A novel separation technique is described that is particularly useful for effecting separations in enzyme immunoassay procedures. A mixture, in an aqueous liquid vehicle, of (1), ligand-enzyme conjugate, and of (2), the conjugate bound through its ligand moiety to a receptor, is brought into contact with an insoluble, immobilized pseudo-substrate material, to which the enzyme normally binds. Free conjugate binds and becomes insoluble. Bound conjugate remains in the liquid phase. The ligand may be an antigen and the receptor, the antibody to the antigen. This separation technique makes feasible several sensitive immunoassay procedures. The material to be assayed may be, for example, rubella virus; hepatitis B surface antigen; gonorrhea antigen; the antibody to any of the foregoing; a general antibody, i.e., an immunoglobulin; a hormone such as choriomammotropin; a steroid, hapten, or the like.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates specifically to a new assay procedure for the 
detection and measurement of certain biologically active substances, 
particularly immunochemical substances. This new assay procedure permits 
rapid qualitative and quantitative determinations to be made in an 
advantageous manner. The invention also relates to a novel separation 
technique that is of general utility, and more particularly, to kits 
useful for assay procedures. 
2. Statement of the Prior Art and Other Information 
There is a continuing need for rapid, accurate qualitative and quantitative 
determinations of many kinds of biologically active substances at 
extremely low concentrations, i.e., at physiological concentrations. 
Today, there is for example a need for determining the presence of drugs or 
narcotics in body fluids, such as saliva, blood or urine. In addition, in 
medical diagnosis, it is frequently important to be able to detect and 
quantify the presence of various substances which are synthesized 
naturally by the body or ingested. These include hormones, both steroidal 
and polypeptides, prostaglandins, and toxins, as well as other materials 
which may be involved in body functions. Frequently, there is concern with 
extremely small amounts and occasionally, with very small differences in 
concentrations. 
Beside these materials, assays effective at extremely low concentrations 
would be desirable for a variety of pesticides, such as insecticides, 
bactericides, fungicides, etc., as well as organic pollutants of other 
kinds, both in the air and water. 
Various methods have been developed in the last two or three decades for 
the determination of a variety of immunochemical substances, including 
antigens, antibodies, haptens, and certain low molecular weight 
substances. Excellent surveys of the field are reported by: Skelley et 
al., Radioimmunoassay, Clinical Chemistry 19: 146-186 (1973); Wisdom, 
Enzyme-Immunoassay, Clinical Chemistry 22: 1243-1255 (1976); and Schuurs 
et al., Clin. Chem. Acta 81: 1-40 (1977). There is also a good description 
of assay techniques in U.S. Pat. No. 4,213,764, issued July 22, 1980. 
Examples of some of the assay methods are: 
1. Radioassay techniques 
a. Competitive protein binding assays 
b. Radioimmunoassay (RIA) 
c. Immunoradiometric assays 
d. Sandwich or 2-site immunoradiometric assays 
2. Fluoroimmunoassays (FIA) 
3. Enzyme immunoassay (EIA) 
4. Latex-particle agglutination (LPA) 
5. Charcoal-particle agglutination (CPA) 
6. Hemagglutination and Hemagglutination Inhibition Assays (HA), (HIA) 
7. Radial Immunodiffusion and Double diffusion (RID) 
8. Viroimmunoassay (VIA); and 
9. Spin immunoassay (SIA), among others. 
Many of the immunochemical assay systems involve the use of labels. There 
are many types of labels that are useful for the detection and measurement 
of biologically important or interesting compounds or substances in serum 
or other media. 
The administration of most of these tests is hampered by one or more of the 
following limitations: (1) lack of sensitivity, (2) complexity of the test 
procedure, (3) instability of reagents, (4) hazardous nature of one or 
more reagents, (5) impure reagents, and (6) expensive equipment required 
to perform quantitative and qualitative analysis of the amount of label 
involved, as in an immunochemical reaction. For a review of the 
development and evaluation of immunological methods and their uses as 
diagnostic tools, reference is made to "Immunology as a Laboratory Tool" 
by Franz Peetoome American Journal of Technology 37: 445-469 (1971). 
In addition to the general limitations mentioned above, it should be 
pointed out that the limitation as to "lack of sensitivity" is a very 
broad term. Some assay procedures have an acceptable sensitivity within 
one molecular weight range, but unacceptable sensitivity outside that 
range. Generally the previously available EIA assay techniques could be 
selected to provide acceptable sensitivity below 1,500 daltons 
(homo-geneous EIA) and above 60,000 daltons (heterogeneous EIA), but have 
not provided fully acceptable sensitivity for antigens, for example, 
having molecular weights between these two figures. 
Both labeled and unlabeled immunochemical assay techniques may employ 
various devices to separate (1), immunochemical constituents which have 
reacted, from (2), nonreacted immunochemical constituents, and from (3), 
substances irrelevant to the test. An excellent survey of separation 
techniques, Separation Techniques in Saturation Analysis, by J. G. 
Ratcliff, appears at Br. Med. Bull. 30: 32-37 (1974). 
For example, some patented EIA techniques require separation through the 
use of one component in the antigen-antibody reaction in an 
"insolubilized" phase for separation; see Schuurs and coworkers, in U.S. 
Pat. Nos. 3,654,090; 3,791,932; 3,850,752; 3,839,153; 3,879,262; 4,016,043 
and Reissue 29,169; see also Ratcliff, supra, at pp. 35-36. The separation 
of bound and unbound antigen is a critical step in some radioimmunoassay 
(RIA) techniques as well as in some enzyme immunoassay (EIA) techniques. 
Marsden, Lab. Management, March: 31-34 (1977). Ratcliffe, supra. Odell et 
al., Proceedings of the Fifth Tenovus Workshop, Wales, U.K. pp. 207-222 
(1975). Collins et al., Proceedings of the Fifth Tenovus Workshop, Wales, 
U.K. p. 223-225 (1975). 
The most widely used separation method in RIA for small antigens is an 
adsorption technique. This system precipitates antibody unbound tracer 
using adsorbent materials, such as dextran coated charcoal, talc, or 
resins, and has advantages in its simplicity and reproducibility. 
EIA techniques so far developed, however, rely mainly on the double 
antibody precipitation method. The double antibody method involves 
precipitation of antibody-bound enzyme-antigen conjugate using a second 
antibody produced against the immunoglobulin of the first animal. It is a 
most reliable and reproducible method. The double antibody method is 
described by Exley et al. in FEBS Letters 79 301-304 (1977) and FEBS 
Letters 91 162-165 (1978), and in Ratcliffe, supra, pp. 34-35. This 
method, however, often requires a long incubation time, frequent washings 
of the precipitates, and involves complex reaction kinetics. The double 
antibody technique was probably developed because it is so difficult to 
find suitable materials that will precipitate only unbound conjugate 
(i.e., enzyme-antigen, enzyme-hapten, or other enzyme-ligand conjugate); 
see Wisdom, supra, and Schuurs et al., supra. 
Those assay techniques that require the presence of a solid phase, as for 
effecting separation, are commonly referred to as heterogeneous. Generally 
assays of this type are considered to have good sensitivity for antigens, 
other substances having high molecular weights, and like ligands. However 
such assays are not readily susceptible to automation, because of the need 
for centrifugation, or other separation step, to separate the solid and 
liquid phases, and because of the need for repeated washings. In a 
different assay technique referred to as the homogeneous technique, all of 
the materials remain in the liquid phase; no solid phase is used. This 
type of assay is generally considered to have good speed and good 
sensitivity, but not for antigens and other substances having high 
molecular weight. The homogeneous type of assay is generally limited as to 
the molecular weights of the ligands with which it is useful. Most types 
of homogeneous assay are considered to be useful with ligands of 1,500 
daltons molecular weight (M.W.) or less. Moreover, homogeneous assays have 
been most successfully applied only above the nanogram/ml. level. They are 
easily adapted to automated clinical equipment and when so adapted, can 
have high sample processing capability (high throughput). 
The homogeneous type of assay method does not require separation of free 
and bound label but rather depends on the inhibition or activation of the 
enzyme label by antibody binding (e.g., the EMIT R-type of Syva 
Corporation of Palo Alto, Calif., for EIA and FRAT, or "free radical assay 
technique", for SIA). Such assay techniques are described in U.S. Pat. 
Nos. 3,880,715; 3,852,157; 3,875,011; 3,935,074; and 3,905,871, and in an 
article by Kenneth S. Rubenstein et al., Homogeneous Enzyme Immunoassay, a 
New Immunochemical Technique, Biochemical and Biophysical Research 
Communications 47: 846-851 (1972). Other homogeneous assays having similar 
but not identical properties to EMIT and FRAT are also known. 
Unfortunately, homogeneous assays or other currently available similar 
assays, where the antibody modulates enzyme activity in the assay, suffer 
from the disadvantage that they are insensitive and unable to measure 
analytes of more than about 1,500 daltons. 
The competitions assay, often used in RIA techniques, is now considered a 
classical and well known technique for detecting immunochemicals such as 
antigens at a very low concentrations. It is based upon the competition 
between labeled and unlabeled antigen for a fixed, limited amount of 
antibody. As applied to RIA, it is described by R. Yalow and S. Berson in 
J. Clin. Invest. 39: 1157 (1960). The amount of unlabeled antigen 
influences the distribution of the labeled antigen between antibody-bound 
(B) labeled antigen and antibody-free (F) labeled antigen. Generally, the 
greater the amount of unlabeled antigen that is present, the smaller the 
amount of labeled antigen that is able to combine with the antibody. In 
order to obtain conclusive results from the distribution, a good 
separation between B and F must be made. Methods used for this purpose 
include, for instance, chromatoelectrophoresis, as described by S. Berson 
and R. Yalow in The Hormones, edited by G. Pincus et al., Academic Press, 
New York (1964), vol IV, 557, or insolubilization of the antibodies. This 
insolubilization can be achieved by chemical means (crosslinking or 
covalent binding to an insoluble carrier) or by physical methods 
(adsorption to an insoluble carrier). 
Of the limitations cited above, a most serious limitation, as reported in 
U.S. Pat. No. 4,213,764, has been lack of adequate sensitivity to detect 
some antigens. In general, three levels of sensitivity are recognizable. 
Low sensitivity techniques, where materials detected and measured exist in 
microgram/milliliter quantities, include RID, CPA, and LPA. Intermediate 
sensitivity techniques, where microgram/milliliter to nanogram/milliliter 
quantities of materials may be measured, include HIA, HA, FIA, SIA, VIA, 
and EIA. Until recently only RIA was able to measure with ultrasensitivity 
the picogram/milliliter to femtogram/milliliter region. 
A great many of the techniques listed above require that some form of 
physically or chemically identifiable label be attached to one or more of 
the reagents in the assay system in order that the result of a test can be 
detected. RIA, FIA, EIA, VIA, and SIA all fall into this category. 
Radioactivity, fluorescent moieties, enzymes, complement, viruses, and 
electron spin labels are used respectively to generate some form of 
endpoint signal. The sensitivity with which these labels can be detected 
directly and fundamentally affects the useful ranges of the test systems 
using them. 
