USOO5268299A

United States Patent (19) 11 Patent Number: 5,268,299

Shih et al. 45) Date of Patent: Dec. 7, 1993
54 DLUENT COMPOSITIONUSEFUL IN THE 5,043,288 8/1991 Motsenbocker .................... 436/518
DETECTION OF ANTBODES IN ASSAYS 5,047,318 9/1991 Snyder et al............................ 435/5
Warren, III et al. ... 435/7.34
75 Inventors: Yihsing Shih, San Mateo, Calif; 5,051,3569/1991 Warren,
Harold C. Warren, III, Rush; 5,077,198 12/1991 Shih et al. ............................ 435/7.9
Margaret J. Smith-Lewis, Pittsford, FOREIGN PATENT DOCUMENTS
both of N.Y.
136798 4/1985 European Pat. Off. .
73 Assignee: Eastman Kodak Company, 0217403 4/1987 European Pat. Off. .
Rochester, N.Y. 023494.1 9/1987 European Pat. Off. .
(21) Appl. No.: 755,403 0294964 12/1987 Japan.
la- 85-04903 11/1985 PCT Int'l Appl. .
22 Filed: Sep. 5, 1991 8703373 6/1987 PCT Int'l Appl. .
0 8703374 6/1987 PCT Int'l Appl. .
Related U.S. Application Data 8703375 6/1987 PCT Int'l Appl. .
62) Division of Ser. No. 181,465, Apr. 14, 1988, Pat. No. 8706005 8/1987 PCT Int'l Appl. .
5,077,198.
5ll Int. Cli............................................... G01N 1/28 OTHER PUBLICATIONS
52 U.S. C. ....................................... 436/18; 436/538 Concise Chemical and Technical Dictionary, H. Bennett,
58) Field of Search................... 252/.364; 436/538, 18 ed. Chemical Publishing Co., New York 1986, p. 1090.
(56) References Cited Ref QD5, B4. e
U.S. PATENT DOCUMENTS Lambden et al., Journal of Immunological Methods, vol.
20, pp. 277-286 (1978).
3,595,755 7/197 Hartel ............................... 436/18 X Nandapalan et al., Journal of Medical Virology, vol. 14,
3,764,478 10/1973 Bergmeyer et al. ... 436/18 X pp. 285-294 (1984).
3,997,470 12/1976 Monte et al. ....... ... 436/18 X Frankel et al., Mol. Immunology, 16, pp. 101-106 (1979).
3,998,973 12/1976 Carlson ............................... 424/357 Morrison-Oncology & Biotechnology News, Apr. 1988.
4,040,785 8/1977 Kim et al. ... ... 436/18 X Primary Examiner-Gary Geist
4,218,490 8/1980 Phillips et al. ...................... 426/549 Attorney, Agent, or Firm-J. Lanny Tucker
4,283,491 8/1981 Dappen........... ... 436/18 X
4,329,448 5/1982 Cox et al. ......................... 424/49 X 57 ABSTRACT
4,468,334 8/1984 Cox et al. .... ... 252/315.3 X Diluent and wash compositions are useful in a rapid and
4,520, 13 5/1985 Gallo et al. ......................... 436/.504 sensitive assay for detecting antibodies, and especially
4,561,985 12/1985 Glass, Jr. ............................ 252/8.51 retroviral antibodies, in a biological specimen. The dilu
4,639,425 1/1987 Baier ............... . 436/S34 X ent composition is buffered to a pH of 6 to 10 and in
4,661,445 4/1987 Saxinger et al. ........................ 435/7 cludes a protein or carbohydrate, a surfactant and a
4,708,818 11/1987 Montagnier et al................... 435/5 negatively-charged organic compound. The wash com
4,725,669 2/1988 Essex . position is buffered to a pH of 5 to 10 and includes a
4,808,524 2/1989 Snyder et al. .................. 436/519 X surfactant. These compositions can be included in a
4,812,414 3/1989 Warren, III et al. ........... 436/534 X
4,828,978 5/1989 Warren, III et al. ........... 435/533 X diagnostic kit. The method of this invention includes
4,828,980 5/1989 Snyder et al. .................. 435/533 X mixing the biological specimen with the diluent compo
4,847,199 7/1989 Snyder et al. .................. 436/SOX sition, forming an immunological complex between
4,868,131 9/1989 Hiratsuka ......................., 436/538 X ligand and antibodies in the specimen and separating
4,870,007 9/1989 Smith-Lewis . ... 435/975 X complexed materials from uncomplexed materials using
4,923,680 5/1990 Nelson ................................ 435/7 X a filtration membrane and a washing step. An enzyme
4,935,339 6/1990 Zahradnik .......... labeled anti-antibody is added to form a ligand-anti
4,965,191 10/1990 Warren, III et al. ................... 435/7 body-antibody complex followed by its detection using
4,997,772 3/1991 Sutton et al........ suitable reagents.
5,017,474 5/1991 McClune et al. .. ... 435/7.5
5,024,935 6/1991 McClune et al. .................... 435/7. 9 Claims, No Drawings
 5,268,299
1. 2
expedient to detect the antibodies produced in the or
DLUENT COMPOST ON USEFUL IN THE ganism.
DETECTION OF ANTBODES IN ASSAYS Human retroviruses, as a family, represent a group of
related exogeneous retroviruses which exert a signifi
This is a division of application Ser. No. 181,465, filed 5 cant proliferative or cytopathic effect upon the target
14 Apr. 1988, now U.S. Pat. No. 5,077,198. T-lymphocytes they infect. The resulting effects of
FIELD OF THE INVENTION
these retroviruses include T-cell proliferation leukemia,
T-cell depletion and immunosuppression in humans
The present invention relates to diluent and wash infected by the viruses. These retroviruses are known as
compositions useful in detecting antibodies, and espe O the HTLV (human T-cell leukemia-lymphoma virus)
cially viral antibodies, in biological specimens. It also and HIV (human immunodeficiency virus) families of
relates to a diagnostic kit containing same and to a rapid T4 tropic retroviruses.
and sensitive method for the detection of those antibod The first human retrovirus discovered, (HTLV-I),
ies. The invention is particularly useful for the detection appears to represent the etiological agent of mature
of retroviral antibodies. 5 T-cell leukemia and lymphomas as typified by adult
T-cell leukemia (see, for example, Poiesz et al, Proc.
