METHODS AND COMPOSITIONS FOR SINGLE CHAIN VARIABLE REGION ENOX2 ANTIBODIES FOR CANCER DETECTION AND DIAGNOSIS

Abstract

Cancers of different cellular or tissue origins express different ENOX2 cancer isoforms or combinations of isoforms and shed these proteins into the circulation. Herein are disclosed methods both for cancer detection and diagnosis of particular origin, based on the patterns and molecular weights of the isoforms which allow the identification of the cell type and or tissue of origin of the neoplasm. Relative ENOX2 amounts are proportional to tumor burden and provide a reliable measure of response to therapy and disease progression. Also provided is the amino acid sequence to which the scFv antibodies bind as the molecular basis for the specificity of the test.

Claims

1. A cell that expressed an antibody that is a single chain variable region ENOX2-specific antibody fusion protein, wherein the antibody is fused with an alkaline phosphatase or other protein for single-step detection or imaging.

2. The cell of claim 1, wherein the cell is a bacterium.

3. The cell of claim 1, wherein the cell is a mammalian cell.

4. A method for detecting cancer comprising: obtaining a sample from a patient suspected of comprising ENOX2 variants associated with a cancer; and detecting isoforms of the ENOX2 protein using a single chain variable region ENOX2 antibody fusion protein, wherein the antibody is fused with an alkaline phosphatase or other single-step protein for detection or imaging, wherein expression of ENOX2 variants associated with cancer are indicative that the subject has or will develop that cancer.

5. A method for producing a single chain variable region ENOX2 antibody comprising introducing into a cell a DNA sequence of SEQ ID NOS:1 and 2, and growing the cells to produce a composition containing a single chain variable region ENOX2 antibody.

6. The method of claim 5, wherein the DNA sequence includes DNA specifically producing the polypeptide linker sequence substantially as shown in SEQ ID NO:5 or 16.

7. The method of claim 5, wherein the DNA sequence comprises the sequence substantially as shown in SEQ ID NO:3.

8. The method of claim 5, wherein the DNA sequence additionally includes DNA for a detectable marker.

9. The method of claim 7, wherein the DNA sequence for alkaline phosphatase.

10. The method of claim 5, wherein the cell is E. coli.

11. The cell of claim 5, wherein the cell is a mammalian cell.

12. A method for detecting cancer comprising detecting isoforms of the ENOX2 protein in comparison with either -antitrypsin inhibitor or serrotransferrin reference values.

13. The method of claim 12, wherein the method detects isoforms of the ENOX2 protein in comparison with both -antitrypsin inhibitor and serrotransferrin reference values.

14. The method of claim 12, wherein the comparison is done by the relative positions on electrophoresis medium, whereby differentiation among different forms of cancer is facilitated.

15. The method of claim 12, wherein the comparison is of the different isoforms of the ENOX2 protein on a 2-D gel.

16. The method of claim 12, further comprising the step of providing an isolated amino acid sequence immediately adjacent to the amino acid sequence providing the critical antigen sequence required for the ENOX2-specific antibody to specifically bind ENOX2 that provides for recognition of two reference proteins a 53 kDa isoelectric point pH 4.1, mostly phosphorylated al trypsin inhibitor (2-HS-glycoprotein; fetuin A) and an isoelectric point reference and a 79-85 kDa, isoelectric point pH 6.8 serotransferrin.

17. An isolated amino acid sequence immediately adjacent to the amino acid sequence providing the critical antigen sequence required for the ENOX2-specific antibody to specifically bind ENOX2 that provides for recognition of two reference proteins a 53 kDa isoelectric point pH 4.1, mostly phosphorylated al trypsin inhibitor (2-HS-glycoprotein; fetuin A) and an isoelectric point reference and a 79-85 kDa, isoelectric point pH 6.8 serotransferrin.

18. A single chain variable region ENOX2 antibody linked to an alkaline phosphatase or other single-step agent for detection or imaging.

19. A nucleic acid vector that expresses a single chain variable region ENOX2 antibody fusion protein, wherein the antibody is fused with an alkaline phosphatase or other single-step protein for detection or imaging.

20. The nucleic acid vector of claim 19, wherein the nucleic acid encoding the single chain variable region ENOX2 antibody fusion protein has SEQ ID NO: 3.

21. The nucleic acid vector of claim 19, wherein the single chain variable region ENOX2 antibody fusion protein when expressed comprises the linker of SEQ ID NOS:5, 16, or both.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is a schematic representation of the utility of the ENOX2 family of cancer-specific, cell surface proteins for early diagnosis and early intervention of cancer. Cancer site-specific transcript variants of ENOX2 are shed into the serum to permit early detection and diagnosis. The ENOX2 proteins of origin at the cell surface act as terminal oxidases of plasma membrane electron transport functions essential to the unregulated growth of cancer. When the ENOX2 proteins are inhibited, as for example through EGCg/Capsicum synergies, the unregulated growth ceases and the cancer cells undergo programmed cell death (apoptosis).

[0017] FIGS. 2A-2B provide a 2-Dimensional gel/western blot of ENOX2 transcript variants comparing pooled non-cancer (FIG. 2A) and pooled cancer representing major carcinomas plus leukemias and lymphomas (FIG. 2B) patient sera. The approximate location of unreactive (at background) albumin is labeled for comparison. ENOX2 reactive proteins are restricted to quadrants I and IV. Detection use recombinant scFv-S (LysGluThrAlaAlaAlaLysPheGluArgGln HisMetAspSer; SEQ ID NO:8) antibody linked with alkaline phosphatase. The approximately 10 ENOX2 transcript variants of the pooled cancer sera are absent from non-cancer (A) and are cancer site-specific.

[0018] FIGS. 3A-3J show western blots of 2-D gel electrophoresis/western blots of sera from cancer patients analyzed individually. Cancer sites are presented in the order of decreasing molecular weight of the major transcript variant present. FIG. 3A. Cervical cancer. FIG. 3B. Ovarian cancer. FIG. 3C. Prostate cancer. FIG. 3D. Breast cancer. FIG. 3E. Non-small cell lung cancer. FIG. 3F. Small cell lung cancer. FIG. 3G. Pancreatic cancer. FIG. 3H. Colon cancer. FIG. 3I. Non-Hodgkins lymphoma. FIG. 3J. Melanoma. The approximate location of unreactive (at background) albumin (Ab) is labeled for comparison. Approximately 180 non-cancer patient sera were analyzed in parallel without evidence of proteins indicative of specific transcript variants.