The sensitivity with which a labeling moiety can be measured depends upon 
the nature of the signal that it generates, the ability to detect that 
signal, and the intensity of signal available per unit amount of marker 
molecule, i.e., its specific activity. The radioimmunoassay (RIA) method 
in its various forms has been recognized as a very sensitive system. The 
RIA method, unfortunately, has several serious, well recognized 
disadvantages. The possibility of replacing the radioactive label with an 
enzyme label was proposed in 1968 by L. E. M. Miles and C. N. Hales; see 
"Labelled Antibodies and Immunological Assay Systems", Lancet, II, 492 
(1968), and Nature 219, 168 (1968). 
Since then the EIA technique has been extensively investigated and 
developed. It is recognized as a potentially extremely sensitive 
technique, because of the inherent amplification potential of the enzyme 
label. That is, one molecule of enzyme can convert many molecules of its 
substrate, to generate the desired signal. Often the signal is a color 
development. 
Among the patents that are representative of the state of the art in the 
detection and measurement of immunochemical substances by the use of an 
enzyme label are U.S. Pat. Nos. 3,654,090, 3,666,421, 3,791,932, 
3,839,153, 3,850,752, 3,879,262, and 4,190,496. 
Each patent and literature item cited in this application is incorporated 
herein by reference. 
The Problem 
From the foregoing remarks, it can be seen that there are recognized 
deficiencies or disadvantages to each type of immunoassay procedure. In 
fact, there is a very well recognized need in the art, of long standing, 
for an immunoassay that is sensitive and accurate for the detection and/or 
determination of ligands of any molecular weight, but especially in the 
range from about 150 daltons to about 150,000 daltons. The need 
particularly exists for such an assay that can be automated effectively. 
This need is particularly acute with respect to ligands in the molecular 
weight range from about 1,000 daltons to about 40,000 daltons, where the 
automation of sensitive and accurate immunoassay procedures has either 
been very difficult or impossible in the past. 
Definitions 
Antibody: usually a gamma globulin or immunoglobulin that will react 
specifically (that is, in an immunochemical reaction) with an antigen or 
hapten. 
Antigen: a substance that can induce the formation of an antibody in vivo, 
and that is capable of an immunochemical reaction with that antibody. 
Double-Antibody: a separation technique which makes use of a second 
antibody produced against the immunoglobulin of a first animal, to 
precipitate an immune complex of an antigen and an antibody. 
Enzyme: generally a proteinaceous material, that acts as a catalyst, often 
for a single specific reaction. Sometimes enzymes will catalyze more than 
one reaction. 
Hapten: an incomplete or fragmentary antigen, which must be coupled to a 
carrier to form an antigen. 
Heterogeneous: an immunoassay system in which a solid phase is employed, 
usually in the separation step. 
Homogenous: an immunoassay system in which all of the reactants and the 
reaction products remain in solution. The detection step is made on the 
solution. 
Homologous: a system in which the same animal or species is used to produce 
a labeled antigen, the unlabeled antigen, and the antibody. 
Heterologous: a system in which the labeled antigen, the unlabeled antigen, 
and the antibody are produced, respectively, in two or more animal 
species. 
Inhibitor: a substance that modifies the activity of an enzyme, by 
increasing or decreasing it. It is customary to distinguish two broad 
class of inhibitors, competitive and noncompetitive, depending on whether 
the inhibition is or is not relieved by increasing concentrations of 
substrate. In practice, many inhibitors do not exhibit the idealized 
properties of purely competitive or purely noncompetitive inhibition. An 
alternate way to classify inhibitors is by their state of action. Some 
bind to the enzyme at the same place or side as does the substrate (the 
catalytic or active site), while others bind at some region (the 
allosteric site) other than the substrate site. The term inhibitor is use 
herein to refer to a substance to which the enzyme binds (if the enzyme is 
not inhibited or hindered as hereinafter described). 
Ligand: the particular molecule to be assayed, or one immunochemically 
equivalent to it. More generally the term refers to the smaller molecule 
in a complex or conjugate in which the smaller molecule is bound to a 
larger molecule or substance. 
Ligand analog: an analog of the ligand molecule that can be bound to an 
enzyme and that binds to its specific receptor molecule in substantially 
the same way as does the ligand. The term "ligand" as used herein is 
intended to embrace ligand analogs and immunochemically equivalent 
materials. 
Pseudo-substrate: a material to which enzyme binds. In the present 
invention, the preferred pseudo-substrate material is what usually would 
be regarded as a competitive inhibitor, when beta-galactosidase is used as 
the enzyme. 
Receptor: a specific binding partner of the ligand (or ligand analog) that 
is bound to the enzyme. Most often the receptor is a substance such as an 
antibody that binds specifically to another antigenic substance used 
herein as a ligand. 
Sensitivity: the sensitivity of an assay refers to the lowest quantity of 
the ligand (immunoreactant, hapten, or other substance) that can be 
reliably detected by the assay. 
Specificity: the degree of freedom from interfering substances. 
The Prior Art 
The present invention is built upon the extensive background described 
above. However, it makes available a powerful assay tool that is entirely 
novel. 
One of the important features of the present invention is the use of an 
insoluble immobilized pseudo-substrate which may be in the form of an 
affinity gel. In one preferred embodiment of the invention, this 
immobilized pseudo-substrate has been designed for use in connection with 
a particular enzyme, beta-galactosidase. The response of that enzyme to 
several different affinity gels is described in an article by Steers et 
al., in Methods in Enzymology 34: 350-358 (1974). 
Steers et al. formed several different affinity gels, each made from 
agarose coupled to one of several different inhibitors for the enzyme. 
Some of these gels, when formed into columns, permitted a solution of the 
enzyme to flow completely through without inhibition, others exhibited 
some retardation of enzyme flow, and still others retained the enzyme. 
The present inventors and three of their colleagues were seeking to develop 
an improved assay for determining choriomammotropin (human placental 
lactogen or HPL) in serum. As a result of their efforts, a competition 
assay was developed. This assay is described in the publication entitled 
Enzyme-Linked Immunosorbent Choriomammotropin Assay in Clinical Chemistry 
25: 227-229 (1979). In the assay as described in that article, unlabeled 
hormone competes, with a conjugate of the hormone with beta-galactosidase, 
for antibody that is bound to polystyrene tubes. The entire assay can be 
performed in 21/2 hours with good precision. The maximum sensitivity of 
this assay was 200 ug/L. The assay made use of a solid phase 
anti-choriomammotropin antibody. In the assay procedure, any 
immunoreaction that occurred caused the hormone or hormone-enzyme 
conjugate to bind to the solid phase antibody and thus become insoluble. A 
curve was drawn, in FIG. 1 of the article, to demonstrate proportional 
displacement of conjugate by unlabeled hormone. 
The inventors themselves published a brief description of some aspects of 
the present immunoassay technology in an article entitled, Steric 
Hindrance Enzyme Immunoassay, in Research Communications in Chemical 
Pathology and Pharmacology 26: 187-196 (published on or after Oct. 18, 
1979).

SUMMARY OF THE INVENTION 
1. The Separation Process 
One important feature of the assay technique of the invention is a 
separation process. This separation process is effective for separating a 
ligand-enzyme conjugate from that conjugate that is bound to a receptor 
for the ligand. 
The separation process involves placing in contact (1), a solution 
containing the ligand-enzyme conjugate and that conjugate bound through 
its ligand moiety to a receptor for the ligand, and (2), an insoluble 
immobilized pseudo-substrate for the enzyme. The enzyme, receptor and 
pseudo-substrate are specially selected materials. The pseudo-substrate is 
selected so that the free enzyme, and the enzyme moiety of the 
ligand-enzyme conjugate, bind to the immobilized pseudo-substrate. In 
addition, the receptor, when bound to the ligand-enzyme conjugate through 
its ligand moiety, must be one that inhibits the binding of the enzyme of 
the conjugate to the immobilized pseudo-substrate, as by steric hindrance. 
When this separation process is practiced, the ligand-enzyme conjugate is 
bound and immobilized to the immobilized pseudo-substrate. However, the 
complex of the receptor to the ligand-enzyme conjugate remains in 
solution. Consequently, by detecting the enzyme activity of either the 
supernatant solution, or of the immobilized solid phase, or both, as 
compared to the original enzyme activity of the conjugate, an assay 
technique is made possible. 
2. Competitive Assay for the Detection/Determination of Ligand Such as an 
Antigen 
In a preferred embodiment of the invention, the assay technique is employed 
for the detection and/or determination of a biologically active substance 
at a physiological concentration. The biologically active substance may 
be, for example, an antigen, hormone, steroid, hapten, or the like. To 
practice the assay, serum suspected of containing the antigen, for 
example, is mixed with a conjugate of that antigen (or its immunochemical 
equivalent) with an enzyme. This mixture is then incubated with the 
antibody for the antigen. After a suitable period of incubation, the 
mixture is brought into contact with an immobilized pseudo-substrate for 
the enzyme. In one preferred embodiment the immobilized pseudo-substrate 
is in a form in which the solution containing the unknown can be passed 
through it in column form. However, discrete granules of the immobilized 
pseudo-substrate are also convenient for use, particularly if they are 
large enough and heavy enough to separate easily from liquid in which they 
are mixed. 
With properly selected reactants according to the present invention, the 
antigen-enzyme conjugate binds to the immobilized pseudo-substrate and 
becomes itself immobilized. However, the complex of antibody bound to the 
antigen-enzyme conjugate (the complex being the immunochemical reaction 
product of antibody with the antigen moiety of the antigen-enzyme 
conjugate) is inhibited, we theorize by steric hindrance, from binding to 
the immobilized pseudo-substrate. This complex therefore remains in 
solution. If no antigen is present in the unknown serum, then all of the 
antigen-enzyme conjugate originally employed will bind to the immobilized 
pseudo-substrate and in effect will be precipitated out of the system. 
However, if antigen is present in the serum, then some of the antibody 
employed will react immunochemically with the antigen in the serum, and 
there is in effect a competitive assay between the antigen in the serum 
and the antigen moiety of the antigen-enzyme conjugate. Consequently, the 
enzyme activity that appears in either the supernatant liquid or in the 
solid phase can be calibrated to indicate the amount of antigen present in 
the serum. It is preferred that the enzyme activity be read on the liquid 
phase, although it is technically feasible to read it on the solid phase. 
In practice, a 100% reaction efficiency is very difficult to obtain. 
Consequently, calibration curves are required to be run on blanks and 
standardized solutions, against which test results can be compared to 
generate assay result figures. 
