WO1995028639A1

WO1995028639A1 - Protective wash for liposome assay

Info

Publication number: WO1995028639A1
Application number: PCT/US1995/004624
Authority: WO
Inventors: Leslie Oppenheimer; Lewis R. Pollack; Josephine Readio; Karl Devore; Harish Kumar; Ramanand H. Nayak; Linda Liu Allison; Veronia H. Burkett; Abdul M. Butt; Marie Sylvestre; Uri Reichman
Original assignee: Becton Dickinson And Company
Priority date: 1994-04-13
Filing date: 1995-04-13
Publication date: 1995-10-26

Abstract

This invention presents protective wash solutions for use in assays, particularly heterogeneous immunoassays, using bound liposomes. The solution permits bound liposomes to be rinsed at least twice during such assays, thereby increasing the precision of assay by enhancing the removal of non-specifically bound, loosely bound, and/or unbound liposomes while not affecting the bound liposomes.

Description

PROTECTIVE WASH FOR LIPOSOME ASSAY

BACKGROUND OF INVENTION

The use of liposomes in binding assays, including immunoassays, is well known in the art. Because of their versatility in conjugation chemistry, and their ability- to encapsulate a wide array of dyes and other compounds, liposomes are frequently employed as the carrier of the signal generating compound.

In such assays, the liposomes encapsulate a signal generating compound, e.g. a dye, and are chemically attached, or conjugated, to the binding molecule, generally an antibody or analyte analog. The resultant conjugates are then admixed with the sample which contains the material being assayed, and the presence of the analyte is determined by measurement of the signal produced by the signal generating compound.

The assays can be formatted in a variety of heterogeneous configurations including direct and indirect, competitive and sandwich. Key to obtaining a usable result, however, is the separation of specifically bound liposomes from non-specifically bound or unbound liposomes. Ordinarily this is accomplished by washing with an aqueous wash solution. The wash solutions, generally water or aqueous buffers, however, often affect the integrity of the liposomes, limiting the number of washes which can be utilized. Since separation is enhanced by multiple washes, wash solutions which do not affect the liposomes are needed.

SUMMARY OF INVENTION

This invention presents protective wash solutions for use in assays, particularly immunoassays. using bound liposomes. The solution permits bound liposomes to be rinsed at least twice during such assays. The multiple washing confers the benefit of increasing the precision by enhancing the removal of non-specifically bound and/or unbound materials while not affecting the bound liposomes.

The solution comprises a standard wash solution containing a liposome wash protectant. These protectants, it has been found, serve to stabilize the liposomes during the wash, permitting multiple washes without loss of bound liposomes.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 presents a graphical representation of the relative fluorescent signal (RFU) versus the number of washes for liposomes of differing sizes in a digoxin assay. Figure 2 presents a graphical representation of RFU versus the number of washes in a theophylline assay, containing varying concentrations of BSA.

Figure 3 presents a graphical representation of RFU versus the number of washes in an FT4 assay containing varying concentrations of BSA.

DETAILED DESCRIPTION OF INVENTION

The wash solution of this invention contains a liposome wash protectant in a solvent. Preferred liposome wash protectants include bovine serum albumin (BSA), ethanol, and polyvinylpyrrolidone (PVP), most preferably PVP and BSA; but generally include many polymeric materials. The use of cryoprotectants is specifically preferred, as such compounds confer a significant protective effect on the liposomes. As used herein, cryoprotectant denotes a material which protects a biological material from freezing damage. Cryoprotectants include materials capable of dissolving in said solvent and which, upon such dissolution, will lower the freezing point of the resultant solution, as well as other materials, such as BSA, which confer freezing damage protection by other mechanisms. However, it is contemplated that any protectant material can be used, the only criterion being its compatibility with the solvent and the liposome. Similarly, any compatible solvent can be used, so long as it does not deleteriously affect the liposome. Preferred solvents include aqueous phosphate buffers and other buffers of inorganic salts.