[0019] FIGS. 4A-4B show analytical gel electrophoresis and immunoblots of patient sera. FIG. 4A. Sera from a patient with non-small cell lung cancer contains a 54 kDa, isoelectric point pH 5.1 transcript variant (arrow). FIG. 4B. Sera from a patient with small cell lung cancer contains a 52 kDa, isoelectric point 4.3 transcript variant (arrow). The reference spots to the right, Mr 52 kDa and isoelectric point pH 4.1 are al-antitrypsin inhibitor. Albumin and other serum proteins are unreactive.

[0020] FIGS. 5A-5E show 2-D gel electrophoretic separations and detection of ENOX2 transcript variants specific for breast cancer by western blotting of patient sera in FIGS. 5A-5E. Arrow=66 to 68 kDa breast cancer specific transcript variant. R=52 kDa, isoelectric point pH 4.1 al-antitrypsin inhibitor reference spot.

[0021] FIG. 6 is an interpretive diagram to illustrate the various stages of cancer progression (estimated to require as long as 20 y) beginning with a cancer-causing event (initiation) through development of a clinically defined malignancy.

DETAILED DESCRIPTION OF THE INVENTION

[0022] 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 ENOX2 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 ENOX2 isoforms which reflect particular cancers.

[0023] Two-dimensional gel electrophoresis separates by displacement in two dimensions oriented at right angles to one another and immunoblotting identifies the ENOX2 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.

[0024] Ecto-Nicotinamide Adenine Dinucleotide Oxidase DisulfideThiol Exchanger 2 (ENOX2) (GenBank accession no. AF207881; Chueh et al., 2002) also known as Tumor Associated Nicotinamide Adenine Dinucleotide Oxidase (ENOX2) is ideally suited as a target for early diagnosis as well as for early preventive intervention (FIG. 1). The proteins are expressed on the cell surface of malignancies and detectable in the serum of patients with cancer (Cho et al., 2002). ENOX2 proteins are terminal hydroquinone oxidases of plasma membrane electron transport. From the standpoint of early intervention, they are important in the growth and enlargement of tumor cells (Morre and Morre, 2003; Tang et al., 2007. Biochemistry 46:12337-12346; 2008. Oncol. Res. 16:557-567). Our approach using ENOX2, as a target for both early detection and for early interventions, is based on these properties (Cho et al., 2002; Morre and Morre, 2003; reviewed by Davies and Bozzo, 2005. Drug News Perspect 19: 223-225). While ENXO2 presence provides a non-invasive approach to cancer detection, without methodology to identify cancer site-specific ENOX2 forms, it did not offer an indication as to cancer type or location.

[0025] The opportunity to simultaneously determine both cancer presence and cancer site emerged as a result of 2-dimensional gel electrophoretic separations where western blots with a pan ENOX2 recombinant single chain variable region (ScFv) antibody carrying an S tag (FIGS. 2A and 2B) was employed for detection (Hostetler et al., 2009. Clin. Proteomics 5: 46-51). The antibody cross reacted with all known ENOX2 forms from hematological and solid tumors of human origin but, of itself, did not differentiate among different kinds of cancers. Analyses using this antibody, when combined with two-dimensional gel electrophoretic separation, revealed specific ENOX2 species subsequently identified as transcript variants, each with a characteristic molecular weight and isoelectric point indicative of a particular form of cancer (Hostetler et al, 2009; Table I).

[0026] ENOX transcript variants of specific molecular weights and isoelectric points are produced uniquely by patients with cancer. The proteins are shed into the circulation and have the potential to serve as definitive, non-invasive and sensitive serum markers for early detection of both primary and recurrent cancer in at risk populations with a low incidence of false positives, as they are molecular signature molecules produced specifically by cancer cells and absent from non-cancer cells.

[0027] As the 2-D-western blot protocol detects cancer early, well in advance of clinical symptoms. The opportunity to combine early detection with early intervention as a potentially curative prevention strategy for cancer by eliminating the disease in its very earliest stages is unique.

[0028] Analytical 2-D gel electrophoresis and immunoblotting of ENOX proteins from a mixed population of cancer patients (cervical, breast, ovarian, lung and colon carcinomas, leukemias and lymphomas) revealed multiple species of acidic proteins of molecular weight between 34 and 100 kDa in quadrants I and IV (FIG. 1), none of which were present in sera of non-cancer patients (FIG. 1) (Hostetler et al., 2009). Separation in the first dimension was by isoelectric focusing over the pH range of 3 to 10 and separation in the second dimension was by 10 percent SDS-PAGE. Isoelectric points of the ENOX2 transcript variants were in the range of 3.9 to 6.3. The principal reactive proteins other than the ENOX2 forms were a 53 kDa isoelectric point pH 4.1, mostly phosphorylated al-antitrypsin inhibitor (2-HS-glycoprotein; fetuin A) (Labeled R in FIGS. 2A and 2B) which served as a convenient loading control and isoelectric point reference and a 79-85 kDa, isoelectric point pH 6.8 serotransferrin which served as a second point of reference for loading and as an isoelectric point reference (Table II). The two cross reactive reference proteins are present in a majority of sera and plasma of both cancer and non-cancer subjects. Albumin and other serum proteins do not react. On some blots, the recombinant scFv was weakly cross-reactive with heavy (ca. 52 kDa) and light (ca. 25 kDa) immunoglobulin chains.

[0029] Sera from individual patients with various forms of cancer were analyzed by 2-D gel electrophoresis and immunoblotting to assign each of the ENOX2 isoforms of FIGS. 2A and 2B to a cancer of a particular tissue of origin (Table 1). Sera of breast cancer patients contained only the 64 to 68 kDa ENOX2 (FIG. 3D) and the al-antitrypsin inhibitor reference protein (FIGS. 3A and 3J). Sera from cervical cancer patients contained the 94 kDa ENOX2 transcript variant (FIG. 3A). Sera from ovarian cancer patients contained ENOX1 transcript variants of 80 kDa and 40.5 b kDa (FIG. 3B). Sera from patients with prostate cancer contained one or more 75 kDa ENOX2 transcript variants resulting in small variations in isoelectric points (FIG. 3C). Sera from patients with non-small cell lung carcinoma contained a 52 kDa ENOX2 transcript variant while sera from non-small cell lung carcinoma patients contained a of 52 kDa ENOX2 transcript variant (FIGS. 3E and 3F and FIGS. 4A and 4B). ENOX2 transcript variants of 50 and 52 kDa characterized sera of pancreatic cancer patients (FIG. 3G) whereas sera of colon cancer patients contained ENOCX2 transcript variants of 52 kDa and 43 kDa (FIG. 3H). FIG. 3I from sera of a patient with non-Hodgkin's lymphoma illustrates the 45 kDa ENOX2 transcript variant of low isoelectric point characteristic of leukemias and lymphomas. Sera of patients with malignant melanoma contained an ENOX2 transcript variant of 38 kDa (FIG. 3J).

