the cancer diagnostic system of the present invention utilizes two - dimensional polyacrylamide gel electrophoretic techniques for the separation of proteins in human sera to generate cancer - specific isoform patterns and compositions indicative of cancer presence , tumor type , disease severity and therapeutic response . the protocol is designed for the detection of at least 20 cancer - specific tnox isoforms which are resolved to indicate cancer presence and disease severity . this specification illustrates the process of the isoform - resolving two - dimensional gel electrophoresis protocol and subsequent immunoanalysis to detect tnox isoforms which reflect particular cancers . two - dimensional gel electrophoresis separates by displacement in two dimensions oriented at right angles to one another and immunoblotting identifies the tnox isoforms . in the first dimension isoforms are separated according to charge ( pi ) by isoelectric focusing ( ief ). the isoforms are then separated according to size ( mr ) by sds - page in a second dimension . the isoforms are then blotted onto a nitrocellulose membrane for further analysis using a pan - cancer specific antibody preparation . rabbits were immunized and sera were prepared against a 33 . 5 kda component of culture medium conditioned by the growth of hela cells that exhibited an antitumor sulfonylurea -( morré et al . ( 1995 ) biochim . biophys . acta 1240 : 11 - 17 ) and capsaicin - inhibited ( morré et al . ( 1995 ) proc . natl . acad . sci . usa 92 : 1831 - 1835 ) nadh oxidase activity and was capable of binding ( 3 h )- ly181984 . these antisera cross - reacted on western blots with the 34 kda tnox protein from hela cells and with the 68 kda and 136 kda multimeric bands of the material from fplc separations enriched in capsaicin - inhibitable nadh oxidase activity . no reactivity was observed with preimmune sera . antibodies specific for the plasma membrane tnox and the shed forms in urine and serum of cancer patients and animals with neoplastic disorders are useful , for example , as probes for screening dna expression libraries or for detecting or diagnosing a neoplastic disorder in a sample from a human or animal . the antibodies ( or second antibodies which are specific for the antibody which recognizes tnox ) can be bound to a substance which provides a cofactor , inhibitor , fluorescent agent , chemiluminescent agent , magnetic particle or other detectable signal . suitable labels include but are not limited to radionuclides , enzymes , substrates , magnetic particles and the like . united states patents describing the use of such detectable moieties ( labels ) include , but are not limited to , u . s . pat . nos . 3 , 817 , 837 ; 3 , 850 , 752 ; 3 , 939 , 350 ; 3 , 996 , 345 ; 4 , 277 , 437 ; 4 , 275 , 149 ; 4 , 331 , 647 ; 4 , 348 , 376 ; 4 , 361 , 544 ; 5 , 444 , 744 ; 4 , 460 , 561 ; 4 , 624 , 846 ; 4 , 366 , 241 , 5 , 716 , 595 ; among others . for use in therapeutic regimens , the antibody of the present invention can be coupled to a therapeutic radionuclide , a chemotherapeutic agent , a ribonucleolytic agent or a toxin . see , among others , u . s . pat . nos . 5 , 541 , 297 , 6 , 395 , 276 . the invention may be further understood by the following non - limiting examples . 1 . add 40 μl of nickel agarose beads to a 0 . 5 ml microfuge tube a . shake beads to disrupt pellet and bring fully into solution b . cut about 2 mm off tip before using a . add 400 μl of deionized distilled - h 2 o to fill tubes b . vortex tubes , fully mixing beads for 2 - 3 seconds c . centrifuge tubes at 1 , 000 g for 30 seconds d . place tubes vertically in holder allowing beads to settle e . discard water / ethanol supernatant 3 . add 400 μl of serum to beads 4 . vortex tubes for 2 - 3 seconds to fully disrupt pellet 5 . incubate tubes on horizontal side at 4 ° c . on shaker overnight 3 . wash beads 3x with deionized distilled - h 2 0 as described above a . add 1 % dithiothreitol ( dtt ) to solution immediately before use ( 0 . 