It is also noted that while the precise amount of liposome wash protectant added to the wash solution is a matter of choice to the artisan, the concentration chosen must be sufficiently high to produce the desired stabilizing effect, yet not so high as to adversely affect the liposomes. Generally, a concentration of 0.005-8%, preferably 0.02-5%, by weight, is sufficient, while the precise concentration will be governed also by solvent effects, and a buffer concentration of at least 25mM. The wash solutions can be used in assays involving bound liposomes, particularly immunoassays, more particularly heterogeneous immunoassays wherein a portion of the liposomes becomes directly or indirectly bound to a separate phase and the bound liposomes are washed to remove unbound and loosely or non-specifically bound material. As unbound material tends to generate a false positive signal, enhanced removal serves to produce an assay having enhanced precision. Repeated washing will aid in this removal.

The wash solutions of this invention also serve to permit multiple washes without a significant loss of signal. Generally, the solutions of this invention permit two or more washes with a signal loss of less than 10%, preferably less than 5%.

The assays with which the wash solutions can be used are preferably immunoassays using liposomes, more preferably heterogeneous immunoassays using liposomes. The format can be either competitive (e.g., wherein the liposome is conjugated to the analyte ot^¬ to an analog of the analyte or contains the analog incorporated in the liposome bilayer) or non-competitive (e.g., wherein the liposome is conjugated to a binding molecule specific for the analyte) and include direct and sandwich assays. The signal can either be radiometric (as in RlA's) or visible, as described in e.g. United States Patent No. 4,703,017 to Campbell et al., incorporated herein by reference, as well as any other signal format known including chemiluminescent, bioluminescent, enzymatic, fluorimetric, and colorimetric. wherein an instrument and/or development steps are required.

EXAMPLES The following examples illustrate certain preferred embodiments of this invention but are not intended to be illustrative of all embodiments.

Example 1 - Standard Wash

To illustrate the effect of multiple washing of liposomes in a binding assay, a digoxin assay was utilized. Briefly, the assay involved the use of a heterogeneous fluorescence immunoassay using liposome conjugated ligand in a competitive binding format with antibody coated tubes.

Specifically, 100 ul of serum is admixed with a premeasured vial of liposomes, and the resultant mixture is added to a tube coated with rabbit anti-digoxin serum. The tube is then incubated at 45°C for 15 minutes, after which it is rinsed with saline (containing 0.1% sodium azide). The tube is then coupled with a lysing vial (containing 1% detergent in deionized water) and the bound liposomes are lysed and fluorescence is measured. The RFUs were measured in the Becton Dickinson IQ analyzer.

To evaluate the effect of multiple washings on the assay performance, samples of the bound device were subjected to up to sixteen washes with standard buffer. The average signal generated in four replicate trials, using liposomes of 100 nm, 166nm, and 23 1 nm, is graphically presented in Figure 1. As shown, the total relative fluorescent signal (RFU) is greatly reduced by such washings, indicating that both bound and unbound liposomes are removed. This is markedly apparent with the larger diameter liposomes. Example 2 - Comparative Washes

To ascertain the effects of multiple washing with various reagents, samples of the digoxin assay of Example 1 were subjected to multiple washes with eight buffers. The results are presented in Table I:

TABLE I: WASH STUDY USING VARIOUS SOLUTIONS

WASH SOLUTION INITIAL RFUs* NUMBER OF WASHES/% INITIAL RFUs

AFTER 1 WASH

I 2 4 6

Control (saline) 521 100 86 22 14 0.2% PVP in saline 496 100 89 64 55 1% PVP in saline 541 100 106 77 56 0.2% BSA in water 398 100 70 49 47 1% BSA in water 467 100 79 58 53 0.1% BSA in 100 mM 476 100 100 99 96

Phosphate 1.0% BSA in lOOmM 481 100 93 88 94

Phosphate 0.5M glycine, 0.03% 578 100 96 54 ANS, 5%ethanol, 0.02% azide

*RFUs = relative fluorescence units

It can be seen that all seven solutions containing at least one liposome protectant (ethanol, BSA, or PVP) resulted in a much higher signal (RFU) than the saline, when subjected to multiple washes.

Example 3 - Effect of Solvent

To ascertain the effect of solvent on the system, a comparative study was run utilizing aqueous BSA and PVP wash solutions, and the same concentrations of protectants in lOOmM phosphate buffer solutions. The results are presented in Table II, below: Table II

No. of Aqueous 100 mM Phosphate Buffet

Washes Saline 1% BSA 0.1% PVP 1% BSA 0.1%PVP

2 446 367 440 479 445

4 1 13 271 319 470 421

6 73 248 274 456 460

8 70 226 203 466 457

As shown, the use of lOOmM phosphate buffer greatly enhances the protection.

