Biochemical avidin-biotin multiple-layer system

Abstract

A multiple-layer process for applying, in alternate, successive layers, the protein, avidin, and a biotin-containing extender material to a solid surface to modify the properties of the surface and to the multiple-layer product so prepared.

Description

of biotin-horse radish peroxidase (BHRPO) to each avidin layer treatment. The BHPRO served as a signal extender. Appropriate washing and control steps and treatment were carried out. The HRPO color at 500 nanometers was measured after each avidin layering step as a measure of the amount of avidin (most specifically, available avidin-binding sites for BHRPO), and the layering process was found to generate increasing amounts of avidin with each avidin layer (amplification layering), one of the three possibilities (constant, decreasing or increasing), cited earlier. The color-vs.-number-of-layers is as shown in Table 1.

TABLE I
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Absorbance 500 mm (color) vs.
Number of Layering Cycles
Absorbance
No. of Layers  Color     Difference
Avidin (n)     Absorbance
Values
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1              .746
2              .832       .086*
3              .964      .132
4              1.124     .160
5              1.379     .255
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*0.832 - 0.746 = 0.086

In order to illustrate more fully the nature of the invention and the manner of practicing the same, the following Example is presented:

EXAMPLE

PAC Materials

1. Affigel-701 from Bio-Rad--an aminoethyl derivative of polyacrylamide in a bead form, 1-3 microns in diameter. The beads were provided in an aqueous suspension at 25±3 μ/mol of amine groups/ml.

2. Phosphate buffered saline (PBS)--an 0.01 M sodium phosphate, 0.15 M sodium chloride, pH 7.4.

3. Avidin--dissolved in PBS at 0.1 mg/ml based on weight.

4. Wash buffer--The buffer used for all washing steps was PBS containing bovine serum albumin (BSA) at 0.02% wt and Tween-20 surfactant at 0.05% wt.

5. HRPO substrate--was freshly prepared by dissolving phenol (100 mg) and 4-aminoantipyrine (16.2 mg) in a solution composed of 0.5 M Na₂ HPO₄ (2 ml), 0.5 M KH₂ PO₄ (18 ml), water (180 ml) and 30% H₂ O₂ (20 μl).

6. Silanized glass tubes--Disposable borosilicate glass tubes (12×75 mm) were silanized by filling with a 2% solution of chlorotrimethylsilane in benzene. The silanizing reagent was decanted after 1/2 hour, the tubes rinsed with acetone and air-dried.

7. Biotin NHS ester (biotin N-hydroxysuccinimide ester)--was prepared as defined in Jasiewicz, M. M., Schoenberg, D. R., and Mueller, G. C., Exp. Cell Res. 100, 213 (1978), hereby incorporated by reference.

8. Caproylamidobiotin-NHS and caproylamidobiotin-RNase (BRNase)--were prepared as defined previously (Costello, S. M. Felix, R. T. and Giese, R. W., Clin. Chem. 25, 1572 (1979), herein incorporated by reference).

9. BHRPO horse radish peroxidase (Worthington Biochemical)--10 mg were dissolved in 1 ml of water. This was added to a solution consisting of 1,6-hexanediamine (116 mg). 0.2 M sodium pyrophosphate (2.0 ml), water (5.0 ml) and sufficient concentrated HCl to bring the pH to 5.5. A solid water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopyroyl) carbodiimide (EDC) was added to the gently mixed solution at room temperature. Three separate additions of 190 mg each were made over a 1-hour period. 11/2 hours after the first addition, the contents of the beaker were placed in a dialysis bag and dialyzed against 4×400 ml of PBS (pH=7.4). An aliquot (10 ml) from the dialysis bag was added to a solution of caproylamidobiotin-NHS ester (4.1 mg) in N,N-dimethylformamide (DMF) (0.1 ml). This solution was allowed to stand at room temperature for 11/2 hours and was then dialyzed against 4×400 ml of PBS (pH=7.4).