014 g / 1 . 4 ml ) 2 . add 150 μl of re - hydration solution to agarose bound sample 4 . remove immobiline drystrips ( amersham pharmacia biotech ) from freezer and allow strips to equilibrate to room temperature for 5 minutes ( but not more than 10 min prior to use ). 5 . label plastic back of strips with black felt tip pen . 6 . centrifuge sample at 1 , 000 g for 1 minute to pellet beads . 7 . place tubes in holder vertically and allow pellet to completely settle . 8 . load 125 μl of sample ( supernatant ) to level tray per 7 cm drystrip . 9 . place drystrips gel - side down over sample 10 . insure sample is evenly spread throughout strip by carefully lifting strip in and out of sample a few times if needed . 11 . if samples are concentrated in one region of the strip , redistribute sample by pipetting . 12 . remove air bubbles by gently pressing down on drystrip with pipette tip . 13 . place lid on tray , and place tray in plastic bag with wet paper towels . 14 . seal bag . 15 . allow sample to re - hydrate overnight ( between 12 - 24 hours ) at room temperature on level surface allowing strips to absorb samples . a . add 1 % dithiothreitol ( dtt ) to solution before use ( 0 . 014 g / 1 . 4 ml ) ( contacting with reducing agent ) 2 . add 135 μl of re - hydration solution per microfuge tube 3 . add 15 μl of sera per microfuge tube 4 . vortex tubes for 15 min 5 . remove immobiline drystrips ( amersham pharmacia biotech ) from freezer and allow strips to equilibrate to rt for 5 minutes ( but not more than 10 min .) 6 . label plastic back of strips 7 . load 125 of sample to level tray per 7 cm . drystrip . 8 . place drystrips gel - side down over sample 9 . insure sample is evenly spread throughout strip by carefully lifting strip in and out of sample a few times if needed . 10 . if samples are concentrated in one region of the strip , redistribute sample by pipetting . 11 . remove air bubbles by gently pressing down on drystrip with pipette tip . 12 . place lid on tray , and place tray in plastic bag w / wet paper towels . 13 . seal bag . 14 . allow sample to re - hydrate overnight at rt on level surface allowing strips to absorb samples . 1 . turn on ipgphor 3 ( amersham ) 2 . remove strips from tray and carefully remove excess sample from strips by blotting both sides with a tissue . 3 . place strips on manifold focusing tray ( amersham ) as follows a . gel side up b . positive ( acidic ) end towards back c . strips are aligned d . between metal strips ( so electrodes fit and touch metal strip ) 4 . employ 2 paper wicks ( amersham pharmacia , # 80 - 6499 - 14 ) per strip 5 . wet wicks with 150 μl distilled water per wick . 6 . place wicks over anodic and cathodic ends of gel ( approx . 0 . 3 cm ). 7 . place electrodes on wicks , but away from gel ( be sure prong is on metal plate ), and lock in place . 8 . cover strips , filling entire lane with drystrip cover fluid ( amersham pharmacia , # 17 - 1335 - 01 ) 9 . close apparatus 10 . ief run with ipgphor 3 ( amersham ) at a maximum amperage of 50 μamps at 20 ° c . as needed , pause run and replace wicks , continuing the run until dye front disappears . 1 . prior to use , wash and scrub plates very well in soap and hot water . 2 . rinse in distilled water 3 . leave the plates to air dry or wipe with methanol - soaked tissues . 4 . assemble plates in protean - plus multi - gel casting chamber 5 . ensure screws are fully tightened . 6 . degas gel solution prior to addition of aps and temed 7 . carefully pour degassed gel solution through gel plates to prevent formation of air bubbles . 8 . stop pouring when gel is about 3 cm from top of glass plates . 9 . gently overlay gels with distilled water . 