BACKGROUND OF THE INVENTION Nat Acad. Sci., U.S.A., 77, 1980 and Yoshida et al, su
The immune systems of humans and animals are pra, 79, 1982). At present, the presence of HTLV-I and
amazingly complex and have mechanisms for protect T-cell malignancies are believed to occur at increased
ing the host from ill effects caused by foreign chemical 20 rates in the populations of certain Caribbean islands and
or biological substances which enter the host in some southern Japan, but HTLV-I is now widely recognized
manner. Such foreign substances include haptens, anti as a worldwide medical concern. People infected with
gens, viruses, microorganisms, drugs, hormones, plant HTLV-I or having come into contact with the virus
lectins and other substances readily apparent to one generally have antibodies directed against HTLV-I in
skilled in the art. In this application, such substances are 25 their body fluids, and especially in their blood. In addi
identified as "ligands'. When a host is invaded by a tion, a significant portion of the patients suffering from
ligand, the host normally produces proteins in response the neurological disorder known as Tropical Spastic
which will complex specifically with the ligand. Such Paraparesis possess antibodies to HTLV-I.
proteins are known as antibodies. It is often desirable to Continuing research has determined that there are
detect the presence of ligands for appropriate diagnosis 30 several additional retroviruses which are of significant
and treatment. medical importance. Besides HTLV-II, a third virus
However, sometimes, the ligand cannot be readily was discovered and identified variously as HTLV-III,
detected, for example where it is masked by other mate Lymphadenopathy Associated Virus (LAV) and HIV-I
rials in the host or is present in very low concentrations (described below).
undetectable by standard assays. Because antibodies
35 Acquired immune deficiency syndrome (AIDS) is a
react specifically with a corresponding ligand to form relatively recently recognized disease evident in several
immunological complexes, the presence of antibodies is parts of the world. It has been observed that the disease
is predominant in certain high-risk segments of the pop
often detectable when the presence of the-ligand is not. ulation of certain countries, including practicing homo
Using immunological reactions, then, it is sometimes sexual men, illegal intravenous drug users, hemophili
possible to design assays to detect the antibodies as an acs, blood transfusion recipients and those having inti
indication of the presence of the ligand. mate heterosexual relationships with these high risk
In other instances, antibodies are present in a host groups. It has been discovered by U.S. and French
organism because of autoimmune responses, that is, in researchers that the disease is spread by the transmission
an abnormal situation when antibodies are produced 45 of the retrovirus HIV-1.
against the host's normal tissues, cells or organs. Detec No cure has been found to date for AIDS, and medi
tion of the antibodies in such instances may help identify cal and scientific observations indicate that it is unlikely
pathological activities in the host. a cure will be found within the near future. Moreover,
Viruses present in humans and animals are a major it is well known that all sufferers from the disease are
health concern today, not only because of the effect 50 likely to die. These tragic consequences have spurred
they have upon the host organism, but also because of a the urgency in medical and diagnostic research associ
continuing need for improved means for detection, ated with this disease.
diagnosis, treatment and contamination prevention. As with other viral diseases, exposure to or infection
Some viruses do not harm the host organism while from HIV-I produces an immunological response, that
others merely cause minor discomfort or temporary 55 is the production of antibodies. More specifically, anti
inconveniences. Still others, however, may cause seri bodies to antigens of the virus have been detected by
ous illness and result in death. researchers and clinicians in many sero-epidemiologic
There are, then, obvious reasons to detect the prese studies. A number of human biological fluids are consid
ence of viruses in host organisms quickly for effective ered vectors for HIV-I infection. Human blood is the
treatment, appropriate health safety measures or for primary biological fluid in which the virus or antibodies
screening biological fluids, tissues or organs which may therefor are found. While people having HIV-I antibod
be used by another human or animal. ies in their blood may not necessarily also carry the
Virus particles and virally infected cells contain spe virus or themselves develop AIDS, there remains a
cific antigenic components which can induce an immu serious concern about viral transmission through
nological response to produce antibodies specific to the 65 contact with or donation of blood by such individuals.
components. In some instances, the antigenic compo Hence, there is continuing research and development to
nents can be detected in vitro using antibodies specific provide rapid and sensitive assays for detecting the
thereto. However, in other instances, it is easier or more presence of HIV-I antibodies in blood samples. Blood
 5,268,299
3 4
banks have an obvious interest in screening donated The assay of this invention is rapid. That is, it gener-
blood to insure a virus-free supply. Medical and dental ally takes less than 20 minutes to carry out. A slcilled
practitioners in hospitals and offices need diagnostics clinician can carry it out in less than 10 minutes. More-
tests to adequately protect other patients as well as over, the assay is sensitive, exhibits very low back-
themselves from viral infection. S ground (that is, very few false positives) and is rela-
Many immunological techniques are known for de- tively inexpensive compared to the complicated and
tection of antigens or antibodies, including HIV-I anti- time-consuming assays known in the art. The present
bodies. For example, U.S. Pat. Nos. 4,520,113 (issued invention is useful to a great advantage to detect anti-
May 28, 1985 to Gallo et al) and 4,708,818 (issued Nov. bodies produced in response to any biological condition
24, 1987 to Montagnier et al) describe radioimmunoas- 10 or ligand (and particularly in response to retroviral
says, Western blot techniques and ELISA (enzyme- antibodies) in situations where rapid testing is needed or
linked immunosorbent assay) for HIV-I antibody detec- where more complicated tests are unavailable or hard to
tion. A competitive ELISA is described in E.P. Publica- use. For instance, the present invention can be packaged
tion 234,941 (published Sep. 2, 1987). as a diagnostic lcit for use in medical and dental offices,
While a number of useful assays are known, they IS hospitals, trauma centers, police stations, immigration
generally require hours to perform (see for example, offices or remote areas of the world.
E.P. Publication 234,941, Example 5) and may also The advantages of the present invention are achieved
require extensive equipment and complicated protocols because of the use of certain diluent and wash composi-
in order to obtain accurate results. This adds to the tions. The diluent composition is buffered at a pH of
expense of the test as well as the likelihood of error. 20 from about 6 to about 10 and comprises a protein or
Moreover, such tedious and time-consuming tests are carbohydrate, a surfactant and a negatively-charged
not suitable for instances where a determination of the organic compound. The aqueous wash composition is
presence of a retrovirus (or any other virus) is needed buffered at a pH of from about S to about 10, and com-
quickly. For example, practitioners or clinicians in po- prises a surfactant. In one embodiment, the wash com-
lice stations, trauma centers, hospital emergency rooms, 2S position also comprises a water-miscible polar organic
immigration offices and dental and medical offices may solvent and an alkali metal or ammonium salt present in
need to know if patients are infected with a given virus an amount to provide an ionic strength of at least about
immediately. A rapid viral test would be a significant 0.25. The diluent and wash compositions can be pack-
advance for such instances. Also, rapid viral tests would aged as a diagnostic lcit.
be highly valuable for use in remote parts of the world 30
where conventional medical or testing facilities are DETAILED DESCRIPTION OF THE
nonexistant. INVENTION
Hence, there is a need in the art for a rapid and sensi­ The lcit and assay of the present invention can be used
tive assay for viral antibody detection in biological to rapidly detect the presence of antibodies in a biologi-
specimens. 35 cal specimen from human or animal hosts. Biological
samples which can be so assayed include, but are not
SUMMARY OF THE INVENTION limited to, whole blood or a component (serum or
The problems of known assays are overcome with a plasma) thereof, saliva, lacrimal fluid, spinal fluid, feces,
method for the determination of antibodies comprising urine, vaginal secretions, seminal fluid, human tissue or
the steps of: 40 organ extracts and human milk. This invention is partic­
A. mixing a biological specimen suspected of contain­ ularly useful for assaying human serum or plasma.
ing antibodies to a specific ligand with a diluent compo­ Antibodies to any chemical or biological ligand can
sition buffered to a pH of from about 6 to about 10 and be detected with this invention. This ligand can be a
comprising a protein or carbohydrate, a surfactant and foreign substance (antigen, virus, hapten, drug, hor­
a negatively-charged organic compound, 45 mone, plant lectin, toxin, microorganism and others
B. contacting the diluted specimen with the ligand to readily apparent in the art) which invades the host.
form an immunological complex of the ligand and any Alternatively, the antibodies can be produced from
antibodies present in the specimen, autoimmune responses in the host.
