Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 

 

 

Title:  Process for preparation of a biomarker specific for O-acetylated sialic acid useful for diagnosing, monitoring treatment outcome, and predicting relapse of lymphoblastic leukemia

United States Patent:  6,693,177

Issued:  February 17, 2004

Inventors:  Mandal; Chitra (Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta, IN 700 032); Pal; Santanu (Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta, IN 700 032); Chatterjee; Mitali (Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta, IN 700 032)

Appl. No.:  453555

Filed:  December 3, 1999

Abstract

The invention relates to a novel process for preparation of a biomarker specific for O-acetylated sialic acid and useful for the diagnosis, monitoring outcome of treatment and prediction of relapse of acute lymphoblastic leukemia, said process comprising the steps of (I) separating serum from the blood of patients of acute lymphoblastic leukemia; (ii) separation of low molecular weight fractions and galactose binding proteins from the serum on affinity matrix; (iii) passing the galactose free protein fraction obtained in step (ii) over another affinity matrix to capture O-acetyl sialic acid specific protein fraction; (iv) eluting specific protein fraction with a buffer at alkaline pH in the range of 8.0-11.0 followed by immediate neutralization of the fraction; (v) passing O-acetyl sialic acid specific protein obtained in step (iv) over Agarose column to get O-acetyl sialic acid specific antibody and eluting the said antibody with an appropriate buffer at acidic pH, followed by immediate neutralization of the fraction and dialyzing the neutralized protein to get purified disease specific antibody as biomarker and a method of diagnosing, monitoring outcome of treatment and prediction of relapse of acute lymphoblastic leukemia using the biomarker obtained by the novel process.

SUMMARY OF THE INVENTION

A main advantage of the present invention is to provide a method for the preparation of antibodies specific for O-acetylated sialic acid, useful for the diagnosis, monitoring outcome of treatment and prediction of relapse of acute lymphoblastic leukemia.

Another advantage of the present invention is to provide a process for the quantification of the anti-antibody specific for O-acetylated sialic acid present in the serum of ALL patient with the help of a simple, specific, sensitive, non-invasive and economical bovine submaxillary mucin ELISA which allows the assessment of the treatment outcome of ALL patients.

Yet another advantage is to provide a method for the diagnosis, monitoring outcome of treatment and prediction of relapse of ALL.

The invention relates to a novel process for preparation of a biomarker specific for O-acetylated sialic acid and useful for the diagnosis, monitoring outcome of treatment and prediction of relapse of acute lymphoblastic leukemia, said process comprising the steps of (I) separating serum from the blood of patients of acute lymphoblastic leukemia; (ii) separation of low molecular weight fractions and galactose binding proteins from the serum on affinity matrix; (iii) passing the galactose free protein fraction obtained in step (ii) over another affinity matrix to capture O-acetyl sialic acid specific protein fraction; (iv) eluting specific protein fraction with a buffer at alkaline pH in the range of 8.0-11.0 followed by immediate neutralization of the fraction; (v) passing O-acetyl sialic acid specific protein obtained in step (iv) over Agarose column to get O-acetyl sialic acid specific antibody and eluting the said antibody with an appropriate buffer at acidic pH, followed by immediate neutralization of the fraction and dialyzing the neutralized protein to get purified disease specific antibody as biomarker and a method of diagnosing, monitoring outcome of treatment and prediction of relapse of acute lymphoblastic leukemia using the biomarker obtained by the novel process.

Additional advantages and novel features of the invention will be set forth in part in the description that follows, and in part will become more apparent to those skilled in the art upon examination of the following or upon learning by practice of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the foregoing advantages, the invention provides a process for preparation of O-acetyl specific immunoglobulin protein useful as a biomarker for the diagnosis, monitoring outcome of treatment and prediction of relapse of ALL comprising the steps of:

(i) separating serum from blood collected from patients of acute lymphoblastic leukemia by known methods;

(ii) removing low molecular weight fractions and galactose binding proteins from the serum by column chromatography on affinity matrix;

(iii) collecting unbound fraction from affinity matrix;

(iv) passing the galactose free protein fraction obtained in step (ii) over another affinity matrix to capture O-acetyl sialic acid specific protein fraction;

