Diagnostic test kit and method for determination of chlamydial or gonococcal antigens using a microporous membrane

Antigens from chlamydial or gonococcal organisms in specimens containing whole blood, mucus or components thereof can be rapidly and sensitively determined using a polyamide microporous membrane which is coated with a surfactant. This determination is accomplished by contacting extracted antigen with the coated polyamide microporous membrane which is substantially free of any particulate matter. The membrane has an average pore size of from about 1 to about 10 .mu.meter. Within about 10 minutes of that contacting, antigen bound to the coated membrane is contacted with chlamydial or gonococcal antibody, respectively, so as to form an immunological complex on the membrane. The presence of the complex on the membrane is then determined as a measure of the amount of chlamydial or gonococcal antigen present in the specimen.

FIELD OF THE INVENTION 
The present invention relates to a surfactant-coated membrane, diagnostic 
kit and method for the detection of chlamydial or gonococcal organisms in 
a biological specimen. More particularly, it relates to the detection of 
such antigens in specimens which contain whole blood, mucus or components 
thereof. 
BACKGROUND OF THE INVENTION 
Immunoassays have been used in recent years to detect the presence of 
infectious diseases. In order for the assay to be useful, it must detect a 
particular organism with a high degree of reliability. In most cases, this 
requires the isolation and reaction of antigens peculiar to the organism 
with corresponding antibodies. For the test to be commercially successful, 
it also needs to be relatively inexpensive, simple to use and rapid. 
One such organism which can be detected by immunoassay is Chlamydia 
trachomatis (herein C. trachomatis) which is one of two microbial species 
of the genus Chlamydiaceae, order Chlamydiales. There are 15 or more 
strains of this species which are the causes of a number of human ocular 
and genital diseases including trachoma, inclusion conjunctivitis, 
lymphogranuloma venereum, nongonococcal urethritis and proctitis. 
Infection from C. trachomatis is pervasive in the general population so 
that it is believed that there are millions of cases each year of 
nongonococcal urethritis alone. 
Gonorrhea is a disease usually transmitted by sexual contact caused by a 
bacterium of the Neisseria genus, especially N. gonorrhoeae. The disease 
has plagued mankind for thousands of years, and although antibiotics have 
helped control its spread, it still persists in epidemic proportions in 
many parts of the world. The importance of detection and treatment of this 
organism is well recognized. N. meningitidis and N. lactamica are also 
species of considerable medical and diagnostic interest. 
Because of the widespread nature of these diseases, there is considerable 
interest in having a rapid, simple and reliable test for detection of 
chlamydial and gonococcal organisms. Considerable research has been 
carried out to find useful ways to extract detectable antigen from 
chlamydial organisms. See for example, U.S. Pat. Nos. 4,427,782 (issued 
Jan. 24, 1984 to Caldwell et al) and 4,663,291 (issued May 5, 1987 to 
Rose) and E.P. Publications Nos. 174,106 (Becton) and 193,431 (Caldwell et 
al). 
Assays for C. trachomatis and N. gonorrhoeae carried out using a solid 
support are described in U.S. Pat. Nos. 4,497,899 and 4,497,900, 
respectively (both issued Feb. 5, 1985 to Armstrong et al and Abram et al, 
respectively). The described assays are performed by extracting antigen 
from the organism and coating it on a bare solid support. The coated 
antigen is then detected with either one or two antibodies, one of which 
is suitably labeled. The critical feature of the assays appears to be the 
use of a solid support for attachment which is untreated or uncoated with 
any material. Attachment of antigen is apparently achieved by incubating 
the coated support for an extended time sufficient to cause adsorption of 
antigen thereon (Col. 2, lines 51-55 of U.S. Pat. No. 4,497,899). From the 
examples of this patent, this time is determined to be at least 30 minutes 
at elevated temperature (37.degree. C.). The entire assay described in 
U.S. Pat. No. 4,497,899 takes at least 3 hours to perform. A similar but 
somewhat quicker assay is described in U.S. Pat. No. 4,497,900 for N. 
gonorrhoeae (see Cols. 4 and 5). 