An aliquot (2 ml) of the above was placed in a dialysis bag and dialyzed against NaHCO₃ (1 M) for 24 hours. The sample (at pH=8.6) was removed from the bag, placed in a small beaker with a magnetic mixer and reacted with 4 5×10 μl aliquots (15 minutes apart) of succinic anhydride (40 mg) in DMF (1 ml). The sample was placed in a dialysis bag 15 minutes after the last addition and dialyzed against 4×400 ml of PBS (pH=7.4).

Assuming 100% recovery of enzyme, the concentration of biotinyl-HRPO (BHRPO) would be approximately 0.8 mg/ml. It migrated electrophoretically (cellulose acetate, pH 8.6 buffer) in a manner similar to native enzyme (although the band was more diffuse).

10. Biotin-beads suspension--Affigel-701 (5.0 ml, about 125 μmol of amine groups) was added to PBS (5.0 ml). This suspension was vortexed 10 seconds, and biotin NHS ester (43 mg, 125 μmol) dissolved in DMF (0.1 ml) was added all at once. The reaction mixture was allowed to mix end over end for 2 hours at room temperature.

The beads were packed by centrifugation and the supernatant discarded. The bead pellet was resuspended in PBS and washed with 4×20 ml of PBS. The beads (biotin beads) were finally suspended in PBS (20 ml) containing NaN₃ (0.02%).

Layering of Biotin Beads

Aliquots (50 ul) of biotin-bead suspension (magnetically mixing) were placed in 12×75 mm silanized glass tubes. Each tube was treated with avidin (0.1 mg in 1 ml PBS) for 10 minutes at room temperature. The beads were then centrifuged and the supernatants collected. The beads were washed X3 with wash buffer.

A layer was applied to the avidin-biotin beads by suspending them in 1 ml of caproylamidobiotin RNase (BRNase approximately 60 μg/ml) for 10 minutes. The beads were then spun and the supernatants collected. The beads were then washed X3 with wash buffer. The newly added biotin residues were next reacted with avidin as above. The sequence of avidin followed by BRNase, with intermittent washing steps, was repeated four more times. This process is set forth in FIG. 2.

Functional biotin binding sites on avidin-biotin beads (or layered beads) were detected by suspending aliquots of the beads after each avidin step in 200 μl of BHRPO (2 μg/ml) in PBS for 30 minutes. Unbound enzyme was removed by threefold washing with wash buffer. Bound enzyme was detected by addition of HRPO substrate (4.5 ml). After 30 minutes at room temperature, the tubes were chilled in an ice bath for 5 minutes and then spun. The supernatants were decanted and diluted with PBS (4.5 ml).

The A₅00 values of the diluted substrate solutions were measured on a Gilford 240 using water as a reference, and are given in Table I. As seen, the amount of functional enzyme on the beads is greater with each cycle of layering, and the rate of increase giving given by the difference values) also is increasing significantly as the layering proceeds; for example, the value 0.255 between layers 4 and 5 is 2.96 times greater than the value 0.086 between layers 1 and 2. This demonstrates the usefulness of layering for placing functional enzyme on a surface, increasing the amount of functional enzyme on a surface, and achieving an increasing rate of layering for the enzyme, that is, a relative increase in the amount of enzyme attached with each successive layer.

Avidin and some of the ligand binding proteins which may be employed in the practice of my invention are set forth in Table II.

TABLE II
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Avidin and Some Other
Ligand-binding Proteins
Usual No.
Protein   Ligand      Affinity (Ka)
of binding sites
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Lectins   Simple sugars
103 -104
4
membrane sites
106 -107
Protein A Fc of IgG
107   4
( S. aureus)
Antibodies
Haptens     105 -1011
2
Antigenic deter-
105 -1011
2
minants
Avidin    Biotin      1015  4
Streptavidin
Biotin      --         4
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Claims

1. A process of modifying the surface properties of a surface, which process comprises:

applying alternative, monomolecular, successive layers of first and and second materials to a surface to be modified, the first material comprising avidin and the second material comprising a noncovalent, biotin-modified extender, one of the materials reacted to the surface, and, thereafter, at least one additional layer of each of the first and second materials alternated, secured and noncovalently reacted to the underlying layer, to provide a surface with the first or second material as the top surface layer thereon.

