Method and apparatus for immunoassays

Disclosed herein is an apparatus and process for conducting immunoassays. The apparatus comprises a first member which is a membrane or a filter to which is bound an antibody, typically a monoclonal antibody, or which is capable of extracting cells from a fluid sample. The apparatus further comprises a second member which is composed of absorbent material which acts when in contact with the first member to induce flow through the first member when a fluid sample is added to it. The apparatus is used to conduct immunoassays by applying a sample to the upper surface of the first member to bind antigen in the sample by means of antibody fixed to the first member or, in certain cases, by extracting cellular material which has antigen associated with it. Addition of the sample is followed by addition of labeled antibody against the antigen being assayed followed by a washing step to remove unbound labeled antibody. The presence of labeled antibody on the first member after washing is indicative of the presence of the antigen in the sample being assayed.

FIELD OF THE INVENTION 
This invention relates to immunoassay processes, particularly those using 
monoclonal antibodies. In another aspect it relates to an apparatus for 
conducting such immunoassays. 
BACKGROUND 
For nearly two decades, immunoassay procedures have provided sensitive 
diagnostic tools for the in vitro detection of a variety of antigens 
associated with disease or other physical conditions of clinical 
significance. Originally such heterogeneous assays used a polyclonal 
antibody preparation bound to the solid phase. In these assays, a solution 
of labeled antigen is allowed to compete with antigen in the sample being 
analyzed for the solid phase antibody. The extent to which the labeled 
antigen is bound to the solid phase or is detected in the liquid phase can 
be used as a measure of the presence and quantity of antigen in the sample 
being analyzed. 
Subsequently, non-competitive immunometric assays became available. In 
these assays, a polyclonal antibody preparation bound to a solid phase was 
also used. The sample containing the suspected antigen was allowed to 
contact the solid phase in order for the antigen to bind to the antibodies 
on the solid phase. Typically, after an incubation step the sample was 
separated from the solid phase which was then washed and incubated with a 
solution of additional polyclonal antibodies which had been labeled, for 
example with a radionuclide, an enzyme, or a fluorescent moiety. 
After this second incubation, the unbound labeled antibody was separated 
from the solid phase and the amount of labeled antibody in either the 
liquid phase or bound to the solid phase in an antibody:antigen:antibody 
sandwich was determined as a measure of the presence and/or concentration 
of antigen in the sample tested. 
More recently, immunoassay procedures have been modified to use monoclonal 
antibodies. For example, U.S. Pat. No. 4,376,110 describes two-site 
immunometric assays using pairs of monoclonal antibodies, one bound to a 
solid phase and the other labeled to permit detection. The use of 
monoclonal antibody pairs which recognize different epitopic sites on an 
antigen has made it possible to conduct simultaneous immunometric assays 
in which the antigen and labeled antibody incubations do not require the 
intermediate washing steps of prior processes. 
In the foregoing processes, the solid phase antibody is typically bound to 
a bead or small particles or coated on a surface. All of these processes 
characteristically require an incubation period with both the solid phase 
and labeled antibodies and, as a result, are time consuming even if 
conducted simultaneously. In fact, it is not unusual for an assay 
procedure to require several hours to complete. Furthermore, the need to 
adhere to timed incubation steps and plural washings with measured 
reagents has largely limited these procedures to large hospital and 
reference clinical laboratories where highly trained personnel and 
sophisticated equipment are available to perform the assays. As a result, 
there has gone unmet a need for a simple and rapid procedure for 
conducting immunoassays which employ a relatively simple apparatus to make 
such assays available for use in the physician's office and even for 
over-the-counter sale to laypersons for use in home health care programs. 
