Test kits for determining at least two specific analytes in foods and other complex matrices

• US 5,958,714 A
• Filed: 10/02/1996
• Issued: 09/28/1999
• Est. Priority Date: 10/02/1996
• Status: Expired due to Fees

• Current Assignee: Saftest, Inc.
• Original Assignee: Safety Associates, Inc.
• Inventors: Mirhashemi, Soheila, Mittelstein, Michael, Sorensen, John T., Elias, John F., Gordon, Virginia C.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): PORTNER, VIRGINIA ALLEN
• Application Number:
  US08/723,636
• Time in Patent Office: 1,091 Days
• Field of Search: 436/516, 435/280, 435/179, 435/7.92, 422/56, 422/58, 422/102, 210/238
• US Class Current: 435/7.92
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: Y10S 210/908 Organic
• CPC Class Codes: Y10S 436/824 Immunological separation techniques
• CPC Class Codes: Y10S 436/825 Pretreatment for removal of interfering factors from sample
• CPC Class Codes: Y10T 436/25125 Digestion or removing interfering materials
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

Methods and apparatus for qualitatively or quantitatively determining one or more analytes in matrices such as foods, biological fluids, etc. An embodiment for determination of a single analyte comprises a sample receiving vessel, a first membrane and a reagent-containing well. The prepared sample passes through the first membrane whereby extraneous matter is removed, and a filtrate enters the reagent-containing well to provide a filtrate-reagent admixture from which the analyte may be determined. An embodiment for determination for multiple analytes includes one or more additional membranes in series with the first membrane, each such additional membrane being operative to capture one or more analytes. Each of the additional analytes may then be eluted from the membrane upon which it has been captured, into a separate reagent-containing well to provide eluant-reagent admixture from which each desired analyte may be determined. Formulations for preparation additives are also included. Additionally, there'"'"'s provided an embodiment of above-described invention for determination of an analyte which is present in a matrix at low (e.g., sub-detectable) levels, wherein the filter of the apparatus is utilized to capture and concentrate the analyte, to provide a filtrate-reagent admixture wherein the analyte is present at detectable concentration.

Claims

1. A test kit for determining the presence of at least first and second analytes in a matrix which contains matter other than said first and second analytes, said test kit comprising:

a first membrane which is operative to prevent some of the matter of said matrix from passing therethrough, while allowing a filtrate containing said analyte to pass therethrough;

a first receiving vessel positioned in relation to said first membrane so as to receive said filtrate therein; and

,

at least one reagent which is combineable with said filtrate in said receiving vessel to provide a reagent-filtrate admixture from which said analyte may be determined;

a second membrane interposed between said first membrane and said first receiving well, said second membrane being operative to capture and hold said second analyte while allowing a sub-filtrate containing said first analyte to pass therethrough and into said first receiving well, said second membrane being subsequently positionable adjacent a second receiving well such that a flush solution may be passed through said second membrane such that an eluant containing said second analyte will be received within said second receiving vessel; and

,

at least one reagent which is combineable with the eluant in said second receiving vessel to provide a reagent-eluant admixture from which said second analyte may be determined;

wherein said first analyte is lipid peroxide and said second analyte is lipid aldehyde, and wherein;

said first membrane is a microporous membrane which will prevent a portion of said matrix from matrix from passing therethrough, while allowing lipid peroxides and lipid aldehydes to pass therethrough in said filtrate;

said second membrane is a membrane which will capture and hold lipid aldehydes, while allowing lipid peroxides to pass therethrough in said sub-filtrate;

said first reagent is xylenol orange such that a xylenol orange-filtrate admixture is provided from wherein the presence of lipid peroxides may be determined by spectrophotometric means; and

,

said second reagent is methyl indole, such that a methyl indole-eluant admixture is provided in which the presence of lipid aldehydes may be determined by spectrophotometric means.

2. The test kit of claim 1 wherein said first membrane is a cellulose ester film having pores of approximately 0.45 microns in size.

3. The test kit of claim 1 wherein said second membrane is a polyamide film having pores of approximately 0.2 microns in size.

