Extraction of test substances

A vessel containing a polymeric acid is used in a method and test kit for extracting a bacterial (e.g., streptococcal) antigen from a test sample, for example, preliminary to an immunoassay. To extract the antigen from bacteria in the sample, a precursor reagent is applied to the vessel acid and incubated with the sample. The kit includes a vial containing the acid and another vial containing the precursor. The kit is produced by including the acid polymer in the vial or vessel, e.g., as a pellet or coating.

BACKGROUND OF THE INVENTION 
This invention relates to the extraction of bacterial antigens, 
particularly for immunoassays. 
Bacterial antigens are bacterial substances which generate, or which 
immunospecifically react with, anti-bacterial antibodies. The presence of 
such an antigen in a sample is standardly detected by an immunospecific 
reaction with anti-bacterial antibodies. 
Some bacterial antigens may be encrypted in larger cell structures which 
interfere with the immunospecific reaction or with its detection. 
Moreover, the sample may include other substances such as cell debris, 
mucus or other flora, which also can infere with the immunospecific 
reaction or with its detection. 
Thus, it is important, e.g., preliminary to an immunoassay, to extract a 
bacterial antigen from cell structures and other sample components that 
surround it. This process is known as extracting the bacterial antigen. 
Rosenstein, Published European Patent Application 153477, discloses a 
diagnostic test for group A streptococci in which an enzymatic extraction 
reagent releases strep A antigen from a swab, and agglutination with 
antibody immobilized on latex beads is detected. 
A more customary rapid extractive medium is a combined solution of acetic 
acid and sodium nitrite. For example, Kholy et al. (1974) Applied 
Microbiology 28(5):836-839 and Hafez et al. (1981) J. Clin. Microbiol. 
14(5):530-533 disclose a method of extracting protein antigens from 
streptococci in which NaNO.sub.2 and glacial acetic acid are added to a 
cell preparation. The extracted antigens are then subjected to 
immunoassay. 
Unfortunately, the above-described nitrous acid extraction solution is 
highly unstable. It is apparent that the acetic acid must be kept separate 
from the nitrite until just before extraction takes place. Otherwise the 
instability of the resulting nitrous acid solution can defeat the 
extraction. Thus if the extractive materials are mixed prematurely there 
is a decay and the resultant solution becomes unsatisfactory after but a 
short delay interval. 
In addition, the required separate packaging and mixing of the precursors 
is prone to leakage, inconvenient, time consuming, and liable to error. 
Once the extraction is completed, the extractive medium may be neutralized, 
e.g., with NaOH as disclosed in Kholy et al. or Hafez et al. cited above, 
and the assay proceeds in conventional fashion. 
Accordingly, it is an object of the invention to facilitate the extraction 
of test substances, as well as the assay of test materials. A related 
object is to facilitate immunoassays, particularly enzyme immunoassays. 
Another object of the invention is to facilitate the extraction of 
nitrogenous substances, such as streptococcal cell walls. A related object 
is to completely eliminate the need for separate packaging of liquid acid 
or precursor prior to extraction. 
Still another object of the invention is to reduce the amount of handling 
required for the extraction of antigens before performing enzymatic 
assays. 
Yet another object of the invention is to eliminate the need for combining 
sodium nitrite with a liquid activator such as acetic acid. A related 
object is to eliminate any adverse consequences from the premature 
combination of an extractive agent with a liquid activator. 
SUMMARY OF THE INVENTION 
In accomplishing the foregoing and related objects the invention provides 
for extraction of a bacterial antigen, by transferring a sample which may 
contain the antigen to the interior of a vessel containing a polymeric 
acid. A precursor reagent is also transferred to the vessel, and the 
sample is incubated for a sufficient time to permit the acid to react with 
the precursor and produce the desired extraction. 
In accordance with one embodiment of the invention, the polymeric acid 
reacts with the precursor to produce a diazotizing medium. For example, 
the precursor is a nitrite salt, such as sodium or potassium nitrite. 
Preferably the bacterial antigen is an antigenic carbohydrate, especially a 
streptococcal antigen. Preferably the polymeric acid is: a polymeric acid 
or an anhydride thereof; a polysulfonic acid such as polystyrene sulfonic 
acid or polyacrylamidosulfonic acid; or a polyphosphoric acid. 
Specifically preferred polymeric acids are polyacrylic acids such as 
polymethacrylic acid. 