It would be desirable to have a much more rapid test for chlamydial or 
gonococcal organisms which has high reliability and can be performed at 
room temperature. Such an improvement is described and claimed in 
copending U.S. Ser. No. 255,923, filed on even date herewith by Pronovost 
and entitled "Determination of a Chlamydial or Gonococcal Antigen Using a 
Positively-Charged Ionically Binding Support". My colleague found that 
ionically charged supports attract chlamydial or gonococcal antigen and 
enable one to quickly and sensitively detect such antigens. However, I 
have found that with some biological specimens, especially those 
containing copious amounts of whole blood, mucus or components thereof, 
that assays using the ionically charged microporous membranes can be 
further improved. 
Thus, while the Pronovost application describes a remarkable advance in the 
diagnostic art, further improvements are needed. 
SUMMARY OF THE INVENTION 
The problems noted above are overcome with a method for the determination 
of a chlamydial or gonococcal antigen comprising: 
A. contacting chlamydial or gonococcal antigen extracted from a specimen 
suspected of containing chlamydial or gonococcal organisms, respectively, 
the specimen further containing whole blood, mucus or components thereof, 
with a surfactant-coated polyamide microporous membrane which is 
substantially free of any particulate matter, and which has an average 
pore size of from about 1 to about 10 .mu.meter, the surfactant coating 
being present in an amount of at least about 20 mg/m.sup.2, 
B. within about 10 minutes of contacting step A, contacting chlamydial or 
gonococcal antigen bound to the coated membrane with chlamydial or 
gonococcal antibody, respectively, so as to form an immunological complex 
on the membrane, and 
C. determining the presence of the complex on the membrane as a measure of 
the presence of chlamydial or gonococcal antigen, respectively, in the 
specimen. 
The membrane useful in the method described above is coated with a 
surfactant in an amount of at least about 20 mg/m.sup.2. This coated 
membrane can be supplied for the method as a part of a diagnostic test kit 
further comprising a reagent composition test kit further comprising a 
reagent composition comprising the chlamydial or gonococcal antibodies 
used in the method. 
The assay of this invention is rapid, reliable and simple to use. For 
example, it can be carried out in less than 30 minutes at room 
temperature. It is highly reliable for detecting extracted chlamydial 
antigen (such as from C. trachomatis), and particularly the 
lipopolysaccharide antigen. It can also be used to rapidly and sensitively 
detect gonococcal antigens (such as proteins IA and IB from N. 
gonorrhoeae). These advantages are achieved with the invention described 
and claimed in U.S. Ser. No. 255,923 of Pronovost (noted above). 
However, the present invention achieves additional advantages because it 
enables one to carry out the assay of biological specimens which may 
contain whole blood, mucus or components thereof. Such materials sometimes 
clog microporous membranes which have ionic charges, and drastically slow 
down the assay. It is uncertain as to what causes the clogging by some 
specimens and not others. 
However, it is clear that commercial assays must be rapid and capable of 
processing specimens no matter what material may be present. The present 
invention is such an assay. It is carried out with a polyamide microporous 
membrane having an average pore size of from about 1 to about 10 
.mu.meter. The membrane is substantially free of any particulate material 
which may be used for antigen capture, and is coated with a a certain 
amount of a surfactant. The present invention can be carried out at room 
temperature with minimal equipment and technical skill.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention comprises a method for determining the presence of C. 
trachomatis (or other chlamydial species), or the presence of N. 
gonorrhoeae (or other gonococcal species) in a biological specimen which 
has been obtained from a patient using any suitable medical or diagnostic 
techniques. Such specimens include, for example, swab specimens obtained 
from the cervix, urethra, throat or anus of a patient, and body fluids 
such as synovial fluid or fluid from lesions. The biological specimens so 
obtained are suspected of containing bacterial organisms which comprise 
the chlamydial or gonococcal antigen (or mixture thereof) to be 
determined. The specimens are particularly likely to contains whole blood 
or mucus, and sometimes large amounts of both. 
While the assay can be carried out to detect antigens from intact 
chlamydial or gonococcal organisms, it is usually desirable to extract the 
antigens from the organisms in order to increase assay sensitivity. 