10 . cover with plastic wrap . 11 . allow gels to polymerize for at least one hour 1 . remove strips from tray and place on whatman filter paper to remove excess oil . 2 . blot both sides of strips with tissue to remove excess oil . 3 . place strips in equilibration plate ( bio - rad ) gel side up ; freeze or equilibrate . if frozen , the plates are wrapped in plastic wrap and stored at − 80 ° c . then , the strips are thawed prior to equilibration ( strips are clear when thawed ). a . equilibrate : cover strips with equilibration buffer , about 1 . 5 ml per strip . 1 . prepare markers by adding 10 μl of standards on whatman 3mm chromatography paper cut to approximately 3 cm × 0 . 5 cm . 2 . decant equilibration buffer . 3 . cover strips in sds running buffer to rinse away excess equilibration buffer . 4 . remove equilibration buffer from strips . 5 . repeat steps 3 and 4 . 6 . carefully place strips gel side out on back plate of second dimensional gel . 7 . overlay strips with 1 % low melting agarose , at room temperature , ensure no air bubbles have formed under the gel . 8 . insert marker next to gel strip &# 39 ; s basic end , ensuring marker is flush to gel strip 9 . allow agarose to solidify 10 . continue for each strip to be loaded in second dimension 11 . place gels in dodeca tank , hinged side down 12 . after all gels have been put in tank , ensure gels are covered entirely by sds second dimension electrophoresis is at 13 ° c ., 250 volts for 1 hour 35 minutes . 1 . fill pyrex tray ( large enough to fit gel ) with transfer buffer 2 . place sponges in bio - rad transblot cell , 2 sponges per gel 3 . fill tank with transfer buffer to allow sponges to saturate with transfer buffer 4 . remove gel from dodeca tank and cut to desired size 5 . soak pre - cut transfer membrane in transfer buffer 6 . assemble transfer cassette as follows a . black side down b . sponge soaked in transfer buffer c . filter paper d . gel e . nitrocellulose membrane — once placed on gel do not move membrane f . filter paper g . sponge 7 . ensure all air bubbles have been removed between gel and membrane 8 . place tray in transblot tank , black side ( gel side ) of tray to black side of tank 9 . transfer at 4 ° c . at 100 volts for 60 minutes . 1 . put 50 ml of silver stain - fix in container add 25 μl 37 % formaldehyde 2 . put gel in solution and allow to shake for 1 hour to fix proteins 3 . carefully remove silver stain - fix 4 . cover gels with 50 ml of silver stain - wash and shake for 20 minutes . 5 . remove silver stain - wash solution 6 . add 50 ml . of silver stain - pretreat and shake by hand for 1 minutes . 7 . carefully remove silver stain - pretreat a . remove distilled water b . repeat rinse two more times 9 . cover gels with 50 ml of silver stain - impregnate and shake for 20 min . 10 . carefully rinse gels in distilled water for 20 seconds . d . remove distilled water e . repeat rinse two more times 13 . cover gels with 50 ml . of silver stain - fix 14 . shake gels to fix proteins for 10 minutes . 7 . add 5 % milk ( enough to cover membrane ) to tray and block for 20 minutes at room temperature 8 . prepare primary antibody solution ( nti scfv mab 12 . 1 ®: ttbs - 1 : 150 ) in container i . cover membrane with ttbs ii . gently shake at room temperature for 10 minutes . adjust ph to 8 . 8 with hcl , and make up to a total volume of 200 ml with distilled water . % t = total percent of acrylamide and piperazine diacrylyl in the 2d acrylamide gel store the buffer at 4 ° c . for no more than 2 weeks . filter ( 0 . 45 μm ), store at 4 ° c . for no more than 2 weeks . weigh 1 . 