# US 5,348,860 A · Screening test and kit for cancerous and precancerous conditions

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- Number: US 5,348,860 A
- Document ID: US5348860A
- Status: Granted
- Kind: A
- Application number: 07/776,786
- Filed: 1991-10-15
- Granted: 1994-09-20
- Earliest claimed: 1988-08-04
- Inventors: Abulkalam M. Shamsuddin
- Web page: https://patentgrep.com/patent/US5348860A

## CPC

- G01N 33/5756: {involving tumour-associated glycolinkage [TAG]}
- G01N 33/5753: of the stomach or small intestine
- Y10S 435/967: Standards, controls, materials, e.g. validation studies, buffer systems
- Y10S 435/97: Test strip or test slide
- Y10S 435/975: Kit
- Y10S 436/813: Cancer
- Y10T 436/143333: Saccharide [e.g., DNA, etc.]
- Y10T 436/144444: Glucose
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

An improved screening test method and kit for cancerous and precancerous conditions which is rapid, employs reagents which can be provided in kit form and which does not give false negatives due to sampling error, immobilizes a sample of a proteinaceous body fluid in a membrane filter, tests for marker carbohydrates therein which have vicinal galactose moieties and which are enzymatically oxidized with galactose oxidase to vicinal aldehydic moieties which then visualizes any thus-produced vicinal aldehydic moieties with Schiff's Reagent, then further oxidizes those samples which test negative for the marker carbohydrates with periodic acid, thereby rendering the sample visualizable with Schiff's Reagent if the sample contains glycoprotein, which applies the galactose oxidase directly to the membrane filter, thereby speeding up color developed, and employs Schiff's Reagent which was refrigerated after preparation until its color faded to a straw shade before being treated with activated charcoal, which treatment renders the solution storage-stable for many months and enhances its color development ability.

## Claims

### Claim 1

A screening method for rapidly testing a human being for a cancerous or precancerous condition and as part of the same testing eliminating the possibility of a false negative, whereby the individual can be immediately retested if a negative assay is due to procedural error, which comprises the steps of (a) obtaining a sample of rectal mucus from an individual; (b) assaying a portion of the sample for the presence therein of glycoprotein containing at least one carbohydrate selected from the group consisting of beta-D-Gal-(1->3)-D-GalNAc, Fuc-alpha-1->2 Gal-Beta(1->4)-Fuc-alpha-1->3-GlcNAc, Fuc-Alpha-1->2 Gal-beta-(1->4)-Fuc-alpha-1->3-GlcNAcbeta-(1->3)-Gal-beta-(1>4)-GlcNAc and Fuc-alpha-1->2-Gal-beta-(1->4)-Fuc-alpha-1->3-GlcNAc-beta-(1->3)-Gal-beta- (1->4)-Fuc-alpha-1->3-GlcNAc, by briefly subjecting the sample to oxidizing conditions which are capable of selectively oxidizing only the cyclic sugar moieties of any said marker carbohydrate present in the glycoprotein in the sample at a hydroxy group-bearing ring carbon atom thereof to form an aldehydic sugar moiety and then visualizing any aldehydic saccharide groups thus formed with a Schiff's base decolorized dye; (c) also assaying a portion of the sample for the presence therein of any glycoprotein by subjecting the sample to the oxidizing action of an oxidizing agent which oxidizes the saccharide moieties of any glycoprotein therein to aldehydic sugar moieties and then visualizing any thus-produced aldehydic sugar moieties, the presence of thereby formed aldehydic sugar moieties confirming the adequacy of the mucus sampling and the absence therein of thereby formed aldehyde sugar moieties establishing that the negative test results were due to mucus sampling error; and (d) either retesting the individual in the same manner with a fresh sample of rectal mucus, if the first sample assays negative in steps (b) and (c), or examining another proteinaceous fluid associated with another organ of the individual in the same manner, if the rectal mucus sample tests positive and no abnormality of the rectum or colon is found.

### Claim 2 (depends on 1)

A method according to claim 1, wherein the assay comprises the steps of adsorbing the mucus sample onto a protein-capturing water-insoluble substrate and then washing the substrate to remove non-immobilized components of the mucus sample from the substrate.