4. A test kit for determining the presence of at least first and second analytes in a matrix which contains matter other than said first and second analytes, said test kit comprising:

a first membrane which is operative to prevent some of the matter of said matrix from passing therethrough, while allowing a filtrate containing said analyte to pass therethrough;

a first receiving vessel positioned in relation to said first membrane so as to receive said filtrate therein; and

,

at least one reagent which is combineable with said filtrate in said receiving vessel to provide a reagent-filtrate admixture from which said analyte may be determined;

a second membrane interposed between said first membrane and said first receiving well, said second membrane being operative to capture and hold said second analyte while allowing a sub-filtrate containing said first analyte to pass therethrough and into said first receiving well, said second membrane being subsequently positionable adjacent a second receiving well such that a flush solution may be passed through said second membrane such that an eluant containing said second analyte will be received within said second receiving vessel; and

,

at least one reagent which is combineable with the eluant in said second receiving vessel to provide a reagent-eluant admixture from which said second analyte may be determined;

wherein said first analyte is lipid peroxide and said second analyte is hexanal, and wherein;

said first membrane is a microporous membrane which will prevent a portion of said matrix from passing therethrough, while allowing lipid peroxides and hexanal to pass therethrough in said filtrate;

said second membrane is a membrane which will capture and hold lipid peroxides, while allowing hexanal to pass therethrough in said sub-filtrate;

said first reagent is a mixture of methyl indole and methane sulfonic acid such that a methyl indole/methane sulfonic acid-filtrate admixture is provided in which the concentration of hexanal may be determined by spectrophotometric means; and

,

said second reagent is xylenol orange such that a xylenol orange-eluant admixture is provided in which the presence of lipid peroxides may be determined by spectrophotometric means.

5. The test kit of claim 4 wherein said first membrane is microporous PVDF having pores of approximately 0.45 microns in size.

6. The test kit of claim 4 wherein said second membrane is silica fused glass having pores of approximately 0.2-0.45 microns in size.

7. A test kit for determining the presence of at least first and second analytes in a matrix which contains matter other than said first and second analytes, said test kit comprising:

a first membrane which is operative to prevent some of the matter of said matrix from passing therethrough, while allowing a filtrate containing said analyte to pass therethrough;

a first receiving vessel positioned in relation to said first membrane so as to receive said filtrate therein; and

,

at least one reagent which is combineable with said filtrate in said receiving vessel to provide a reagent-filtrate admixture from which said analyte may be determined.

a second membrane interposed between said first membrane and said first receiving well, said second membrane being operative to capture and hold said second analyte while allowing a sub-filtrate containing said first analyte to pass therethrough and into said first receiving well, said second membrane being subsequently positionable adjacent a second receiving well such that a flush solution may be passed through said second membrane such that an eluant containing said second analyte will be received within said second receiving vessel; and

,

at least one reagent which is combineable with the eluant in said second receiving vessel to provide a reagent-eluant admixture from which said second analyte may be determined.

wherein said first analyte is lipid peroxide and said second analyte is conjugated linoleic acid, and wherein;

said first membrane is a microporous membrane which will prevent a portion of said matrix from passing therethrough, while allowing lipid peroxides and conjugated linoleic acid to pass therethrough in said filtrate;

said second membrane is a membrane which will capture and hold conjugated linoleic acid while allowing lipid peroxides to pass therethrough in said sub-filtrate;

said first reagent is xylenol orange such that a xylenol orange-sub-filtrate admixture is provided in which the presence of lipid peroxides may be determined by spectrophotometric means; and

,

said second reagent is fat red dye, such that a fat red dye-eluant admixture is provided in which the presence of conjugated linoleic acid may be determined by enzyme immunoassay.

8. The test kit of claim 7 wherein said first membrane is microporous nylon having pores of approximately 0.45 microns in size.

9. The test kit of claim 7 wherein said second membrane is nitrocellulose film coated with an antibody to conjugated linoleic acid.