C. prior to, simultaneously with or subsequent to This inventiop is particularly useful to detect antibod­
complex formation, insolubilizing the ligand by attach- SO ies to human or animal viruses including, but not limited
ment to a solid substrate, to, antibodies to retroviruses (described in detail above),
D. simultaneously with or subsequent to the insolubi­ influenzas, herpes, hepatitis, cytomegalavirus, Epstein
lization of step C, separating the complex from uncom­ barr virus and rubella. In particular, antibodies to any of
plexed materials using a microporous filtration mem- HTLV-1 and -II, HIV-I and -II, individually or to-
brane which retains the complex thereon, S5 gether, can be detected, with antibodies to HIV-I being
E. prior to, simultaneously with or subsequent to the of greatest interest.
separating step, contacting the complex with enzyme­ Because a biological specimen (such as serum or
labeled antibodies directed to the antibodies to form a plasma) obtained from a patient may contain a ligand
ligand-antibody-antibody complex, (such as a virus, which would present a health hazard to
F. washing the ligand-antibody-antibody complex 60 those carrying out the assay) as well as its antibodies, it
retained by the membrane with an aqueous wash solu­ may be desirabl.e to treat the specimen in a suitable
tion to separate uncomplexed materials from the com­ manner to inactivate the ligand without affecting the
plex, and antibodies to be detected. For example, useful viral
G. adding a reagent composition capable of provid­ inactivation techniques include heat treatment for suit­
ing a detectable species in the presence of the enzyme, 65 able times at suitable temperatures, sonication or treat­
and determining the presence of the species as an indica­ ment with surfactants, alcohols, peroxides, dextran sul-
tion of the presence of antibodies to the ligand in the fate, bleach or other reagents which kill the virus. Other
specimen. techniques would be readily apparent to one skilled in
 5,268,299
5 6
the art. The virus can be inactivated before use in the been modified in some manner to provide a compound
assay, or during the early steps of the assay of this in with a low p. These compounds generally have a pI of
vention. less than or equal to about 5. A mixture of proteins or
Detection of antibodies to a specific ligand is accom carbohydrates having the desired pI can also be used.
plished by firstly mixing the specimen with the aqueous The term p (or isoelectric point) is known as the pH at
diluent composition of this invention. This composition which there is an equal number of positive and negative
includes one or more proteins or carbohydrates which charges in a molecule so that the molecule is neutral in
are in their unmodified or underivatized state, as op charge. The p can be measured using standard materi
posed to the proteins and carbohydrates described als and procedures. For example, it can be measured by
below which are modified in some manner to put addi 10 isoelectric focusing using an LKB Ampholine PAG
tional negative charges thereon. These proteins or car plate (available from LKB-Produkter AB, Bromma,
bohydrates can be used individually or in mixtures. Sweden), having a pH range 3.5-9.5, and standard cali
Representative materials include, but are not limited to, brators.
gum arabic, dextran, gelatin, and human or animal Useful modified proteins include casein derivatives or
serum or components thereof (for example, bovine 15 other protein derivatives which are negatively charged
serum albumin, fetal calf serum, rabbit serum, human (for example, derivatives obtained from acylation, alky
serum or goat serum). Gum arabic and bovine serum lation or sulfonation of casein, such as succinylated
albumin are preferred. This protein or carbohydrate is casein, succinylated bovine serum albumin, carboxy
generally present in an amount of at least about 0.5, and methyl casein, and succinylated collagen). These mate
preferably from about 0.5 to about 10 weight percent 20 rials are readily prepared by acylating, alkylating or
(based on total composition weight). sulfonating a protein having available amine groups
The specimen and diluent composition are mixed in a using suitable conditions. Useful acylating agents in
suitable vessel generally at a temperature of from about clude, but are not limited to, those described in U.S. Pat.
10' to about 40 C., and preferably at from about 15° to No. 4,591,571 (issued May 27, 1986 to Kuboyama et al),
about 25 C. The mixing time is generally a few seconds 25 such as anhydrides, acyl halides and esters derived from
but longer times can be used if desired. dicarboxylic and polycarboxylic acids. Alkylation and
The composition also includes one or more buffers sulfonation are generally described in copending U.S.
which maintain the pH of the composition at from about Ser. No. 98,432 filed Sep. 18, 1987 by Warren III et al.
6 to about 10, and preferably at from about 7.5 to about The preparation of succinylated casein is described
8.5. Useful buffers are well known in the art and in 30 below.
clude, but are not limited to, tris(hydroxymethyl Alkylating and sulfonating agents useful in modifying
)aminomethane, phosphate, glycine, morpholinopro proteins include, but are not limited to, bromoacetic
panesulfonic acid, morpholinoethanesulfonic acid and acid, chloroacetic acid, fluoronitrobenzene, m
others known in the art. (chlorosulfonyl)benzoic acid, bromomalonic acid,
Further, the diluent composition contains one or 35 bromopropionic acid and p-(chlorosulfonyl)benzoic
more water-soluble or water-dispersible surfactants. acid.