5 g of agarose , add 150 ml sds running buffer - add trizma and nacl to 3800 ml deionized distilled - h 2 o nox - enriched serum proteins ( approximately 4 - 6 mg ) from sera pooled from cancer patients ( breast , ovarian , lung and colon ) were resolved by 2 - d gel electrophoresis with detection by recombinant anti - ecto - nox antibody ( single chain variable region scfv ) carrying an s - tag followed by alkaline phosphatase - linked anti - s with western blue nbt alkaline phosphatase substrate yield several proteins present in the cancer sera ( fig1 ) but absent from sera of non - cancer patients or healthy volunteers . the tnox proteins were enriched and concentrated from the sera by nickel agarose precipitation . this step results in removal of approximately 90 % of serum proteins prior to analysis . where sera specific for tnox or ecto - nox are used , typically the dilution is from 1 : 10 to 1 : 1000 , monoclonal antibody ( e . g ., 12 . 1 produced by hybridoma on deposit as atcc accession no . pta - 4206 ) is from 1 ; 100 - 1 : 10 , 00 , desirably about 1 : 100 , or ascites fluid from growth of the same hybridoma can be from about 1 : 10 to about 1 : 1000 , desirably about 1 : 100 . results combined from a total of eight different pooled cancer sera were similar to those of fig1 and the total of the findings are summarized in fig2 for quadrant i of the 2 - d gels . some additional tnox proteins were located in quadrant iv and the total of the findings are summarized in fig3 . 2 - d gel analysis as in fig4 when applied to serum from a patient with ovarian cancer yielded ovarian cancer specific tnox proteins of about 80 and 40 . 5 kda in quadrants i and iv ( fig4 ). 2 - d gel analysis as in fig5 when applied to sera of a patient with small cell lung cancer contained a 40 . 5 kda tnox protein in quadrant iv and a 52 kda tnox protein in quadrant i ( fig5 ). example 5 . analysis of non small cell lung cancer patient serum 2 - d gel analysis as in fig1 when applied to plasma from a patient with non - small cell lung cancer revealed a non - small cell cancer specific tnox isoform at 54 kda in quadrant 1 ( fig6 ). 2 - d gel analysis as in fig1 when applied to sera from a patient with breast cancer revealed a breast cancer - specific tnox protein of 68 kda in quadrant i ( fig7 ). 2 - d gel analysis as in fig1 when applied to sera from a patient with prostate cancer revealed prostate cancer - specific tnox isoforms at 75 kda ( fig8 ) ) and isoelectric points of ph 5 . 8 ( α ) and 5 . 2 ( β ). the β - form is most evident with plasma and present infrequently in sera of prostate cancer patients . 2 - d gel analysis as in fig1 when applied to serum from a patient with cervical cancer revealed a cervical cancer - specific tnox isoform at 94 kda ( fig9 ). 2 - d gel analysis as in fig1 when applied to serum from a patient with colon cancer revealed colon cancer - specific tnox isoforms at 43 and 52 kda ( fig1 ). for each kind of cancer there appears to be a tnox isoform ( ovarian , breast , cervical , colon , non - small cell lung , prostate small cell lung ) or combination of tnox isoforms that is specific to the tissue or cell type of origin for the cancer ( fig1 ). the 2 - d gel of fig1 is from a patient with cancer where the primary tumor was unknown . the presence of 40 . 5 , 52 and ca . 80 kda tnox isoforms indicates the primary cancer is ovarian cancer . in more than 25 randomly selected outpatient sera and sera of healthy volunteers , both quadrants i and iv of the 2 - d gels were devoid of tnox isoforms ( fig1 ), confirming previous observations that tnox proteins are absent from non - cancer patients or sera of healthy volunteers . the diagnostic strategy of the invention combines one - and two - dimensional polyacrylamide gel electrophoretic separations of human sera to generate cancer specific isoform patterns and compositions indicative of cancer presence , tumor type , disease severity and therapeutic response . at least 20 cancer - specific tnox isoforms are resolved indicative of cancer presence and disease severity . the system consists of a concentration step where ecto - nox proteins are separated from the bulk of the albumin and other serum proteins using a nickel - agarose precipitation . detection uses a recombinant single chain antibody ( scfv ) that cross reacts with all known ecto - nox isoforms of human origin ; this antibody also has an s - tag that reacts with a second antibody i ( anti - s ). the scfv antibody is described in u . s . provisional application 60 / 824 , 398 , filed sep . 