In more general terms, and expressed in stepwise fashion, an enzyme 
immunoassay procedure for the detection or determination of a ligand in a 
liquid sample according to this preferred embodiment of the invention 
comprises: 
(a) mixing a solution containing the ligand to be detected with a 
predetermined amount of conjugate comprising an enzyme that is bound to a 
ligand that is the same as or the immunochemical equivalent of the ligand 
that is to be detected; 
(b) mixing and incubating with the mixture produced from step (a) a 
predetermined amount of a receptor comprising an immunochemical bending 
partner for both of said ligands, which predetermined amount of receptor 
is less than that required for complete immunochemical reaction with the 
ligand moiety of the conjugate, so that the receptor binds to the ligand 
moiety of the conjugate but only a fraction of said conjugate is so bound 
as receptor-bound conjugate; 
(c) mixing and incubating with the solution from step (b) an amount of 
insoluble immobilized pseudo-substrate for the enzyme, which immobilized 
pseudo-substrate is capable of binding to the enzyme moiety of the 
conjugate that is not bound to receptor, but is incapable of binding to 
said receptor-bound conjugate, and which amount of immobilized 
pseudo-substrate is at least sufficient to bind to all of the enzyme 
moiety of the conjugate of step (a), to form a solid phase and a liquid 
phase, and 
(d) detecting or determining the presence or amount of conjugate in either 
the liquid phase or the solid phase. 
For simply detecting an antigen, the assay procedure is used as a 
qualitative test. The present of the conjugate is detected through its 
enzyme activity, as through a color reaction with a chromagenic substrate. 
To determine the presence of the ligand in the sample in quantitative 
fashion, initially a standard curve is constructed in the usual fashion, 
as hereafter described in an exemplary way. The observed value of enzyme 
activity is then read against the curve, in the manner described in the 
examples below, to produce a value figure based on the standard curve. 
3. Direct Assay for the Detection and/or Determination of Antibody 
In another preferred embodiment of the invention, the assay procedure is 
one for detecting and/or determining the presence of a specific antibody 
in a medium suspected of containing it. The assay technique for detecting 
an antibody involves bringing together in an aqueous liquid vehicle, to 
form a mixture, (1), the medium to be tested, and (2), soluble conjugate 
of an enzyme with an antigen or immunochemical equivalent thereof, that is 
immunoreactant with said antibody, and then bringing into contact with 
this mixture an insoluble, immobilized pseudo-substrate for the enzyme, to 
which pseudo-substrate the enzyme normally bonds. The immobilized 
pseudo-substrate may be in the form of granules that are simply mixed into 
the liquid mixture and then physically separated, or they may be in column 
form, through which the mixture is passed. 
In this assay procedure, the antibody is characterized by the ability, when 
immunochemically bound to the antigen moiety of the conjugate, to inhibit 
the binding of the enzyme moiety of the conjugate to the immobilized 
pseudo-substrate. The conjugate is employed in an amount such that there 
is always, after incubation of said mixture, an excess of the 
immunochemically reactant moiety of the conjugate over that needed for 
complete immunochemical reaction with the amount of antibody present. 
The next step in this procedure involves separating the insoluble 
pseudo-substrate from the liquid. The separated insoluble pseudo-substrate 
has bound to it free conjugate that is not bound to the antibody. The 
enzyme activity of the remaining liquid is then detected as an indicator 
of the presence, if any, of the antibody in the initial medium. The 
detection step could also be applied to the solid phase. In either case, 
for a quantitative determination of the amount of antibody in the sample, 
a standard curve is constructed initially, and then the observed value of 
enzyme activity is read against the standard curve to generate a value for 
antibody concentration in the sample. 
4. Competitive Assay for the Detection and/or Determination of Antibody 
In still another preferred embodiment of the invention, the assay procedure 
is one for detecting the presence of a general antibody in a medium that 
is suspected of containing it. In this case a mixture is formed in an 
aqueous liquid vehicle of (1), the medium that is suspected of containing 
the general antibody; (2), soluble conjugate of said antibody, or the 
immunochemical equivalent thereof, with an enzyme; and (3), an 
immunochemical receptor for the general antibody consisting of antiserum 
to said antibody. The antiserum must be capable of binding to the antibody 
and to the antibody moiety of the conjugate. 
The next step involves bringing into contact this mixture and insoluble 
immobilized pseudo-substrate for the enzyme, to which pseudo-substrate the 
enzyme normally binds. The antiserum is characterized by the ability, when 
bound to the antibody moiety of the conjugate, to inhibit the ability of 
the enzyme moiety of the conjugate to bind to the immobilized 
pseudo-substrate. The immobilized material is then separated from the 
liquid, and the enzyme activity of the liquid is detected as an indicator 
of the presence, if any, of general antibody in the original medium. As 
with other competitive assays, this may be made a quantitative assay 
through the use of a standard curve. 
5. General 
All of these preferred assay procedures of the invention may be conducted 
to be either qualitative assays, quantitative assays, or both. 
In each of these assay procedures, the amount of immobilized 
pseudo-substrate employed should be in excess over that needed for 
complete binding with all of the enzyme of the conjugate. The amount of 
the excess may be slight, as for a single assay of the batch kind using 
the immobilized pseudo-substrate in the form of granules, or very 
substantial, as where it is used in the form of a packed column. For 
practical reasons, generally a substantial excess will be used. 
Also, in each assay procedure, it is technically feasible to make the 
observation of the result on either the solid phase pseudo-substrate or on 
the liquid phase that is separated from it. However, observations taken on 
the liquid phase are simpler and represent the preferred technique. 
DETAILED DESCRIPTION OF THE INVENTION 
In its broader aspects, the invention is concerned with a separation 
technique based on the inhibition of enzyme binding to an immobilized 
pseudo-substrate, as by steric hindrance. 
Based upon the use of our novel separation technique, this invention 
provides a sensitive, automatable method for detecting or determining 
extremely low concentrations of a wide variety of organic materials, by 
relating the presence of a particular unknown to enzymatic activity. The 
assay procedure of the invention is applicable to ligands having a wide 
spectrum of molecular weights, including for example antigens in the 
molecular weight range from 150 to about 150,000. 
The invention is also concerned with a kit that is useful for those who may 
wish to practice the assay. The kit includes supplies of those materials 
needed for use in the assay, at concentrations and in amounts useful in 
the assay procedure, and printed instructions. 
Measurements of the kind permitted by the assay of the invention are 
valuable in the detection and diagnosis of disease, and also in monitoring 
the effectiveness of pharmacologic agents and other treatments. 
As those in the art understand, an amplification is inherent in the assay 
since one molecule of enzyme can transform a large number of molecules of 
substrate. The amplification is achieved by bonding the ligand to be 
assayed, or its immunological equivalent, such as a ligand analog, to an 
enzyme. This combination is referred to in the art and herein as a 
ligand-enzyme conjugate. 
The particular molecule to be assayed is referred to herein as a ligand. 
For some purposes, a ligand analog may be employed in preparing the 
ligand-enzyme conjugate, as will hereafter be described. A ligand analog 
will include either a ligand that is modified by replacing a proton with a 
linking group, to effect covalent coupling to the enzyme, or it may be a 
ligand or analog that is covalently coupled to the enzyme. A ligand analog 
is a ligand modified by some means other than simple replacement of a 
proton that provides a linking site for coupling to the enzyme. 
Competitive Assay Procedures for Antigens and the Like 
Both the ligand and the ligand moiety of a ligand-enzyme conjugate are 
capable of binding to specific sites in a receptor molecule. In conducting 
the assay, reliance is placed on the fact that the ligand and the ligand 
moiety of the ligand-enzyme conjugate compete for these sites. 
Compounds of very similar structures may serve to compete for reactive 
sites on an immunochemical binding partner, e.g., morphine glucuronide and 
codeine will compete with enzyme-bound-morphine for binding to certain 
types of morphine antibodies. In most instances, this is advantageous in 
permitting one to assay for a class of physiologically closely related 
compounds, rather than just for a single specific ligand. 
Normally, the ligand, ligand-enzyme conjugate and receptor will be soluble 
in the aqueous ligand medium employed. The substrate(s) for the enzyme may 
or may not be soluble in the medium. In some situations it may be 
desirable to provide a synthetic substrate which is not soluble or to 
employ an insoluble natural substrate. 
In carrying out the assay, the unknown solution containing the ligand, 
which solution may be a serum, is combined with a known quantity of a 
ligand-enzyme conjugate. Ordinarily buffer is employed in this initial 
mixture. The receptor is then added to and incubated with this mixture. 
Generally the quantities of conjugate and of receptor are accurately known 
and are carefully controlled so that the ligand moiety of the conjugate is 
present in an approximately 50% immunochemical excess relative to the 
active sites of the receptor. The enzymatic activity of the resulting 
solution, after these three substances have been incubated in the 
solution, is derived from the amount of conjugate originally employed. 
For greatest sensitivity in the assay, the amount of ligand (if any) in the 
unknown being assayed is at least equal to and preferably is many times 
the immunochemical equivalent of that present in the conjugate. To the 
extent that such amounts can be predetermined prior to the assay, based 
upon expectation, a ratio on the order of about 100 to 1 is preferred. 
During incubation, the receptor combines immunochemically with any ligand 
present from the unknown or serum, and in addition, combines with the 
ligand moiety of the ligand-enzyme conjugate. Because of the proportions 
employed, all of the receptor becomes bound, but only a part of the ligand 
available becomes bound. In this system, often, one mole of receptor can 
be expected to bind with one mole of the ligand-enzyme conjugate. As those 
skilled in the art will recognize, however, the one-to-one relationship is 
not the only possible relationship; each immunochemical binding partner 
may have several potentially reactive binding sites. The ligand in the 
unknown, if present, will often be present within an expected or known 
concentration range. If the ligand-enzyme conjugate is employed in a given 
or known amount, and a given or known, less than immunochemically 
equivalent amount of receptor is added, then there is a resulting 
competition for receptor sites between the ligand and the ligand-enzyme 
conjugate. 
The incubated solution is then brought into contact with affinity gel made 
up of immobilized pseudo-substrate for the enzyme. This may be done by 
adding discrete granules of the affinity gel to the solution then 
incubating the mixture in a shake flask. Separation can then be done at an 
appropriate time by filtration, centrifugation, or the like. If the 
pseudo-substrate is immobilized to large beads, settling may be adequate 
to cause separation to occur. However, it is preferred to use a column of 
the affinity gel, through which the solution is passed. In either case, 
the amount of pseudo-substrate made available should preferably be in 
substantial excess over that required for binding with all of the 
available enzyme. 
The immobilized pseudo-substrate is selected so that the enzyme moiety of 
the free ligand-enzyme conjugate binds to the immobilized pseudo-substrate 
and is effectively insolubilized or precipitated. The complex of receptor 
with the ligand-enzyme conjugate, however, is, we believe, sterically 
hindered by reason of the presence of the receptor moiety in the 
receptor-enzyme-ligand complex, so that the enzyme moiety of this complex 
cannot bind to the immobilized pseudo-substrate. 
Knowing the amount of receptor added and the amount of initial conjugate 
present, a measurement of the enzyme activity in either the liquid phase 
or the solid phase can be used to reflect the amount of ligand present in 
the serum as an unknown. It is believed that better results are obtained 
when the measurement is made on the liquid phase. 
