COMPOSITION FOR PATIENT SAMPLE PREPARATION

COMPOSITION POUR UNE PREPARATION A BASE D'UN ECHANTILLON PRELEVE SUR UN PATIENT

English Abstract

The object of the present invention is to claim a composition which when added
to a patient sample containing a target antigen in an acidic or formalin-
containing transport medium neutralizes the acid and formalin while preserving
immunoreactivity of the antigen allowing its detection. The composition
comprises an amino glycol buffer, an amino acid, a salt, and a non-ionic
detergent. A further object of the present invention is to claim a method of
processing a sample by adding a composition comprising an amino glycol buffer,
an amino acid, a salt, and a non-ionic detergent.

French Abstract

La présente invention porte sur une composition qui, ajoutée à un échantillon prélevé sur un patient et contenant un antigène cible dans un milieu de transport acide ou formolé, neutralise l'acide et le formol tout en préservant la réactivité immunologique de l'antigène permettant sa détection. Ladite composition comprend un tampon amino-glycolique, un acide aminé, un sel et un détergent non ionique. La présente invention porte également sur un procédé permettant de traiter un échantillon en y ajoutant une composition contenant un tampon amino-glycolique, un acide aminé, un sel et un détergent non ionique.

Claims

THE INVENTION CLAIMED IS:

1. A composition comprising an amino glycol buffer, an amino
acid, a salt and a non-ionic detergent.

2. The composition of claim 1, wherein the amino glycol is selected
from the group consisting of 2-amino-2hydroxymethyl-1,3-propanediol, 2-
amino-2-methyl-1,3-propanediol, and mixtures thereof.

3. The composition of claim 1, wherein the amino acid is selected
from the group consisting of glycine, lysine, arginine, and mixtures thereof.

4. The composition of claim 1, wherein the salt is selected from the
group consisting of sodium salt, potassium salt, and mixtures thereof.

5. The composition of claim 1, wherein the non-ionic detergent is
selected from the group consisting of t-octylphenoxypolyethoxyethanol (Triton
X-100)(octylphenoxyl)-polyethoxyethanol (Nonidet P-40), and mixtures thereof.

6. The composition of claim 1, the composition further comprising
a serum.

7. A method of processing a patient sample comprising the steps
of:
(a) obtaining the patient sample containing a formaldehyde or an
acidic transport medium;
(b) adding a composition comprising an amino glycol buffer, an
amino acid, a salt and a non-ionic detergent.

5




8. The composition of claim 7, wherein the amino gycol is selected
from the group consisting of 2-amino-2hydroxymethyl-1,3-propanediol, 2-
amino-2-methyl-1,3-propanediol, and mixtures thereof.

9. The composition of claim 7, wherein the amino acid is selected
from the group consisting of glycine, lysine, arginine and mixtures thereof.

10. The composition of claim 7, wherein the salt is selected from
the group consisting of sodium salt, potassium salt, and mixtures thereof.

11. The composition of claim 7, wherein the non-ionic detergent is
selected from the group consisting of t-octylphenoxypolyethoxyethanol (Triton
X-100), (octylphenoxyl)-polyethoxyethanol (Nonidet P-40) and mixtures thereof.

12. The composition of claim 7, the composition further comprising
a serum.

6

Description

CA 02368007 2001-09-14
WO 00/60353 PCT/US99/07573
COMPOSITION FOR PATIENT SAMPLE PREPARATION
Background of the Invention
Enteric stool specimens are frequently collected in a 10% formalin or
sodium acetate-acetic acid-formalin (SAF) transport medium which serves to
1 o inactivate the infectivity and preserve the morphology of parasitic
organisms in
the sample. These media may have adverse effects on the function of antibodies
in diagnostic immunoassays.
Formalin, also known as formaldehyde, binds to amino groups on
proteins, potentially altering their structure and functional activity. Low
pH,
15 such as from the acetic acid in SAF, is inhibitory to antigen-antibody
binding.
The development of an effective diagnostic immunoassay for enteric stool
specimens collected in formalin or SAF, therefore, depends on the ability to
counteract these adverse effects. Although some microwell and flow-through
enzyme immunoassays appear to accomplish this through sample dilution,
2o simpler and more effective approaches are needed.
Summary of the Invention
The object of the present invention is to claim a composition which when
added to a patient sample containing a target antigen in an acidic or formalin-

containing transport medium neutralizes the acid and formalin while preserving
2s immunoreactivity of the antigen allowing its detection. The composition
comprises an amino glycol buffer, an amino acid, a salt, and a non-ionic
detergent.
A further object of the present invention is to claim a method of
processing a sample by adding a composition comprising an amino glycol buffer,
3o an amino acid, a salt, and a non-ionic detergent.
Detailed Description of the Invention
The present invention is based on the discovery that a composition
comprising an amino glycol buffer, an amino acid, a salt, and a non-ionic
detergent can, when added to a patient sample containing a target antigen in
an