1 , 2006 and below . monoclonal antibody generated against tnox nadh oxidase tumor cell specific was produced in sp - 2 myeloma cells ; however , the monoclonal antibody slowed the growth of sp - 2 myeloma cells that were used for fusion with spleen cells after 72 h . this phenomenon made it difficult to produce antibody in quantity . to overcome this problem , the coding sequences of the antigen - binding variable region of the heavy chain and the light chain ( fv region ) of the antibody cdna were cloned and linked into one chimeric gene , upstream of the s - tag coding sequence . the fv portion of an antibody , consisting of variable heavy ( v h ) and variable light ( v l ) domains , can maintain the binding specificity and affinity of the original antibody ( glockshuber et al . 1990 . biochemistry 29 : 1262 - 1367 ). for a recombinant antibody , cdnas encoding the variable regions of immunoglobulin heavy chain ( v h ) and light chain ( v i ), are cloned by using degenerative primers . mammalian immunoglobulins of light and heavy chain contain conserved regions adjacent to the hypervariable complementary defining regions ( cdrs ). degenerate oligoprimer sets allow these regions to be amplified using pcr ( jones et al . 1991 . bioltechnology 9 : 88 - 89 ; daugherty et al . 1991 . nucleic acids research 19 : 2471 - 2476 ). recombinant dna techniques have facilitated the stabilization of variable fragments by covalently linking the two fragments by a polypeptide linker ( huston et al . 1988 . proc . natl . acad . sci . usa 85 : 5879 - 5883 ). either v l or v h can provide the nh 2 - terminal domain of the single chain variable fragment ( scfv ). the linker should be designed to resist proteolysis and to minimize protein aggregation . linker length and sequences contribute and control flexibility and interaction with scfv and antigen . the most widely used linkers have sequences consisting of glycine ( gly ) and serine ( ser ) residues for flexibility , with charged residues as glutamic acid ( glu ) and lysine ( lys ) for solubility ( bird et al . 1988 . science 242 : 423 - 426 ; huston et al . 1988 . supra ). total rna was isolated from the hybridoma cells producing tnox - specific monoclonal antibodies by the following procedure modified from chomczynski et al . ( 1987 ) anal . biochem . 162 : 156 - 159 and gough ( 1988 ) anal . biochem 176 : 93 - 95 . cells were harvested from medium and pelleted by centrifugation at 450 × g for 10 min . pellets were gently resuspended with 10 volumes of ice cold pbs and centrifuged again . the supernatant was discarded and cells were resuspended with an equal volume of pbs . denaturing solution ( 0 . 36 ml of 2 - mercaptoethanol / 50 ml of guanidinium stock solution - 4m guanidinium thiocyanate , 25 mm sodium citrate , ph 7 . 0 , 0 . 5 % sarkosyl ) 10 ml per 1 g of cell pellet was added prior to use and mixed gently . sodium acetate ( ph 4 . 