The concentrations of the reagents, i.e., the enzyme-bound-ligand and the 
receptor, may be varied widely. Normally, the concentration of receptor 
(based on active sites) and enzyme-bound-ligand will be from about 
10.sup.-4 M to 10.sup.-14 M, more usually from 10.sup.-6 M to 10.sup.-12 
M. The lower limit for the concentration of ligand-enzyme conjugate is 
predicated on the necessary immunochemical excess of ligand moiety, and 
also upon the minimum amount of enzyme which can be detected. This will 
vary with different enzymes as well as different detection systems. 
The amount of receptor employed is normally calculated based on its 
available immunochemically reactive sites and will vary with the 
concentration and amount of ligand-enzyme conjugate, the ratio of ligand 
to enzyme in the ligand-enzyme conjugate, and the affinity of the receptor 
for the ligand. The immunochemical equivalents employed of receptor and of 
ligand-enzyme conjugate are such that the amount of conjugate employed 
furnishes approximately a 50% immunochemical excess of ligand, at least. 
The ratio may vary to a degree depending on binding constants and the 
amount of ligand suspected of being present in the unknown. 
Competitive Assay Procedures for General Antibodies 
For detecting a general antibody rather than an antigen-type of ligand, the 
amount of conjugate of the general antibody with enzyme employed with be 
selected to optimize the sensitivity of the assay in the concentration 
range of interest. The concentration ratio of antibody-enzyme conjugate to 
immunochemical receptor for the general antibody will be selected based 
upon the minimum and maximum values of the general antibody concentration 
range of interest. 
Conjugate Preparation 
In preparing the enzyme conjugate, the ratio of the ligand (for a 
competitive assay, the ligand will be an antigen or the like, or a general 
antibody of immunochemical equivalent) to the enzyme in the conjugate will 
depend upon the identities of the two moieties of the conjugate. 
In the preferred embodiment of the invention that is illustrated in detail 
in Example 1, the conjugate is formed by coupling choriomammotropin to 
beta-galactosidase through a maleimidobenzoyl bridge. Choriomammotropin is 
a hormone having a molecular weight above 20,000. It is a single 
polypeptide. The enzyme beta-galactosidase is a large molecule with a 
molecular weight of about 550,000. It appears to be a tetramer, with a 
total of about 12 reactive sites per molecule. The enzyme can be 
underconjugated or overconjugated. It is difficult to determine the exact 
amount of choriomammotropin and the exact amount of enzyme in reactant 
solutions, and very difficult to determine their respective proportions in 
the conjugate that is formed. Consequently, hormone-to-enzyme molar 
ratios, when described herein, refer to estimated values calculated from 
the results obtained by Kitagawa and Aikawa, J.Biochem. 79: 233-236 
(1976). 
If the conjugate is made up so that the enzyme is underconjugated, that is, 
the molar ratio of hormone to enzyme is less than about 8:1, then less 
than desired results are obtained in the assay. When the molar ratio is in 
the range from about 8:1 to 12:1, best results are obtained in the assay 
procedure. The ratio that is best will be different for each enzyme and 
ligand (or antibody) pair. 
All other things being equal, the greater the number of enzyme molecules 
per large ligand, the greater the sensitivity of the assay. However, the 
enzyme molecules may interfere with receptor recognition, affect 
solubility, and be deleterious in other ways. 
Competitive Assay for the Determination of Choriomammotropin 
The invention can be illustrated further as applied to a particular ligand. 
The determination of choriomammotropin (formerly called human placental 
lactogen, HPL) in serum is useful for monitoring the progress of high risk 
pregnancies. The number of samples required in this clinical use is such 
that a more rapid and economical method is needed than those now presently 
available. See for example the publication of Dittmar et al in Clinical 
Chemistry 25: 227-229 (1979), and their appended bibliography. 
Our inhibited enzyme immunoassay, or steric hindrance enzyme immunoassay 
(SHEIA), is particularly useful for assays of serum for choriomammotropin 
(hereafter for convenience, HPL). To perform the assay, the essentials 
include serum that is to be assayed, HPL-enzyme conjugate, antibody to 
HPL, the affinity gel, and of course, substrate for enzyme detection. 
The HPL-enzyme conjugate is prepared by bonding HPL to beta-galactosidase 
through a maleimidobenzoyl bridge. The molar ratio employed for the 
conjugation should be such that the hormone to enzyme ratio in the 
conjugate is in the range from 8 to 1 to 12 to 1. The molar ratios can be 
estimated from the results obtained according to Kitagawa and Aikawa, 
supra. The production and characterization of rabbit anti-HPL antibody are 
reported in the article by Castro et al., Clinical Chemistry 22: 1655-1658 
(1976). 
In addition to the reactants that are employed in the assay, insoluble 
immobilized pseudo-substrate must be prepared for use in the separation 
step. For this particular assay, it is preferably prepared by immobilizing 
a pseudo-substrate for the enzyme beta-galactosidase. Thus, in Ex. 1 
hereafter, the 6-aminocaproyl derivative of galactosylamine is attached to 
agarose. (For convenience, this immobilized pseudo-substrate combination 
of carrier and pseudo-substrate is referred to hereafter as affinity gel.) 
It is known that this particular enzyme will not bind to a 
pseudo-substrate unless a bridge of at least 6 carbons is employed to 
increase the distance between the pseudo-substrate and the agarose carrier 
backbone, Steers et al., supra. The affinity gel may be used either in the 
batch method or in an affinity column. 
To practice this embodiment of the invention, serum, conjugate, and 
something substantially less than the theoretical amount (for reaction 
with the HPL moiety of the conjugate) of antibody to HPL are incubated 
together. The mixture is then brought into contact with the affinity gel. 
Free conjugate binds to the affinity gel, and antibody-bound conjugate 
remains in solution. By developing a standard curve for the assay, and 
controlling the relative amounts of the several reactants, the enzyme 
activity of the supernatant liquid can be determined and, using the 
standard curve, will indicate the amount of the hormone present in the 
serum sample. 
Measurement of the beta-galactosidase activity remaining in the solution 
may be done by the method of Dray et al., Biochem. Biophys. Acta. 403: 
131-138 (1975), using o-nitrophenyl-beta-D-galactopyranoside (ONGP) as the 
enzyme substrate. 
Construction of the Standard Curve 
To make up the standard curve, the concentration of receptor and enzyme 
will be related to the expected range of concentration of the liquid (HPL 
in the sample) to be assayed. The sample is used directly. If a relatively 
high concentration of the unknown ligand is expected to be present, then 
the unknown solution may be diluted so as to provide a convenient 
concentration. However, in many biological systems of interest, the amount 
of material being assayed will be relatively small and dilution of the 
unknown substrate will often not be required. 
To prepare the standard curve for the HPL assay, a series of assay 
procedures is carried out in which all concentrations of reagents are kept 
constant, except HPL. In this operation, several standard solutions of HPL 
are prepared and employed, at different concentrations. In this manner, a 
series of observed values of enzyme activity in the supernatant liquid is 
generated. These values can be graphically represented in the form of a 
standard curve, plotting observed values of enzyme activity against the 
several different, known concentration values of the HPL standard 
solutions. 
This standardized data can then be used when a serum containing an unknown 
amount of HPL is employed in the assay, and all of the other conditions 
are those employed for generating the standard curve. The standardized 
system can be used to determine rapidly, accurately, and efficiently the 
amount of HPL in the unknown. This same approach is applicable, of course, 
for assays for other liquids in other unknowns. 
In addition to concentration, other parameters must be fixed for developing 
the standard curve, and then for conducting assays that will make use of 
the standard curve. This applies for example to the enzyme assay, 
temperature, and pH. 
The manner of assaying for enzyme activity in the solution or supernatant 
liquid may take a variety of forms, depending on the enzyme and to some 
degree upon the particular ligand and the medium in which the ligand is 
obtained. Preferably, spectrophotometric measurements are employed where 
the product of a substrate for the enzyme absorbs light. However, other 
observation techniques may be employed including fluorimetry, measurements 
of luminescence, and the other techniques that are well known in the art. 
This assay is generally carried out at room temperature, that is, at about 
20.degree. C., but can be carried out over a broad range of temperatures. 
Generally ambient temperatures of 15.degree. C. to 40.degree. C. are 
prevalent under most assay conditions and the assay is operative in this 
range as well as outside of it, although the ambient temperature obviously 
affects reaction rates. 
The pH at which the assay is carried on is generally in the range from 
about 5 to about 10, more often from pH 6 to pH 9. Useful buffers for 
achieving a desired pH include phosphate, citrate-phosphate, borate, amine 
salts, and the like. 
Ligands 
Turning now to a general consideration of the reagents, any ligand may be 
employed for which an appropriate receptor may be found having 
satisfactory specificity for the ligand. The recent literature contains an 
increasing number of reports of receptors for an increasingly wide variety 
of biologically active materials. So long as there is an available 
specific binding partner, the ligand (unknown) to be assayed may be an 
immunochemical reactant such as an antigen, a specific antibody, a general 
antibody, or a variety of other materials. Specific compounds for which 
receptors can be provided range from simple phenyl alkylamines, e.g., 
amphetamine, to very high molecular weight polymers, e.g., proteins. 
Among drug ligands are compounds which act as narcotics, hypnotics, 
sedatives, analgesics, antipyretics, anaesthetics, psychotogenic drugs, 
muscle relaxants, nervous system stimulants, anticholinesterase agents, 
parasympathomimetic agents, sympathomimetic agents, alpha-adrenergic 
blocking agents, antiadrenergic agents, ganglionic stimulating and 
blocking agents, neuromuscular agents, histamines, antihistamines, 
5-hydroxytryptamine and antagonists, cardiovascular drugs, antiarrhythmic 
drugs, antihypertensive agents, vasodilator drugs, diuretics, pesticides 
(fungicides, antihelminthics, insecticides, ectoparasiticides, etc.), 
antimalarial drugs, antibiotics, antimetabolites, hormones, vitamins, 
sugars, thyroid and antithyroid drugs, corticosteroids, insulin, oral 
hypoglycemic drugs, tumor cells, bacterial and viral proteins, toxins, 
blood proteins, and their metabolites. 
Included among such drugs and agents are alkaloids, steroids, polypeptides 
and proteins, protaglandins, catecholamines, xanthines, arylakylamines, 
heterocyclics, e.g., thiazines, piperazines, indoles, and thiazoles, amino 
acids, etc. 
Other ligands of interest besides drugs are industrial pollutants, 
flavoring agents, food additives, e.g., preservatives, and food 
contaminants. 
Generally, the ligands and receptors to be detected by the use of the 
present invention will be organic compounds whose molecular weights fall 
in the range of from about 150 to about 150,000 daltons. However, the 
assay of the present invention is particularly useful for ligands and 
receptors with molecular weight within the range from about 20,000 to 
about 50,000 daltons. The ligand or receptor may be a simple organic 
compound or a compound having one or more recurring groups, referred to 
for simplicity as polymers. With care, the present invention can be used 
for the detection of certain blood proteins whose molecular weight 
generally is in excess of 100,000 daltons. 
Ligands may be divided into three different categories based on their 
biological relationship to the receptor. The first category is antigens, 
which when introduced into the bloodstream of a vertebrate, result in the 
formation of antibodies. The second category is haptens, which when bound 
to an antigenic carrier, and then introduced into the bloodstream of a 
vertebrate, elicit the formation of antibodies specific for the hapten. 