Standard techniques can be used for lysing the organism to release antigen 
including, for example, solvent dilution or high pH lysing solutions, 
enzyme treatment and physical agitation such as sonication or 
centrifugation. Heating is described as a lysing technique in E.P. 
Publication No. 183,383 (published June 4, 1986). The use of anionic 
detergents or salts such as sodium dodecyl sulfate and deoxycholate is 
described in U.S. Pat. Nos. 4,497,899, 4,497,900 (both noted above) and 
4,663,291 (issued May 5, 1987 to Rose). 
In a preferred embodiment, the present invention can be used to detect the 
chlamydial lipopolysaccharide (glycolipid group) antigen (as described, 
for example, in E.P. Publication No. 193,431, published Sept. 3, 1986). 
Extraction procedures are also described therein. In another embodiment, 
the detected antigen can be the chlamydial major outer membrane protein of 
the organism which comprises about 60% of the total associated outer 
membrane protein. This antigen and methods of extraction are described in 
U.S. Pat. No. 4,427,782 (issued Jan. 24, 1984 to Caldwell et al). In some 
instances, a mixture of these chlamydial antigens will be detected using 
the present invention. In still other embodiments, the invention is used 
to detect one or more gonococcal antigens (IA or IB protein), or mixtures 
of antigens from individual gonococcal strains. 
A preferred extraction composition is described in detail in copending U.S. 
Ser. No. 255,928 filed on even date herewith by Pronovost, Mauck, 
Sullivan, Greer and Gilbert and entitled "High pH Extraction Composition 
and Its Use to Determine a Chlamydial, Gonococcal or Herpes Antigen". The 
central feature of that composition is the presence of an alcoholamine or 
salt thereof and its high pH. Further details of this preferred 
composition are provided below in relation to the examples. 
In addition, it may be desirable to use a protease in the extraction 
procedure to break down whole blood and mucus. This is described in 
copending U.S. Ser. No. 255,922 filed on even date herewith by Gilbert, 
Mauck and Stowers and entitled "Use of a Protease in the Extraction of 
Chlamydial, Gonococcal and Herpes Antigens". 
Once antigen is extracted from the organism, it is desirable, although not 
essential, that the specimen be prefiltered to remove cell debris, 
particulate matter and other unwanted materials prior to further handling. 
Prefiltering can be carried out in a suitable container having a filter of 
some type. While prefiltering may be carried out, it does not always 
remove all of the extraneous materials so as to facilitate the assay. 
Thus, the present invention overcomes the failures of any prefiltering 
after extraction. 
Extraction can be carried out in any suitable container, including devices 
specially designed for extraction of antigen. Useful devices are known in 
the art, including U.S. Pat. No. 4,746,614 (issued May 24, 1988 to 
Devaney, Jr. et al). 
Extracted antigen is contacted with a polymeric microporous membrane having 
an average pore size of from about 1 to about 10 .mu.meter, and preferably 
of about 5 .mu.meter. The membrane is prepared from a polyamide, that is a 
long-chain synthetic polymer having recurring amide groups in the polymer 
backbone. They are generally copolymers of a diamine and a dicarboxylic 
acid, or homopolymers of a lactam of an amino acid. Representative 
materials include, but are not limited to, polyhexamethylene 
dodecanediamide (nylon 612), polyhexamethylene adipamide (nylon 66), 
poly-.epsilon.-caprolactam (nylon 6), polyhexamethylene sebacamide (nylon 
610) and poly-7-aminoheptanoamide (nylon 7), and mixtures thereof. 
Polyhexamethylene adipamide (nylon 66) is preferred. 
Further details of useful membrane materials and details of their 
preparation are found in various published references including U.S. Pat. 
No. 4,340,479 (issued July 20, 1982 to Pall) and Pall Corp. trade 
literature brochures PSD-750a (March, 1983, pp. 1-20) and NM-900c 
(September, 1984, pp. 1-28). A preferred polyamide microporous membrane is 
the nylon 66 membrane manufactured and sold by Pall Corp. as 
Biodyne.RTM.-A membrane. 