0 , 1 ml of 2m ), 10 ml of phenol saturated water and 2 ml of chloroform : isoamyl alcohol ( 24 : 1 ) mixtures were sequentially added after each addition . the solution was mixed thoroughly by inversion . the solution was vigorously shaken for 10 sec , chilled on ice for 15 min and then centrifuged 12 , 000 × g for 30 min . the supernatant was transferred and an equal volume of 2 - propanol was added and placed at − 20 ° c . overnight to precipitate the rna . the rna was pelleted for 15 min at 12 , 000 × g , and the pellet was resuspended with 2 - 3ml of denaturing solution and 2 volumes of ethanol . the solution was placed at − 20 ° c . for 2 h , and then centrifuged at 12 , 000 × g for 15 min . the rna pellet was washed with 70 % ethanol and then 100 % ethanol . the pellet was resuspended with rnase - free water ( depc - treated water ) after centrifugation at 12 , 000 × g for 5 min . the amount of isolated rna was measured spectrophotometrically and calculated from the absorbance at 280 nm and 260 nm . the poly ( a ) mrna isolation kit was purchased from stratagene . total rna was applied to an oligo ( dt ) cellulose column after heating the total rna at 65 ° c . for 5 min . before applying , the rna samples were mixed with 500 μl of 10 × sample buffer ( 10 mm tris - hcl , ph 7 . 5 , 1 mm edta , 5 m nacl ). the rna samples were pushed through the column at a rate of 1 drop every 2 sec . the eluates were pooled and reapplied to the column and purified again . preheated elution buffer ( 65 ° c .) was applied , and mrna was eluted and collected in 1 . 5 ml of centrifuge tubes on ice . the amount of mrna was determined at od 260 ( 1 od unit = 40 μg of rna ). the amounts of total rna and mrna obtained from 4 × 10 8 cells were 1328 μg and 28 μg , respectively . mrna ( 1 - 2 μg ) dissolved in depc - treated water was used for cdna synthesis . mrna isolated on three different dates was pooled for first - strand cdna synthesis . the cdna synthesis kit was purchased from pharmacia biotech . mrna ( 1 . 5 μg / 5 μl of depc - treated water ) was heated at 65 ° c . for 10 min . and cooled immediately on ice . the primed first strand mix containing mulv reverse transcriptase ( 11 μl ) and appropriate buffers for the reaction were mixed with mrna sample . dtt solution ( 1 μl of 0 . 1 m ) and rnase - free water ( 16 μl ) also were added to the solution . the mixture was incubated for 1 h at 37 ° c . degenerate primers for light chain and heavy chain ( novagen , madison , wis .) were used for pcr . pcr synthesis was carried out in 100 μl reaction volumes in 0 . 5 ml microcentrifuge tubes by using robocycler ( stratagene , la jolla , calif .). all pcr syntheses included 2 μl of sense and anti - sense primers ( 20 pmoles / μl ), 1 μl of first - strand cdna as a template , 2 μl of 10 mm of dntps , 1 μl of vent polymerase ( 2 units / μl ), 10 μl of 10 × pcr buffer ( 100 mm tris - hcl , ph 8 . 8 at 25 ° c ., 500 mm kcl , 15 mm mgcl 2 , 1 % triton x - 100 ), 82 μl of h 2 o . triton x - 100 is t - octylphenoxypolyethoxyethanol . all pcr profiles consisted of 1 min of denaturation at 94 ° c ., 1 min of annealing at 55 ° c ., and 1 min of extension at 72 ° c . this sequence was repeated 30 times with a 6 - min extension at 72 ° c . in the final cycle . pcr products were purified with qiaex ii gel extraction kit from qiagen , valencia , calif . pcr amplification products for heavy and light chain coding sequences were analyzed by agarose gel electrophoresis and were about 340 base pair ( bp ) long and 325 bp long , respectively . total rna or dna was analyzed by agarose gel electrophoresis ( 1 % agarose gels ). agarose ( 0 . 5 g in 50 ml of tae buffer , 40 mm tris - acetate , 1 mm edta ) was heated for 2 min in a microwave to melt and evenly disperse the agarose . the solution was cooled at room temperature , and ethidium bromide ( 0 . 5 μg / ml ) was added and poured into the apparatus . each sample was mixed with 6 × gel loading buffer ( 0 . 25 % bromophenol blue , 0 . 25 % xylene cyanol ff , 40 % ( w / v ) sucrose in water ). tae buffer was used as the running buffer . voltage ( 10 v / cm ) was applied for 60 - 90 min . according to the proper size for heavy and light chain cdnas , the bands were excised from the gels under uv illumination , and excised gels were placed in 1 ml syringes fitted with 18 - gauge needles . gels were crushed to a 1 . 