The third category of ligands includes those which have naturally 
occurring receptors in a living organism, where the receptor can be 
isolated in a form specific for the ligand. 
Antigens are for the most part protein or polysaccharide in nature and 
generally are foreign to the animal into which they are injected. 
In the third group of ligands, i.e., those which have naturally occurring 
receptors, the receptors may be proteins, nucleic acids, such as 
ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or membranes 
associated with cells. Illustrative ligands which have naturally occurring 
receptors are thyroxine, many steroids, such as the estrogens, cortisone, 
corticosterone, and estradiol; polypeptides such as insulin and 
angiotensin, as well as other naturally occurring biologically active 
compounds. See Murphy, et al., J. Clin. Endocr., 24: 187 (1964); Murphy 
ibid, 27: 973 (1967) ibid, 28: 343 (1968); BBA 176: 626 1969); McEwen, et 
al., Nature, 226: 263 (1970) Morgan, Diabetes, (1966); Page et al., J. 
Clin. Endocr. 28: 200 (1969). 
The ligands may also be categorized by the chemical families which have 
become accepted in the literature. In some cases, included in the family 
for the purpose of this invention, will be those physiomimetic substances 
which are similar in structure to a part of the naturally occurring 
structure and either mimic or inhibit the physiological properties of the 
natural substances. Also, groups of synthetic substances will be included, 
such as the barbiturates and amphetamines. In addition, any of these 
compounds may be modified for linking to the enzyme at a site that may 
cause all biological activity to be destroyed. Other structural 
modifications may be made for the ease of synthesis or control of the 
characteristics of the antibody. These modified compounds are referred to 
as ligand counterfeits. 
A general category of ligands of particular interest includes drugs and 
chemically altered compounds, as well as the metabolities of such 
compounds. The interest in assaying for drugs varies widely, from 
determining whether individuals have been taking a specific illicit drug, 
or have such drug in their possession, to determining what drug has been 
administered or the concentration of the drug in a specific biological 
fluid. 
The ligand analogs of drugs of interest herein generally have molecular 
weights in the range from about 150 to 1500. They include for example the 
opiates such as morphine and heroin, meperidine, and methadone, and 
epinephrine-like drugs such as amphetamine, narceine, ephedrine and 
L-dopa. 
Polypeptide and protein ligands are of great interest. Polypeptides usually 
encompass from about 2 to 100 amino acids units (usually less than about 
12,000 molecular weight). Larger polypeptides are arbitrarily called 
proteins. Proteins are usually composed of from 1 to 20 polypeptide 
chains, called subunits, which are associated by covalent or non-covalent 
bonds. Subunits are normally of from about 100 to 400 amino acid groups 
(10,000 to 50,000 molecular weight). 
Individual polypeptides and protein subunits will normally have from about 
2 to 400, more usually from about 2 to 300 recurring amino acid groups. 
Usually, the polypeptides and protein subunits of interest will be not 
more than about 50,000 molecular weight. Because of the wide variety of 
functional groups which are present in the amino acids and frequently 
present in the various naturally occurring polypeptides, in making a 
ligand-enzyme conjugate from such as ligand, the enzyme can be bonded to 
any convenient functionality of the ligand. Polypeptides of interest are 
ACTH, oxytocin, lutenizing hormone, insulin, Bence-Jones protein, 
chlorionic gonadotropin, pituitary gonadotropin, growth hormone, rennin, 
thyroxine bonding globulin, bradykinin, angiotensin, follicle stimulating 
hormone, etc. 
The invention is also useful in assays for steroids. These compounds have a 
wide range of functionalities depending on their function in the body. In 
addition to the steroids, are the steroidmimetic substances, which while 
not having the basic polycyclic structure of the steroids, do exhibit some 
of the same physiological effects. 
The steroids have been extensively studied and derivatives prepared which 
have been bonded to antigenic proteins for the preparation of antibodies 
to the steroids. Illustrative compounds include: 17 
beta-estradiol-6-(o-carboxymethyl-oxime)-BSA (Exley et al, Steroids 18: 
593 (1971)); testosterone-3-oxime derivative of BSA (Midgley, et al., Acta 
Endocr. 64 supplement 147, 320 (1970)); and progesterone-3-oxime 
derivatives of BSA (Midgley, et al. ibid.) 
These steroids find use as hormones, male and female (sex) hormones, which 
may be divided into oestrogens, gestogens, androgens, adrenocortical 
hormones (glucocorticoids), bile acids, cardiotonic glycosides and 
aglycones, as well as saponins and sapogenins. 
Steroid mimetic substances, particularly sex hormones, are illustrated by 
diethyl stilbestrol. The sex hormones of interest may be divided into two 
groups; the male hormones (androgens) and the female hormones 
(oestrogens). Illustrative androgens, with which the invention may be 
employed, include testosterone, androsterone, isoandrosterone, 
etiocholanolone, methyltestosterone and dehydroisoandrostone. 
Illustrative compounds which may be linked to an enzyme to form an 
androgen-enzyme conjugate include N-carboxymethoxy testosteroneimine, 
17-monotesteroyl carbonate, androsteronyl succinate, testosteronyl 
maleate, 0.sup.3 -carboxymethyl 0.sup.17 -methyl 
androst-5-ene3beta,17beta-diol, testosterone 0-carboxypropyl oxime and 
androsteronyl carbonate. 
Illustrative oestrogen compounds which may be detected by the invention 
include 3-carboxymethyl estradiol, 2-chloromethylestrone estrone 
glutarate, 0-carboxymethyloximine of 6-ketoestradiol, equilenyl 
N-carboxymethyl thiocarbamate. 
Illustrative gestogen compounds which may be detected include progesterone, 
pregnenolone, allopregnane-3a:20a-diol and allopregnan-3a-ol-20-one. 
Illustrative compounds which may be linked to an enzyme to form a conjugate 
include 20-progesterone 0-carboxymethyl oxime, 0-carboxymethyl 
progesterone 3-oxime, pregnenolonyl lactic acid, and 
allopreganane-3-carboxymethyl-20-ol. 
Another important group of steroids is the corticosteroids which includes 
both the mineral corticoids and the glucocorticoids. 
Illustrative compounds of this type which may be bonded to an enzyme to 
form a conjugate are 17-hydroxydioxycortiocosterone (compound S), 
deoxycorticosterone, cortisone, corticosterone, 11-dihydrocortisone 
(Compound F), cortisol, prednisolone and aldosterone. 
Illustrative compounds of this type which may be linked to an enzyme to 
form a conjugate include 0.sup.21 carboxymethyl corticosterone, 
N-carboxymethyl 21-carbamate cortisol, 21-cortisone succinate, 
21-deoxocorticosterone succinate, and 0.sup.17 -methyl, 0.sup.21 
-carboxymethyl cortisone. 
The assay of the invention is also useful for the detection of antibiotics 
such as penicillin, chloromycetin, actinomycetin, tetracycline, 
terramycin, and nucleic acid or derivatives, such as nucleosides and 
nucleotides. It is also useful for the determination of serotonin, which 
is 3-(2'-aminoethyl)-5-hydroxyindole. 
Enzymes 
Enzymes vary widely in their substrates, cofactors, specificity, 
ubiquitousness, stability to temperature, pH optimum, turnover rate, and 
the like. Other than interest factors, there are also the practical 
considerations, that those enzymes selected for use have been 
characterized extensively, have accurate reproducible assays already 
developed, and be commercially available. 
In addition, for the purpose of this invention the enzymes should either be 
capable of specific labelling or allow for efficient substitution, so as 
to be useful in the subject assays. By specific labelling is intended 
selective labelling at a site in relationship to the active site of the 
enzyme, so that upon binding of the receptor to the ligand moiety of the 
conjugate, the enzyme is satisfactorily sterically hindered or otherwise 
inhibited with respect to the specific immobilized pseudo-substrate that 
is to be used. When the receptor is bound to the ligand moiety of the 
conjugate, the degree of substitution should not unreasonably diminish the 
turnover rate for the enzyme moiety of the conjugate nor substantially 
change the solubility characteristics of the conjugate. 
From the standpoint of operability, a very wide variety of enzymes is 
available for use. As a practical matter, there are a number of groups of 
enzymes which are preferred. Employing the International Union of 
Biochemists (I.U.B.) classification, the oxidorreductases (1.) and the 
hydrolases (3) are of greatest interest. 
Of the oxidoreductases, the ones acting on the CHOH group, the aldehyde or 
keto group, or the CH-NH.sub.2 group as donors (1.1, 1.2, and 1.4 
respectively) and those acting on hydrogen peroxide as acceptor (1.11) are 
preferred. Also, among the oxidoreductases those preferred include those 
which employ nicotinamide adenine dinucleotide, or its phosphate or 
cytochrome as an acceptor, namely 1..times.0.1 and 1..times.0.2, 
respectively, under the I.U.B. classification. 
Of the hydrolases, of particular interest are those acting on glycosyl 
compounds, particularly glycoside hydrolases, and those acting on ester 
bonds, both organic and inorganic esters, namely the 3.1 and 3.2 groups 
respectively, under the I.U.B. classification. Other groups of enzymes 
which might find use are the transferases, the lyases, and isomerases, and 
the ligases. 
In choosing an enzyme, there are several important criteria. These can be 
tabulated as follows: 
Table 1 
Criteria for the Choice of a Preferred Enzyme Label 
1. Available cheaply in high purity. 
2. High specific activity. 
3. Stable under assay and storage conditions. 
4. Soluble. 
5. Known assay method that is simple, sensitive, rapid, and cheap. 
6. Absent from biological fluids. 
7. Substrates, inhibitors, and disturbing factors absent from biological 
fluids. 
8. Capable of retaining activity while undergoing appropriate linkage 
(conjugating) reactions. 
9. Capable of being sterically hindered when antibody or other receptor 
binds to the ligand-enzyme conjugate. 
10. Assay conditions compatible with ligand-receptor binding. 
In more detail, the enzyme should be stable when stored for a period of at 
least three months, and preferably at least six months at temperatures 
which are convenient for storage in the laboratory, normally -20.degree. 
C. or above. 
The enzyme should have a satisfactory turnover rate at or near the pH 
optimum for binding of the ligand, or of the ligand moiety of the 
ligand-enzyme conjugate, to the receptor. This is normally at about pH 
6-10, usually 6.0 to 8.0. Preferably, the enzyme will have the pH optimum 
for its turnover rate at or near the pH optimum for binding of the 
receptor to the ligand. 
The enzyme should have a substrate (including cofactors) which has a 
molecular weight in excess of 300, preferably in excess of 500, there 
being no upper limit. A product should be either formed or destroyed as a 
result of the enzyme action on its substrate. The reaction product 
obtained should be one which absorbs light in the ultraviolet region or 
the visible region, that is, in the range of about 250-750 nm, preferably 
300-600 nm, to facilitate optical or visual detection of enzymic activity. 