These surfactants can be nonionic, anionic or ampho Useful low p carbohydrates include water-soluble
teric. Any suitable surfactant can be used that does not cellulose derivatives, such as carboxymethyl cellulose,
adversely affect the complexation of viral antigen and carboxyethyl cellulose and others which would be
corresponding antibodies or the ability of the other readily apparent to one skilled in the art. Such materials
components in the diluent composition to provide de are generally commercially available.
sired sensitivity. Preferred low p proteins and carbohydrates include
Particularly useful surfactants include nonionic sur succinylated casein, carboxymethyl cellulose, suc
factants including, but not limited to, polyoxyethylene cinylated bovine serum albumin and succinylated colla
derivatives, for example ethoxylated fatty esters or mix 45 gen. Succinylated casein is most preferred.
tures thereof. Representative examples include poly The low p protein or carbohydrate is present in an
oxyethylene sorbitan monolaurate, polyoxyethylene amount of at least about 0.1, and preferably from about
sorbitan monopalmitate, polyoxyethylene sorbitan 1 to about 3, weight percent (based on total composition
monostearate. Representative ethoxylated fatty esters weight).
are marketed under the Tween tradename (ICI Ameri SO The diluent can also comprise one or more viral inac
cas, Inc.) and T-MAZ (trademark of Mazer Chemicals). tivation agents, such as a peroxide or dextran sulfate,
Tween-20 is a particularly useful nonionic surfactant. which will not interfere or deleteriously affect the other
The surfactant is generally present in an amount of at components of the diluent composition.
least about 0.01, and preferably from about 0.1 to about Once the specimen has been mixed with the diluent
3, weight percent. 55 composition, the diluted specimen is then contacted
The diluent composition also comprises a negatively with the appropriate ligand reactive with the antibodies
charged organic compound, or mixture thereof. The in the specimen. If antibodies are present, this contact
compound provides a highly negative environment for produces an immunological complex of ligand and anti
the assay so that non-specific interactions by the anti bodies directed to the ligand. This contact and forma
bodies and antigens are significantly reduced. This envi tion of complex takes place quickly, usually within 5
ronment can be provided by chemically modified prote minutes and preferably within 1 minute. The diluted
ins or carbohydrates, anionic surfactants, negatively specimen and immunological reagents are generally
charged buffers or other chemical or biological con mixed together for a suitable period of time and incu
pounds having the requisite negative charges to effec bated at a temperature of from about 15 to about 30
tively repel other negatively charged materials within 65 C., and more likely at 18 to 25 C.
the diluent and biological specimen. In one embodiment, the ligand and antibodies react to
Particularly useful negatively-charged compounds form a water-soluble complex which is further reacted
are water-soluble proteins or carbohydrates which have to form an insoluble complex. For example, the ligand
 5,268,299
7
could be biotinylated for attachment to avidin on an Preferred activated 2-substituted ethylsulfonyl and
insoluble substrate. Other attachment means are known vinylsulfonyl monomers can be represented by the for
in the art. The soluble complex could also be further mula (I):
reacted with other antibodies which are specific for the
ligand or anti-antibodies specific for the antibodies R O (I)
which are specific for the ligand in the specimen. CHac-L-S-R1
In a preferred embodiment, the ligand is attached to
an insoluble substrate so that the resulting complex is O
insolubilized by the complexation reaction. Suitable
substrates include, but are not limited to magnetic parti 10 wherein R is hydrogen or substituted or unsubstituted
cles, glass or polymeric particles, glass or polymeric alkyl (generally of 1 to 6 carbon atoms, such as methyl,
fibers, membranes, the sides of test tubes, test devices, ethyl, isopropyl or hexyl. Preferably, R is hydrogen or
microtiter wells and others readily apparent to one methyl.
skilled in the art. Attachment of ligand to the substrate R1 is -CH=CHR2 or -CH2CH2X wherein X is a
can be accomplished in any suitable manner, for exam 15 leaving group which is displaced by a nucleophile or is
ple by absorption or covalent attachment through reac eliminated in the form of HX by treatment with a base
tive groups, avidin-biotin linkages, protein or other (such as halo, acetoxy, alkylsulfonyloxy such as methyl
linking compounds or other techniques known in the sulfonyloxy, arylsulfonyloxy such as p-tolylsul
it. fonyloxy, trialkylammonio, for example, a trimethylam
More preferably, the ligand is part of an immunologi 20 monio salt or pyridinio salt), R2 is hydrogen, substituted
cal reagent comprising polymeric particles to which the or unsubstituted alkyl (generally of 1 to 6 carbon atoms
ligand is suitably attached. The reagent can be formed as defined for R), or substituted or unsubstituted aryl
from polymeric particles of any suitable composition. (generally of 6 to 12 nuclear carbon atoms, such as
Many suitable particles are known in the art which are phenyl, naphthyl, xylyl or tolyl). Preferably, R is
prepared from polyamides, polycarbonates, polyvinyl 25 -CH2CH2X. This group, which is an activated 2-sub
aromatics, polyesters or other polymers. The exact stituted ethyl group, can be substituted with any group
composition may be dependant upon the method of which does not impair the displacement of the leaving
attachment of the ligand and the particular ligand den group X.
sity desired. Generally, the surface density of the ligand L is a linking group which can be a substituted or
on the particles is sufficient to provide acceptable sensi 30 unsubstituted alkylene generally having 1 to 20 carbon
tivity in the assay. It will vary with the ligand being and hetero atoms in the backbone. This definition of
attached. If the method of ligand attachment is adsorp alkylene is meant to include alkylene groups interrupted
tion, the type of useful polymeric particles may be com or terminated with oxy, thio, -NR3- wherein R is
posed of certain materials whereas other materials are hydrogen, substituted or unsubstituted alkyl of 1 to 6
more useful for covalent attachment, which is pre 35 carbon atoms (such as methyl, chloromethyl or 2
ferred. Methods of attachment, such as those noted hydroxyethyl) or substituted or unsubstituted aryl of 6
above, are well known in the art. Covalent attachment to 10 carbon atoms (such as phenyl, naphthyl or xylyl)),
of ligand is usually accomplished using surface reactive ester (-COO-), amide (-CONH-), urylene
groups which are capable of reacting directly with free
amine or sulfhydryl groups of the ligand or through 40
linkages (for example avidin-biotin or proteins). Such
surface reactive groups include, but are not limited to,
carboxy, epoxy, aldehyde, active halo atoms, activated
2-substituted ethylsulfonyl and other groups known in sulfonyl (-SO2-), carbonate, sulfonamide, azo, phos
the art. The following discussion regarding preferred 45 phono or other similar groups. Representative alkylene
embodiments is for exemplification only, and is not groups include methylene, ethylene, isobutylene, hexa
meant to be limiting. methylene, carbonyloxyethyleneoxycarbonylethylene,
Particularly useful polymeric particles are those de methylenebis(ininocarbonyl)ethylene, carbonylox
scribed and claimed in copending U.S. Ser. No. 136,165, ydodecylenecarbonyloxyethylene, carbonyliminome
filed Dec. 18, 1987 by Sutton et al. Such particles are 50 thyleneiminocarbonyliminoethylene, Car
generally water-insoluble latex particles having an aver bonyliminomethyleneiminocarbonylethylene and other
age particle size greater than about 0.01 micrometers. groups described or suggested by U.S. Pat. Nos.