SUMMARY OF THE INVENTION 
The present invention provides a process for simply and rapidly performing 
immunoassays which uses a simple apparatus and which does not require 
lengthy incubation steps. The apparatus of the invention comprises, as a 
first member, a porous member such as a membrane or filter to which is 
bound antibody, preferably a monoclonal antibody against the target 
antigen, or which is capable of separating from the sample being analyzed 
cells or cellular debris with which the antigen being assayed is 
associated to thereby fix the antigen to the porous membrane. The 
apparatus further comprises, as a second member, an absorbent member 
having capillary pathways therethrough generally transverse to its upper 
and lower surfaces. As used herein, the term "capillary" includes a 
capillary or other channel or pathway which permits a liquid to traverse 
the absorbent member. The second member is in capillary communication with 
the porous first member and is selected to have a capillary pore size so 
as to induce flow of liquid through the first member without the use of 
external means when the hydrostatic pressure of the sample and subsequent 
addends used in the assay are not sufficient to induce flow through the 
first member. The second member may also provide support for the first 
member. 
The assay of the present invention comprises the steps of adding a liquid 
sample to the porous member whereby, as the liquid flows through the 
member, antibody bound to the member binds antigen in the sample at a rate 
that is substantially faster than the rate observed in the absence of flow 
through the member. If the antigen is on the surface of cellular material, 
the cellular material is either bound by antibody bound to the member or 
is entrapped by the member as the sample flows through. The addition of 
sample is followed by addition of an antibody solution, preferably a 
solution of a monoclonal antibody, in which the antibody is labeled to 
permit detection. The preferred label is an enzyme although other labels, 
for example, a radionuclide or a fluorescent label may also be used. The 
antibody binds to the antigen previously extracted from the sample, either 
by the bound antibody or by entrapment of cellular material. The addition 
of labeled antibody may be followed immediately, or after a brief 
incubation to increase sensitivity by permitting greater binding of 
antigen and labeled antibody, by a washing step to remove unbound labeled 
antibody. The presence of labeled antibody on the porous member is then 
determined as an indication of the presence of the target antigen in the 
sample. In the case of an enzyme label this is done by addition of a 
solution of a color forming substrate to the member which reacts with the 
enzyme as the solution passes through it.

DESCRIPTION OF PREFERRED EMBODIMENTS 
As noted above, the apparatus of the present invention comprises, as a 
first member, a porous membrane or filter to which is bound antibody or 
which is capable of filtering cellular material from a sample being 
assayed if the antigen is associated with the cellular material. In the 
latter case, the membrane or filter is selected to have a pore size which 
permits this separation. Any of a variety of filtering members may be used 
including glass fiber filters and filters of various synthetic or natural 
materials. 
In a preferred embodiment the first member is a membrane or filter to which 
an antibody preparation is covalently bound. Preferably the antibody 
preparation comprises a monoclonal antibody even though polyclonal 
antibodies from antisera may be used. Techniques for polyclonal and 
monoclonal antibody preparation are now well known and require no citation 
here. The material of the member is selected from a material to which the 
antibody can be bound. A preferred material is nylon which has amino group 
residues or into which such groups have been introduced by chemical means, 
which permit antibodies to be coupled to it by the well known 
glutaraldehyde method. Antibodies can be coupled to glass fibers through 
aminosilanes. Other natural or synthetic materials which can be coupled 
directly or through intermediates to an antibody may also be used. 
The second member is an absorbent member having capillary passageways 
generally transverse to the upper and lower surfaces. The second member is 
assembled with the first in a manner which permits direct communication 
between the pores or interstices of the first member and the capillaries 
of the second. Thus, as a liquid is applied to the first member and 
saturates it, the liquid is drawn into the absorbent member. As a result, 
flow can be induced through the first member when a liquid sample is 
applied to the upper surface of the first member even though the 
hydrostatic pressure of the fluid is so low that unaided it could not flow 
through the first member without the application of pressure to force it 
through or a vacuum to draw it through. 
The selection of material for the second member is not critical and a 
variety of fibrous filter materials can be used. A useful material is 
cellulose acetate fibers oriented as in a cigarette filter. Those skilled 
in the art will appreciate that other absorbent members made of polyester, 
polyolefin or other materials may be used in place of cellulose acetate. 