The membrane is substantially free of particulate material, such as 
polymeric particles, which may be used for antigen capture, for example as 
described in E.P. Publication No. 264,036 (published Apr. 20, 1988). 
In the practice of this invention, the membrane is coated or treated with 
one or more surfactants in an amount of at least about 20, and preferably 
from about 50 to about 150, mg/m.sup.2. Useful surfactants include, but 
are not limited to, anionic, amphoteric or nonionic surfactants, with 
nonionic surfactants being preferred. There are many useful surfactants, 
and a worker skilled in the art can consult the standard resource, 
McCutcheon's Emulsifiers and Detergents, 1986 Ed., McCutcheon Division 
Publishing Co., Glen Rock, N.J. to find useful surfactants. For example, 
useful anionic surfactants include, but are not limited to sodium dodecyl 
sulfate, lithium decyl sulfate, ammonium dodecyl sulfate, sodium decyl 
sulfate and others known in the art. Useful amphoteric surfactants include 
Zonyl.TM. FSK and others known in the art. 
Particularly useful nonionic surfactants include fluorinated nonionic 
surfactants such as perfluoroalkylpoly(ethylene oxide) alcohols, for 
example commercially available as Zonyl.TM. FSN (DuPont), Nonidet P-40 
(Sigma Chemical) or as Fluowet.TM. OT (American Hoechst). 
Coating the membrane with a surfactant or mixture thereof can be 
accomplished in any suitable way, for example, by dipping the membrane in 
a solution (aqueous or organic) of the surfactant, or by applying the 
surfactant by a coating technique. Once coated, the membrane is dried 
under suitable conditions. 
The surfactant-coated membrane described herein can be used in combination 
with other equipment (bottles, test tubes, swabs, beakers or cups) in 
order carry out the assay. Alternatively and preferably, it is fitted into 
a disposable test device in which the assay can be carried out and all 
fluids accommodated. Useful configurations of test devices are known in 
the art including U.S. Pat. Nos. 3,825,410 (issued July 23, 1974 to 
Bagshawe), 3,888,629 (issued June 10, 1975 to Bagshawe), 3,970,429 (issued 
July 20, 1976 to Updike) and 4,446,232 (issued May, 1984 to Liotta). 
Particularly useful devices are described and claimed in copending and 
commonly assigned U.S. Ser. Nos. 19,810 (filed Feb. 27, 1987 by Hinckley) 
and 98,248 (filed Sept. 18, 1987 by Hinckley). 
Almost immediately upon contact of the antigen with the coated membrane, 
the antigen is bound thereto. The antigen is preferentially bound to the 
membrane as opposed to other proteins, cell components, whole blood or 
mucus or other debris which may be present in the test specimen or 
reagents used in the assay. 
Within about 10 minutes, and preferably within 1 to 5 minutes, of the 
contact, the bound antigen is contacted with a reactive composition 
comprising a chlamydial or gonococcal antibody so as to form an 
immunological complex bound to the support. Fluid and unbound materials 
may be removed quickly at the same time. If the assay is carried out using 
a disposable test device, fluid and unbound materials (such as whole blood 
and mucus components) in the specimen are allowed to flow through the 
membrane and collected in a suitable compartment during the time the 
antigen is bound to the membrane. 
The antibody used in this assay is specifically immunoreactive with one or 
more chlamydial or gonococcal strains (depending upon what organism is of 
interest). It can be polyclonal or monoclonal. If polyclonal, it is 
commercially available or prepared in various animals using known 
techniques employing an antigen common to the strain of organism to be 
detected. A single antibody or mixture thereof can be used. For example, 
antibody to either the chlamydial lipopolysaccharide or major outer 
membrane protein antigen, or antibodies to both antigens can be used in 
the assay. Preferably, the antibodies are monoclonal which are either 
commercially available or prepared using standard hybridoma technology. 
Useful procedures for preparing antibodies are described, for example, in 
E.P. Publication No. 193,431 and U.S. Pat. No. 4,427,782 (noted above). 
In one embodiment, the antibody to the antigen is labeled for detection. 