[0030] Particularly relevant are observations where the 64 to 68 kDa ENOX2 transcript variant (pH 4.5) of sera correlated with disease presence in both late (Stage IV) (FIG. 5A) and early (Stage I) (FIG. 5F) disease and in Stage IV recurrence (FIG. 5C) but was absent from sera of non-cancer (normal) volunteers (FIG. 5B) or in survivors free of disease for one to five years (FIG. 5D). Additionally, the 64 to 68 kDa breast cancer-specific transcript variant does not apply to a subset of breast cancer patients but appears to be universally present. Analyses of sera of more than 60 patients with active disease including 20 Stage I and Stage II breast cancer patients all tested positive.

[0031] Unlike most published cancer markers, cancer-specific ENOX2 variants are not simply present as elevated levels of a serum constituent present in lesser amounts in the absence of cancer. The cancer-specific ENOX2 transcript variants result from cancer-specific expression of alternatively spliced mRNAs (Tang et al., 2007; 2008). Neither the splice variant mRNAs nor the ENOX2 isoform proteins are present in detectable levels in non-cancer cells or in sera of subjects without cancer (Table I).

[0032] Findings from a separate study with small cell and non-small cell lung cancer suggest that the 2-D-western blot test detects cancer presence 5 to 7 years in advance of the appearance of clinical symptoms. This supposition is based mainly on our analysis of two special cancer panels of sera obtained through the Early Detection Research Network (EDRN) of the National Cancer Institute. One panel consisted of about 20 known lung cancer patient sera and 35 control patient sera. Using the 2-D-western blot protocol to identify specific ENOX2 isoforms, we successfully identified all 20 of the known lung cancer patient sera. However, unexpectedly a high incidence of ENOX2 presence was encountered in sera from the control group which were obtained from a community screening study. From additional information obtained through the EDRN, 16 of the 17 positive control subject samples where our findings specifically indicated lung cancer (the lung cancer ENOX2 markers were found) were smokers with smoking histories in the range of 15 to 88 pack-years. However, the anticipated incidence of undetected lung cancers in such a population would be in the order of 10% or less rather than nearly 50%. Since the aberrant ENOX2 transcript variants associated with lung cancer, are single molecular species produced only by lung cancer, the possibility was raised that lung cancer was being detected much earlier than was currently possible by other methods. The indications might be as early as 5 to 7 years before clinical symptoms, based on the estimated 20 year development time for lung cancer expression between carcinogen exposure and a clinically evident cancer (Petro et al., 2000. Br. Med. J. 231: 323-329) as diagrammed in FIG. 6.

[0033] Similar results were obtained with a panel of female subjects at risk for breast and ovarian cancer. An analysis of a panel of 127 sera in a Biomarker Reference Set for Cancers in Women also provided through the Early Detection Research Network of the National Cancer Institute support our indications that the 2-D gel-western blot system is able to detect cancer presence 5 to 7 years in advance of clinical symptoms. The panel consisted of samples pooled form 441 women in 12 different gynecologic and breast disease categories plus 115 sera from age-matched control women. Of the 127 sera samples in the panel 29 tested positive for breast cancer and another 16 tested positive for ovarian cancer. Since the aberrant transcript variants are single molecular species produced by specific cancers such as lung, breast or ovarian, the findings suggest that cancer was being detected in the control population much earlier than is currently possible by other methods. As estimated for lung cancer, the indications might be as early as 5 to 7 years before clinical symptoms based on the estimated development time estimated for breast as well as lung cancer expression between a cancer causing event and clinically evident disease (Weinberg, 2007. The Biology of Cancer, Garland Science).

[0034] Many cancers are detected only after clinical symptoms present and often after the cancer has spread leaving chemotherapy as perhaps the only resource for treatment. Tomographic or x-ray methods may detect before clinical symptoms present but only after a tumor mass has already formed. There appear to be few, if any, on-going indications of opportunities either for early cancer detection or for early intervention. Various genomic, transcriptomic and/or proteomic analyses, while of potential utility for tissue analyses of biopsy material, have thus far failed to provide new and reliable non-invasive serum indicators of cancer occurrence (Goncalves and Bertucci, 2011. Med. Prin. Pract. 204: 4-18) despite continued promise offered by circulating microRNAs (Wu et al., 2011. J. Biomed. Biotechnol. Article ID 597145). A relatively small percentage of all cancers can be attributed to predisposing genes such as BRACA1, BRACA2 and less frequently p53 and PTEN (Lee et al., 2010. Breast Cancer Res. Treat. 122: 11-25) for 5 to 10% of all breast cancers. While indicative of cancer risk, predisposing genes do not necessarily signal cancer presence.

[0035] Table I shows that sera from patients with different cancers exhibit distinct patterns of ENOX2 isoforms with characteristic molecular weight and isoelectric points (pH). Updated from Hostetler et al. (2009).

TABLE-US-00001 Sera Molecular Isoelectric Cancer analyzed weight point, pH Cervical 18 94 kDa 5.4 Ovarian 41 80 and 40.5 kDa 4.2 and 4.1 Prostate 70 75 kDa 6.3 Breast/Uterine 55 64 to 68 kDa 4.5 Non-small cell lung 83 54 kDa 5.1 Small cell lung 22 52 kDa 4.3 Pancreatic 24 50 kDa 4.3 Colon 55 52 and 34 kDa 4.3 and 3.9 Lymphoma, Leukemia 16 45 kDa 3.9 Melanoma 12 38 kDa 5.1

[0036] Table II provides protein sequence similarity between ENOX2 and the two reference proteins 1-anti-trypsin inhibitor and serrotransferrin reactive with the pan ENOX2 scFv recombinant antibody. Regions of similarity are restricted to a 7 amino acid sequence (underlined) adjacent in ENOX2 to the EEMTE SEQ ID NO:20 quinone inhibitor-binding site which serves as the antigen sequence to which the specific scFv antibodies bind.