Turning now to FIG. 1, there is shown in cross-section a device which can 
be used with the apparatus of this invention to perform immunoassays. 
Thus, in FIG. 1, a cylindrical container 10 although it may have any other 
appropriate shape is provided having an upper opening 12 defined by 
sidewall 14. The container may be made of glass or a suitable plastic 
material. As shown in FIG. 1, container 10 also has a lower opening 16, in 
which is inserted a removable plug 18, to permit insertion of the porous 
member 20, a circular membrane or filter disc, and an optional member 21, 
whose function is described below, which rest on cylindrical absorbent 
member 22, which is also inserted through opening 16. 
A portion of container 10 is constricted as shown in FIG. 1 by reference 
numeral 24 to provide an integral funnel to direct sample onto the member 
20 and to assure that effective washing of sample and other addends onto 
the member 20 is accomplished. 
The size of member 22 and, therefore, the volume of the portion of 
container 10 below the constriction is preferably selected so that all of 
the liquid to be added to the apparatus during an assay can be received in 
and retained in absorbent member 22. Means for venting air (not shown in 
FIG. 1), for example, small ports, is provided in container 10, near the 
bottom, to allow displaced air to escape. Optionally, the bottom of 
container 10 can be eliminated and liquid allowed to pass through members 
20 and 22 and exit the container through the bottom. However, since the 
article is intended to be disposable and to facilitate the disposal of 
sample in a simple and hygienic fashion, it is preferred to use a 
structure shown in FIG. 1. 
As previously noted, member 20 may be used to either filter cellular 
material from a sample or as a support for bound antibody against the 
antigen being assayed. In either case, the liquid sample is applied to the 
member 20 by introduction through opening 12. After it permeates the 
member 20 and the liquid is drawn therethrough by and into absorbent 
member 22, a solution of labeled antibody, preferably a monoclonal 
antibody, is added through opening 12 to member 20. 
The labeled antibody then binds either to antigen bound to antibody on the 
member 20 or associated with cellular material trapped on the surface of 
20. If member 20 has a monoclonal antibody bound to it, and the labeled 
antibody is also a monoclonal antibody, the two antibodies are selected to 
bind to non-interfering antigen binding sites as described in U.S. Pat. 
No. 4,376,110 and application Ser. No. 323,498 filed June 6, 1981, the 
disclosures of which are incorporated by reference. 
Preferably the soluble antibody is labeled with an enzyme although other 
conventional immunoassay labels may be used in appropriate circumstances. 
For example, a fluorescent label or a radionuclide can be used. 
After the labeled antibody solution has passed through the member 20, a 
washing liquid is applied to member 20 to flush unbound labeled antibody 
from member 20 and into member 22. The sloping structure of the walls 24 
provides an integral funnel to facilitate application of the washing 
liquid to the walls to remove adhered residue of the labeled antibody 
solution. 
The addition of labeled antibody solution and washing liquid to the member 
20 may be preceded by brief incubation periods to permit more extensive 
binding by antibody or antigen in solutions trapped on or in the 
interstices of member 20 and, thereby, increase the sensitivity of the 
assay. We have found, however, that such incubation steps are either 
unnecessary or may be very brief, i.e., on the order of 60 seconds or 
less. The flow of solutions containing antigen or labeled antibody through 
the member 20 results in a substantially faster rate of binding than is 
observed in the absence of flow. 
If the antibody label is an enzyme, after washing to remove unbound 
antibody from member 20, a solution of the enzyme substrate is added to 
member 20. If the target antigen is bound either to antibody bound to 
member 20 or to cellular material on member 20, the antigen will have 
bound to it a portion of labeled antibody. The enzyme will cause the 
substrate to react and generate, if properly selected, a visual color 
change. 
We have found that the use of cellulose acetate material for the absorbent 
member 22 may bind labeled antibody non-specifically at its upper surface. 