They are composed of polymers prepared from one or 4,161,407 and 4,548,870, noted above.
more ethylenically unsaturated polymerizable mono L can also be substituted or unsubstituted arylene
ners at least one of which has active halo atoms or 55 generally having 6 to 12 nuclear carbon atoms. Repre
activated 2-substituted ethylsulfonyl or vinylsulfonyl sentative arylene groups include phenylene, tolylene,
groups. naphthylene and others noted in the patents mentioned
Monomers having an active halogen atom include above. Also included in this definition of L are divalent
vinyl chloroacetate, vinyl bronoacetate, haloalkylated groups which are combinations of one or more of each
vinyl aromatics (for example, chloromethylstyrene or of the alkylene and arylene groups defined above (for
bronomethylstyrene), haloalkyl acrylic or methacrylic example, arylenealkylene, alkylenearylenealkylene and
esters (for example, chloroethyl methacrylate, 3-chloro others readily determined by one of ordinary skill in the
2-hydroxypropyl methacrylate and 3-chloropropyl ac art), as well as such combinations which are interrupted
rylate) and others known to one skilled in the art. The or terminated by one or more amide or ester groups (for
haloalkylated vinyl aromatics, for example those having 65 example, carbonyliminoarylenealkylene). Preferably, L
active haloalkyl groups of 1 to 3 carbon atoms, are is substituted or unsubstituted phenylenealkylene for
preferred when the active halogen atom is used as the example, substituted with one or more alkyl groups (as
reactive group. Chloromethylstyrene is most preferred. defined for R), alkoxy groups (generally of 1 to 6 car
 5,268,299
9 O
bon atoms, for example, methoxy, propoxy or butoxy) For example, a preferred manner of obtaining HIV-I
or halo groups), carbonyliminoarylenealkylene antigen is to culture the virus in a suitable cell line fol
(wherein arylene and alkylene are defined above), or lowed by removal of the virus from the cell. The re
carbonyliminoalkyleneiminocarbonylalkylene (wherein moved virus, or component thereof, is immobilized as
alkylene are defined above). described above on the polymeric particles to form a
Representative useful monomers include m & p-(2- reagent. For example, HIV-I viral antigen can be ob
chloroethylsulfonylmethyl)styrene, m & p-2-(p-tolyl tained by detergent lysis of HIV-I viral particles iso
sulfonyloxy)ethylsulfonylmethylstyrene, m & p-vinyl lated from a suitable host cell line. Such cell lines,
sulfonylmethylstyrene, N-m & p-(2-chloroethylsul which are permissive to the growth of HIV-1, include
fonylmethyl)phenyl)acrylamide, and N-(2-(2-chloroe O but are not limited to: Hut 78 (ATCC TIB 161), H9
thylsulfonyl)ethylformamidomethylacrylamide. The (ATCC No. CRL 8543), Molt 3, CEM, OKT4+, Tit.4,
first monomer is preferred. HT and clones thereof, and others as described, for
One or more of the monomers described above can be example, in U.S. Pat. Nos. 5,647,773 (issued Mar. 3,
polymerized individually or in combination to form 1987 to Gallo et al) and 4,652,599 (issued Mar. 24, 1987
homo- or copolymers. Alternatively, and preferably, 15 to Gallo et al). Viral particles can also be isolated from
one or more of them are copolymerized with at least new cell lines established from patients having AIDS or
one other ethylenically unsaturated polymerizable mon what is known as "pre-AIDS” (chronic generalized
omer. Generally such monomers provide various desir lymph-adenopathy which often precedes AIDS). In
able properties such as hydrophobicity, dispersibility or addition antigenic material can be obtained from HIV
other features. Particularly useful comonomers are de 20 infected persons. Cultivation of the cells and isolation of
scribed in copending U.S. Ser. No. 136,165 (noted viral particles are carried out using known methods.
above). The viral particles can be obtained from multiplied cells
Representative useful polymers include the follow as well as the supernatant by lysis with a detergent or
ing: poly(m & p-chloronethylstyrene), poly(styrene-co surfactant, The Hut 78 cell line, once infected with
m & p-chloromethylstyrene-co-2-hydroxyethyl acry 25 HIV-I, is preferred in obtaining HIV-I antigens.
late) (67:30:3 molar ratio), poly(styrene-co-m & p A preferred method for obtaining HTLV-I antigen is
chloroethylsulfonylmethylstyrene) (95.5:4.5 molar ra by lysing. HTLV-I viral particles isolated from a suit
tio), poly(styrene-co-N-m & p-(2-chloroethylsulfonyl able host cell line. Such cell lines include, but are not
methyl)phenyl)acrylamide) (99.3:0.7 molar ratio), po limited to Hut 102 and MT-2 and clones thereof. The
ly(m & p-chloromethylstyrene-co-methacrylic Hut 102 cell line, which is available from the American
acid)(95:5, 98:2 and 99.8:0.2 molar ratio), poly(styrene Type Culture Collection (ATCC TIB 162), is preferred.
co-m & p-chloroethylsulfonylmethylstyrene-co-metha HTLV-I can also be isolated from new cell lines estab
crylic acid)(93.5:4.5:2 molar ratio), poly(styrene-co-N- lished from peripheral blood T-cells or tissues obtained
m & p-(2-chloroethylsulfonylmethyl)phenylacryla from cutaneous T-cell leukemia/lymphoma patients.
mide-co-methacrylic acid) (97.3:07:2 molar ratio), and 35 Cultivation of the cells and isolation of antigen are car
poly(styrene-co-m & p-chloromethylstyrene)(70:30 ried out using known procedures. HTLV-II antigens
molar ratio). can be similarly obtained.
In preparing an immunological reagent, ligand (such Once the immunological complex has been formed
as viral antigen) is generally mixed with the particles and insolubilized in some manner, it is separated from
under suitable conditions depending upon the method the uncomplexed materials using a microporous filtra
of attachment (absorption or covalent). For attachment tion membrane which has pores large enough to allow
to the preferred particles described above having reac uncomplexed materials to pass through while retaining
tive halo atoms, activated 2-substituted ethylsulfonyl or the complex. Complex insolubilization can occur sub
vinylsulfonyl groups, the ligand is generally mixed with stantially simultaneously with separation, or prior
the particles for up to 24 hours at a temperature of from 45 thereto. Such membranes can be constructed of any
about 20 to about 40 C. The particle suspension is suitable water-insoluble material which will maintain its
generally buffered to a pH of from about 7 to about 10. integrity during the assay. Such materials include, but
In the mixture, particles are generally present in an are not limited to, filter papers, porous particulate struc
amount of at least about 0.2 weight percent, and ligand tures, porous polymeric films, cellulose, glass fibers,
is generally present in an amount of at least about 1% 50 woven and nonwoven fabrics, polymeric filters and
(based on total weight of particles). others known in the art. Particularly useful membranes
Viral antigen useful in the practice of preferred em are the polyamides (for example nylon) marketed by
bodiments can be prepared in any suitable manner. For Pall Corp., for example the membrane marketed under
example, it can be isolated from biological sources such the trademark LOPRODYNE. The membrane useful in
as cell cultures or infected animal or human hosts, or 55 this invention can be further treated or coated if desired
synthesized using various biotechnological techniques. (for example with surfactants, polymers or proteins) to
In preferred embodiments, retroviral antigen can be enhance separation or fluid flow or to reduce nonspeci
obtained in any suitable manner. It can be naturally fic interactions.
occurring viral substances or components thereof, or The membrane can be used as a separate substrate
synthetic peptides, or polypeptides produced using re with suitable containers for carrying out the assay of
combinant DNA technology as described, for example, this invention. Preferably, however, it is mounted as
in PCT Publications 86/01834 (University of California, part of a test device. Various test devices are known in
Berkeley) and 86/02930 (Harvard University) and U.S. the art including those described in U.S. Pat. Nos.
