Polymer coated solid matrices and use in immunoassays

• US 4,921,809 A
• Filed: 09/29/1987
• Issued: 05/01/1990
• Est. Priority Date: 09/29/1987
• Status: Expired due to Fees

• Current Assignee: Bostik Findley Incorporated
• Original Assignee: Findley Adhesives Incorporated
• Inventors: Wasserman, Charles J., Garlock, Henrietta M., Schiff, Robert, Lehnus, Gary
• Primary Examiner(s): Richman, Barry S.
• Assistant Examiner(s): Johnston, Jill
• Application Number:
  US07/105,136
• Time in Patent Office: 945 Days
• Field of Search: 436/531, 436/524, 436/532, 422/73
• US Class Current: 436/531
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• CPC Class Codes: G01N 33/545 Synthetic resin
• CPC Class Codes: G01N 33/6854 Immunoglobulins

Abstract

Certain polyethyleneimines are used to coat solid carriers used in immunoassays to improve the sensitivity of the assay over what is obtained without the coating. Use of the invention in enzyme immunoassays yields sensitivities equal to that of radioimmunoassays. A post-coating is also used after the solid carrier has had a layer of capture molecule applied and before the assay is performed, to improve even further the sensitivity, reliability and dynamic range of the assay results.

Claims

1. In an immunoassay solid support carrying a capture molecule directly or through an interposing layer of a material facilitating covalent or adsorptive bonding of the capture molecule to the support, the improvement which comprises a layer of a polyethyleneimine on said support intermediate said support and said capture molecule and wherein there is a blocking agent present on the polyethyleneimine capture antibody composite comprising a mixture of ovalbumin and dithiothreitol.

2. The immunoassay solid support of claim 1 wherein the capture molecule is an antibody or antigen.

3. The immunoassay solid support of claim 2 wherein the polyethyleneimine has a molecular weight of from 600 to 60,000.

4. The immunoassay solid support of claim 3 wherein the solid support is polystyrene.

5. The immunoassay solid support of claim 4 wherein said interposing layer is glutaraldehyde.

6. The immunoassay solid support of claim 4 wherein the polyethyleneimine has a molecular weight of 800 to 1800.

7. The immunoassay solid support of claim 1, wherein the blocking agent additionally contains one or more of lactalbumin, lactalbumin enzymatic hydrolyzate, para-aminobenzoic acid and pyridoxyl phosphate.

8. The product of claim 7 wherein the capture antibody is for beta-HCG.

9. In a method for producing a solid support substrate useful in performing an immunoassay wherein a capture molecule is attached to said solid support directly or through an interposing layer of a material facilitating covalent or adsorptive bonding of the capture molecule to said layer, the improvement which comprises applying a layer of a polyethyleneimine onto said support intermediate said support and said capture molecule and then treating the resulting product with a mixture comprising ovalbumin and dithiothreitol sufficient in amount to provide a blocking layer of the polyethyleneimine layer.

10. The method of claim 9 wherein the capture molecule is an antibody or antigen.

11. The method of claim 10 wherein the polyethyleneimine has a molecular weight of from 600 to 60,000.

12. The method of claim 11 wherein the solid support is polystyrene.

13. The method of claim 12 wherein said interposing layer is glutaraldehyde.

14. The method of claim 13 wherein the support layer is polystyrene.

15. The method of claim 13 wherein the capture antibody is for beta-HCG.

16. The method of claim 13 wherein the blocking mixture further comprises one or more of the following:

para-aminobenzoic acid, lactalbumin, enzymatic hydrolysate of lactalbumin, and pyridoxyl phosphate.

17. The method of claim 16 wherein the blocking mixture comprises, by weight, 45-80% ovalbumin, 0.7-1.5% dithiothreitol, 16-50% lactalbumin or lactalbumin enzymatic hydrolysate, 1.3-2.2% para-aminobenzoic acid, and 15-18% pyridoxyl phosphate in aqueous solution.

18. In a method for conducting an immunoassay wherein a solid support carrier having a capture antibody attached thereto either directly or through an interposing layer of a material facilitating covalent or adsorptive bonding of the capture antibody to the layer, and said antibody is contacted with a liquid in which the presence and/or concentration of an antigen is to be determined, and said contacted antibody is contacted with a labeled form of a binding partner of said antigen and the extent of attachment of such labeled form is measured to measure the presence and/ or concentration of said antigen, the improvement which comprises applying a layer of a polyethyleneimine to said support carrier intermediate said support and said capture antibody and then treating the resulting product with a mixture comprising ovalbumin and dithiothreitol sufficient in amount to provide a blocking layer on the polyethyleneimine layer.

19. The method of claim 18 wherein the polyethyleneimine has a molecular weight of from 600 to 60,000.

20. The method of claim 19 wherein the solid support is polystyrene.

21. The method of claim 20 wherein a layer of glutaraldehyde is applied to said layer of polyethyleneimine to facilitate covalent bonding of the capture antibody thereto.

22. The method of claim 21 wherein the support layer is polystyrene.