(v) eluting specific protein fraction with a buffer at alkaline pH in the range of 8.0-11.0 followed by immediate neutralization of the fraction;

(vi) passing O-acetyl sialic acid specific protein obtained in step (iv) over Protein G-agarose or protein A agarose or protein A Sepharose or protein G Sepharose or anti-human immunoglobulin or only IgG/IgM coupled to Sepharose or agarose column to get O-acetyl sialic acid specific antibody i.e. immunoglobulin and eluting the said antibody with a buffer at acidic pH in the range of 2.0-6.5;

(vii) immediate by neutralizing the fraction and subjecting the neutralized protein and dialyzing to get purified O-acetyl specific immunoglobulin protein as disease specific antibody or biomarker.

In an embodiment, a disease specific antibody or immunoglobulin i.e. the biomarker specific for O-acetylated sialic acid is purified from the serum of patients suffering from acute lymphoblastic leukemia at different stages of the disease.

In another embodiment, whole blood is collected in a container in presence or absence of any available anticoagulants such as Elseiver's solution or heparin or dextrose.

In yet another embodiment, blood is incubated for clotting at room temperature for a short period of time or plasma may be collected from blood.

In a further embodiment, nonspecific proteins are removed from specific affinity matrix such as bovine submaxillary mucin--Sepharose 4B by extensive washing of unbound non O-acetyl sialic acid specific fractions with buffer selected from phosphate buffer saline or Tris buffer saline.

In still another embodiment, specific protein is eluted with buffer of alkaline pH in the range of 8.0-11.0 from bovine submaxillary mucin--Sepharose 4B.

In one more embodiment, eluted specific protein is immediately neutralized with sodium acetate or monosodium phosphate for better stability of the eluted protein.

The invention preferably provides a method of diagnosing, monitoring the outcome, treatment and prediction of relapse of acute lymphoblastic leukemia in patients, which method comprises the steps of:

(i) collecting an anticoagulated blood sample from the patient,

(ii) removing low molecular weight fractions and galactose binding proteins from the serum by column chromatography on affinity matrix,

(iii) collecting unbound fraction from affinity matrix,

(iv) passing the galactose free protein fraction obtained in step (ii) over another affinity matrix to capture O-acetyl sialic acid specific protein fraction,

(v) eluting specific protein fraction with a buffer at alkaline pH in the range of 8.0 -11.0 and immediately neutralizing the fraction,

(vi) passing O-acetyl sialic acid specific protein obtained in step (iv) over Protein G--agarose or protein A agarose or protein A Sepharose or protein G Sepharose or anti-human immunoglobulin or only IgG/IgM coupled to Sepharose or agarose column, and

(vii) determining the quantity of biomarker specific for O-acetylated sialic acid in the serum of the patient by immunodetection assay in relation to the blast cells in the peripheral blood wherein the quantity of the biomarker is indicative of the different phases or stages of ALL wherein a decrease in the antibody level with the chemotherapeutic response is related directly to the decrease in relapse of the disease.

In the above preferred method, whole blood is collected in a container in presence or absence of any available anticoagulants such as Elseiver's solution or heparin, dextrose.

In another embodiment, blood is incubated for clotting at room temperature for a short period of time or plasma may be collected from blood.

In yet another embodiment, nonspecific proteins are removed from specific affinity matrix such as bovine submaxillary mucin--Sepharose 4B by extensive washing of unbound non O-acetyl sialic acid specific fractions with buffer such as phosphate buffer saline or Tris buffer saline.

In still another embodiment, specific protein is eluted with buffer of alkaline pH in the range of 8.0-11.0 from bovine submaxillary mucin--Sepharose 4B.

In another embodiment, eluted specific protein is immediately neutralized with sodium acetate or monosodium phosphate for better stability of the eluted protein.

In an embodiment, the IgG1 and IgG2 specific for O-Acetyl sialic acid in the patient's serum are determined by an isotype ELISA to diagnose phases or stages of acute lymphoblastic leukemia.

In an embodiment the serum is separated from the blood of patients afflicted with acute lyphoblastic leukemia wherein the blood is collected in the absence or in the presence of any anti-coagulants such as Elsevier's solution, potassium oxalate, ammonium oxalate, heparin, dextrose, etc.