Pat. No. 4,725,669 (issued Feb. 16, 1988 to Essex et al). 3,825,410 (issued Jul. 23, 1974 to Bagshawe), 3,888,629
Other technical and patent references describing syn (issued Jun. 10, 1975 to Bagshawe), 3,970,429 (issued
thetic methods for producing HTLV-I, HTLV-II, Jul. 20, 1976 to Updike) and 4,446,232 (issued May 1,
HIV-I, HIV-II and other retroviral antigen are too 1984 to Liotta). Particularly useful devices are de
numerous to mention here. scribed in copending U.S. Ser. No. 98.248 (filed Sep. 18,
 5,268,299
11 12
1987 by Hinckley etal now U.S. Pat. No. 4,921,677) and gate composition is drained through the filtration mem
in copending U.S. Ser. No. 136,211 (filed Dec. 18, 1987 brane immediately.
by Smith-Lewis now U.S. Pat. No. 4,870,007). The insoluble complex thereby formed is washed to
More specifically, the test device comprises a water remove uncomplexed materials with an aqueous wash
insoluble substrate having one or more test zones composition which is buffered at a pH of from about 5
therein each of which can accommodate a sample of a to about 10 use or more suitable buffers, and comprises
biological specimen and appropriate reagents. a surfactant. Generally, only a few drops of wash con
The substrate can be prepared from any useful water position are needed to remove the uncomplexed materi
insoluble material such as glass, polymeric materials, als through the filtration membrane. Preferably, the pH
fibrous materials, cellulosic materials and other materi 10 of the wash composition is from about 6.5 to about 8.5.
als known in the art. In one embodiment, the wash composition comprises
In a preferred embodiment, the test device has three at least about 0.01 weight percent of a surfactant com
test zones or wells designed for providing a test result prising a dodecyl sulfate anion and an alkali metal or
and positive and negative control results. Each test well ammonium cation, as described, for example in copend
has a microporous membrane mounted therein. An 15 ing U.S. Ser. No. 155,670, filed Feb. 12, 1988 by
other test device is described and claimed in copending McClune et al. In another embodiment, the wash con
and commonly assigned U.S. Ser. No. 19,810 (filed Feb. position comprises at least about 1.5 weight percent of a
27, 1987 by Hinckley). Other variations of useful test surfactant comprising a lower alcohol sulfate anion
devices would be within the purview of a worker of having from 6 to 10 carbon atoms and an alkalimetal or
ordinary skill in the art. The immunological reagent ammonium cations as described for example, in copend
described above can be incorporated into the disposable ing U.S. Ser. No. 155,441,filed Feb. 12, 1988 by Warren
test device, if desired, for example as a coating on the III et al now U.S. Pat. No. 4,965,191. Particularly useful
membrane. Alternatively, the reagent can be separately wash compositions are described below.
added during the assay. In still another embodiment, a novel wash composi
Separation of the immunological complex from un 25 tion Includes a water-miscible polar organic solvent
complexed materials occurs virtually simultaneously which generally has a molecular weight less than about
with contacting the membrane with the specimen. In 200. Useful solvents include amides (for example 1
other words, it takes only a few seconds, and at most a methyl-2-pyrrolidinone, N,N-dimethylformamide and
minute or two for the specimen to drain through the N,N-dimethylacetamide), ketones (for example acetone,
membrane while the immunological complex is retained 30 methyl ethyl ketone and 3-pentanone), alcohols (for
thereon. example methanol, ethanol, t-butanol and isopropanol).
The immunological complex formed between the Tert-butanol and 1-methyl-2-pyrrolidone are preferred.
ligand and any antibodies in the specimen is contacted Useful surfactants in this wash composition are non
with an enzyme-labeled conjugate of antibodies di ionic, anionic or amphoteric surfactants. Useful non
rected to the ligand antibodies to form a complex of 35 ionic surfactants include the polyoxyethylene deriva
labeled antibody-antibody-ligand. This contact can tives described above, octylphenoxy polyethoxyethanol
occur prior to, simultaneously with or subsequent to the surfactants, fluorocarbon surfactants and others readily
separation step described above. In addition, this com apparent to one skilled in the art. Anionic surfactants
plex can be water-soluble. That is, it is formed prior to include, for example, deoxycholic acid and derivatives
insolubilizing the ligand. In preferred embodiments, 40 thereof, alkyl sulfate esters and others readily apparent
however, the ligand is insolubilized prior to forming the to one skilled in the art.
ligand-antibody-antibody complex. Also, in preferred One or more simple water-soluble salts are also pres
embodiments, separation of uncomplexed materials ent in the novel wash composition, including alkali
from the ligand-antibody complex occurs prior to form metal and ammonium salts, such as lithium chloride,
ing the ligand-antibody-antibody complex. The insolu 45 sodium chloride, potassium chloride, sodium iodide,
ble complex is collected on the filtration membrane for annonium chloride, ammonium sulfate and others
subsequent detection. known in the art. The salts are present in amount suffi
Conjugates of enzymes and anti-antibodies are pre cient to provide an ionic strength of at least about 0.25,
pared using standard starting materials and well known and preferably from about 0.25 to about 1. A representa
preparatory procedures. Some conjugates are commer SO tive wash composition is described below.
cially available. Useful enzyme labels include, but are The insoluble complex retained on the membrane is
not himited to, peroxidase, glucose oxidase, alkaline contacted with a reagent composition which comprises
phosphatase, urease and 3-galactosidase. Peroxidase (in one or more reagents for providing a detectable species
any of its forms and from any source) is preferred. The upon reaction with the enzyme label. The reagents will
antibodies can be monoclonal or polyclonal, or whole 55 then depend upon the enzyme, and for each enzyme
antibodies or parts of antibodies as long as they react there are many known reagents for this purpose. One or
with the antibodies to the ligand in the assay. For exam more reactions may be necessary to form the detectable
ple, they can be goat or mouse anti-human antibodies. species.