TABLE-US-00002 (SEQIDNO:17) ENOX2 EEMTETKETEESALVS (SEQIDNO:18) -antitrypsininhibitor GTDCVAKEATEAAKCN (SEQIDNO:19) Serrotransferrin CLDGTRKPVEEYANCH

Details of Method

A. Sample Preparation

[0037] 1. Prepare/thaw re-hydration solution (20, 1.5 mL tube labeled RB)

[0038] a. Add 1% Dithiothreitol (DTT) to solution before use (0.01 g/1.0 mL)

[0039] 2. Add 120 L of Rehydration Buffer to a 1.7 ml tube

[0040] 3. Add 30 L of sera to tube

[0041] 4. Vortex solution until fully mixed

[0042] 5. Remove Immobiline DryStrips from freezer (20 C., pH 3-10) and allow strips to equilibrate to RT for 5 minutes.

[0043] a. Do not leave strips at RT for more than 10 min.

[0044] 6. Record ID# from strip

[0045] 7. Load 130 L of sample to tray per 7 cm DryStrip. Ensure tray is level.

[0046] 8. Place DryStrips gel-side down over sample

[0047] 9. Ensure sample is evenly spread throughout strip by carefully lifting strip in and out of sample a few times if needed.

[0048] 10. If samples are concentrated in one region of the strip, redistribute sample by pipetting.

[0049] 11. Remove air bubbles by gently pressing down on DryStrip with pipette tip.

[0050] 12. Place lid on tray and place tray in plastic bag with ddi-H.sub.2O soaked paper towels.

[0051] 13. Seal bag.

[0052] 14. Allow sample to re-hydrate overnight at RT on a level surface allowing strips to absorb sample for 12-24 hrs.

B. Isoelectric Focusing (First Dimension)

[0053] 1. Turn on IPGphor (ensure proper startup of machine)

[0054] 2. Place strips on Manifold focusing tray as follows

[0055] a. Gel side up

[0056] b. Positive (acidic) end towards back

[0057] c. Strips are aligned

[0058] d. Between metal strips (so electrodes fit and touch metal strip)

[0059] 3. Obtain 2 Paper Wicks per strip

[0060] 4. Wet wicks with 150 L ddi-H.sub.2O per wick.

[0061] 5. Place wicks over anodic and cathodic ends of gel (approx. 0.3 cm).

[0062] 6. Place electrodes on wicks, but away from gel (be sure prong is on metal plate), and lock in place.

[0063] 7. Cover strips with DryStrip Cover fluid

[0064] a. Fill strips entire lane with oil

[0065] b. Ensure strips are fully covered

[0066] 8. Close lid

[0067] 9. IEF run with IPGphor II

[0068] a. Maximum amperage: 50 Amps

[0069] b. Temperature: 20 C.

[0070] c. Ensure correct assembly by checking initial voltage

[0071] d. As needed, pause run and replace wicks, continue run until dye front disappears.

TABLE-US-00003 Step Voltage Time/Vhrs 7 cm Strip pH 3-10 1 - Stp. 250 V 250 Vhrs. (Run #1) 2 - Stp. 500 V 500 Vhrs. 3 - Stp. 1000 V 1000 Vhrs. 4 - Grd. 4000 V 3 hrs. 5 - Stp. 4000 V 25,000 Vhrs. 6 - Stp. 500 V Hold

C. Prepare SDS-Page Gels (for Second Dimension)

[0072] 1. Prior to use, wash and scrub plates very well in soap and hot water.

[0073] 2. Rinse in diH.sub.2O.

[0074] 3. Leave the plates to air dry or wipe with ethanol-soaked Kimwipes.

[0075] 4. Order plates in Protean-plus Multi-Gel casting Chamber (Bio-Rad) as per manual (with a spacer between each plate and block).

[0076] 5. Ensure screws are fully tightened.

[0077] 6. Add gel solution

[0078] 7. Stop pouring when gel is about 1-1.5 cm from top of glass plates.

[0079] 8. Gently overlay gels with ethanol

[0080] 9. Cover with Saran Wrap.

[0081] 10. Allow gels to polymerize for at least one hour (best if overnight)

D. Equilibration (First Dimension)

[0082] 1. Remove strips from tray and place on Kimwipe to remove excess oil.

[0083] 1. Place strips gel side up on Kimwipe

[0084] 2. Overlay strips with a second Kimwipe and gently blot to remove oil.

[0085] 2. Place strips in equilibration plate gel side up; freeze or equilibrate.

[0086] 1. Freeze: Wrap plate in plastic wrap, store at 80 C.

[0087] i. Thaw strips prior to equilibration (clear when thawed).

[0088] 2. Equilibrate: continue to next step

[0089] 3. Cover strips with equilibration buffer, about 1.5 mL per strip.

[0090] 4. Heat up Agarose until it is liquefied

[0091] 5. Shake 20 min at RT

E. SDS-PAGE (Second Dimension)

[0092] 1. Prepare left (pH 10 side) markers by adding 8 L of standards on Whatman 3MM chromatography paper cut to about 3 cm.times.0.75 cm.

[0093] 1. Standards should be added to bottom of paper, about 1 cm high.

[0094] 2. Prepare right (pH 3 side) markers by adding 8 L of standards on Whatman 3MM chromatography paper cut to approximately 3 cm.times.0.75

[0095] 1. Standards should be added to bottom of paper, about 1 cm high

[0096] 3. Pour off Equilibration Buffer.

[0097] 4. Cover strips in SDS Running buffer to rinse away excess Equilibration Buffer.

[0098] 5. Remove SDS Running buffer from strips.

[0099] 6. Repeat SDS Running buffer rinse.

[0100] 7. Carefully place strips gel side out on back plate of SDS-PAGE gel.

[0101] 8. Overlay strips with 1% low melting agarose once it has cooled enough to touch skin

[0102] 1. Ensure no air bubbles have formed under the gel.

[0103] 2. Use ruler to tap gel and remove air bubbles.

