Immunological determination using lectin

• US 4,289,747 A
• Filed: 12/26/1978
• Issued: 09/15/1981
• Est. Priority Date: 12/26/1978
• Status: Expired due to Term

• Current Assignee: E-Y Laboratories, Inc.
• Original Assignee: E-Y Laboratories, Inc.
• Inventors: Chu, Albert E.
• Primary Examiner(s): Padgett, Benjamin R.
• Assistant Examiner(s): Nucker, Christine M.
• Application Number:
  US05/972,696
• Time in Patent Office: 994 Days
• Field of Search: 424/1, 424/12, 23/230 B, 435/7
• US Class Current: 435/7.8
• CPC Class Codes: G01N 2470/04 Sandwich assay format
• CPC Class Codes: G01N 33/537 with separation of immune complex from unbound antigen or antibody
• CPC Class Codes: G01N 33/5375 by changing the physical or chemical properties of the medium or immunochemicals, e.g. temperature, density, pH, partitioning

Abstract

A method for the determination of one or more components of an immunological conjugate, e.g., antigens, of a fluid sample in a competitive or sandwich technique in which the conjugate is labelled and separated from its reactive mixture by reversible attachment to a solid surface. In a preferred embodiment, the solid surface comprises insolubilized sugar which reversibly bonds to a lectin covalently bonded to one member of the conjugate. After separation of such solid surface from the remainder of the reaction mixture, the insolubilized sugar-lectin bond is broken by contact with a sugar solution which displaces the labelled lectin compound. The immunological components including label and lectin may be preincubated in a homogeneous solution prior to reversible attachment to the sugar solid surface. For a competitive system, a sample containing antigen is incubated with a known quantity of labelled antigen and lectin-bound antibody. In the sandwich technique, the sample antigen is incubated with lectin-bound antibody and further with labelled antibody and this reaction mixture is contacted with insolubilized sugar. Either the competitive or sandwich technique are adaptable to a sequential flowthrough system with sufficient residence time to eliminate the preliminary incubation steps.

Claims

1. A method for the determination of one component of an immunological conjugate in a fluid sample comprising the steps of

(a) forming an immunological conjugate reaction product including said one component from a fluid sample immunologically bound to its corresponding component, a bound label compound, and a bound lectin, said lectin being bound to said corresponding component,

(b) contacting said reaction product with insolubilized sugar selectively and reversibly reactive with said bound lectin to reversibly bind the reaction product through the lectin to the insolubilized sugar,

(c) separating the insolubilized sugar-bound lectin product from the remainder of said reaction mixture, and

(d) determining the amount of label compound in either said insolubilized sugar-bound lectin product or said remainder as a measure of the quantity of said one component in said sample.

2. A method for the determination of one component of an immunological conjugate in a fluid sample comprising the steps of

(a) contacting fluid sample containing said one component with its corresponding immunological component bound to a lectin and with a labelled or an anti(corresponding component) to form an immunological reaction product,

(b) incubating the reaction mixture of step (a) for a sufficient time to cause an immunological reaction,

(c) contacting said incubated reaction mixture with insolubilized sugar selectively and reversibly reactive with said bound lectin to reversibly bind the lectin to the insolubilized sugar, and

(d) separating the insolubilized sugar-bound lectin reaction product from the remainder of said reaction mixture.

3. A method for the determination of one component of an immunological conjugate in a fluid sample comprising

(a) mixing said sample containing said one component with a known amount of said one component bound to a label compound in soluble form and with the corresponding component of said immunological conjugate bound to a lectin to form a fluid reaction mixture,

(b) incubating the reaction mixture of step (a) to cause a competitive immunological reaction,

(c) contacting said incubated reaction mixture with insolubilized sugar selectively and reversibly reactive with said bound lectin to reversibly bind the lectin to the insolubilized sugar,

(d) separating the insolubilized sugar-bound lectin reaction product from the remainder of said reaction mixture, and

(e) determining the amount of label compound in either said insolubilized sugar-bound lectin product or said remainder as a measure of the quantity of said one component in said sample.