The conjugate can be used in admixture with other In a preferred embodiment where the enzyme is per
materials including buffers, surfactants, proteins and oxidase, the reagent composition comprises suitable
peroxidase rate enhancers. A particular conjugate com reagents for forming a detectable species (chromogen
position is described in detail below. or fluorogen) in the presence of hydrogen peroxide and
Forming the ligand-antibody-antibody complex gen peroxidase. This reagent composition includes appro
erally occurs upon incubating the materials for up to 5 priate reagents, one of which acts as a substrate for
minutes at a temperature between about 15' C. to about 65 peroxidase, which are capable of providing a detectable
30' C. Preferably, the incubation is carried out at from dye in the presence of peroxidase and hydrogen perox
about 15 C. to about 25 C. for about one minute. When ide. The substrate itself can be a dye-forming com
the complex is insolubilized, any fluid from the conju pound, such as benzidine, tetramethylbenzidine or other
 5,268,299
13 14
benzidine derivatives, 2,2'-azino-di-(3-ethyl-benzthiazo molar NaCl, 0.001 molar ethylenedianinetetraacetic
lone-6-sulfonic acid), phenol red, o-phenylenediamine, acid) layered over a 1300 ml linear 22-65% sucrose
pyrogallol, 4-aminoantipyrine, bromopyrogallol red gradient in the buffer with a conventional zonal rotor
and others known in the art. Alternatively, a hydrogen and ultracentrifuged overnight at about 30,000 x g. The
donor and an electron acceptor can be combined to gradient was fractionated into about 110 fractions (12
provide a detectable species (for example, see the com ml each) and all fractions with densities between 1.14
pounds described in U.S. Pat. No. 4,260,679). and 1.18 g/ml were pooled, diluted 3- to 4-fold with the
Preferably, the reagent composition includes a leuco buffer and centrifuged at about 50,000x g for two hours
dye which provides a dye in the presence of hydrogen to recover the purified HIV-I viral particles. The parti
peroxide and peroxidase for example, a triarylimida O
zole leuco dye as described in U.S. Pat. Nos. 4,089,747 cles were then suspended in 10 ml of a solution contain
(issued May 16, 1978 to Bruschi) or a triarylmethane ing 0.6 molar KCl and 0.5% of a nonionic octylphenoxy
leuco dye as described in 4,670,385 (issued Jun. 2, 1987 polyethoxyethanol surfactant, sonicated with three 5
to Babb et al). A preferred dye-providing composition second bursts, incubated for one hour at 37 C. and
is described and claimed in copending U.S. Ser. No. 15 centrifuged at 80,000Xg for one hour to remove debris.
136,166, filed Dec. 18, 1987 by McClune. The solubilized HIV-I preparation was then extracted
Once a dye has formed in the presence of the insolu twice with an equal volume of anhydrous ether and the
ble complex, it can be evaluated visually or using spec resulting aqueous phase was used as the the source of
trophotometric equipment to determine if the assay HIV-I antigen in the following example.
indicates the presence of ligand antibodies in the speci 20 Diluent Compositions:
men. Both positive and negative control tests may be Two diluent compositions of this invention were
desirably carried out with the specimen test. Appropri prepared:
ate reagents would be used for each control test to give (1) succinylated casein (1%), gum arabic (1%),
the desired result.
The kit of this invention can include the diluent and 25 Tween 20 nonionic surfactant (0.05%) and Thimerosal
wash compositions described herein which are pack (0.01%) in 0.1 molar Tris buffer (pH 8), and
aged in a suitable manner and included in a carrier of (2) succinylated casein (1%), bovine serum albumin
some type which can be compartmentalized to receive (4%), Tween 20 nonionic surfactant (0.05%) in 0.1
one or more containers holding the compositions. In molar Tris buffer (pH 8).
addition, it can also include one or more of the follow 30 Wash Compositions:
ing which are useful in carrying out the method: dispos Two wash compositions of this invention were also
able test device containing a filtration membrane, rea prepared:
gent composition, enzyme-labeled antibody composi (i) 1-methyl-2-pyrrolidinone (10%), Tween 20 non
tion, and an immunological reagent. Reagents can be ionic surfactant (0.25%), Nonidet P-40 nonionic surfac
provided in dry form or in appropriate solutions. Non 35 tant (0.1%), sodium chloride (0.5 molar) in 0.05 molar
reactive components of the kit can include instructions, sodium phosphate buffer (pH 7.4), and
mixing vessels, stirring means, pipettes and the like. (2) sodiurn decyl sulfate (2.4%) in 0.1 molar sodium
The following compositions were used in carrying phosphate buffer (pH 7.2).
out the assay described in the illustrated example below. Two peroxidase-antibody compositions were pre
All percentages are by total weight of the composition. pared:
Immunological reagent:
This reagent comprised particles of polystyrene-co (l) horseradish peroxidase conjugated to rabbit anti
m & p-(2-chloroethylsulfonylmethyl)styrene) (95.5:4.5 human antibodies, diluted 1:3000, 4% bovine serum
molar ratio) onto which were covalently attached albumin and Thimerosal preservative (0.01%) in 0.5
HIV-I antigen. 45 molar phosphate buffered saline, and
HIV-I antigen was prepared by culturing HIV-I in (2) the same conjugate with succinylated casein (1%), .
the Hut 78 cell line in Reswell Park Memorial Institute 4'-hydroxyacetanilide (0.15%), bovine serum albumin
1640 medium in the presence of 10% bovine serum (1%) in 0.1 molar Tris buffer (pH 8).
albumin and gentamycin (50 g/ml). The cell density Dye-Providing Reagent Composition:
was maintained at approximately 105-106 cells/ml, and 50 A composition for providing a dye in the presence of
the cells were subcultured by the addition of fresh me hydrogen peroxide and peroxidase was prepared as
dium to maintain this density. The viral particles were follows: a leuco dye solution was prepared by dis
isolated in a closed-system stainless steel filtration/con solving 2-(4-hydroxy-3,5-dimethoxyphenyl)-4,5-bis(4-
centration apparatus by pooling the cultures to be har methoxyphenyl)imidazole (to make a 0.1% solution) in
vested in a holding tank which permits the cell culture 55 a solution of 20% poly(vinyl pyrrollidone) in sodium
fluid to be pumped through a filter housing fitted with phosphate buffer (5 mmolar). This solution was then
a 0.45um filter. This first filtration step removed whole added to a solution containing hydrogen peroxide (10
cells and cell debris. The cell-free supernatant was then mmolar), 4'-hydroxyacetanilide electron transfer agent
pumped through a second filter housing fitted with a 0.2 (5 mmdlar) and diethylenetriaminepentaacetic acid che
um filter in order to eliminate any residual cell debris lating agent (10 umolar) in sodium phosphate buffer to
not removed by the 0.45 um filter. The second superna produce a final concentration of 1% poly(vinyl pyrrol
tant was pumped through a concentration cassette fitted
with a 100,000 dalton cutoff membrane to concentrate idone) and 0.005% leuco dye.
the preparation to a suitable volume. The crude viral Preparation of Succinylated Casein:
particles thus obtained were pelletized by centrifuga 65 Succinylated casein was prepared by reacting casein
tion for two hours at about 50,000 x g and resuspended with an equal weight of succinic anhydride for four
in about 40 ml of a buffer solution pH 7.8, 0.01 molar hours at 25 C. in 0.5 molar phosphate buffer (pH 8.5),
tris(hydroxymethyl)aminomethane hydrochloride, 0.01 then purifying the product by dialysis.