A test kit in accordance with the invention includes a first vial with a 
polymeric acid and a second vial that includes an extractive precursor. 
The polymeric acid may be a pellet within the first vial, or it may be a 
coating on an interior wall of the first vial. Components that may be 
included with the polymeric acid include a surfactant to assure wetting of 
the interior wall surfaces, a plasticizer to avoid brittleness, and a 
fluorescent compound to test for presence and distribution of the acid. In 
addition, the first vial is an open or stoppered tube, advantageously of 
plastic. The second vial desirably includes sodium nitrite solution. The 
activator coating reacts with a sodium nitrite solution to produce an 
extractive medium for the bacterial antigen. Preferably the polymeric acid 
in the kit is one of the acids described above. 
In a method of producing a structure for the extraction of a test antigen, 
a vessel is provided and an acidic polymer is included within the vessel. 
The acidic polymer may be a thin coating or it may be applied to the 
interior of the vessel as a pellet. 
The acidic acid polymer coating may be created by applying the coating 
applied to the interior of the vessel as a coating solution, and the 
vessel is continuously rotated to deposit a symmetrical coating on the 
interior of the vessel as the solvent is evaporated. If a fluorescent 
tracer is included with the acid, the method of producing may include 
illuminating the acid in the vial to verify its presence and distribution.

DETAILED DESCRIPTION I. The Process of Extraction 
FIG. 1A and FIG. 1B show a test arrangement for the extraction of a 
bacterial antigen from a substance in a test swab 1 (or a first vial not 
shown). The swab 1 carries a test substance 2, which also may include a 
bacterial antigen, such as strep antigen. 
In order to conduct an assay for the strep antigen, it is preliminarily 
necessary to expose the antigen from the test substance 2. For that 
purpose first and second vials 3 and 4, respectively, are provided. The 
first vial 3 contains a precursor reagent of sodium nitrite solution 5. 
The second vial 4 of the extractive test assemblage contains a polymeric 
acid, e.g., polyacrylic acid coating 6. 
To accomplish the extraction the sodium nitrite solution 5 from the first 
vial 3 is poured into the second vial 4 as indicated by an arrow. The test 
substance 2 from the swab 1 is inserted or poured into the second vial 4 
as indicated by another arrow. This may take place simultaneously, 
following or preceding the entry of the sodium nitrite into the second 
vial 4. 
The extractive procedure in accordance with FIG. 1A and FIG. 1B contrasts 
with the typical antigen extraction procedure for streptococcus samples in 
accordance with the prior art where the reagents are sodium nitrite 
solution and dilute acetic acid. The instability of the acetic 
acid-nitrous acid combination has forced users to form the final reagent 
immediately prior to use. The standard commercial practice is to have the 
user dispense acetic acid solution and sodium nitrite solution from 
individual vials for mixing in the extraction vial or tube 4. 
By contrast with the prior art, FIG. 1A, and FIG. 1B depict a precoating of 
the extraction tube 4 with a dry film 6 of polymeric acid. This allows the 
acetic acid vial to be completely eliminated from the extraction kit, 
along with the accompanying manual operation. This simplifies not only the 
final configuration of the assay kit but also the assay protocol, without 
compromising assay performance. 
One suitable polymer is polyacrylic acid MW 5000 (Goodrite 732 from the B. 
F. Goodrich Company). This polymer forms a clear, colorless, continuous 
film upon evaporation of its carrier solvent, which can be water. Another 
suitable polymer is poly(2-acrylamido-2-methyl-1-propane sulfonic acid 
(available from Aldrich Chemical Co., Milwaukee, Wis.). 
In order to promote a uniform coating of the polymer or the interior of the 
tube 4, an aliquot of a polymer coating solution is dispensed to the 
bottom of the receiving tube 4. When the tube is hydrophobic, a surfactant 
is included to assure wetability and uniform coverage. The tube 4 is then 
rotated continuously in a 70.degree. C. drying oven so that the loading 
material deposits and concentrates symmetrically along the inner wall of 
the tube 4, which is desirably inclined during this process to restrict 
the coating to the bottom of the tube. This ensures that a small volume of 
sodium nitrite solution will be able to dissolve the entire amount of 
polyacrylic acid as rapidly as possible and yield the final concentration 
levels that are needed for the extraction. 