4. The method of claim 3 in which said one component of said immunological conjugate is selected from the group consisting of antigen, antibody, or portions and equivalents thereof.

5. The method of claim 4 in which said one component is an antigen and said corresponding component is an antibody.

6. The method of claim 3 in which said one component is an antibody and said corresponding component is an antigen.

7. The method of claim 3 in which said label compound is selected from the group consisting of enzyme, radioactive molecules, and fluorescent molecules.

8. The method of claim 7 in which said label compound is an enzyme and the amount of said enzyme is determined by measuring its enzyme activity.

9. The method of claim 3 in which step (e) is performed by contacting said insolubilized sugar-bound lectin product with soluble sugar of the same type as said insolubilized sugar to cause the bound lectin product to react with said soluble sugar for conversion to a solubilized form, then separating the soluble and insoluble components into different fractions and thereafter measuring the amount of label compound in the soluble fraction.

10. The method of claim 3 in which the insolubilized sugar comprises sugar covalently bound to a solid surface.

11. The method of claim 3 in which said sample contains glycoprotein reactive with lectin and prior to step (c) said unbound lectin is added to said sample of the same type as said bound lectin, said unbound lectin being in excess of said glycoprotein reactive therewith in the sample, so that said unbound lectin blocks the lectin-reactive sites of said glycoprotein.

12. The method of claim 3 in which said sample contains glycoprotein reactive with lectin and prior to step (c) said sample is passed through a bed of insolubilized lectin with glycoprotein-reactive sites and of the same type as said bound lectin so that said glycoprotein is retained on said column and thereby removed from said sample.

13. A method for the determination of one component of an immunological conjugate in a fluid sample comprising the steps of

(a) incubating said sample containing said one component with the corresponding component of said immunological conjugate bound to a lectin to cause said immunological reaction to occur,

(b) incubating the one component with anti(one component) bound to a label compound to cause an immunological reaction between said one component and said anti(one component),

(c) contacting the reaction mixture of step (b) with insolubilized sugar selectively and reversibly reactive with said bound lectin to reversibly bind the lectin to the insolubilized sugar,

(d) separating the insolubilized sugar-bound lectin reaction product from the remainder of said reaction mixture, and

(e) determining the amount of label compound in either said insolubilized sugar-bound lectin product or said remainder as a measure of the quantity of said sample component.

14. The method of claim 13 in which said one component is an antigen and said corresponding component and anti(one component) are different antibodies immunologically reactive with said antigen.

15. A method for the determination of at least a first and second antigen or antibody in a fluid sample comprising

(a) mixing and incubating said sample containing said first and second antigens (or antibodies) with (1) a known amount of said first soluble antigen (or antibody) bound to a first label compound and a soluble second antigen (or antibody) bound to a different second label compound and (2) first and second antibodies (or antigens) specific for the sample first and second antigens (or antibodies), respectively, and bound to lectin to form the respective antigen-antibody pairs,

(b) contacting said incubation reaction mixture with insolubilized sugar selectively and reversibly reactive with said lectin to reversibly bind the lectin to the insolubilized sugar,

(c) separating the insolubilized sugar-lectin products from the remainder of said reaction mixture, and

(d) determining the amount of first and second label compounds in either said insolubilized sugar-lectin products of said remainder as a measure of the quantity of said first and second antigens.

16. The method of claim 15 in which step (d) is performed by contacting said insolubilized sugar-bound lectin products with soluble sugars of the same type as said insolubilized sugar to cause the bound lectin products to react with said soluble sugar for conversion to a solubilized form, then separating the soluble and insoluble components into different fractions and thereafter measuring the amount of first and second label compound in the soluble fraction.