[0104] 9. Insert marker's next to appropriate end of IEF strip, ensuring marker is flush to the gel on the strip

[0105] 10. Allow polymerization of agarose

[0106] 11. Continue for each strip to be loaded in 2.sup.nd dimension

[0107] 12. Place gels in Dodeca tank, HINGED SIDE DOWN

[0108] 13. After all gels have been put in tank, ensure gels are covered in entirety by SDS running buffer

[0109] 14. 2.sup.nd dimension run is done at 13 C.

[0110] 1. 250 V

[0111] 2. 1-1.5 hr. (allow gel to run until gel front approaches tubing in lid of tank).

F. Protein Transfer (for Western Blot)

[0112] 1. Remove gel from Dodeca tank

[0113] 2. Cut gel to desired size

[0114] 3. Fill tray (large enough to fit gel) with transfer buffer.

[0115] 4. Place sponges in transblot cell2 sponges per gel

[0116] 5. Fill tank with transfer buffer to allow sponges to saturate with transfer buffer

[0117] 6. Soak pre-cut transfer membrane

[0118] 7. Assemble transfer cassette as follows

[0119] 1. Black side down

[0120] 2. Sponge soaked in transfer buffer

[0121] 3. Filter paper

[0122] 4. Gel

[0123] 5. Nitrocellulose membraneonce placed on gel do not move membrane

[0124] 6. Filter Paper

[0125] 7. Sponge

[0126] 8. Ensure all air bubbles have been removed between gel and membrane

[0127] 9. Place tray in Transblot tank, black side (gel side) of tray to black tank side

[0128] 10. Transfer at 4 C. and following conditions (transfer can be done in an ice bath if needed)

[0129] 1. 90 V for 50 min.

[0130] 2. Membrane can be left in tank overnight at 4 C. after transfer.

G. Immunological Analysis for Western Blot Using ScFv with S-Tag Linked to Alkaline Phosphatase as Antibody

[0131] 1. Remove membrane from transfer

[0132] 2. Rinse membrane in 1% milk (enough to cover membrane) and block, 10 min, RT

[0133] 3. Prepare antibody solution (According to Titer instructions on Ab)

[0134] 4. Remove blocking solution (save at 4 C.)

[0135] 5. Place membrane into container with antibody solution

[0136] 6. Incubate at 4 C. overnight (usually 8-12 hr)

H. Development of Western Blot and Scanning

[0137] 1. Remove 1 antibody

[0138] 2. Wash membrane 4

[0139] 1. Cover membrane with TBST

[0140] 2. Gently shake at RT for 5 min.

[0141] 3. Cover membrane with Western Blue

[0142] 4. Allow to develop until reference spots reach maximum intensity

[0143] 5. Stop develop by rinsing with ddi-water

[0144] 6. Dry membrane

[0145] 7. Scan membrane

Solutions Used for First Dimension

I. Rehydration Buffer pH 7 (25 mL):

[0146]

TABLE-US-00004 Amount Molar Mass Final Chemical Added (g/mol) Value Urea 10.51 g 60.06 7M Thiourea 3.81 g 76.12 2M CHAPS 0.5 g 614.88 2% asb-14 0.125 g 434.68 0.5% 40% Ampholytes 330 L N/A 0.5% IPG Buffer 125 L N/A 0.5% ddi-H.sub.2O To 25 mL 18.002 N/A Bromophenol Blue 3 mg 669.96 0.012% Dissolve Urea in minimal ddi-H2O(do not heat over 30 C.). Dissolve Thiourea in Urea/ddi-H.sub.2O solution, and then add remaining chemicals. Q.S. with to 25 mL and aliquot to 1 mL tubes and store at 80 C. Add 1% DTT - 10 mg (0.01 g) before use.

Solutions Used for Second Dimension

Tris Buffer (1.5 M, pH 8.8) (1 L):

[0147]

TABLE-US-00005 Amount Molar Mass Final Chemical Added (g/mol) Value Trizma 181.65 g 121.14 1.5M HCl pH to 8.8 36.46 N/A ddi-H.sub.2O To 1 L 18.02 N/A Dissolve Trizma in 750 mL and adjust pH to 8.8 with HCl. Q.S. to final volume of 1 L with ddi-H.sub.2O and store at 4 C.

Equilibration Buffer (400 mL):

[0148]

TABLE-US-00006 Amount Molar Mass Final Chemical Added (g/mol) Value Tris-Buffer 134 mL N/A 0.5M (1.5M, pH 8.8) Urea 144.14 g 60.06 6M Glycerol 120 mL (150 g) 92.09 30% SDS 10 g 288.38 2.5% ddi-H.sub.2O To 400 ml 18.02 N/A Bromophenol Blue Trace amount 669.96 N/A Q.S. to final volume of 4 L with ddi-H.sub.2O. Add Bromophenol Blue (add with pipette tip to give trace of blue). Aliquot to 15 mL tubes and store at 20 C. Acrylamide Gel (20.times.20; 1-mm Thick)

TABLE-US-00007 Tris Buffer 1.5M, 30% Gel # pH 8.8 Acrylamide ddi-H.sub.2O 10% APS TEMED % gels (mL) (mL) (mL) (mL) (mL) 10 6 100 133.33 166.67 4 0.4 8 125 166.67 208.33 5 0.5 10 150 200 250 6 0.6 12 175 233.33 291.67 7 0.7 Formulations for Protean II gels.

10% APS

[0149]

TABLE-US-00008 Amount Molar Mass Final Chemical Added (g/mol) Value APS 2 g 228.18 10% ddi-H.sub.2O To 20 mL 18.02 N/A Q.S. to final volume of 20 mL with ddi-H.sub.2O.

Agarose Solution (1%):

[0150]

TABLE-US-00009 Amount Molar Mass Final Chemical Added (g/mol) Value Agarose 1.5 g N/A 1% SDS-Running Buffer 150 mL N/A N/A Bromophenol Blue Trace amount 669.96 N/A Combine agarose and SDS-Run Buffer. Microwave to heat and dissolve. Add Bromophenol Blue (add with pipette tip to give trace of blue).

10SDS Running Buffer (4 L):

[0151]

TABLE-US-00010 Amount Molar Mass Final Chemical Added (g/mol) Value Trizma 121.2 g 121.14 0.25M Glycine 576 g 75.07 1.92M SDS 40 g 288.38 1% ddi-H.sub.2O To 4 L 18.02 N/A Q.S. to final volume of 4 L with ddi-H.sub.2O.