### Claim 3 (depends on 2)

A method according to claim 2, wherein the insoluble substrate is a membrane filter.

### Claim 4 (depends on 1)

A method according to claim 1, wherein the marker carbohydrate sugar moieties are assayed by selectively oxidizing the glycoprotein so as to selectively oxidize the primary hydroxy groups of any galactose moieties thereof to aldehydic groups and the thus-oxidized glycoprotein is then assayed for oxidized vicinal galactose moieties.

### Claim 5 (depends on 4)

A method according to claim 4, wherein the galactose moieties are oxidized with galactose oxidase.

### Claim 6 (depends on 4)

A method according to claim 4, performed simultaneously on a plurality of samples obtained from a plurality of individuals rectal mucus as part of a field screening for cancer.

### Claim 7 (depends on 5)

A method according to claim 5, wherein the oxidized vicinal galactose moieties are visualized with basic fuchsin.

### Claim 8 (depends on 1)

A method according to claim 1, wherein step (c) is conducted concurrently with step (b) on a different portion of the same mucus sample; and wherein immediately thereafter, when a negative result is obtained in both of steps (b) and (c), immediately thereafter another sample of rectal mucus is collected from the individual steps (a), (b) and (c) are repeated.

### Claim 9 (depends on 8)

A method according to claim 8, wherein any aldehydic sugar moieties in a mucus sample further oxidized with periodic acid are visualized with basic fuchsin.

### Claim 10 (depends on 1)

A method according to claim 1, wherein prior to the assaying thereof the rectal mucus sample is adsorbed onto a protein-capturing water-insoluble substrate and the substrate is then washed to remove the non-immobilized components of the mucus sample from the substrate; wherein the sample is assayed in step (a) by selectively oxidizing any glycoprotein in the sample so as to oxidize the primary hydroxy group of the galactose sugar moiety of any said carbohydrate therein to an aldehydic group and the thus-oxidized galactose moiety is then visualized with basic fuchsin; and wherein a mucus sample which tests negative in that assay is further oxidized with periodic acid and the aldehydic sugar moiety in the thus-oxidized sample is visualized with basic fuchsin.

### Claim 11 (depends on 10)

A method according to claim 10, wherein the insoluble substrate is a membrane filter and wherein the galactose sugar moieties are oxidized with galactose oxidase.

### Claim 12 (depends on 10)

A method according to claim 10, wherein the oxidizing agent for the galactose moieties is present in the insoluble substrate or is applied directly thereto after the mucus sample is applied thereto.

### Claim 13 (depends on 12)

A method according to claim 12, wherein the insoluble substrate is a membrane filter, wherein the galactose sugar moieties are oxidized with galactose oxidase and wherein the galactose oxidase is applied directly to the membrane filter after the mucus sample is applied thereto.

### Claim 14

A method of eliminating a false negative as a result of mucus sampling error in a rectal mucus method for screening for a cancerous or precancerous condition in a human being, which comprises conducting, within about a half hour period after collecting a sample of rectal mucus, the steps of (a) subjecting a first portion of the sample of rectal mucus in an assay for the presence therein of at least one marker carbohydrate selected from the group consisting of beta-D-Gal-(1->3)-D-GalNAc, Fuc-alpha-1->2 Gal-Beta(1->4)-Fuc-alpha-1->3-GlcNAc, Fuc-Alpha-1->2Gal-beta-(1->4)-Fuc-alpha-1->3-GlcNAc-beta-(1->3)-Gal-beta-( 1>4)-GlcNAc and Fuc-alpha-1->2-Gal-beta-(1->4)-Fuc-alpha-1->3-GlcNA-beta-(1->3)-Gal-beta-( 1->4)-Fuc-alpha-1->3-GlcNAc, and (b) either concurrently subjecting another portion of the same mucus sample or, if the first portion tests negative in step (a), thereafter subjecting the same portion, to oxidizing conditions which are capable of oxidative ring opening of the cyclic sugar moieties of any glycoprotein present in the sample at a hydroxy group-bearing ring carbon atom thereof to form aldehydic sugar moieties and then assaying the thus-oxidized sample for the presence therein of any aldehydic sugar moieties thus formed, the presence of thereby formed aldehydic sugar moieties confirming the adequacy of the mucus sampling and additionally, when the assay for marker carbohydrates is negative, that none of the marker carbohydrates are present in the mucus sample.