17. The method of claim 15 in which the insolubilized sugar is covalently bound to a solid surface.

18. The method of claim 15 in which prior to step (d), the sugar-lectin products are separated into different fractions for independent measurement of the amounts of first and second labels.

19. A method for the determination of at least a first and second antigen or antibody in a fluid sample comprising the steps of

(a) incubating said sample containing said first and second antigens or antibodies with first and second antibody or antigen, specific for said sample antigens or antibodies, and bound to lectins to form the respective antigen-antibody pairs,

(b) incubating the reaction product of step (a) with a third antibody or antigen bound to a first label component and a fourth antibody or antigen bound to a second label compound to cause said third and fourth antibodies ot antigens to immunologically react with said first and second sample antigens or antibodies,

(c) contacting the reaction mixture of step (b) with insolubilized sugar selectively and reversibly reactive respectively with said lectin to reversibly bind the lectin to the insolubilized sugar,

(d) separating the insolubilized sugar-lectin products from the remainder of said reaction mixture, and

(e) determining the amount of first and second label compounds in either said insolubilized sugar-lectin product or said remainder as a measure of the quantity of said first and second antigens.

20. The method of claim 19 in which prior to step (e) the sugar-lectin products are separated into different fractions for independent measurement of the amounts of first and second labels.

21. The method of claim 19 in which step (e) is performed by contacting said insolubilized sugar-lectin product with soluble sugar of the same type as said insolubilized sugar to convert the lectin product to solubilized form, separating the soluble and insoluble components into different fractions, and thereafter measuring the first and second label compounds in the soluble fraction.

22. A method for the determination of one component of an immunological conjugate in a fluid sample, comprising the steps of

(a) passing the corresponding component of said immunological conjugate bound to a lectin through a contained volume of insolubilized sugar selectively and reversibly reactive with said bound lectin to reversibly bind the corresponding component bound lectin to the insolubilized sugar,

(b) passing said sample containing said one component and a known amount of said one component bound to a label compound in soluble form through said contained volume to cause a competitive immunological reaction between the components of said immunological conjugate,

(c) separating the insolubilized sugar-bound lectin reaction product from the remainder of said reaction mixture, and

(d) determining the amount of label compound in said insolubilized sugar-bound lectin product as a measure of the quantity of said one component in said sample.

23. The method of claim 22 in which step (d) is performed by contacting said insolubilized sugar-bound lectin product with soluble sugar of the same type as said insolubilized sugar to cause the bound lectin product to convert to a solubilized form, separating the soluble and insoluble components into different fractions and thereafter measuring the amount of label compound in the soluble fraction.

24. The method of claim 22 in which said sample contains glycoprotein reactive with lectin and lectin unbound to said corresponding component and of the same type as said bound lectin is present in said sample during step (b) said unbound lectin being in excess of glycoprotein reactive therewith in the sample, so that said unbound lectin blocks the lectin-reactive sites of said glycoprotein.

25. The method of claim 22 in which said sample contains glycoprotein reactive with lectin and prior to step (b) said sample is passed through a bed of insolubilized lectin with glycoprotein-reactive sites and of the same type as said bound lectin so that said glycoprotein is retained on said column and thereby removed from said sample.

26. A method for the determination of one component of an immunological conjugate in a fluid sample, comprising the steps of

(a) contacting the corresponding component of said immunological conjugate bound to a lectin with a contained volume of insolubilized sugar selectively and reversibly reactive with said bound lectin to reversibly bind the lectin to said insolubilized sugar,

(b) passing said sample containing said one component through said contained volume under conditions to cause immunological reaction with said corresponding component,

(c) passing anti(one component) bound to a label compound through said contained volume to cause an immunological reaction between said one component and said anti(one component),

(d) separating the insolubilized sugar-bound lectin reaction product from the remainder of said reaction mixture, and

(e) determining the amount of label compound in said insolubilized sugar-bound lectin product as a measure of the quantity of said sample component.