Useful labels are known in the art and include chemical or biological 
compounds which are directly detectable using suitable procedures and 
equipment, as well as compounds which can be detected through further 
chemical or specific binding reactions to provide a detectable species. 
Examples of useful labels include radioisotopes, enzymes, fluorescent 
compounds, chemiluminescent compounds, phosphorescent compounds, biotin or 
its derivatives, avidin or its derivative, ferritin, magnetizable 
particles, dyed particles, gold sols, dye sols, colored Staphylococcus 
aureus cells and others readily apparent to one skilled in the art. 
Radioisotopes or enzymes are preferred labels. The labels can be attached 
to antibodies using known techniques. Where the label is not directly 
detectable, further reagents or compounds are needed to render the 
reaction or specific binding product detectable. For example, if the label 
is biotin, it can be reacted with avidin which is conjugated with an 
enzyme to provide a detectable species. Where the label is an enzyme, such 
as glucose oxidase, urease, peroxidase, alkaline phosphatase and others, 
substrates in dye-providing compositions are also needed. 
In a particularly preferred embodiment, the label is peroxidase, and at 
some point in the assay, hydrogen peroxide and a suitable dye-providing 
composition is added to provide a detectable dye. For example, useful 
dye-providing reagents include leuco dyes, such as triarylimidazole leuco 
dyes (as described in U.S. Pat. Nos. 4,089,747, issued May 16, 1978 to 
Bruschi), or other compounds which react to provide a dye in the presence 
of peroxidase and hydrogen peroxide (that is, compounds which react to 
provide a dye upon catalytic action of peroxidase). 
In a preferred embodiment, the chlamydial or gonococcal antibody is not 
labeled, and detection of the antibody-antigen complex formed and bound to 
the membrane is accomplished using a second antibody (described below) 
which is specific to the unlabeled antibody and is appropriately labeled. 
The chlamydial or gonococcal antibody can be contacted with the bound 
antigen in a reagent composition (also known as a blocking composition) 
further comprising one or more proteins which reduce nonspecific 
interactions on the support. Useful proteins are well known and include, 
for example, casein, .alpha.-casein, fetal bovine serum and porcine gamma 
globulin. A particularly useful reagent composition comprises a protein 
and an amphoteric surfactant, as described and claimed in copending U.S. 
Ser. No. 255,925 filed on even date herewith by Pronovost and entitled 
"Immunological Reagent Composition and Its Use in the Determination of 
Chlamydial or Gonococcal Antigens." 
Once the bound antigen has been contacted with the chlamydial or gonococcal 
antibody, a bound immunological complex is formed on the coated membrane. 
To hasten the formation of this complex, the antibody and antigen are 
generally incubated at a temperature of from about 15.degree. to about 
30.degree. C. for up to 10 minutes. Preferably, the incubation is carried 
out at from about 18.degree. to about 25.degree. C. (that is, room 
temperature) for from 1 to 5 minutes. These mild incubation conditions are 
in sharp contrast to the 30 minutes at 37.degree. C. described as 
necessary for adsorption of chlamydial antigen to bare supports in U.S. 
Pat. No. 4,497,899 (noted above). 
After the incubation and within about 10 minutes of the antibody-antigen 
contact, the bound complex is washed one or more times with a wash 
solution which generally has a pH of from about 7 to about 12. The 
solution preferably contains one or more surfactants to aid in separating 
unbound materials from the bound complex. Particularly useful surfactants 
are cationic surfactants, as described in copending U.S. Ser. No. 255,924, 
filed on even date herewith by Pronovost and Gilbert and entitled "Wash 
Composition Containing a Cationic Surfactant and Its Use in Chlamydial and 
Gonococcal Determinations." 
In the embodiment described above where the chlamydial or gonococcal 
antibody is labeled, the assay procedure after washing is to detect the 
label directly or indirectly after addition of the appropriate reagents. 
This is done relatively quickly after washing the bound complex, that is 
generally within about 10 minutes, and preferably within about 1 to about 
5 minutes. If desired, label detection can be hastened with incubation if 
the reagents warrant it. The label is then detected using standard 
equipment and procedures. 