10. A test kit for determining the presence of at least first and second analytes in a matrix which contains matter other than said first and second analytes, said test kit comprising:

a first membrane which is operative to prevent some of the matter of said matrix from passing therethrough, while allowing a filtrate containing said analyte to pass therethrough;

a first receiving vessel positioned in relation to said first membrane so as to receive said filtrate therein; and

,

at least one reagent which is combineable with said filtrate in said receiving vessel to provide a reagent-filtrate admixture from which said analyte may be determined;

a second membrane interposed between said first membrane and said first receiving well, said second membrane being operative to capture and hold said second analyte while allowing a sub-filtrate containing said first analyte to pass therethrough and into said first receiving well, said second membrane being subsequently positionable adjacent a second receiving well such that a flush solution may be passed through said second membrane such that an eluant containing said second analyte will be received within said second receiving vessel; and

,

at least one reagent which is combineable with the eluant in said second receiving vessel to provide a reagent-eluant admixture from which said second analyte may be determined;

wherein said first analyte is all chloroacetanilide herbicides and said second analyte is alachlor herbicide and wherein;

said first membrane is a microporous membrane which will prevent a portion of said matrix from passing therethrough while allowing all chloroacetamide herbicides including alachlor to pass therethrough in said filtrate;

said second membrane is a membrane which will capture and hold alachlor while allowing chloroacetanilide herbicides other than alachlor to pass therethrough in said sub-filtrate;

said first reagent is ethylchloroformate such that an ethylchloroformite-sub-filtrate admixture is provided in which the concentration of chloroacetamide herbicides other than alachlor may be determined by spectrophotometric means; and

,

said second reagent is ethylchloroformite such that an ethylchloroformite-eluant admixture is provided in which the concentration of alachlor may be determined by spectrophotometric means.

11. A method for determining the presence of first and second analyses within a matrix, said method comprising the steps of:

a) passing the matrix through a first membrane to remove extraneous matter therefrom, such that a filtrate containing said first and second analytes will pass through the first membrane;

b) passing the filtrate through a second membrane to capture within said second membrane said first analyte, such that a sub-filtrate containing said second analyte will pass through said second membrane;

c) collecting the sub-filtrate which has passed through said first and second membranes, and combining said sub-filtrate with at least one reagent to provide a sub-filtrate-reagent admixture from which the presence of said first analyte may be determined;

d) eluting the second analyte from the second membrane, and combining such eluant with at least one reagent to provide an eluant-reagent admixture from which the presence of said second analyte may be determined;

e) determining the presence of said first analyte in said sub-filtrate-reagent admixture; and

,

f) determining the presence of said second analyte in said eluant-reagent admixture;

wherein said first analyte is lipid peroxide and said second analyte is lipid aldehyde, and further wherein;

said first membrane is a microporous membrane which will prevent a portion of said matrix from matrix from passing therethrough, while allowing lipid peroxides and lipid aldehydes to pass therethrough in said filtrate;

said second membrane is a membrane which will capture and hold lipid aldehydes, while allowing lipid peroxides to pass therethrough in said sub-filtrate;

said first reagent is xylenol orange such that a xylenol orange-filtrate admixture is provided from wherein the presence of lipid peroxides may be determined by spectrophotometric means; and

,

said second reagent is methyl indole, such that a methyl indole-eluant admixture is provided in which the presence of lipid aldehydes may be determined by spectrophotometric means.

12. A method for determining the presence of first and second analyses within a matrix, said method comprising the steps of:

a) passing the matrix through a first membrane to remove extraneous matter therefrom, such that a filtrate containing said first and second analytes will pass through the first membrane;

b) passing the filtrate through a second membrane to capture within said second membrane said first analyte, such that a sub-filtrate containing said second analyte will pass through said second membrane;

c) collecting the sub-filtrate which has passed through said first and second membranes, and combining said sub-filtrate with at least one reagent to provide a sub-filtrate-reagent admixture from which the presence of said first analyte may be determined;

d) eluting the second analyte from the second membrane, and combining such eluant with at least one reagent to provide an eluant-reagent admixture from which the presence of said second analyte may be determined;

e) determining the presence of said first analyte in said sub-filtrate-reagent admixture; and