27. The method of claim 26 in which said sample contains glycoprotein reactive with lectin and lectin unbound to said corresponding component and of the same type as said bound lectin is present in said sample during step (b), said unbound lectin being in excess of glycoprotein reactive therewith in the sample, so that said unbound lectin blocks the lectin-reactive sites of said glycoprotein.

28. The method of claim 26 in which said sample contains glycoprotein reactive with lectin and prior to step (b) said sample is passed through a bed of insolubilized lectin with glycoprotein reactive sites and of the same type as said bound lectin so that said glycoprotein is retained on said column and thereby removed from said sample.

30. The method of claim 26 in which step (d) is performed by contacting said insolubilized lectin-bound sugar product with soluble lectin of the same type as said insolubilized lectin to cause the bound sugar product to convert to soluble form, then separating the soluble and insoluble components into different fractions and thereafter measuring the amount of label compound in the soluble fraction.

29. A method for the determination of one component of an immunological conjugate in a fluid sample, comprising the steps of

(a) passing the corresponding component of said immunological conjugate bound to sugar through a contained volume of insolubilized lectin selectively and reversibly reactive with said bound sugar to reversibly bind the sugar to the insolubilized sugar,

(b) passing said sample containing said one component and a known amount of said one component bound to a label compound in soluble form through said contained volume to cause an immunological reaction between the components of said immunological conjugate,

(c) separating the insolubilized lectin-bound reaction product from the remainder of said reaction mixture, and

(d) determining the amount of label compound in said insolubilized sugar-bound lectin product as a measure of the quantity of said one component in said sample.

31. A method for the determination of one component of an immunological conjugate in a fluid sample, comprising the steps of

(a) contacting the corresponding component of said immunological conjugate bound to a sugar with a contained volume of insolubilized lectin selectively and reversibly reactive with said bound sugar to reversibly bind the sugar to said insolubilized lectin,

(b) passing said sample containing said one component through said contained volume to cause said immunological reaction to occur,

(c) passing anti(one component) bound to a label compound through said contained volume to cause an immunological reaction between said one component and said corresponding component,

(d) separating the insolubilized sugar-bound lectin reaction product from the remainder of said reaction mixture, and

(e) determining the amount of label compound in said insolubilized sugar-bound lectin product as a measure of the quantity of said sample component.

32. The method of claim 31 in which during step (b) said sample contains lectin unbound to said corresponding component and of the same type as said bound lectin in excess of extraneous glycoprotein reactive in the sample so that at least a portion of said unbound lectin blocks said lectin reactive sites of said glycoprotein.

33. A method for the determination of one component of an immunological conjugate in a fluid sample comprising

(a) mixing said sample containing said one component with a known amount of said one component bound to a label compound in soluble form, and the corresponding component of said immunological conjugate bound to a sugar to form a fluid reaction mixture,

(b) incubating the reaction mixture of step (a) to cause an immunological reaction between the components of said immunological conjugate,

(c) contacting said incubated reaction mixture with insolubilized lectin selectively and reversibly reactive with said bound sugar to reversibly bind the sugar to the unsolubilized lectin,

(d) separating the insolubilized lectin-bound sugar reaction product from the remainder of said reaction mixture, and

(e) determining the amount of label compound in said insolubilized lectin-bound sugar product as a measure of the quantity of said one component in said sample.

34. The method of claim 33 in which the insolubilized lectin comprises lectin covalently bound to a solid surface.

35. A method for the determination of one component of an immunological conjugate in a fluid sample comprising the steps of

(a) incubating said sample containing said one component with the corresponding component of said immunological conjugate bound to a sugar to cause said immunological reaction to occur,

(b) incubating the reaction product of step (a) with anti(one component) bound to a label compound to cause an immunological reaction between said one component and said anti(one component),

(c) contacting the reaction mixture of step (b) with insolubilized lectin selectively and reversibly reactive with said bound sugar to reversibly bind the sugar to the insolubilized lectin,

(d) separating the insolubilized lectin-bound sugar reaction product from the remainder of said reaction mixture, and

(e) determining the amount of label compound in said insolubilized lectin-bound sugar product as a measure of the quantity of said sample component.