5 ml eppendorf tube . the barrel of each syringe was washed with 200 μl of buffer - saturated phenol ( ph 7 . 9 ± 0 . 2 ). the mixture was thoroughly centrifuged and frozen at − 70 ° c . for 10 min . the mixture was centrifuged for 5 min , and the top aqueous phase was transferred to a new tube . the aqueous phase was extracted again with phenol / chloroform ( 1 : 1 ). after centrifuging for 5 min , the top aqueous phase was transferred to a clean tube , and chloroform extraction was performed . sodium acetate ( 10 volumes of 3 m ) and 2 . 5 volumes of ice - cold ethanol were added to the top aqueous phase to precipitate dna at − 20 ° c . overnight . purified heavy and light chain cdnas were ligated into plasmid pstblue - 1 vector and transfected into novablue competent cells ( stratagene ). colonies containing heavy and light chain dnas were screened by blue and white colony selection and confirmed by pcr analysis . heavy and light chain dnas were isolated and sequenced using standard techniques . tables 2a and 2b show the dna sequences of heavy and light chain dnas of scfv . see also seq id no : 3 and seq id no : 4 . pcr amplification and the assembly of single scfv gene was according to davis et al . ( 1991 ) bio / technology 9 : 165 - 169 . plasmid pstblue - 1 carrying v h and v l genes were combined with all four oligonucleotide primers in a single pcr synthesis . following first pcr synthesis , one tenth of the first pcr product was removed and added to a second pcr reaction mixture containing only the primer a ( v h sense primer ) and primer d ( v l antisense primer ). the product of the second pcr synthesis yielded single scfv gene . the single scfv gene was ligated to plasmid pt - adv ( clontech , palo alto , calif .). pt - adv carrying scfv gene was used for dna sequencing . the complete scfv gene was assembled from the v h , v l and linker genes to yield a single scfv gene by pcr ( tables 2a and 2b ). the dna sequence encoding the linker was 45 nucleotides long ( ggaggcggtggatcgggcggtggcggctcgggtggcggcggctct ; seq id no : 6 ), which translates to a peptide of 15 amino acids ( glyglyglyglyserglyglyglyglyserglyglyglyglyser ; seq id no : 5 ). primers for pcr amplification are shown in tables 2a and 2b . s - peptide was linked to the c - terminus of scfv [ scfv ( s )]. s - peptide binds to s - protein conjugated to alkaline phosphatase for western blot analysis . the dna sequence of the s - peptide is aaagaaaccgctgctgctaaattcgaacgccagcacatggacagc ( seq id n 0 : 7 ) which translates to s - peptide ( lysgluthralaalaalalysphegluargglnhismetaspser ; seq id no : 8 ). recombinant scfv ( s ) was expressed in e . coli . first , oligo nucleotides encoding s - peptide were linked to the 3 ′ end of the open reading frame ( orf ) of scfv dna by pcr amplification . incorporation of s - peptide enables to detect expressed scfv protein by s - protein conjugated to alkaline phosphatase . the tnox - specific scfv ( s ) coding sequence was then subcloned to plasmid pet - 11a , a plasmid designed for protein expression in e . coli ( stratagene , calif .). for pcr amplification , two primers were designed to amplify orf of scfv ( s ) containing endonuclease restriction sites ( ndel and nhel ) and s - peptide residues . plasmid pet - 11a and orf of scfv ( s ) were digested with restriction enzymes ndel and nhel and ligated to produce plasmid pet11 - scfv ( s ). e . coli bl21 ( de3 ) was transformed with pet11 - scfv ( s ) and grown at 37 ° c . for 12 h in lb medium containing ampicillin ( 100 μg / ml ). scfv was expressed by addition of 0 . 