Solutions for Western Blot

Western Transfer Buffer (4 L):

[0152]

TABLE-US-00011 Amount Molar Mass Final Chemical Added (g/mol) Value Trizma 121.2 g 121.14 0.025M Glycine 57.6 g 75.07 0.192M Methanol 480 mL 32.04 0.12% SDS 3 g 288.38 .075% ddi-H.sub.2O To 4 L 18.02 N/A Q.S. to final volume of 4 L with di-H.sub.2O.

Blocking Buffer (5% BSA)

[0153]

TABLE-US-00012 Molar Mass Final Chemical Mass (g/mol) Value BSA 5 g 66500 5% N.sub.3Na 0.2 g 65.01 0.2% TBST To 100 mL 18.02 N/A Q.S. to final volume of 100 mL with TBST.

10TBST (4 L)

[0154]

TABLE-US-00013 Amount Molar Mass Final Chemical Added (g/mol) Value Trizma 48.4 g 121.14 100 mM NaCl 350.6 g 58.44 1.5M Tween 20 20.6 g 1227.54 0.5% HCl pH to 7.5 36.46 N/A ddi-H.sub.2O To 4 L 18.02 N/A Add Trizma and NaCl to 3.5 L ddi-H.sub.2O. pH to 7.5 by HCl. Add Tween 20. Q.S. to 4 L with ddi-H.sub.2O.

Summary of 2-D Gel Electrophoresis Western Blot Early Detection Protocol

[0155] Serum was prepared from 5 ml of blood collected by venipuncture (with tourniquet) in standard B & D 13.times.100 (7 ml) vacutainer clot tubes (or equivalent) with or without hemoguard closure. After approximately 30 min at room temperature to allow for clotting, the clot was pelleted by centrifugation for 5 to 10 min at 2,500 to 3,000 rpm. Clot-free serum was decanted into a clean tube, labeled and analyzed fresh or stored frozen.

[0156] For western blot analysis, 30 l of sera was added to 120 l of Rehydration Buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS [(3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), a nondenaturing zwitterionic detergent], 0.5% (w/v) ASB-14 (amidosulfobetaine-14, a zwitterionic detergent), 0.5% (v/v) ampholytes pH 3-10 (Bio-Rad), 0.5% (v/v) immobilized pH gradient (IPG) buffer pH 3-10 (Amersham-Pharmacia Biotech) and 65 mM dithiothreitol). The samples were quickly vortexed to mix sera with Rehydration Buffer. Four to six mg of protein were loaded for analysis. The samples were electrophoresed in the first dimension by using a commercial flatbed electrophoresis system (Ettan IPGphor 3, Amersham-Pharmacia Biotech) with IPG dry strips (Amersham). A linear pH range of 3 to 10 on 7 cm IPG strips was used. The IPG strips were rehydrated with the samples overnight at room temperature. The strips were then focused at 50 mA per strip and at increasing voltage of 250 V for 250 Vhrs, 500 V for 500 Vhrs, 1,000 V for 1,000 Vhrs and 4,000 V for 3 hrs. The samples were then focused at a constant 4,000 V for 28,000 Volt-hours. After isoelectric focusing, the IPG strips were re-equilibrated for 20 min in 2.5% (w/v) SDS, 6 M urea, 30% (v/v) glycerol, 100 mM Tris-HCl (pH 8.8). The strips were placed onto linear SDS-PAGE gels (10% (w/v) polyacrylamide) and electrophoresed at a constant 250 V for 75 min. The samples were then transferred to nitrocellulose membranes by electroblotting using the Bio-Rad Trans-Blot Electrophoretic Transfer Cell. The membranes were blocked using milk protein (1% low fat dry milk) at room temperature for 10 min. Detection was with recombinant anti-ENOX2 single chain variable region of antibody (scFv) that was alkaline phosphatase-linked overnight at 4 C. After washing, detection was performed with Western Blue nitrotetrazolium (NBT) substrate (Promega, Madison, Wis.; Cat. No. 53841) at room temperature. Images were scanned and processed using Adobe Photoshop. Quantitation utilized an algorithm developed for this purpose. Reactive proteins appeared reddish blue. For interpretative purposes, the blots were divided into quadrants I-IV with unreactive serum albumin at the center (FIGS. 2A and 2B).

EXAMPLES

Example 1

[0157] Analysis of Sera Pooled from Cancer Patients

Example 1A

[0158] 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. Detection was 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 (FIG. 1) but absent from sera of non-cancer patients or healthy volunteers. Example 1B. The same procedure can be followed as in Example 1A, except that detection can be by recombinant anti-ECTO-NOX variable region single chain (scFv) using the scFv linked directly as described above to alkaline phosphatase (overnight at 4 C.). By using the directly linked antibody the process is less expensive and one day faster than using an S-tag followed by an anti-S tag antibody linked to alkaline phosphatase.

Example 2

Analysis of Small Cell Lung Cancer Patient Serum

Example 2A

[0159] 2-D gel analysis when applied to sera of a patient with small cell lung cancer contained a 52 kDa, pH 4.2 ENOX2 protein in quadrant I with detection using the S-tag procedure in Example 1A above. Example 2B. The same procedure can be followed as in Example 2A, except that detection can be by recombinant anti-ECTO-NOX variable region single chain (scFv) using the scFv linked directly as described above to alkaline phosphatase (overnight at 4 C.). By using the directly linked antibody the process is less expensive and one day faster than using an S-tag followed by an anti-S tag antibody linked to alkaline phosphatase.

Example 3

Analysis of Non Small Cell Lung Cancer Patient Serum

Example 3A

[0160] 2-D gel analysis as in FIG. 1 when applied to plasma from a patient with non-small cell lung cancer revealed a non-small cell cancer specific ENOX2 isoform at 54 kDa, pH 5.1 in quadrant 1 with detection using the S-tag procedure in Example 1A above. Example 3B. The same procedure can be followed as in Example 3A, except that detection can be by recombinant anti-ECTO-NOX variable region single chain (scFv) using the scFv linked directly as described above to alkaline phosphatase (overnight at 4 C.). By using the directly linked antibody the process is less expensive and one day faster than using an S-tag followed by an anti-S tag antibody linked to alkaline phosphatase.