In another embodiment the solvent used for removal of albumin and other low molecular weight proteins is saturated ammonium sulfate solution in the range of 15% to 40%.

In another embodiment the galactose binding proteins is removed by using any galactose containing glycoproteins such as asialo bovine submaxillary mucin, asialo sheep submaxillary mucin, asialo human chronic gonadotropin, asialo equine gastric mucin or hog gastric mucin etc. coupled with Sepharose or agarose as an affinity matrix.

In another embodiment the affinity matrix, for capturing O acetyl sialic acids specific fractions, used may be any available sialoglycoconjugates having terminal O acetyl sialic acids such as bovine submaxillary mucin or any gangliosides having terminal O acetyl sialic acids such as GD3.

In another embodiment the solvent used for eluting O acetyl sialic acids specific proteins may be a buffer selected from as ammonium hydroxide, borate buffer, carbonate bicarbonate buffer and ammonium bicarbonate buffer with high pH from 8 to 11 even expensive sugar such as O-acetylated sialic acid (as a monocaccharide or disaccharides or higher oligosaccharides with subterminal galactose of N acetyl galactosamine) may be used as eluting sugar. Molarity of buffer may be in the range of 0.02-0.5M.

In another embodiment neutralization may be carried out with sodium acetate or monosodium phosphate for better stability of the eluated protein. If sugars are used as eluting solution it may be removed simply by dialysis. Dialysis may be carried out at cold temperature between 4-25oC. for 24 to 72 hours to get disease specific antibody as a novel biomarker.

Accordingly, the present invention provides a disease specific antibody or immunoglobulin as biomarker by the process of present invention and used the same for diagnosis, monitoring outcome of treatment and prediction of relapse using a bovine submaxillary mucin-Enzyme linked immunosorbent assay (ELISA).

The novelty of this process is to capture the disease specific antibody as a novel biomarker specific for O-acetylated sialic acid by using bovine submaxillary mucin as an affinity matrix after careful removal of albumin, other low molecular weight fractions and galactose binding proteins from ALL serum. Characterization of this antibody as biomarker by several biochemical, immunochemical techniques it reveals that (i) biomarker is specific to leukemic blast cells in acute lymphoblastic leukemia (ii) irrespective of the lymphocytic origin of cancer cells such as acute lymphoblastic leukemia of B lymphocytes or acute lymphoblastic leukemia of T lymphocytes, this biomarker shows equal specificity, therefore, it may be considered as common biomarker and used for diagnosis of both types of acute lymphoblastic leukemia (iii) the amount of this biomarker specific for O-acetylated sialic acid is quantitated and has been exploited to correlate the status of patients with regard to their blast leukemic blast cells which stay in peripheral blood even after chemotherapy. The quantity of this disease specific antibody as the biomarker, specific for O-acetylated sialic acid, has been established to be directly proportional to presence of leukemic blast cells i.e., cancer cells, therefore, by simply measuring the amount of antibody as the biomarker by newly developed ELISA, it is possible to know the status of different stages of the disease (iv) thus its correlation with the status of the disease is assessed and confirmed that the disease specific antibody as the biomarker has both diagnostic and prognostic potential (v) The antibody i.e. immunoglobulin G (IgG) subclass distribution reacting with O-AcSA in comparison to antibody present in normal healthy individual has been characterized by the ELISA. Amongst the four human IgG subclasses, IgG1, and IgG2 are significantly increased in patients as compared to normal individuals. However, their IgG3 and IgG4 levels are unchanged and comparable to normal healthy individuals. Therefore, measuring both IgG1 and IgG2 specific for O-Acetyl sialic acid in patients by an isotype ELISA is very much beneficial. The disease specific IgM antibody has also been found to be potentially important.