5 mm iptg and incubation for 4 h . cells were harvested and lysed using a french pressure cell ( french pressure cell press , slm instruments , inc .) ( three passages at 20 , 000 psi ). cell extracts were centrifuged at 10 , 000 × g for 20 min . pellets containing denatured inclusion bodies of scfv were collected . renaturation of the inclusion bodies of the scfv was according to goldberg et al . ( 1995 ) folding & amp ; design 1 : 21 - 27 . dna sequence of heavy chain scfv ( v h ), seq id no : 1 dna sequence of light chain scfv ( v l ), seq id no : 2 standard techniques for cloning , dna isolation , amplification and purification , for enzymatic reactions involving dna ligase , dna polymerase , restriction endonucleases and the like , and various separation techniques are those known and commonly employed by those skilled in the art . a number of standard techniques are described in sambrook et al . ( 1989 ) molecular cloning , second edition , cold spring harbor laboratory , plainview , n . y . ; maniatis et al . ( 1982 ) molecular cloning , cold spring harbor laboratory , plainview , n . y . ; wu ( ed .) ( 1993 ) meth . enzymol . 218 , part i ; wu ( ed .) ( 1979 ) meth . enzymol . 68 ; wu et al . ( eds .) ( 1983 ) meth . enzymol . 100 and 101 ; grossman and moldave ( eds .) meth . enzymol . 65 ; miller ( ed .) ( 1972 ) experiments in molecular genetics , cold spring harbor laboratory , cold spring harbor , n . y . ; old and primrose ( 1981 ) principles of gene manipulation , university of california press , berkeley ; schleif and wensink ( 1982 ) practical methods in molecular biology ; glover ( ed .) ( 1985 ) dna cloning vol . i and ii , irl press , oxford , uk ; hames and higgins ( eds .) ( 1985 ) nucleic acid hybridization , irl press , oxford , uk ; setlow and hollaender ( 1979 ) genetic engineering : principles and methods , vols . 1 - 4 , plenum press , new york ; fitchen , et al . ( 1993 ) annu . rev . microbiol . 47 : 739 - 764 ; tolstoshev , et al . ( 1993 ) in genomic research in molecular medicine and virology , academic press ; and ausubel et al . ( 1992 ) current protocols in molecular biology , greene / wiley , new york , n . y . abbreviations and nomenclature , where employed , are deemed standard in the field and commonly used in professional journals such as those cited herein . antibody vaccines are described in dillman r . o . ( 2001 ) cancer invest . 19 ( 8 ): 833 - 841 . durrant l . g . et al . ( 2001 ) int j . cancer 1 ; 92 ( 3 ): 414 - 20 and bhattacharya - chatterjee m , ( 2001 ) curr . opin . mol . ther . feb ; 3 ( 1 ): 63 - 9 describe anti - idiotype antibodies . many of the procedures useful for practicing the present invention , whether or not described herein in detail , are well known to those skilled in the arts of molecular biology , biochemistry , immunology , and medicine . monoclonal , polyclonal antibodies , peptide - specific antibodies or single chain recombinant antibodies and antigen binding fragments of any of the foregoing , specifically reacting with the tnox isoform proteins described herein , may be made by methods known in the art . see e . g ., harlow and lane ( 1988 ) antibodies : a laboratory manual , cold spring harbor laboratories ; and goding ( 1986 ) monoclonal antibodies : principles and practice , 2d ed ., academic press , new york . all references cited in the present application are incorporated by reference herein to the extent that there is no inconsistency with the present disclosure . such references reflect the skill in the arts relevant to the present invention . the examples provided herein are for illustrative purposes , and are not intended to limit the scope of the invention as claimed herein . any variations in the exemplified antibodies , epitopes , purification methods , diagnostic methods , preventative methods , treatment methods , and other methods which occur to the skilled artisan are intended to fall within the scope of the present invention . 1 . morré , d . j . 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