### Claim 15 (depends on 14)

A method according to claim 14, wherein prior to the assaying thereof the rectal mucus sample is adsorbed onto a protein-capturing water-insoluble substrate and the substrate is then washed to remove the non-immobilized components of the mucus sample from the substrate; wherein a first portion of the sample is assayed for the marker carbohydrates by selectively oxidizing any glycoprotein in the sample with galactose oxidase so as to selectively oxidize the primary hydroxy groups of any galactose sugar moieties thereof to aldehydic sugar moieties; the thus-oxidized is then assayed for oxidized vicinal galactose moieties; and wherein a separate second portion of the rectal sample is concurrently oxidized in Step (b) with periodic acid and any of the cyclic sugar moieties in the thus-oxidized second portion of the sample are visualized with basic fuchsin.

### Claim 16

In a process for producing an aqueous solution of basic fuchsin wherein a basic solution of fuchsin and sodium bisulfate is treated with activated charcoal and is stored at below room temperature, the improvement which comprises storing the basic solution at below room temperature until the color thereof fades to a straw color before the activated charcoal is added thereto, thereby rendering the solution storage-stable and enhancing its color development ability.

### Claim 17 (depends on 16)

The process according to claim 16, wherein the solution is stored at about 0.degree.-15.degree. C. for at least one day.

### Claim 18 (depends on 16)

The process according to claim 16, wherein the basic fuchsin solution is Schiff's Reagent.

### Claim 19

A method for screening for a cancerous or precancerous condition in an organ other than the large intestine of a human being, which comprises subjecting a sample of proteinaceous secretion other than rectal mucus associated with that organ, to an assay for the presence therein of a marker carbohydrate selected from the group consisting of beta-D-Gal-(1->3)-D-GalNAc, Fuc-alpha-1->2 Gal-Beta(1->4)-Fuc-alpha-1->3-GlcNAc, Fuc-Alpha-1->2 Gal-beta-(1->4)-Fuc-alpha-1-22 3-GlcNAc-beta-(1->3)-Gal-beta-(1>4)-GlcNAc and Fuc-alpha-1->2-Gal-beta-(1->4)-Fuc-alpha-1->3-GlcNAc-beta-(1->3)Gal-beta-( 1->4)-Fuc-alpha-1->3-GlcNAc.

### Claim 20 (depends on 19)

A method according to claim 19, wherein prior thereto the proteinaceous secretion is adsorbed onto a protein-capturing water-insoluble substrate and the substrate is then washed to remove the nonimmobilized components of the mucus sample from the substrate; wherein the sample is assayed for the marker carbohydrates by selectively oxidizing any glycoprotein in the sample with galactose oxidase so as to selectively oxidize the primary hydroxy group of any galactose sugar moiety of any said marker carbohydrate therein to an aldehydic group and the thus-oxidized galactose moiety is then visualized with basic fuchsin; and wherein a portion of the sample which tests negative in that assay is further oxidized with periodic acid and the aldehydic sugar moiety in the thus-oxidized sample is visualized with basic fuchsin.

### Claim 21 (depends on 20)

A method according to claim 20, which comprises the portion of the sample of proteinaceous secretion which is oxidized with periodic acid is a different portion of the same sample which is oxidized with galactose oxidase and is oxidized concurrently therewith.

### Claim 22 (depends on 19)

The method of claim 19 wherein the human being is female and the fluid is vaginal or endocervical mucus.

### Claim 23 (depends on 19)

The method of claim 19, wherein the human being is female and the secretion is discharge from a breast thereof.

### Claim 24 (depends on 19)

The method of claim 19 wherein the human being is male and the fluid is prostatic secretion or semen.

### Claim 25 (depends on 19)

The method of claim 19, wherein the human being is a patient exhibiting abnormal respiratory symptoms and the secretion is sputum.