CA 02368007 2001-09-14
WO 00/60353 PCT/US99/07573
acidic or formalin-containing transport medium, allow for the neutralization
of
the acid and formalin while preserving immunoreactivity of the antigen
allowing
its detection.
The composition of the present invention includes an amino glycol buffer.
Suitable amino gylcol buffers include 2-amino-2hydroxymethyl-1,3-propanediol
to (Tris buffer), 2-amino-2-methyl-1,3-propanediol.
The present composition further includes an amino acid. Suitable amino
acids include glycine, lysine and arginine.
The composition further includes a salt. Suitable salts include
sodium salts such as sodium chloride and potassium salts such as
1s potassium chloride.
The composition of the present invention further includes a non-ionic
detergent. Suitable non-ionic detergents include polyoxyethylenes such as t-
octylphenoxypolyethoxyethanol (Triton X-100) and (octylphenoxyl)-
polyethoxyethanol (Nonidet P-40).
2o The composition of the present invention may further include serum. It is
believed that serum serves to prevent or reduce non-specific binding of
antibodies that may be subsequently used in an immunoassay when non-specific
binding is a problem. Suitable serums include normal mammalian serum of any
kind, such as that derived from humans. Other mammalian serums include
2s bovine, porcine, equine and murine.
Formulation:
The key elements of the treatment buffer formulation are the use of an
amino acid in a basic buffer medium. We have selected glycine as the amino
acid, and Tris base as the basic buffer . Also included in the treatment
buffer is
3o some detergent, Triton X-100, which appears to help extract antigens
relevant to
our specific assay from the stool matrix, sodium chloride to help mimic
physiological salt conditions which are theoretically ideal for antigen-
antibody
binding to occur, and sodium azide, which is added as an anti-microbial



CA 02368007 2001-09-14
WO 00/60353 PCT/US99/07573
preservative. In addition, normal bovine serum was added to block non-specific
binding.
The final formulation consists of 1 M Tris, 1 M glycine, 0.9% sodium
chloride, 0.5% Triton X-100, and 0.01% sodium azide. The sources of the
chemicals we have used to develop the treatment buffer are the Sigma Chemical
to Company (St. Louis, Mo USA) and the J.T. Baker Chemical Company
(Phillipsburg, NJ USA). The vendor catalog numbers are: Tris Base (also known
as Trizma Base), Sigma T-1503; Glycine, Sigma G-7126; Sodium chloride, Baker
3624-07; Triton X-100, Sigma T-6878; and Sodium azide, Sigma S-2002. These
chemicals are standard grade materials and we have no reason to believe that
is other sources of these chemicals would not be equally as effective.
Eff~:
The need for a treatment buffer became apparent during our early
development work on lateral flow immunoassays where a stool specimens was
the patient sample being tested. It was noted that 10% formalin or SAF when
2o run directly in our lateral flow assays severely weakened or completely
eliminated the development of test and control lines.
Therefore, tests were run in an attempt to eliminate the interference
caused by formalin using a Group A Streptococcus assay (Rapid Strep A test,
Genzyme Diagnostics, San Carlos, California) and an Infectious Mononucleosis
25 assay (Rapid Mono test, Genzyme Diagnostics), both lateral flow assays. We
discovered that formalin or SAF did not cause an antibody-specific inhibition
of
signal, but was a more generalized phenomenon that would be applicable to
many different immunoassays of this type.
Initially, after demonstrating that adding various types of carrier proteins
3o was relatively ineffective in counteracting formalin, glycine was tried and
shown
to be effective. However when SAF-treated stool samples were tested,
significant inhibitory effects on signal were still observed. It appears that
this
inhibition was caused by the presence of acetic acid, which made the samples



CA 02368007 2001-09-14
WO 00/60353 PCT/US99/07573
rather acidic in pH. When Tris base was added to such samples, signal was
restored.
The data shown in the table below illustrates the suppressive effects of
SAF on control line signal development in a lateral flow assay. These assays
utilize goat anti-mouse IgG antibody striped onto a membrane, which in turn
to captures a particulate-labeled mouse IgG antibody to generate a visible
signal.
As shown, the use of the sample treatment buffer (STB) restores the control
line
signal to approximately the same intensity as the control level.
Table
Sample Device Control Line Intensity


Control (water) ++++



Without STB (SAF + water) -



With STB (SAF + STB ++++


In the above experiments, no formal incubation time was used. The treatment
buffer was added immediately prior to pipeting the sample into the test
devices.
The chemical reactions, which neutralize formalin and acid occur quickly and
functionally, can be thought of as instantaneous. It is possible that
additional
2o neutralization may take place as the sample is flowing in the device.