In the preferred embodiment, the chlamydial or gonococcal antibody is 
unlabeled, and after washing the bound complex, it is contacted with an 
antibody directed to the unlabeled antibody. This second antibody (that 
is, an anti-antibody) is appropriately labeled with any of the labels 
described above. The antibody can be monoclonal or polyclonal and either 
purchased or prepared using known techniques. In a chlamydial assay, the 
anti-antibody is preferably a polyclonal antibody which is reactive with 
either of the lipopolysaccharide or major outer membrane protein 
antibodies. 
After this contact, the resulting antigen-antibody-antibody complex which 
is bound to the coated membrane is incubated for up to about 10 minutes at 
a temperature of from about 15.degree. to about 30.degree. C., and 
preferably for about 1 to about 5 minutes at from 18.degree. to 25.degree. 
C. 
Further washing is carried out to remove uncomplexed materials, and 
suitable enzyme substrates or other needed reagents are added to provide a 
detectable species. The bound antigen-antibody-labeled antibody complex is 
then detected on the membrane using standard radiometric, colorimetric, 
fluorescent or other detection techniques. 
The diagnostic test kit of the present invention comprises the coated 
membrane described herein and one or more other component compositions, 
solutions or devices for carrying out the assay. For instance, it 
generally includes a reagent composition comprising an antibody (labeled 
or unlabeled) to a chlamydial or gonococcal antigen. Additional optional 
materials in the kit include wash solutions, dye-providing compositions, 
labeled anti-antibody compositions, extraction compositions, extraction 
devices, swabs or other specimen collecting means, disposable test devices 
and others known to one skilled in the art. Preferably, the kit includes 
the coated membrane mounted as part of a disposable test device. 
The following example is provided to illustrate, but not limit the scope 
of, the present invention. 
EXAMPLE 
Comparison of Charged and Surfactant-Coated Uncharged Microporous Membranes 
in Chlamydial Determinations 
This example illustrates the practice of ths present invention and compares 
it to the invention described and claimed in U.S. Ser. No. 255,923 of 
Pronovost (noted above) wherein a positively charged microporous membrane 
is used in the assay. 
Materials Used 
The following materials and compositions were used in the assay. 
The microporous membrane used in the practice of this invention was an 
uncharged nylon 66 membrane purchased from Pall Corp. as Biodyne.TM. A and 
which was coated with about 50 mg/m.sup.2 of Zonyl.TM. FSN nonionic 
surfactant (DuPont). The Control charged membrane (according to U.S. Ser. 
No. 255,923, noted above) was purchased from Pall Corp. as Biodyne.TM. B. 
These membranes were mounted in each of the three test wells of disposable 
test devices like those described in U.S. Ser. No. 98,248 (noted above). 
Specimens for testing were obtained from female patients using endocervical 
swabs. The specimens contained large amounts of whole blood or mucus, and 
were also tested using standard culture techniques. Each specimen was 
tested using separate test devices having the Control membrane or the 
surfactant-coated membrane of the present invention. 
An extraction device like those described in U.S. Pat. No. 4,746,614 (noted 
above) was used to extract chlamydial antigen from the specimens by drying 
at separate locations on the inside thereof: (1) a coating of Trizma.TM. 
(Sigma Chemical) buffer (20 .mu.l of 1.65 molar solution, pH 11.1) and 
thimerosal preservative (0.01 weight %), and (2) 2-(N-morpholino)ethane 
sulfonic acid (10 mmolar, 50 .mu.l solution), sodium azide (1.54 mmolar), 
ethylenediaminetetraacetic acid (5.4 mmolar), 
5,5-dimethyl-1,3-cyclohexanedione (21.4 mmolar), dithiothreitol (0.188 
molar) and poly(acrylamide)(6.35 weight %). 
Composition 1: contained Amideck.TM. protease (4 .mu.g/ml, 170 units/mg, 
available form BioProducts Division, Eastman Kodak Co.) in 10 mmolar 
2-(N-morpholino)ethane sulfonic acid buffer (pH 6), sodium chloride (50 
mmolar), calcium chloride (5 mmolar), 1,2-propanediol (10% w/v) and 
preservative (0.01 weight %). 