Although a thin-film coating is desirable, it is not required. The assay 
protocol prescribes mechanical agitation of the test specimen. Because the 
total quantity of polymer for one assay is so small, this provides enough 
mixing to dissolve even a concentrated pellet of acid polymer. In an 
alternative fabrication procedure, the coating material is introduced as a 
dry pellet, or it is introduced in a solvent, at the bottom of the tube, 
and there is no need for special orientation of the tube during the drying 
stage. 
Trials have been conducted using polystyrene plastic, as well as 
borosilicate and pyrex glass tubes. When plastic tubes are used, it is 
desirable to use a surfactant in the coating solution in order to ensure 
wetting. 
To avoid brittleness and chipping of the coating, an inert non-volatile 
hydrophilic plasticizer such as ethylene glycol can be included in the 
coating formulation. Preferably the plasticizer constitutes 10-80% (most 
preferably about 25%) of the final dry coating by weight. 
A fluorescent tracer, such as a stilbene derivative, also can be added to 
the coating, so that coating presence or coating integrity can be verified 
easily and non-destructively using an ultraviolet inspection lamp. 
After the antigens are extracted in accordance with FIG. 1A and FIG. 1B, 
the reagent is neutralized, for example by a solution from a vial 7 as 
shown by an additional arrow. An assay can be performed with any of a wide 
variety of test kits, as described below. 
Polymeric acid coating and extraction of bacterial antigen will now be 
illustrated with specific examples, which are offered as illustrative and 
not as limitations. 
EXAMPLE 1 
Polyacrylic Acid Coating 
To prepare an acid tube-coating solution, 50microliters of 50% polyacrylic 
acid MW 5000 were diluted with 0.50 mL (milliliters) of water. 0.50 mL of 
Tween 20 surfactant (from Sigma Chemical Company of St. Louis, Mo. were 
then diluted with 500 mL of water. 0.20 mL of the resulting mixture were 
combined with the diluted polyacrylic acid by vortex mixing. 50 
microliters of this coating solution were next dispensed directly to the 
bottom of a three mL, conical polystyrene tube (Sarstedt Model No. 73.646 
of Princeton, N.J.). The tubes were then coaxially attached to a motorized 
rotator and placed in a 70.degree. F. convection oven at an incline of 
20.degree. for three hours until thoroughly dry. 
EXAMPLE 2 
Polyacrylic Acid Coating with Plasticizer 
In order to avoid brittleness and increase coating adherence, a 
non-volatile hydrophilic plasticizer was added to the coating solution. 
For example 8.4 g Goodrite 732 polyacrylic acid (B. F. Goodrich, 
Cleveland, OH), 50% w/w solution (Mw 5,100), was combined with 11 mL 
H.sub.2 O, 0.30 mL Tween 20 (Sigma Chemical Co., St. Louis, MO), 1.05 mL 
Carbowax 350 (Union Carbide, Danbury, CT), and 0.1 mL Intrawhite diluted 
1:100 (Crompton & Knowles, Rumford, RI). The coating was applied to a vial 
as described in Example 1. 
EXAMPLE 3 
Polyacrylamidosulfonic Acid Coating (P-AMPS) 
A pAMPS coating was prepared by combining and thoroughly mixing 10 g poly 
(2-acrylamido-2-methyl-1-propane sulfonic acid), available as 10% w/w 
aqueous solution from Aldrich Chemical Co., Milwaukee, Wis., with 10 mL 
H.sub.2 O and 0.3 mL Tween 20. The coating was applied to a vial as 
described in Example 1. 
EXAMPLE 4 
Polystyrene Sulfonic Acid Coating 
A polymeric acid coating was prepared by mixing 5 g Versa TL 72 (20% w/w 
polystyrene sulfonic acid sold by National Starch co., Bridgewater, N.J.) 
with 11.4 mL water and 0.30 g Tween 20. 50 microliters of this solution 
was dispensed to 12.times.55 polystyrene test tubes then dried at 
70.degree. C. for 3 hours. 
EXAMPLE 5 
Bacteria Extraction Using Polyacrylic Coating 
Acid coated vials prepared in accordance with Example 1 were used in an 
assay for Group A streptococcus. The extraction performance for the acid 
coated vials was compared with that for the conventional generation of 
nitrous acid and tested in an enzyme immunoassay. 