(a) By fluorescence activated cell sorter (FACS) analysis using flurescin (FITC) conjugated purified antibody, leukemia blasts show 50-90% binding. Both B and T blasts showed similar binding with FITC-antibody (leukemic purified disease specific antibody);

(b) In BSM-ELISA also Patient serum, both from B & T ALL Patient showed binding with BSM. Detection of this disease specific antibody as a biomarker specific for O-acetylated sialic acid, in patients serum has been carried out by bovine submaxillary mucin--ELISA for estimation of this antibody as a biomarker specific for O-acetylated sialic acid:

Preparation of Bovine Submaxillary Mucin

Bovine submaxillary mucin was prepared according to the method of Murphy & Gottschalk, Biochimica et Biophysica Acta, vol. 52, p 349, 1961. Briefly, tissues were homogenized and extracted thrice with an equal amount of water by centrifugation at 10,000 g for 15 minutes at 4oC. The supernatant was collected, pH adjusted to 4.5 and the resulting precipitate removed by centrifugation at 5000 g for 20 minutes. The supernatant was then neutralized (pH 6.0) and dialyzed against water. Barium acetate was slowly added to the dialysate to make it 0.1 M followed by precooled methanol to give an alcohol concentration of 64% (v/v) and incubated overnight at 4oC. The precipitate formed was retrieved by centrifugation, dissolved in 0.1 M EDTA, dialyzed extensively against water and stored at -20oC. until use. Bovine submaxillary mucin and asialo-bovine submaxillary mucin were separately coupled to sepharose 4B using the method of Kohn and Wilchem. Asialo-bovine submaxillary mucin was prepared by acid hydrolysis of bovine submaxillary mucin with 0.05 M H2 SO4 at 80oC. for one hour. De-O-acetylated Bovine submaxillary mucin was prepared by incubation with 0.2N NaOH for 45 minutes at 4oC. followed by immediate neutralization.

Bovine submaxillary mucin, having high percentage of 9-O-acetyl sialic acid, has been used to successfully capture a disease specific antibody as a biomarker, specific for O-acetylated sialic acid from the patients serum which in turn can be detected by horse radish peroxidase conjugated goat anti-human IgG and detected by using a substrate and optical density measured in an ELISA reader. Briefly, in bovine submaxillary mucin coated plate, wells were coated with bovine submaxillary mucin. Following three washes with buffer the wells were blocked. Purified antibody i.e., O-acetylated sialic acid specific biomarker or patient sera was incubated for some time at cold and its binding to the coating material was measured colorimetrically using horse radish peroxidase conjugated goat anti human antibodies and azino-bis thio-sulfonic acid as the substrate. The optical density was measured in an ELISA reader.

The amount of antibody as a biomarker, specific for O-acetylated sialic acid was determined and has been exploited to correlate the status of patients with regards to their blast cells which stay in peripheral blood even after chemotherapy. Thus, its correlation with the status of the disease was assessed.

The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

EXAMPLE 1

Approximately 2-3 ml of blood was collected from T-acute lymphoblastic (T-ALL)leukemia patients, blood was allowed to clot and the serum was separated by centrifugation.

Purification of a biomarker, the antibody specific for O-acetylated sialic acid: Patient serum (5 ml) pooled from two T-ALL patients containing 184 mg of total protein was used to purify the polyclonal antibody fraction with preferential affinity for O-AcSA. Briefly, 100% saturated solution of ammonium sulphate solution was added slowly to the patient's serum at 4oC. in such a way so that final concentration of ammonium sulphate in the solution become 33%. The solution was kept at cold for overnight for complete precipitation. Next day solution was centrifuged and dialyzed extensively against phosphate buffer saline to remove trace amount of ammonium sulphate fractionation. Now the total content of proteins is 138 mg. This protein (138 mg) was passed over an asialo-bovine submaxillary mucin--Sepharose 4B (3.6 mg/ml) column to remove galactose binding proteins. The galactose specific fractions remain bind to the column and unbound fractions do not have affinity for galactose. Therefore, they do not bind to the column. These unbound free fractions (70 mg) was then loaded onto an another column, namely bovine submaxillary mucin--Sepharose 4B column (5.7 mg/ml) which had been previously equilibrated with phosphate buffered saline (PBS, pH 7.2). Following removal of non-specifically bound proteins by extensive washings in PBS, specific protein was eluted with 0.1 M NH4 OH, pH11.0 and immediately neutralized with 0.2N acetic acid. The eluted protein fraction (4 mg) was then passed over a Protein G-agarose column (2 ml, Pierce), previously equilibrated with PBS, eluted with 0.1M citric acid from pH 6.5 was carried out and the fractions immediately neutralized with 2M Tris followed by extensive dialysis against PBS. The yield the biomarker of the invention, anti-O-acetyl sialic acid antibody, IgG1 was 0.140 mg.

