genotypically same and had strong cytocidal activity against saccharomyces and candida species. the scfv k had strong reactivity toward nmab-kt, like hm- as judged by spr analysis.  the scfv antibodies generated after modified subtractive panning appear to have superior binding properties and cytocidal activity than regular panning. a simple modification of the elution condition in the phage-display panning protocol makes a large difference in determining success. our method offers an attractive platform to discover potential therapeutic candidates. 