Preferably, the enzyme which is employed, or other enzymes with like 
activity, will not be present in the fluid on which the assay is carried 
out, or if present, can be easily removed or deactivated prior to the 
addition of the assay reagents. Also, there should not be naturally 
occurring inhibitors for the enzyme present in fluid to be assayed. 
Also, although enzymes of up to 600,000 molecular weight can be employed, 
usually relatively low molecular weight enzymes will be employed of from 
10,000 to 300,000 molecular weight, more usually from about 10,000 to 
150,000 molecular weight, and frequently from 10,000 to 100,000 molecular 
weight. Where an enzyme has a plurality of subunits the molecular weight 
descriptions refer to the enzyme and not to the subunits. 
For synthetic convenience, it is preferable that there be a reasonable 
number of groups to which the ligand may be covalently coupled or bound, 
particularly amino groups. However, other groups to which the ligand may 
be bound include hydroxyl groups, thiols, and activated aromatic rings, 
e.g., phenolic. 
Accordingly, the enzyme selected for use will be one that is already 
sufficiently characterized so as to assure the availability of sites for 
linking either in positions which allow for steric hindrance or other form 
of inhibition of the enzyme when the ligand is bound to the antibody or 
other receptor, or there should exist a sufficient number of positions as 
to make this occurrence likely. 
The following enzymes are among those often used in EIA, which may have the 
capability of being used in accordance with the present invention. 
TABLE 2 
______________________________________ 
Enzymes Suitable For Use 
Enzyme Source 
______________________________________ 
acetylcholinesterase (EC 4.2.1.1) 
-- 
alkaline phosphatase (EC 3.1.3.1) 
calf intestinal mucosa and 
E. coli 
carbonic anhydrase (EC 4.2.1.1) 
Rhizopus nivens 
beta-D-galactosidase (EC 3.2.1.23) 
E. coli 
glucoamylase (EC 3.2.1.3) 
A. oryzae 
glucose oxidase (EC 1.1.3.4) 
fungal 
glucose-6-phosphate 
dehydrogenase (EC 1.1.1.49) 
Leuconostoc mesenteroides 
horse-radish peroxidase (EC 1.11.1.7) 
horse-radish 
lysozyme (EC 3.2.1.17) 
egg white 
malate dehydrogenase (EC 1.1.1.37) 
pig heart mitochondria 
______________________________________ 
All of these are available commercially. Many other enzymes that are useful 
in EIA techniques are identified in U.S. Pat. No. 4,190,496, cols. 32-38. 
The Ligand-Enzyme Conjugate 
The ligand-enzyme conjugate consists of the ligand covalently linked to one 
or more enzyme molecules. Such linking can be achieved either by direct 
condensation or by using external bridging molecules, in accordance with 
methods known to those skilled in the art. 
Thus, the production of enzyme conjugate employing a covalent bond can be 
effected by difunctional reagents such as carbodiimides, diisocyanates, 
glutaraldehyde, and bisdiazolbenzidine. 
Preferably, the ligand or ligand analog is bonded either directly to the 
enzyme, by a single or double bond, or to a linking group. For those 
ligands which are haptens, and for which the receptors are antibodies, the 
ligand will have been bound to a protein. Since the antibodies will 
recognize that portion of the ligand molecule which extends from the 
protein, ordinarily the same linking group will be attached on the same 
site on the ligand, as was used in bonding the ligand to the protein to 
provide the antigenic substance. 
The functional groups which will be present in the enzyme for linking are 
amino (including guanidino), hydroxy, carboxy, and mercapto. In addition, 
activated aromatic groups or imidazole may also serve as a site for 
linking. 
Amino acids having amino groups available for linking include lysine, 
arginine, and histidine. Amino acids with free hydroxyl groups include 
serine, hydroxypropyline, tyrosine and threonine. Amino acids which have 
free carboxyl groups include aspartic acid and glutamic acid. 
In most instances, the preferred linking group will be the amino group. 
Making conjugate from an enzyme requires knowledge of the morphology of 
the enzyme molecule and, as well, generally some amount of 
experimentation. 
The ligand, of course, may have a great diversity of functionalities. In 
addition, the functionalities which are present may be modified so as to 
form a different functionality, e.g., keto to hydroxy, or an olefin to 
aldehyde or carboxylic acid. To that extent, the choice of groups for 
linking to the ligand may be varied much more widely than the choice of 
groups for linking to the enzyme. In both cases, however, a wide variety 
of different types of functionalities have been developed, specifically 
for linking various compounds to proteins and particularly to enzymes. 
When a cross-linking agent is used in forming the conjugate, the bonds 
formed must be stable under the conditions of the assay. When bonding the 
ligand to the enzyme by a cross-linking agent, the enzyme must retain at 
least a portion of its activity upon isolation. The enzyme must not 
prevent binding of the ligand to the receptor. The functionalities should 
permit some selectivity, so that bonding can be directed to specific 
groups or types of groups in both the ligands and enzymes. 
Receptors 
The receptor is always a specific binding partner for the ligand in the 
enzyme conjugate. For the most part, the receptors will be macromolecules 
which have sites which recognize specific structures. The recognition of 
the specific structures may be based on van der Waals forces, which 
provide a specific spatial environment which maximizes the van der Waals 
forces; diple interactions, either by permanent or induced dipoles; 
hydrogen and ionic bonding; coordinate covalent bonding; and hydrophobic 
bonding. For a detailed discussion of such binding mechanisms see 
Goldstein et al., Principles of Drug Action, Harper and Rowe, New York, 
1968. 
A macromolecule is generally essential in order that the 
receptor-ligand-enzyme complex have sufficient bulk or blocking (because 
of site) contributed by the receptor so that the desired steric hindrance 
or other form of enzyme inhibition occurs. 
The macromolecules of greatest interest are proteins and nucleic acids 
which are found in cell membranes, blood, and other biological fluids. 
These compounds include antibodies, ribonucleic acid (RNA) and 
deoxyribonucleic acid (DNA), and natural receptors. 
The most convenient groups of proteins for use in the subject invention are 
antibodies. These materials are conveniently used in the analysis of the 
category of ligands referred to as haptens, as well as for antigens. 
Antibodies are produced by introducing an immunogenic substance into the 
bloodstream of a living animal. The response to the introduction of the 
immunogenic substance or antigen is the production of antibodies which act 
to coat the antigen and detoxify it or precipitate it from solution. The 
protein forms a coat which is geometrically arranged so as to have the 
antigen fit the spatial arrangement of the protein. This may be analogised 
to a lock and key. The interaction is normally reversible, in that the 
antigen is subject to displacement or removal by various means without 
destruction of the receptor site of the antibody. 
There are many materials which are antigens and will produce an immunogenic 
response by being introduced into the bloodstream of a vertebrate. 
However, a number of materials of interest are not antigens, but are 
haptens, and in that case, an extra step in preparing the antibody is 
required. This method of preparing antibodies with materials other than 
antigens is well known, Microbiology, Hoeber Medical Division, Harper and 
Rowe, 1969. See also, Landsteiner, Specificity of Serological Reactions, 
Dover Publications, N.Y. 1962; Kabat et al., Experimental Immunochemistry, 
Charles C. Thomas, Springfield, Ill., 1969; and Williams et al., Methods 
in Immunology and Immunochemistry, Vol. 1, Academic Press, New York 1967. 
The material which is to be assayed is bonded to a protein by any 
convenient means and the modified protein introduced into the blood 
stream. The same type of bonding groups used with the enzyme attachment to 
the ligand may be employed. 
The antibodies which form will include groups of antibodies which are 
shaped to fit the foreign moiety (hapten) bonded to the protein. 
Therefore, antibodies are obtained which are specific to the compound or 
moiety bonded to the protein. By careful separation techniques, the 
antibodies primarily concerned with the moiety in question can be 
concentrated so as to provide an antibody composition which is primarily 
related to the specific moiety which was bonded to the protein. 
To illustrate this method, para-aminobenzene arsonate is diazotized to form 
the diazo salt. By combining the diazo salt with rabbit globulin, the 
rabbit globulin may be labeled with para-azobenzene arsonate. By 
introducing this composition into the blood stream of an animal other than 
a rabbit, for example a sheep, antibodies can be formed which will have a 
spatial arrangement which accepts solely the azobenzene arsonate. 
In addition to antibodies, there are a number of naturally occurring 
receptors which are specific to compounds of biological interest. 
Compounds for which receptors are naturally occurring include thyroxine, 
corticosterone, cortisone, 11-desoxycortisol, 11-hydroxyprogesterone, 
estrogen, insulin and antigen. See, for example, Vonderhaar et al., 
Biochem. Biophysics Acta., 176, 626 (1969). All of these ligands have been 
studied and reported upon in the literature in connection with studies on 
their binding with specific receptors. 
Immobilized Pseudo-Substrate 
The immobilized pseudo-substrate consists, preferably, of two or three 
components. One of the essential components is an inert carrier. The word 
"inert" is employed to define the role of the carrier with respect to the 
assay materials with which it comes in contact, rather than with respect 
to the pseudo-substrate. 
A second component is the pseudo-substrate. In conventional parlance, many 
of the preferred materials, that can serve as the pseudo-substrate, would 
be called competitive, reversible inhibitors. For the purposes of the 
present invention, the pseudo-substrate employed must be one of the type 
that forms a complex with either free enzyme or with the enzyme moiety of 
enzyme that is conjugated to ligand. Preferably, but not essentially, the 
pseudo-substrate-enzyme complex that forms is reversible, so that after 
the complex has formed and has served its purpose, the complex can be 
uncoupled and the enzyme released, to permit eventual reuse of the 
immobilized pseudo-substrate. 
These are the two essential components of the immobilized pseudo-substrate, 
that is, the carrier and the pseudo-substrate itself. In some cases, the 
pseudo-substrate may be immobilized directly to the carrier. However, 
preferably, a third component is present in the form of a covalent 
coupler, that chemically couples the pseudo-substrate to the carrier. 
A most important property of the immobilized pseudo-substrate is its 
ability to form a complex with enzyme, so that free enzyme-ligand 
conjugate is bound to the immobilized pseudo-substrate to effect a 
separation for assay purposes. The manner in which the immobilized 
pseudo-substrate is prepared will depend upon the identity of the enzyme 
that is to form the complex. Those skilled in enzymology are sensitive to 
the spatial requirements that will permit the enzyme-pseudo-substrate 
complex to form. See for example, Steers et al., Methods in Enzymology, 
supra, where several different types of affinity gel are described, some 
of which were effective in forming complexes and some which were not. The 
authors concluded that the enzyme (beta-galactosidase) with which they 
were working required that the pseudo-substrate be connected to the 
particular carrier, that they were using, by a cross-linking molecule 
containing at least a certain minimum number (6) of carbon atoms, to 
permit the enzyme-pseudo-substrate complex to be formed. 