,

f) determining the presence of said second analyte in said eluant-reagent admixture;

wherein wherein said first analyte is lipid peroxide and said second analyte is hexanal, and further wherein;

said first membrane is a microporous membrane which will prevent a portion of said matrix from passing therethrough, while allowing lipid peroxides and hexanal to pass therethrough in said filtrate;

said second membrane is a membrane which will capture and hold lipid peroxides, while allowing hexanal to pass therethrough in said sub-filtrate;

said first reagent is methyl indole in combination with methane sulfonic acid such that a methyl indole/methane sulfonic acid-filtrate admixture is provided in which the concentration of hexanal maybe determined by spectrophotometric means; and

,

said second reagent is xylenol orange such that a xylenol orange-eluant admixture is provided in which the presence of lipid peroxides may be determined by spectrophotometric means.

13. A method for determining the presence of first and second analyses within a matrix, said method comprising the steps of:

a) passing the matrix through a first membrane to remove extraneous matter therefrom, such that a filtrate containing said first and second analytes will pass through the first membrane;

b) passing the filtrate through a second membrane to capture within said second membrane said first analyte, such that a sub-filtrate containing said second analyte will pass through said second membrane;

c) collecting the sub-filtrate which has passed through said first and second membranes, and combining said sub-filtrate with at least one reagent to provide a sub-filtrate-reagent admixture from which the presence of said first analyte may be determined;

d) eluting the second analyte from the second membrane, and combining such eluant with at least one reagent to provide an eluant-reagent admixture from which the presence of said second analyte may be determined;

e) determining the presence of said first analyte in said sub-filtrate-reagent admixture; and

,

f) determining the presence of said second analyte in said eluant-reagent admixture;

wherein said first analyte is lipid peroxide and said second analyte is conjugated linoleic acid, and further wherein;

said first membrane is a microporous membrane which will prevent a portion of said matrix from passing therethrough while allowing lipid peroxides and conjugated linoleic acid to pass therethrough in said filtrate;

said second membrane is a membrane which will capture and hold conjugated linoleic acid while allowing lipid peroxides to pass therethrough in said sub-filtrate;

said first reagent is xylenol orange such that a xylenol orange-sub-filtrate admixture is provided in which the presence of lipid peroxides may be determined by spectrophotometric means; and

,

said second reagent is fat red dye, such that a fat red dye-eluant admixture is provided in which the presence of conjugated linoleic acid may be determined by enzyme immunoassay.

14. A method for determining the presence of first and second analyses within a matrix, said method comprising the steps of:

a) passing the matrix through a first membrane to remove extraneous matter therefrom, such that a filtrate containing said first and second analytes will pass through the first membrane;

b) passing the filtrate through a second membrane to capture within said second membrane said first analyte, such that a sub-filtrate containing said second analyte will pass through said second membrane;

c) collecting the sub-filtrate which has passed through said first and second membranes, and combining said sub-filtrate with at least one reagent to provide a sub-filtrate-reagent admixture from which the presence of said first analyte may be determined;

d) eluting the second analyte from the second membrane, and combining such eluant with at least one reagent to provide an eluant-reagent admixture from which the presence of said second analyte may be determined;

e) determining the presence of said first analyte in said sub-filtrate-reagent admixture; and

,

f) determining the presence of said second analyte in said eluant-reagent admixture;

wherein said first analyte is all chloroacetanilide herbicides and said second analyte is alachlor herbicide, and further wherein;

said first membrane is a microporous membrane which will prevent a portion of said matrix from passing therethrough while allowing all chloroacetamide herbicides including alachlor to pass therethrough in said filtrate;

said second membrane is a membrane which will capture and hold alachlor while allowing chloroacetanilide herbicides other than alachlor to pass therethrough in said sub-filtrate;

said first reagent is ethylchloroformate such that an ethylchloroformite-sub-filtrate admixture is provided in which the concentration of chloroacetamide herbacides other than alachlor may be determined by spectrophotometric means; and

,

said second reagent is ethylchloroformite such that an ethylchloroformite-eluant admixture is provided in which the concentration of alachlor may be determined by spectrophotometric means.