Example 4 - Effect of Varying BSA Concentration

To ascertain the effect of varying the concentration of BSA, a basic wash solution (containing 20 mM phosphate, 50mM NaC l, 0. 1% NA Azide. 0.005% gentamycin, (i.e. without BSA), was mixed with various amounts of BSA (ranging from 0-1%, by weight) and was then used as the wash solution in an assay for theophylline. Briefly, the assay is conducted using a competitive assay format with theophylline analog conjugate incorporated into the liposome bilayer and antibody coated tubes. The assay procedure was basically the same as Example 1, except that the 45 °c incubation was for 20 rather than 15 minutes

Samples were washed 1-6 times, the results are graphically presented in Figure 2. As shown, a concentration of 0.01% BSA was sufficient to prevented signal loss as compared with the BSA deficient solution.

Example 5 - Effect of Varying BSA Concentration In a Free T4 Assay

To Demonstrate the versatility of the invention, a wash solution of BSA in a 0. 15 M phosphate buffer was used in an assay for free T4. Briefly, the assay was conducted using a competitive assay format of thyroxin analog conjugate incorporated into the liposome bilayer and antibody coated tubes. The assay procedure was the same as in Example 1.

The results are presented in Figure 3. Again, a concentration of 0.01% BSA in the buffer prevented signal loss as compared with the BSA deficient solution.

It is apparent that many modifications and variations of this invention as herein set forth may be made without departing from the spirit and scope hereof. The specific embodiments described are given by way of example only and the invention is limited only by the terms of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A wash solution for use in assays employing liposomes comprising:

(a) water or an aqueous solution containing at least one inorganic salt and (b) a liposome wash protectant

2. The wash solution of Claim 1 wherein the liposome wash protectant is a cryoprotectant.

3. The solution of Claim 1 wherein the liposome wash protectant is selected from the group consisting of bovine serum albumin, ethanol, and polyvinylpyrrolidone.

4. The solution of Claim 1 wherein the cryoprotectant is at a concentration of 0.005-8%, by weight, in buffer.

5. The solution of Claim 1 wherein the inorganic salt concentration is at least 25mM.

6. The solution of Claim 1 wherein the inorganic salt is selected from the group consisting of metallic chlorides, metallic phosphates, metallic azides. and mixtures.

7. The solution of Claim 6 wherein the metallic chloride is sodium chloride.

8. The solution of Claim 6 wherein the metallic azide is sodium azide.

9. The solution of Claim 6 wherein the metallic phosphate is sodium phosphate.

10. In a heterogeneous assay for an analyte which employs liposomes containing a signal generating compound, which liposomes are either conjugated to, or contain incorporated in the liposome bilayer, the analyte or an analog of the analyte. or a binding molecule specific for the analyte or analog of the analyte, such that a portion of said liposomes becomes bound to a separate phase, the improvement comprising washing the bound liposomes at least twice with a wash solution which contains:

(a) water or an aqueous solution of at least one inorganic salt and

(b) a liposome wash protectant.

11. The assay of claim 10 wherein the liposome wash protectant is a cryoprotectant.

12. The assay of Claim 10 wherein the liposome wash protectant is selected from the group consisting of bovine serum albumin, ethanol, and polyvinylpynolidone.

13. The assay of Claim 10 wherein the liposome wash protectant is at a concentration of 0.005-8%, by weight.

14. The assay of Claim 10 wherein the inorganic salt concentration is at least 25mM.

15. The assay of Claim 10 wherein the inorganic salt is selected from the group consisting of metallic chlorides, metallic phosphates, metallic azides. and mixtures.

16. The assay of Claim 15 wherein the metallic chloride is sodium chloride.

17. The assay of Claim 15 wherein the metallic azide is sodium azide.

18. The assay of Claim 15 wherein the metallic phosphate is sodium phosphate.

19. The assay of Claim 10 wherein the signal-generating compound contained within the liposome is selected from the group consisting of radiometric compounds, colored compounds chemiluminescent compounds, bioluminescent compounds, enzymes, and fluorescent compounds.

20. The assay of Claim 10 wherein the binding molecule is an antibody specific for the analyte.