The carrier may be either organic or inorganic. In either case, it is 
preferably in the form of particles that remain discrete whether used by 
admixture with the assay liquid, or packed in a column through which the 
assay liquid is passed. Generally the same techniques that are employed 
for immobilizing enzymes are useful for immobilizing pseudo-subtrate, 
subject to the qualification that the bridge must be one that permits the 
complex to form between the immobilized pseudo-substrate and the free 
conjugate. Thus the carrier may be in the form of alumina granules, 
polystyrene beads, porous glass beads, charcoal granules, glass fibers, 
and the like. Similarly, organic carriers such as Sephadex and agarose are 
useful. 
In one preferred form of immobilized pseudo-substrate, 
agarose-aminocaproyl-beta-D-galactosylamine is employed as a solid phase 
enzyme affinity complex. 
Enzyme Assay 
Turning now to a consideration of the determination of the amount of active 
enzyme, assaying for enzymatic activity is well established for a wide 
variety of enzymes. A wide diversity of media, conditions and substrates 
have been determined for measuring enzymatic activity. See, for example, 
Bergmeyer, Methods for Enzymatic Analysis, Academic Press, New York, 1965. 
Since there are differences, not only between assays for different 
enzymes, but even in the variety of assays for a particular enzyme, no 
general description of the assay techniques is possible, but none is 
needed for those skilled in the art. 
Specific Examples 
The invention will now be described further by detailed descriptions of 
several specific demonstrations of assays conducted in accordance with the 
invention. In these examples, and elsewhere throughout this application, 
all references to parts are to parts by weight, and all temperatures are 
in degrees Celsius, unless specifically stated otherwise. 
EXAMPLE 1 
Steric Hindrance Enzyme Immunoassay for Choriomammotropin (HPL) 
A. Introduction 
The theoretical basis for this assay is illustrated in FIGS. 1A-1D 
inclusive, and these figures are self-explanatory. FIG. 1E is a 
diagrammatic representation of the assay that was demonstrated in this 
example, except that the ligand is identified as an "antigen", whereas in 
this example, it was in fact the hormone, choriomammotropin. For 
simplicity of illustration and ease of comprehension, FIG. 1E is an 
over-simplified representation. For example, the reaction mixture would 
actually contain some antigen-antibody complex, but this is not shown in 
the drawing, since in essence it plays no active part in the assay because 
of the use of a standard curve, as explained hereafter. 
B. Materials 
Commercial materials were used if available. This was the case with the 
hormone choriomammotropin (HPL), the enzyme beta-galactosidase from E. 
coli, and certain chemicals whose use is described hereafter: 
meta-aminobenzoic acid, benzaldehyde, and maleic anhydride. 
The rabbit anti-HPL antiserum was prepared and characterized as reported in 
Castro et al., Clin Chem. 22: 1655-1658 (1976), and following the 
procedure described there, its affinity constant was calculated to be 
3.1.times.10.sup.9. 
C. Conjugate Preparation 
To make the hormone-enzyme conjugate, 
m-maleimidobenzoyl-N-hydroxysuccinamide (MBS) was first synthesized by the 
procedure of Kitagawa et al., supra, for use in coupling HPL to the enzyme 
through a maleimidobenzoyl bridge. 75 .mu.l of solution of MBS (5 mg/ml, 
1.2 moles) in tetrahydrofuran was added to 2 ml phosphate buffer (pH 7.0, 
0.05 M, buffer A) containing 15.4 mg of HPL (0.66 moles). The mixture was 
incubated for 30 minutes at room temperature. One ml of citrate-phosphate 
buffer (pH 5.0, 1 M) was then added and the precipitate formed was 
collected by centrifugation at 2,000.times.g for 15 minutes. The 
precipitate was washed twice with 0.01 M citrate buffer (pH 5.3, 2 
ml.times.2). 
The washed precipitate was then redissolved in buffer A. One ml of 
beta-galactosidase dissolved in buffer A was then added to the HPL-MBS 
solution and incubated for 2 hours at room temperature. No appreciable 
reduction in enzyme activity was observed during the incubation. The 
reaction mixture was then directly chromatographed on a Sephadex G-75 
column (1.8.times.33 cm) using buffer A as the eluting buffer. 
The fractions of eluate containing the peak of enzyme activity were further 
purified by affinity chromatography, to remove overconjugated enzyme and 
enzyme which had not bound to inhibitor. The enzyme-conjugate solution was 
passed through a column (1.times.3 cm) containing agarose-6-aminocaproyl 
beta-D-galactosylamine (immobilized pseudo-substrate) which had been 
washed extensively with buffer A. After passing the conjugate solution 
into the column, the column was again washed extensively with buffer A. 
When no more enzyme activity was detected in the eluate, the column was 
eluted with borate buffer (pH 10.0, 0.1 M). The column contents consisted 
of particles of the immobilized pseudo-substrate to which the 
hormone-enzyme conjugate was immobilized. 
The fractions containing the major peak of enzyme activity were collected 
and then dialyzed overnight in one liter of buffer A. After dialysis, 
0.05% NaN.sub.3 and 2 .mu.l/ml of 1 M MgCl.sub.2 were added and stored at 
4.degree. C. Measurement of beta-galactosidase activity was done by the 
method of Dray et al., Biochem. Biophys. Acta 403, 131-138 (1975) using 
o-nitrophenyl-beta-D-galactopyranoside (ONGP) as the enzyme substrate. 
To determine the relative amount of enzyme that was conjugated with HPL, 
the conjugate was mixed with an excess of rabbit anti-HPL antibody. Goat 
anti-rabbit gamma-globulin (Calbiochem, La Jolla, Calif. 92037) was then 
added in excess. The resulting precipitate contained more than 90% of the 
enzyme activity initially present. A control with normal rabbit serum in 
place of the antibody gave no detectable enzyme activity in the 
precipitate. 
D. Affinity Gel Preparation 
One of the best inhibitors for beta-galactosidase isolated from E. coli is 
beta-D-galactosylamine (K.sub.I =0.225 mM), Lai, et al., Biophys. Res. 
Comm. 54: 463-468 (1973). This inhibitor is a potent competitive 
inhibitor. Agarose attached 6-aminocaproyl derivative of galactosylamine 
has been used widely in affinity chromatography for the purification of 
beta-galactosidase from various sources, Harpaz, et al., Biochem. Biophys. 
Acta. 341: 213-221 (1974). 
It is known that beta-galactosidase will not bind to an inhibitor unless a 
long arm (at least 6 carbon chain) is used to increase the distance 
between the beta-D-galactosylamine and the agarose back bone, indicating 
rather strict physicochemical requirement for their bindings, Steers, et 
al., supra. We utilized the hindrance of this rigid steric requirement by 
antibody to develop the enzyme immunoassay. We have employed the batch 
method to separate antibody bound conjugate and unbound or free conjugate 
in this example. 
E. Assay Procedure 
To each of an array of glass test tubes (16.times.10 mm) 150 .mu.l of 
buffer A was added, followed by addition of 5 .mu.l of each of several 
standard solutions containing respectively 0, 2, 4, 6 and 10 mg/l of HPL 
in 5% BSA solution. One hundred .mu.l each of the HPL-enzyme conjugate of 
a dilution characterized by absorbance of 0.6-0.8 at 420 nm and of 0.5% 
BSA were then added successively. After mixing, 100 .mu.l of 1:1,000 
dilution of anti-serum was added to each tube, which was then incubated 
for 1 hour at 4.degree. C. 
One hundred .mu.l of buffer A containing 5 .mu.l (wet volume) of washed 
agarose-6-aminocaproyl beta-D-galactosylamine particles (the affinity gel) 
was then added to each tube. The tubes were then placed in a shaker 
operated at 100 rpm, for 60 minutes. 
Following incubation in the shaker, the test tubes were centrifuged for 10 
minutes at 2,000.times.g and 250 .mu.l of the supernatant liquid was taken 
from each tube and assayed for enzyme activity, by adding the substrate 
ONGP and observing the intensity of any color change. 
The assay showed a maximum sensitivity of 4 ng/tube. In a standard curve 
produced from the data generated by the procedure described above, as 
represented in FIG. 2 of the drawings, the assay system was selected to 
cover a range of 0-10 mg/l with maximum sensitivity between 0-4 mg/l using 
the 5 .mu.l sample size. 
The observations were as follows: 
TABLE 3 
______________________________________ 
Standardized Solution 
of HPL Containing, 
Observation, Plotted In FIG. 2 
in mg/l % B/B.sub.o as Point 
______________________________________ 
0 100 K 
2 65 L 
4 40 M 
6 25 N 
10 20 O 
______________________________________ 
In the center column of the caption in Table 3 above, % B/B.sub.o is a 
percentage ratio. B is the amount of conjugate that is bound to anti-HPL 
antibody in the presence of the different respective amounts of the 
standards, as determined by absorbance at 420 nm. B.sub.o is the value 
determined by absorbance at 420 nm with the sample to which no HPL was 
added. Actually, each point plotted in FIG. 2 represents the mean 
value.+-.S.E. from four replicate runs. 
To use the standard curve to make assays on serum containing an unknown but 
generally suspected amount of HPL, best results are obtained if the 
unknown contains an amount of HPL that generates a reading on the most 
sensitive part of the curve, i.e., preferably that portion of the curve 
falling between points K and M of FIG. 1. 
Generally the amount of HPL to be found in a blood sample of an expectant 
mother can be predicted in a rough way. If necessary the blood sample can 
be adjusted in concentration by dilution, so that the assay result will 
fall within the desired section of the standard curve. 
As those skilled in the art will understand, other standard curves can be 
generated by using different concentrations. With each standard curve, 
however, the assay procedure is the same. The assay is run to establish a 
B value for the unknown, a % B/B.sub.o value is calculated, and the 
standard curve is then used to read off a concentration value of HPL for 
the unknown. Generally such an assay can be completed in 21/2-3 hours. 
The effect of incubation time upon the formation of an insoluble complex 
between the HPL-enzyme conjugate and the affinity gel is illustrated in 
the several curves in the graph of FIG. 3. 
The ratio of HPL to the enzyme was not directly determined. The ratios 
reported hereafter are the expected values calculated from the results 
obtained by Kitogawa et al., supra. The conjugates in each case were 
purified by affinity chromatography prior to use. 
In order to precipitate the enzyme from solution, particles of affinity gel 
were added to the solution and incubated in a shaker (100 rpm) for 30, 60, 
90 and 120 minutes. The solid phase was then precipitated by 
centrifugation, and the enzyme activity in the supernatant was examined. 
The results showed that only 5% of the added enzyme had become bound to 5 
.mu.l (wet volume) per tube of affinity gel with 60 minutes or more of 
incubation (FIG. 3). 
In FIG. 3, Curve A reports the results obtained with a conjugate above, 
with an expected 2:1 ratio of HPL to enzyme; Curve B, with a conjugate 
with a 2:1 ratio, incubated for one hour with antibody to HPL prior to the 
separation step; Curve C, with a conjugate, alone, with an 8:1 ratio, and 
Curve D, with a conjugate with an 8:1 ratio, incubated for one hour with 
antibody to HPL, prior to the separation step. 