Example 4

Analysis of Breast Cancer Patient Serum

Example 4A

[0161] 2-D gel analysis as in FIG. 1 when applied to sera from a patient with breast cancer revealed a breast cancer-specific ENOX2 protein of 68 kDa, pH 4.5 in quadrant 1 with detection using the S-tag procedure in Example 1A above. Example 4B. The same procedure can be followed as in Example 4A, except that detection can be by recombinant anti-ECTO-NOX variable region single chain (scFv) using the scFv linked directly as described above to alkaline phosphatase (overnight at 4 C.). By using the directly linked antibody the process is less expensive and one day faster than using an S-tag followed by an anti-S tag antibody linked to alkaline phosphatase.

Example 5

Analysis of Prostate Cancer Patient Serum

Example 5A

[0162] 2-D gel analysis as in FIG. 1 when applied to sera from a patient with prostate cancer revealed prostate cancer-specific ENOX2 isoforms at 75 kDa and isoelectric points of pH 6.3 with detection using the S-tag procedure in Example 1A above. Example 5B. The same procedure can be followed as in Example 5A, except that detection can be by recombinant anti-ECTO-NOX variable region single chain (scFv) using the scFv linked directly as described above to alkaline phosphatase (overnight at 4 C.). By using the directly linked antibody the process is less expensive and one day faster than using an S-tag followed by an anti-S tag antibody linked to alkaline phosphatase.

Example 6

Analysis of Cervical Cancer Patient Serum

Example 6A

[0163] 2-D gel analysis as in FIG. 1 when applied to serum from a patient with cervical cancer revealed a cervical cancer-specific ENOX2 isoform at 94 kDa, pH 5.4 with detection using the S-tag procedure in Example 1A above. Example 6B. The same procedure can be followed as in Example 6A, except that detection can be by recombinant anti-ECTO-NOX variable region single chain (scFv) using the scFv linked directly as described above to alkaline phosphatase (overnight at 4 C.). By using the directly linked antibody the process is less expensive and one day faster than using an S-tag followed by an anti-S tag antibody linked to alkaline phosphatase.

Example 7

Analysis of Colon Cancer Patient Serum

Example 7A

[0164] 2-D gel analysis as in FIG. 1 when applied to serum from a patient with colon cancer revealed colon cancer-specific ENOX2 isoforms at 38 and 52 kDa, pH 4.3 and 3.9 with detection using the S-tag procedure in Example 1A above. Example 7B. The same procedure can be followed as in Example 7A, except that detection can be by recombinant anti-ECTO-NOX variable region single chain (scFv) using the scFv linked directly as described above to alkaline phosphatase (overnight at 4 C.). By using the directly linked antibody the process is less expensive and one day faster than using an S-tag followed by an anti-S tag antibody linked to alkaline phosphatase.

Example 8

Cancer Specific Isoforms

[0165] For each kind of cancer there appears to be a ENOX2 isoform (ovarian, breast, cervical, colon, non-small cell lung, prostate small cell lung) or combination of ENOX2 isoforms that is specific to the tissue or cell type of origin for the cancer. This test is preferably done with recombinant anti-ECTO-NOX variable region single chain (scFv) using the scFv linked directly as described above to alkaline phosphatase. By using the directly linked antibody the process is less expensive and one day faster than using an S-tag followed by an anti-S tag antibody linked to alkaline phosphatase.

Example 9

[0166] Analysis of Patient Serum where Cancer of Unknown Origin

[0167] The 2-D gel of sera from a patient with cancer where the primary tumor was unknown revealed the presence of 40.5 and 80 kDa, pH 4.2 and 4.1 ENOX2 isoforms to indicate that the primary cancer was ovarian cancer (not shown).

[0168] 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 ENOX2 isoforms, confirming previous observations that ENOX2 proteins are absent from non-cancer patients or sera of healthy volunteers.

[0169] 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 ENOX2 isoforms are resolved indicative of cancer presence and disease severity. Detection uses a recombinant single chain antibody (scFv) that reacts with all known ECTO-NOX isoforms of human origin. While the technique can be used with an antibody that has an S-tag, the process is less expensive and faster by using the scFv linked directly to alkaline phosphatase or another suitable detection aid.

[0170] Monoclonal antibody generated against ENOX2 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 (VH) and variable light (VL) domains, can maintain the binding specificity and affinity of the original antibody (Glockshuber et al. 1990. Biochemistry 29:1262-1367).

[0171] For a recombinant antibody, cDNAs encoding the variable regions of immunoglobulin heavy chain (VH) and light chain (VI), 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. Bio/Technology 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 VL or VH can provide the NH2-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).

[0172] Total RNA was isolated from the hybridoma cells producing ENOX2-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.times.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.times.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.times.g, and the pellet was resuspended with 2-3 ml of denaturing solution and 2 volumes of ethanol. The solution was placed at 20 C. for 2 h, and then centrifuged at 12,000.times.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 12,000g for 5 min. The amount of isolated RNA was measured spectrophotometrically and calculated from the absorbance at 280 nm and 260 nm.

[0173] 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 l of 10sample 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 OD260 (1 OD unit=40 g of RNA). The amounts of total RNA and mRNA obtained from 4.times.10.sup.8 cells were 1328 g and 28 g, respectively.

[0174] mRNA (1-2 g) 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 g/5 l 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 l) and appropriate buffers for the reaction were mixed with mRNA sample. DTT solution (1 l of 0.1 M) and RNase-free water (16 l) also were added to the solution. The mixture was incubated for 1 h at 37 C.

[0175] Degenerate primers for light chain and heavy chain (Novagen, Madison, Wis.) were used for PCR. PCR synthesis was carried out in 100 l reaction volumes in 0.5 ml microcentrifuge tubes by using Robocycler (Stratagene, La Jolla, Calif.). All PCR syntheses included 2 l of sense and anti-sense primers (20 pmoles/1), 1 l of first-strand cDNA as a template, 2 l of 10 mM of dNTPs, 1 l of Vent polymerase (2 units/1), 10 l of 10PCR buffer (100 mM Tris-HCl, pH 8.8 at 25 C., 500 mM KCl, 15 mM MgCl2, 1% Triton X-100), 82 l of H2O. 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.

[0176] 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 g/ml) was added and poured into the apparatus. Each sample was mixed with 6gel 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.

[0177] 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 l of buffer-saturated phenol (pH 7.9.+0.0.2). 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.

[0178] 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.

[0179] 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 VH and VL 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 (VH sense primer) and primer d (VL 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.