The biological activity and affinity of this purified biomarker, specific for O-acetylated sialic acid was confirmed by bovine submaxillary mucin--ELISA and its specificity for O-acetylated sialic acids validated using de-O-acetylated Bovine submaxillary mucin a coating agent in bovine submaxillary mucin--ELISA.

EXAMPLE 2

The use of this biomarker for the diagnosis of acute lymphoblastic leukemia was carried out by developing a bovine submaxillary mucin--ELISA. Diagnosis of T-ALL:

In Bovine submaxillary mucin- ELISA, ELISA plate was coated with bovine submaxillary mucin (5 .mu.g/ml, 100 .mu.l/well) in 0.02M phosphate buffer, pH 7.4 and left it for overnight at 4oC. Following three washes with phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T), the wells were blocked with 2% bovine serum albumin for 2 hours at 25oC. Patient sera from T-ALL patients having 90% leukemic blasts or purified O-acetylated sialic acids specific antibody as a biomarker was incubated overnight at 4oC. in 1:10 dilutions and its binding to bovine submaxillary mucin was measured colorimetrically using horse radish peroxidase (HRP) conjugated protein A (1:5000, Sigma, St. Louis, Mo., USA) and azino-bis thio-sulfonic acid (ABTS) as the substrate.

EXAMPLE 3

Diagnosis of B-ALL by developing a bovine submaxillary mucin--ELISA

In Bovine submaxillary mucin--ELISA, ELISA plate was coated with bovine submaxillary mucin (5 .mu./ml, 100 .mu.l/well) in 0.02M phosphate buffer, pH 7.4 and left overnight at 4oC. Following three washes with phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T), the wells were blocked with 2% dry milk powder for 2 hours at 25oC. Patient from B ALL having 80% leukemic blasts or purified O-acetylated sialic acids specific antibody as the biomarker was incubated overnight at 4oC. in 1:10 dilutions and its binding to bovine submaxillary mucin was measured colorimetrically using horse radish peroxidase (HRP) conjugated goat anti human IgG1 (1:5000, Sigma, St. Louis, Mo., USA) and azino-bis thio-sulfonic acid (ABTS) as the substrate.

EXAMPLE 4

Diagnosis of ALL having both T and B leukemic blasts by developing a bovine submaxillary mucin ELISA

In bovine submaxillary mucin--ELISA, ELISA plate was coated with bovine submaxillary mucin (5 .mu.g/ml, 50 .mu.l/well) in 0.02M phosphate buffer, pH 7.4 and left for 6 hours at 4oC. Following three washes with phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T), the wells were blocked with 2% bovine serum albumin for 1 hours at 37oC. Patient sera of mixed lineage having 80% leukemic blasts in both B and T lymphocytes or purified O-acetylated sialic acids specific biomarker was incubated overnight at 4oC. in 1:10 dilutions and its binding to bovine submaxillary mucin was measured calorimetrically using horse radish peroxidase (HRP) conjugated goat anti human IgG2 (1:5000, Sigma, St. Louis, Mo., USA) and azino-bis-sulfonic acid (ABTS) as the substrate.

Claim 1 of 13 Claims

What is claimed is:

1. A novel process for preparation of O-acetyl specific immunoglobulins as a biomarker specific for O-acetylated sialic acid for diagnosing, monitoring treatment outcome and predicting relapse of acute lymphoblastic leukemia, said process comprising the steps of:

(i) separating serum from whole blood from a patient suffering from acute lymphoblastic leukemia;

(ii) removing galactose-binding proteins from said serum by column chromatography on an asialo-bovine submaxillary mucin affinity matrix;

(iii) collecting the unbound galactose-free protein fraction from the affinity matrix;

(iv) passing said galactose-free protein fraction over another affinity matrix having terminal O-acetyl sialic acid moiety to capture the O-acetyl sialic acid specific protein fraction;

(v) eluting the O-acetyl sialic acid specific protein fraction with a buffer at alkaline pH in the range of about 8.0-11.0 followed by neutralization of said fraction; and

(vi) purifying from the O-acetyl sialic acid specific protein fraction, the O-acetyl sialic acid specific immunoglobulins.




____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]