Composition 2: contained ethanolamine (0.47 molar), sodium chloride (0.27 
molar), preservative (30 mmolar) ethylenediaminetetraacetic acid (50 
mmolar), Emcol.TM. CC-36 cationic surfactant (0.45 weight % from Witco 
Chemical) and sodium hydroxide (0.66 normal, pH 13.4). 
Composition 3: contained hydrogen peroxide (12 weight % in water), 
diethylenetriaminepentaacetic acid (10 .mu.molar) and preservative (0.01 
weight %). 
Composition 4: contained 3-cyclohexylamino-2-hydroxy-1-propanesulfonic acid 
buffer (0.05 molar, pH 10), Emcol.TM. CC-9 cationic surfactant (0.75 
weight %) and preservative (0.01 weight %). 
Composition 5: contained creatine kinase-MB antibody (5 .mu.g/ml), casein 
(0.01 weight %), Lonzaine.TM. C amphoteric surfactant (0.01 weight % from 
Lonza Corp.) and preservative (0.01 weight %) in phosphate buffered saline 
solution (pH 7.2). 
Composition 6: contained chlamydial lipopolysaccharide monoclonal 
antibodies prepared using standard hybridoma technology and a mouse cell 
line (4 .mu.g/ml) and the non-antibody components of Composition 5. 
Composition 7: contained goat anti-mouse IgG antibodies conjugated to 
horseradish peroxidase (from BioRad Labs.) (1:700 dilution) in the 
non-antibody components of Composition 5, along with 4'-hydroxyacetanilide 
(10 mmolar). 
Composition 8: contained 
2-(4-hydroxy-3-methoxyphenyl)-4,5-bis(4-methoxyphenyl)imidazole leuco dye 
(0.008 weight %), poly(vinyl pyrrolidone) (1 weight %), sodium phosphate 
(10 mmolar, pH 6.8) diethylenetriaminepentaacetic acid (10 .mu.molar), 
4'-hydroxyacetanilide (2 mmolar) and hydrogen peroxide (10 mmolar). 
Assay 
In performing the assays of each specimen, Composition 1 (7 drops) was 
added to the extraction device and a patient swab containing each specimen 
was placed into the device, rotated 5-10 seconds, followed by incubation 
of the device for 3 minutes at room temperature (18.degree.-25.degree. 
C.). Composition 2 was then added to the device containing the swab which 
was rotated again for 5-10 seconds, and incubation was again carried out 
for 3 minutes at room temperature. Composition 3 was then added, the swab 
was rotated again followed by a third incubation. 
The resulting solution containing extracted lipopolysaccharide antigen was 
removed from the device using a pipette, prefiltered, and transferred to 
each well of a disposable test device (4 drops per well). Fluid was 
allowed to drain through the microporous membranes in the wells. Each well 
was then washed with Composition 4 (4 drops per well). 
Composition 5 (2 drops) was then added to one of the wells of each test 
device (considered a negative control well). Composition 6 (2 drops) was 
added to each of the two remaining wells of each device. One of those two 
wells (considered a positive control well) contained dried chlamydial 
lipopolysaccharide antigen on the membrane. 
The wash step was repeated after fluid drainage, and Composition 7 (2 
drops) was added to each well followed by incubation at room temperature 
for 5 minutes. After a wash step was carried out, Composition 8 (2 drops) 
was added to each well. After 5 minutes incubation at room temperature, 
the resulting dye on the membranes of each test device was visually graded 
on a scale of 0 to 10 (no density to highest density). 
It was observed that the test devices having the surfactant-coated, 
uncharged membrane provided better sensitivity (93.8%) than the Control 
devices having the charged membrane (73.3%). In addition, the Control 
devices exhibited poor drainage of fluid due to the large amounts of whole 
blood and mucus in the specimens, poorer spot quality and higher 
backgrounds than the devices used in the present invention. 
The invention has been described in detail with particular reference to 
preferred embodiments thereof, but it will be understood that variations 
and modifications can be effected within the spirit and scope of the 
invention.