[0180] The complete ScFv gene was assembled from the VH, VL and linker genes to yield a single ScFv gene by PCR (Tables 2A and 2B). The DNA sequence encoding the linker was 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 NO:7) which translates to S-peptide (LysGluThrAlaAlaAlaLysPheGluArgGln HisMetAspSer; SEQ ID NO:8).

[0181] 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 ENOX2-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 (NdeI and NheI) and S-peptide residues.

[0182] Plasmid pET-11a and ORF of ScFv(S) were digested with restriction enzymes NdeI and NheI 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 g/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 10,000g 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 & Design 1:21-27.

TABLE-US-00014 TABLE1A DNASequenceofHeavychainScFv(V.sub.H), SEQ.IDNO:1 1 gaggtcaagctgcaggagtcaggaactgaagtggtaaagc ctggggcttc 51 agtgaagttgtcctgcaaggcttctggctacatcttcaca agttatgata 101 tagactgggtgaggcagacgcctgaacagggacttgagtg gattggatgg 151 atttttcctggagaggggagtactgaatacaatgagaagt tcaagggcag 201 ggccacactgagtgtagacaagtcctccagcacagcctat atggagctca 251 ctaggctgacatctgaggactctgctgtctatttctgtgc tagaggggac 301 tactataggcgctactttgacttgtggggccaagggacca cggtcaccgt 351 ctcctca

TABLE-US-00015 TABLE1B DNASequenceofLightchainScFv(V.sub.L), SEQ.IDNO:2 1 gaaaatgtgctcacccagtctccagcaatcatgtctgcat ctccagggga 51 gagggtcaccatgacctgcagtgccagctcaagtatacgt tacatatatt 101 ggtaccaacagaagcctggatcctcccccagactcctgat ttatgacaca 151 tccaacgtggctcctggagtcccttttcgcttcagtggca gtgggtctgg 201 gacctcttattctctcacaatcaaccgaatggaggctgag gatgctgcca 251 cttattactgccaggagtggagtggttatccgtacacgtt cggagggggg 301 accaagctggagctgaaagcg

TABLE-US-00016 TABLE2A DNASequenceforScFv,SEQ.IDNO:3 1 gtggtaaagcctggggcttc 51 gaggtcaagctgcaggagtcaggaactgaacatcttcaca agttatgata 101 agtgaagttgtcctgcaaggcttctggctagacttgagtg gattggatgg 151 tagactgggtgaggcagacgcctgaacaggaatgagaagt tcaagggcag 201 ggccacactgagtgtagacaagtcctccagcacagcctat atggagctca 251 ctaggctgacatctgaggactctgctgtctatttctgtgc tagaggggac 301 tactataggcgctactttgacttgtggggccaagggacca cggtcaccgt 351 ctcctcaggaggcggtggatcgggcggtggcggctcgggt ggcggcggct 401 ctgaaaatgtgctcacccagtctccagcaatcatgtctgc atctccaggg 451 gagagggtcaccatgacctgcagtgccagctcaagtatac gttacatata 501 ttggtaccaacagaagcctggatcctcccccagactcctg atttatgaca 551 catccaacgtggctcctggagtcccttttcgcttcagtgg cagtgggtct 601 gggacctcttattctctcacaatcaaccgaatggaggctg aggatgctgc 651 cacttattactgccaggagtggagtggttatccgtacacg ttcggagggg 701 ggaccaagctggagctgaaagcgaaagaaaccgctgctgc taaattcgaa 751 cgccagcacatggacagc

TABLE-US-00017 TABLE2B AminoAcidSequenceforScFv,SEQ.IDNO:4 1 EVKLQESGTEVVKPGASVKLSCKASGYIFTSYDIDWVRQT PEQGLEWIGW 51 IFPGEGSTEYNEKFKGRATLSVDKSSSTAYMELTRLTSED SAVYFCARGD 101 YYRRYFDLWGQGTTVTVSSGGGGSGGGGSGGGGSENVLTQ SPAIMSASPG 151 ERVTMTCSASSSIRYIYWYQQKPGSSPRLLIYDTSNVAPG VPFRFSGSGS 201 GTSYSLTINRMEAEDAATYYCQEWSGYPYTFGGGTKLELK AKETAAAKFE 251 RQHMDS

TABLE-US-00018 TABLE3 PrimersforPCRamplificationofscFv(s)gene 1.Primersforcloningofvariableregionsof heavychainandlightchainofantibody (A)Primersforheavychain(VH) Forwardprimer:5-GGCCCAGCCGGCCGAGGTCAAGCTGCAGGAG TCAGGA-3 (SEQIDNO:9) Reverseprimer:5-CTCGGAACCTGAGGAGACGGTGACCGTGGTC CC-3 (SEQIDNO:10) (B)Primersforlightchain(VL) Forwardprimer:5-TCCAAAGTCGACGAAAATGTGCTCACCCAGT CTCCA-3 (SEQIDNO:11) Reverseprimer:5-AGCGGCCGCTTTCAGCTCCAGCTTGGTCCCC CC-3 (SEQIDNO:12) 2.PrimersforsubcloningofScFv(s)geneinto pET-11aexpressionvector (A)Primersforheavychain(VH)andlinker amplification Forwardprimer:5-GTCAAGCTGCAGGAGTCAGGA-3 (SEQ IDNO:13) Reverseprimer:5-AGAGCCGCCGCCACCCGAGCCGCCACCGCCC GATCCACCGCCTCCTGAGGAGACGGTGACCGTGGT-3 (SEQID NO:14) (B)Primersforlightchain(VL),linkerandS-tag amplification Forwardprimer:5-GGAGGCGGTGGATCGGGCGGTGGCGGCTCGG GTGGCGGCGGCTCTGAAAATGTGCTCACCCAGTCT-3 (SEQIDNO: 15) Reverseprimer:5-AGTCAGGCTAGCTTAGCTGTCCATGTGCTGG CGTTCGAATTTAGCAGCAGCGGTTTCTTTCGCTTTCAGCTCCAGCTT-3 (SEQIDNO:16)

[0183] 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. February; 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.

[0184] Monoclonal, polyclonal antibodies, peptide-specific antibodies or single chain recombinant antibodies and antigen binding fragments of any of the foregoing, specifically reacting with the ENOX2 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.

[0185] 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.

[0186] 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.