36. The method of claim 35 in which said immunological conjugate is an antigen-antibody pair.

37. The method of claim 36 in which said one component is an antigen and said corresponding component and anti(one component) are different antibodies immunologically reactive with said antigen.

48. The reaction product of claim 35 reversibly bonded through said sugar to an insolubilized lectin on a solid surface, said lectin being selectively reactive with said sugar.

38. A method for the determination of at least first and second antigens or antibodies in a fluid sample, comprising the steps of

(a) passing through a contained volume of insolubilized sugar, first and second antibodies or antigens specific for the sample first and second antigens or antibodies, respectively, each specific antibody or antigen being bound to a lectin, said sugar being selectively and reversibly reactive with said bound lectin, to reversibly bind the lectin to the insolubilized sugar,

(b) passing said sample containing said first and second antigens or antibodies and a known amount of said first and second antigens or antibodies bound respectively to first and second label compounds in soluble form through said contained volume to cause an immunological reaction between the first and second antigens or antibodies and said first and second sugar-bound antibodies or antigens, respectively,

(c) separating the insolubilized sugar-bound lectin reaction products from the remainder of said reaction mixture, and

(d) determining the amount of first and second label compounds in said insolubilized sugar-bound lectin products as a measure of the of said first and second antigens or antibodies in said sample.

39. The method of claim 38 in which step (d) is performed by contacting said insolubilized sugar-bound lectin products with soluble sugar of the same type as said insolubilized sugar to cause the bound lectin products to react with said soluble sugar for conversion to a solubilized form, separating the soluble and insoluble components into different fractions and thereafter measuring the amount of first and second label compound in the soluble fraction.

40. A method for the determination of at least a first and second antigen or antibody in a fluid sample, comprising the steps of

(a) passing through a contained volume of insolubilized sugar, first and second antibodies or antigens, specific for said sample first and second antigens, respectively, each antibody or antigen being bound to lectin, said insolubilized sugar being selectively and reversibly reactive with said bound lectin to reversibly bind the lectin to said insolubilized sugar,

(b) passing said sample containing said first and second antigens or antibodies through said contained volume to cause an immunological reaction to occur with said first and second sugar-bound antibodies or antigens,

(c) passing third and fourth antibodies to said sample antigens or antibodies bound to first and second label compounds, respectively, through contained volume to cause an immunological reaction with said first and second antigens or antibodies,

(d) separating the insolubilized sugar-bound lectin reaction product from the remainder of said reaction mixture, and

(e) determining the amount of first and second label compounds in said insolubilized sugar-bound lectin product as a measure of the quantity of said first and second antigen in said sample.

41. A reaction product for use in an immunological assay comprising a labelled immunological reaction product in which one member of the reaction product is irreversibly bonded to a lectin and another member of the reaction product is irreversibly bonded to a label compound.

42. The reaction product of claim 41 in which said label compound is selected from the group consisting of enzyme, radioactive molecules, and fluorescent molecules.

43. The reaction product of claim 42 in which said label compound is an enzyme and the amount of said enzyme is determined by measuring its enzyme activity.

44. The reaction product of claim 41 reversibly bonded through said lectin to an insolubilized sugar on a solid surface, said sugar being selectively reactive with said lectin.

45. A reaction product for use in an immunochemical assay comprising a labelled immunological reaction product in which one member of the reaction product is irreversibly bonded to a sugar and another member of the reaction product is irreversibly bonded to a label compound.

46. The reaction product of claim 45 in which said label compound is selected from the group consisting of enzyme, radioactive molecules, and fluorescent molecules.

47. The reaction product of claim 46 in which said label compound is an enzyme and the amount of said enzyme is determined by measuring its enzyme activity.