2. The process of claim 1 wherein the top surface of the process comprises the second extender material.

3. The process of claim 1 wherein the first material comprises a modified avidin adapted to react with the second extender material through the avidin portion of the modified material.

4. The process of claim 1 which includes pretreating the surface with a monomolecular layer of biotin, and wherein the first avidin material is applied over and secured to the biotin layer.

5. The process of claim 1 which includes:

(a) applying a monomolecular layer of biotin and covalently binding the biotin to the surface;

(b) applying and reacting a monomolecular layer of avidin to the biotin layer; and

(c) applying and reacting a monomolecular layer of a biotin extender material to the avidin layer.

6. The process of claim 5 which includes:

(a) applying another layer of avidin; and

(b) applying another layer of the biotin extender material to the other layer of avidin.

7. The process of claim 1, which process includes varying the concentration of the first or second material in the alternate, successive layers.

8. The process of claim 7 which includes increasing the concentration of the alternate, successive layers of the first and second materials.

9. The process of claim 7 which includes decreasing the concentration of the alternate, successive layers of the first and second materials.

10. The process of claim 1 which includes applying alternate, successive layers of approximately the same stochiometric concentration.

11. The process of claim 1 wherein the surface comprises a polymeric surface.

12. The process of claim 11 wherein the surface comprises the surface of finely-divided, polyacrylamide, polymer particles.

13. The process of claim 11 wherein the surface comprises erythrocytes.

14. The process of claim 1 wherein the surface comprises the surface of amino polyacrylamide particles, and the process comprises applying a layer of biotin-nhs esters to the surface of the particles, and, thereafter, applying successive, alternate, monomolecular layers of avidin and biotin-ribonuclease material.

15. The process of claim 1 wherein the first and second materials comprise three or more layers.

16. The process of claim 1 wherein the first and second materials comprise two monomolecular layers.

17. The process of claim 1 wherein each layer of the materials is monomolecular or monoparticulate in thickness.

18. The process of claim 1 which includes reacting at least one of the avidin layers with a biotin-horse radish peroxidase or a biotin ribonuclease as a signal extender.

19. The layering system produced by the process of claim 1.

20. The layering system produced by the process of claim 14.

21. A monomolecular-layering process of modifying the surface properties of a substrate surface of a polymer, which process comprises:

(a) applying a layer of a biotin-N-hydroxysuccinimide ester as a biotin-extender material to the surface of a polymer, to react covalently the biotin-N-hydroxysuccinimide to the surface of the polymer;

(b) washing the polymer surface to remove unreacted biotin-N-hydroxysuccinimide;

(c) applying a layer of avidin to the washed polymer surface, to react noncovalently the avidin with the biotin-N-hydroxysuccinimide extender material;

(d) washing the reacted surface to remove unreacted avidin;

(e) applying to the washed avidin surface a noncovalent layer of a caproylamidobiotin nhs or rnase as a biotin-extender material;

(f) washing the reacted surface to remove unreacted biotin-extender material; and

(g) recovering the polymer having multiple layers, with the top layer composed of a biotin-extender material.

22. The process of claim 21 which includes:

(a) applying avidin to the washed polymer surface, to react another layer of avidin with the biotin-extender material;

(b) washing the reacted surface to remove unreacted avidin; and

(c) recovering a polymer having multiple layers, with the top layer composed of avidin.

23. The process of claim 22 which includes repeating the successive application of biotin-extender material and avidin, with intermittent wash steps, to provide a polymer surface with successive monomolecular layers of avidin and biotinextender biotin-extender material, with the top monomolecular layer being either avidin or a biotin-extender material.

24. The process of claim 21 wherein the polymer comprises an aminoalkyl polyacrylamide polymer.

25. The process of claim 21 which includes reacting a small amount of a biotin-peroxidase or -ribonuclease material, as a signal extender for the avidin, with at least one of the applications of the avidin.

26. The polymeric-layering system produced by the process of claim 21.

27. The polymeric-layering system produced by the process of claim 22.

28. The polymeric-layering system produced by the process of claim 23.