Accordingly, some visual color change may occur at this surface just under 
the member 20. To avoid this color change being visualized through member 
20, a separating member (designated 21 in FIG. 1) of porous polyethylene 
or other material which does not bind antibody non-specifically is 
preferably disposed between members 20 and 22. 
Turning now to FIG. 2, there is shown a top view of member 20. The phantom 
line 26 represents the outer circumference of the area 28 in which 
antibody, preferably a monoclonal antibody, is bound in a preferred 
embodiment. This area has a diameter less than the diameter of the 
restriction formed by walls 24 at its narrowest point. Thus, when an 
enzyme is used as the antibody label the following results may occur: (1) 
the development of more color in the area 28 than in the periphery of 
member 20 will be read as a positive result; (2) if no color development 
is observed in member 20, a negative result is read; (3) if after washing 
some labeled antibody remains in the member 20, a modest color change 
which is uniform over the entire visible surface may occur. Such result is 
also interpreted as negative. 
The foregoing is a general description of the apparatus and process of the 
invention. We have found it useful in performing immunoassays from 
introduction of sample to reading a positive result in less than five 
minutes. Thus, in a specific example, a monoclonal antibody against human 
choriogonadotropin (HCG), an antigen which is elevated in the urine of 
pregnant women, is bound to a porous nylon membrane using the 
glutaraldehyde technique and placed in a container such as 10 in FIG. 1 
and supported there by an absorbent member of cellulose acetate but 
separated therefrom by a disc of porous polyethylene. 
Samples of urine (4 ml containing 0 and 50 mIU/ml of HCG) were added to the 
apparatus described and drawn through members 20 and 21 into the absorbent 
material 22. Three (3) drops of a solution of second monoclonal antibody 
against HCG to which is bound alkaline phosphatase were then added. After 
a brief incubation, about 1 minute, during which time the conjugate is 
drawn through member 20, 4 ml of water was added to remove unbound 
antibody from member 20. This addition was followed by three drops of a 
solution containing indoxyl phosphate, a substrate for alkaline 
phosphatase. After two minutes no color developed in the device used to 
test the sample containing no HCG (0 mIU/ml). For the 50 mIU/ml HCG sample 
a distinct blue color developed in the center of the disc within thirty 
seconds which became dark blue within two minutes. No color developed in 
the periphery of the disc. The entire assay consumed about five (5) 
minutes. It will be appreciated that the sensitivity of the assay may be 
adjusted by varying the volume or incubation times. 
Although the invention has been described using an assay for HCG as an 
example, it will be appreciated that a similar assay for other antigens 
may be constructed. The entire list of target antigens is too lengthy to 
be listed, but antigens such as IgE, prostatic acid phosphatase, prostate 
specific antigen, alphafetoprotein, carcinoembryonic antigen, leutenizing 
hormone, creatine kinase MB and other antigens in serum, plasma, urine, or 
other liquid media may be detected. Additionally, liquid samples 
containing material having antigens associated therewith such as antigens 
associated with bacteria, parasites, fungi, or viruses including, for 
example, group A and B streptococcus, Neisseria gonorrhea, Gardnerella 
vaginalis, Trichomonas vaginalis, Candida albicans, Chlamydia trachomatis, 
hepatitis B, and cytomegalovirus can be detected by using a filter which 
will trap the cells or a filter to which antibody specific for the antigen 
is bound as member 20. Addition of a solution of a monoclonal antibody 
labeled, for example, with an enzyme, will result in binding of the 
antibody to the antigen. Washing and substrate addition will result in the 
color change associated with presence of the labeled antibody on the 
cells, which can be detected visually or by aid of an instrument. 
If a label other than an enzyme is used, the procedure may be varied. 
Fluorescence of the membrane could be measured if a fluorescing label is 
used. If a radionuclide label such as .sup.125 I is used, the membrane can 
be removed and counted. These and other variations may be made by persons 
skilled in the art without departure from the spirit of the invention.