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US 8,911,951 B2Granted
Plasma kallikrein fragments as diagnostic biomarkers for lung cancers
AssigneeProtan Bio Co., Ltd.
InventorsJe-Yeol Cho, Jae-Yong Park, Seung-Jin Lee
Life of this patent
Estimating the term…
Abstract
Disclosed herein are diagnostic markers for lung cancer, isolated from serum glycoproteins. The disclosed diagnostic markers for lung cancer are specifically expressed only in the sera of lung cancer patients at high levels, and thus will be very useful for diagnosing lung cancer and estimating disease progression and treatment.
Claims11
A method for diagnosing lung cancer in a subject, the method comprising:
- (i) isolating and concentrating glycoproteins from a biological sample of the subject by using a multi-lectin affinity column packed with ConA (concanavalinA), LCH (lentil lectin), GNA (snowdrop lectin, WGA (wheat germ agglutinin), SNA (elderberry lectin), MAL (Maackia amurensis lectin), AIL (Jacalin), and PNA (peanut agglutinin);
- (ii) denaturing the isolated and concentrated glycoproteins in a buffer containing mercaptoethanol and octyl beta-D-glucopyranoside;
- (iii) deglycosylating the denatured glycoproteins in a reaction buffer comprising peptide-N-glycosidase F (PNGase F);
- (iv) separating the deglycosylated glycoproteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE);
- (v) transferring the SDS-PAGE separated glycoproteins onto a Western blot membrane;
- (vi) treating the Western blot membrane containing the transferred glycoproteins with a primary antibody and a secondary antibody to form an antigen-antibody complex wherein the glycoproteins from the biological sample comprise a plasma kallikrein fragment (KLKB1) which comprises SEQ. ID NO:
- 1 and is present on the Western blot membrane,
- wherein the primary antibody is an anti-KLKB1 antibody that binds an epitope within amino acid residues of 361-400 of the plasma kallikrein fragment, and
- the secondary antibody binds the primary antibody;
- and
- (vii) diagnosing lung cancer in the subject when a 15-20 kDa mass fragment band on the Western blot membrane which corresponds to the plasma kallikrein fragment is detected at a level higher than a normal level found in a healthy person.
8 more elements
A method for diagnosing lung cancer in a subject, the method comprising:
- (i) isolating and concentrating glycoproteins from plasma of the subject by using a multi-lectin affinity column packed with ConA (concanavalinA), LCH (lentil lectin), GNA (snowdrop lectin, WGA (wheat germ agglutinin), SNA (elderberry lectin), MAL (Maackia amurensis lectin), AIL (Jacalin), and PNA (peanut agglutinin);
- (ii) denaturing the isolated and concentrated glycoproteins in a buffer containing mercaptoethanol and octyl beta-D-glucopyranoside at about 100° C. for about 10 minutes;
- (iii) deglycosylating the denatured glycoproteins in a reaction buffer comprising peptide-N-glycosidase F (PNGase F);
- (iv) separating the deglycosylated glycoproteins using a 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (15% SDS-PAGE);
- (v) transferring the 15% SDS-PAGE separated glycoproteins onto a Western blot nitrocellulose membrane;
- (vi) treating the Western blot membrane containing the transferred glycoproteins with a primary antibody and a secondary antibody to form an antigen-antibody complex wherein the glycoproteins from the biological sample comprise a plasma kallikrein fragment (KLKB1) which comprises SEQ. ID NO:
- 1 and is present on the Western blot membrane,
- wherein the primary antibody is an anti-KLKB1 antibody that binds an epitope within amino acid residues of 361-400 of the plasma kallikrein fragment, and
- the secondary antibody binds to the primary antibody;
- and
- (vii) diagnosing lung cancer in the subject when a 15-20 kDa mass fragment band on the Western blot membrane which corresponds to the plasma kallikrein fragment is detected at a level higher than a normal level found in a healthy person.
8 more elements
Dependencies are inferred from the claim text.