 5,268,299
15 16
EXAMPLE 1. The diluted specimen (100 l) was then added to each
of the test wells and incubated at room temperature for
Assay of Blood Serum Specimen for HIV-I Antibodies about three minutes to allow formation of an insoluble
A human serum specimen was assayed for the pres antibody-antigen immunological complex, followed by
ence of antibodies to HIV-I in the following manner fluid drainage.
and using a disposable test device like that described in The peroxidase-labeled-anti-antibody composition
U.S. Ser. No. 98,248 (noted above). (50 ul) was added and an insoluble labeled-antibody
This test device had three test wells, each with a antibody-antigen complex was allowed to form without
LOPRODYNE filtration membrane commercially
O
fluid drainage during a one minute incubation period at
available from Pall Corp. The membrane had been room temperature. Upon draining the liquid, the result
coated with FC 134 surfactant (0.05 g/m2, available ing complex was washed twice with the decyl sulfate
from DuPont). A positive control test well contained: a wash composition described above (first with 240 ul,
sample (0.23 mg) of the immunological reagent de then with 60 ul) to separate uncomplexed materials
scribed above and heat treated serum containing inacti from the complex.
vated HIV-I. A negative control test well contained the 15 A sample (40 ul) of the dye-providing composition
polymeric particles as used in the test well, but the described above was added. After one minute incuba
particles were coated with casein only. The test well tion at room temperature, a red dye was observed in the
designed for receiving the test specimen contained only wells and visually graded by comparison to a color
the immunological reagent described above. aradient chart (values 0-10, with 10 representing the
A human serum specimen (50 ul) was mixed with a densest color).
sample (5 ml) of the first diluent composition described Test and Control compositions are shown in Table I
above for a 100:1 dilution. The diluted specimen (100 and the results are shown in Table II.
ul) was then added to each of the test wells and incu
bated at room temperature for about two minutes to TABLE I
allow formation of an insoluble immunological com 25
Test A: diluent composition 1, wash composition 1 and
plex, followed by fluid drainage. peroxidase-antibody composition 1.
The peroxidase labeled-antiantibody composition (40
ul) was added and the insoluble labeled antibody-anti Test B: diluent composition 2, wash composition 1 and
body-antigen complex was allowed to form without peroxidase-antibody composition 1.
fluid drainage during a one minute incubation period at 30 Test C: diluent composition 2, wash composition 2 and
room temperature. Upon draining the liquid, this result peroxidase-antibody composition 2.
ing complex was washed twice with the first wash com Control A: wash composition 1, peroxidase-antibody
position described above (first with 240 ul, then with 60 composition , and diluent composition 1 without
ul) to separate uncomplexed materials from the com gum arabic, succinylated casein and surfactant.
plex. 35 Control B: wash composition 1, peroxidase-antibody
A sample (40 ul) of the dye-providing composition composition 1, and diluent composition 2 without
described above was added. After two minutes incuba succinylated casein and surfactant.
tion at room temperature, a red dye was observed in the Control C: wash composition 2, peroxidase-antibody
specimen test well which indicated the presence of composition 2 and diluent composition 2 without
HIV-1 antibodies in the serum specimen. The negative 40 succinylated casein and surfactant.
control well showed little background, and the positive Control D: peroxidase-antibody composition 1, diluent
control well showed a positive test as well. composition 1, and wash composition 1 without sur
EXAMPLE 2 factant and 1-methyl-2-pyrrolidinone.
Control E: peroxidase-antibody composition 2, diluent
Assay for HIV-I Antibodies Using Different Diluent composition 2, and wash composition 2 without sur
and Wash Compositions factant.
A human serum specimen was assayed for the pres TABLE I
ence of antibodies to HIV-I using test assays which Negative Sample Positive
were similar to that of Example 1 except for different Control Well Wel Control Wel
diluent and wash compositions, and a number of other 50 Test A: 2 3.3
minor changes. Disposable test devices like that de Test B: 2.7 6
scribed in U.S. Ser. No. 98,248 (noted above) were used. Test C: 2.3 5 6
In addition, a number of comparative Control assays Control A: 4. 5 6.3
were performed. These assays are outside the scope of Control
Control
B;
C:
4.
3.3
4.
4.
7.7
7.3
this invention. 55
Control D: 6. 5.7 6.
Each test device had three test wells, each with a Contro E: 7.7 7.7 7.7
nylon filtration membrane commercially available from
Pall Corporation. A positive control test well con
tained: a sample (0.15 mg) of the immunological reagent The tests showed low background (negative control
described above and heat treated serum containing inac 60 well) and adequate sensitivity (difference of at least one
tivated HIV-I. A negative control test well contained between negative control well and sample well), while
the polymeric particles as used in the test well, but the the Controls showed high background and poor sensi
particles were coated with casein only. The test well tivity.
designed for receiving the test specimen contained only The invention has been described in detail with par
the immunological reagent described above. 65 ticular reference to preferred embodiments thereof, but
A human serum specimen was mixed with a sample of it will be understood that variations and modifications
the second diluent composition (containing BSA in can be effected within the spirit and scope of the inven
stead of gum arabic) described above for a 80:l dilution. tion.
 5,268,299
17 18
We claim: 6. An aqueous composition buffered to a pH of from
1. An aqueous composition buffered to a pH of from about 6 to about 10 and consisting essentially of an
about 6 to about 10 and comprising an unmodified pro unmodified protein or carbohydrate, a nonionic poly
tein or carbohydrate, a nonionic polyoxyethylene sur oxyethylene surfactant and a chemically modified,
factant and a chemically modified, water-soluble pro water-soluble protein having a pi less than or equal to
tein having a pI less than or equal to about 5. about 5.
2. The composition of claim 1 wherein said chemi 7. The composition of claim 6 further including a
cally modified protein is succinylated casein, carbox viral inactivation agent.
ymethylated casein, succinylated bovine serum albumin 8. An aqueous composition buffered to a pH of from
or succinylated collagen. O about 6 to about 10 and consisting of an unmodified
protein or carbohydrate, a nonionic polyoxyethylene
3. The composition of claim 2 wherein said chemi surfactant and a chemically modified, water-soluble
cally modified protein is succinylated casein. protein or carbohydrate having ap less than or equal to
4. The composition of claim 1 wherein said chemi about 5.
cally modified protein or carbohydrate is present in an 15 9. The composition of claim 8 wherein said chemi
amount of at least about 0.1 weight percent. cally modified protein or carbohydrate is succinylated
5. The composition of claim 1 wherein said unmodi casein, carboxymethyl cellulose, carboxymethylated
fied protein or carbohydrate is gum arabic, gelatin, casein, succinylated bovine serum albumin or suc
dextran, human or animal serum or a component cinylated collagen.
x : x :
thereof. 20

25

30

35

45

50

55

65