Positive sample swabs were seeded with a solution of 10.sup.6 organisms per 
milliliter of strep A in a Phosphate Buffered Saline (PBS) solution. 
Extraction tubes were used in accordance with Table 1 below. Tubes of Type 
A were prepared as noted above for Example 1 and tubes of Type B are the 
same tube without an acid coating and served as a control. 
TABLE 1 
______________________________________ 
EXTRACTION REAGENT FORMULAE 
ADDED 
REAGENTS TUBES OF TYPE A TUBES OF TYPE B 
______________________________________ 
Sodium Nitrite 
0.30 mL at 2.7 Molar 
0.20 mL at 4 Molar 
Acetic Acid 
None 0.10 mL at 1.5 Molar 
______________________________________ 
Swabs were incubated in the individual tubes for 3 minutes and 0.30 mL of 
neutralization buffer added to each. 
The neutralization buffer was one molar 
N-hydroxyethyl-piperazine-N'-ethanesulphonic acid (available from Research 
Organics Inc., Cincinnati, Ohio), adjusted to pH 8 with 0.25% w/w bovine 
serum albumin and 0.2% w/w Tween 20. 
EXAMPLES 6-8 
Bacterial Extraction with Other Polymeric Acids 
Tubes containing polymeric acid as described in each of Examples 2-4 were 
subjected to test swabs generally using the procedure of Example 5. After 
extraction, the extract was neutralized. 
II. Assaying The Extract 
The extracts prepared according to the invention are useful in immunoassays 
which are generally well-known. For example, the extract can be assayed 
with sandwich ELISA system utilizing an anti-strep antibody-coated 
dipstick. Specifically, one antibody is immobilized to a polystyrene 
paddle, and a second enzyme-labelled antibody is added to the solution to 
be assayed. 
The following examples illustrate one use for antigen extracted by the 
technique of part I, above. 
EXAMPLE 9 
Sandwich ELISA Paddle Assay 
In one example of the sandwich ELISA paddle assay described, 0.45 mL of 
extract from Example 5 was mixed with 0.050 mL of antibody conjugate 
(having a horseradish peroxidase label, in the molar ratio of 1:1.5 
antibody to label). The assay can then be performed with a polystyrene 
paddle coated with rabbit anti-strep immunoglobulin. The resultant 
immunocomplex on the paddle, Ab-Ag-AbE, is formed by incubation of the 
paddles in the extract/conjugate mixture for 20 minutes, followed by 
removal and washing for 3 seconds under running tap water. The paddle was 
subsequently incubated for 10 minutes in chromogen solution which was 
previously prepared by combining 3 parts of tetramethylbenzidine solution 
(1.5 grams per liter in methanol) with 7 parts citrate-phosphate buffer 
(0.05 molar at pH 5.0) containing 0.007% hydrogen peroxide. 
The results obtained using the above-described ELISA paddle method are 
summarized in Table 2. Units are in optical density at a wavelength of 630 
nanometers. 
TABLE 2 
______________________________________ 
Swabs 
Tube Type 
Pos. No. 1 
Pos. No. 2 
Neg. No. 1 
Neg. No. 2 
______________________________________ 
A (with 0.763 0.796 0.021 0.021 
dry acid) 
B (with liquid 
0.595 0.769 0.015 0.025 
acid) 
______________________________________ 
As noted above, the results are in optical density units at 630 nanometers. 
At the indicated wavelength, an optical density greater than 0.05 
indicates a positive result. It is to be noted that the invention gives 
uniform results of equal sensitivity or greater than does the standard 
extraction of the prior art. 
EXAMPLES 10-12 
Extraction and Assay Using Vials Prepared as in Examples 6-8 
The extracts prepared in Examples 6-8 were assayed with a paddle assay 
generally using the procedure in Example 9. 
An alternative to the paddle assay described in the above examples is a 
flow-through ELISA assay, in which a first antibody is immobilized on a 
membrane and the extract is poured through the membrane, the antigen being 
trapped by the first antibody. To detect the antigen, it is reacted 
(either before or after reaction with the membrane) with a second antibody 
that is enzyme labeled. Depending on the enzyme labeling system used, the 
membrane-immobilized antibody may also be co-immobilized with an enzyme, 
so that the two enzymes catalyze separate reactions which together 
generate color from a suitable substrate and chromogen. 
Other aspects of the invention will be apparent to those of ordinary skill 
in the art.