Prior to purification of the enzyme conjugate using the affinity column, 
when the conjugation of HPL to beta-galactosidase was carried out at 
different molar ratios, the enzyme activity remaining in the supernatant, 
following incubation with excess affinity gel and centrifugation, was 
found to depend on the molar ratio employed of enzyme to HPL, in preparing 
the conjugate. With an expected HPL to enzyme ratio in the conjugate of 
8:1 or 12:1, about 30% or 50%, respectively, of the total enzyme activity 
remained in the supernatant, while a reduction in the expected molar ratio 
to 4:1 or less resulted in about 10% remaining in the supernatant (FIG. 
4). This inhibition of the binding of conjugate to the affinity gel 
suggested a possible steric hindrance on binding, by the HPL itself. When 
these conjugates were purified by the affinity column, however, the enzyme 
activity remaining in the supernatant was reduced to about 5%, suggesting 
the removal of overconjugated enzyme (FIG. 4). 
The effect of the molar ratio of HPL to enzyme, on complex formation with 
the affinity gel, is illustrated in the bar chart of FIG. 4. Bars R, 
R.sub.1, R.sub.2 and R.sub.3 illustrate the situations observed in cases 
where the enzyme conjugate was incubated with the affinity gel without 
having undergone purification by affinity chromatography. Bars T and 
T.sub.1 illustrate the observations made when the conjugate had been 
purified by affinity chromatography. Bars S, S.sub.1, S.sub.2 and S.sub.3 
illustrate the results observed when the conjugate, without purification 
by affinity chromatography, was incubated for one hour with antibody to 
HPL, then with the affinity gel. The readings on enzyme activity in each 
case were taken on the supernatant liquid, after separation from the 
affinity gel. Finally, bars V and V.sub.1 illustrate observed results 
where the conjugate was purified by affinity chromatography, then 
incubated for one hour with antibody to HPL, prior to separation of the 
supernatant from the affinity gel. 
In summary, we have developed, and demonstrated in this example, a new 
method for the separation of antibody bound and unbound enzyme conjugates. 
The technique as applied to the assay of choriomammotropin involves the 
use of beta-D-galactosylamine bound to agarose to separate the unbound 
choriomammotropin-beta-galactosidase conjugates from antibody bound 
conjugates. When beta-galactosidase was conjugated with choriomammotropin 
using the N-hydroxy-succinamide ester of m-maleimidobenzoic acid, the 
affinity of the enzyme conjugate to beta-D-galactosylamine attached to 
agarose diminished markedly following incubation with antibody. In a 
typical enzyme immunoassay of choriomammotropin, as described in this 
example, 5 .mu.l of swelled affinity gel per tube was required to 
precipitate unbound enzyme following one hour of gentle shaking at room 
temperature. Choriomammotropin antibody was used at a titer of 1:1,000. 
The standard curve for the assay (FIG. 2) was developed to cover a range 
of 0-10 mg/l with maximum sensitivity between 1-4 mg/l. 
EXAMPLE 2 
Assay for Thyroxine (T-4) 
A. Conjugate Preparation 
As an initial step, a solution of beta-galactosidase in 0.05 M phosphate 
buffer was dialyzed to remove ammonium sulfate from the enzyme. 
A solution was prepared at 0.2 mg/ml of m-maleimidobenzoyl-L-thyroxine 
methyl ester (MBTM) in the dialyzed enzyme solution, and this was 
subjected to further dialysis. 
This dialyzed material was subjected to chromatography with Sephadex G-25, 
with the recovery of several aliquots. Tests on each of these aliquots 
with the substrate ONGP in BSA buffer at 0.8 mg/ml indicated those 
aliquots containing enzyme activity. The purpose of the chromatography was 
to eliminate the unbound MBTM from the enzyme-MBTM conjugate solution. 
The T.sub.4 -enzyme conjugate was further purified by affinity 
chromatography using the same technique described in Ex. 1, to remove 
enzyme which had not bound to inhibitor. 
B. Assay Procedure 
To each of several glass test tubes (12 mm.times.75) was added 150 .mu.l of 
buffer A, followed by the addition to each 0.1 ml of the T-4-enzyme 
conjugate, followed in turn by the addition to each of 0.1 L ml of a 
different standard solution respectively. The standard solutions contained 
0, 2.5, 5.0, 10 and 20 .mu.g/dl of T-4 in 5% BSA solution. 
After mixing, 0.1 ml of a 1:400 dilution of anti-T-4 antiserum was added to 
each test tube, followed by 0.4 ml of phosphate buffer. The assay mixture 
was then incubated for one hour. 
A suspension of the same immobilized pseudo-substrate as in Ex. 1 then 
added to each tube. Each tube was then shaken for 60 minutes. The tubes 
were then centrifuged for 10 minutes at 2,000 rpm, and the supernatant 
liquids respectively were assayed for enzyme activity. 
The observations were as follows: 
TABLE 4 
______________________________________ 
Standardized Solution of T.sub.4 
Containing, in ug/dl 
Observation, % B/B.sub.o 
______________________________________ 
0 100 
2.5 87 
5.0 72 
10 63 
20 53 
______________________________________ 
These data were used to generate the standard curve shown in FIG. 2 of the 
drawings. It could then be used for individual sample assays in the same 
fashion as the HPL standard curve in Ex. 1. 
EXAMPLE 3 
Assay for Hepatitis B Surface Antigen 
The hepatitis B surface antigen is a component of hepatitis B virus and its 
associated antibodies. It is a relatively large molecule. Its detection 
and determination are important because the transmission of the hepatitis 
virus via blood donors constitutes a significant public health risk. 
One recent proposal for an assay for hepatitis antigens and their 
associated antibodies is discussed in U.S. Pat. No. 4,016,043. This assay 
is based on a competition reaction where one of the reactants is bonded to 
an insoluble carrier material. 
Our procedure is different and parallels that used in Exs. 1 and 2 hereof 
and makes use of our novel separation technique. The assay procedure 
should involve adding the test sample, i.e., human serum or plasma, to a 
solution of a given amount of an antigen-enzyme conjugate. The given 
amount should be selected so that it is preferably at least 50% in excess 
over that required to react fully with a known quantity of antibody 
against hepatitis B surface antigen, which should also be added. As in 
Exs. 1 and 2, the conjugate reactant should be carefully purified, and a 
standard curve developed. 
After incubation, passage of the resulting reaction mixture over the 
affinity gel, or admixture with it if granules or beads are used, will 
precipitate out conjugate that is not bound to antibody. After separation 
from the precipitate, a determination of enzyme activity on the liquid 
will produce a value that can be applied to the standard curve to provide 
an indication of the presence and concentration of the antigen in the 
serum. 
EXAMPLE 4 
Direct Assay for Antibodies Against Hepatitis B 
Surface Antigen in Human Serum or PLasma 
To check serum after vaccination, for example, a different assay procedure 
is needed than that of Exs. 1, 2 and 3. It makes use of our novel 
separation process. 
In this procedure, for example, in the detection of antibodies against 
hepatitis B surface antigen, the serum or plasma sample will be suspected 
of containing antibody against hepatitis B surface virus. The conjugate 
employed should be a conjugate of (1), the antigen that is immunoreactant 
with this antibody, with (2), enzyme, preferably betagalactosidase. A 
standard curve should be prepared from a serum sample containing a given 
or known amount of antibodies against hepatitis B antigens using just 
these two reactants, i.e., the sample (containing the antibodies) and the 
antigen-enzyme conjugate. Once the standard curve has been constructed, it 
is used in the same fashion as in Ex. 1. However, in making the assay, the 
only reactants that are essential are: the unknown, containing antibody 
material; the antigen-enzyme conjugate, furnishing a given amount of 
antigenic activity, i.e., a given amount of immunochemical reactivity as 
to its binding partner, the antibody material in the unknown; the affinity 
gel; and whatever is needed to detect and/or determine enzyme activity in 
either the supernatant or in the precipitate on the affinity gel. 
In this case, the immunochemical reactant activity in the conjugate should 
be selected always to be greater than that expected in the unknown. Then, 
the activity found in the supernatant should indicate, by differing from 
the original given amount, the amount of antibody material present in the 
unknown. 
This simplified assay procedure may be used wherever the antibody material 
to be detected and/or determined is very specific, as is the case with the 
antibody for each of rubella virus, hepatitis B surface antigen, and 
gonorrhea. 
EXAMPLE 5 
Competitive Assays for More General Immunoglobulins 
The assay technique for use in detecting immunoglobulins may be a simple 
variation on the assay procedures of Exs. 1 and 2. Thus, to detect and 
determine IgG, IgM, IgE, and IgA, the assay procedure of Ex. 1 should be 
modified. 
The conjugate employed should be a conjugate of the particular 
immunoglobulin with enzyme. The receptor added to the incubated mixture of 
serum sample and conjugate should be antibody to the particular 
immunoglobulin to be detected. The affinity gel employed may be the same 
as in Ex. 1. 
A standard curve must be prepared as in Ex. 1, but adjusted for appropriate 
standard solution concentrations. The standard curve may then be used in 
the same way as that in Ex. 1. Also, the procedural steps to be followed 
are essentially the same as those of Ex. 1. This provides a very sensitive 
assay. 
General 
The assay procedures of the present invention exhibit good separation, 
provide good sensitivity, and have unusual reliability for both small and 
large molecules, and for viruses as well. They can be applied to 
infectious disease detection and will be useful in providing and in 
developing new assays for parasites which will be important in developing 
countries. 
Commercially the assay procedure will be made available through the 
manufacture and sale of assay kits with instructions, as well as through 
commercial laboratories. 
Kit for Competitive Assay Procedures 
In one such embodiment of the invention, the kit will be one for detecting 
the presence of ligand such as an antigen, hormone, steroid, or hapten, in 
a medium suspected of containing it. Such a kit will include all necessary 
supplies of the following components, preferably at concentrations and in 
amounts useful in carrying out the assay procedure, or easily dilutable to 
be such: 
(1), ligand-enzyme conjugate, where the ligand is the same as or the 
immunochemical equivalent of that for which the assay procedure is to be 
conducted; 
(2), receptor for the ligand, that is, an entity that can specifically bind 
to the ligand moiety of the conjugate and also to the ligand in the sample 
of the liquid on which the assay is to be conducted; and 
(3), immobilized, insoluble pseudo-substrate for said enzyme, to which the 
enzyme normally binds. 
The receptor must be characterized by the ability, when bound to the ligand 
moiety of the conjugate, to inhibit the ability of the enzyme moiety of 
the conjugate to bind to the insoluble, immobilized pseudo-substrate. 
The enzyme will preferably be selected from the group consisting of 
horse-radish peroxidase, alkaline phosphatase, beta-D-galactosidase, 
glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, 
lysozyme, malate dehydrogenase, and glucose-6-phosphate dehydrogenase. The 
most preferred enzyme is beta-galactosidase. Generally, to form the 
enzyme-ligand conjugate, the enzyme will be bound to the ligand by a 
cross-linker. When the enzyme is beta-galactosidase, the immobilized 
pseudo-substrate will preferably be formed from beta-D-galactosylamine 
that is covalently coupled to insoluble particles of a carrier substance, 
preferably agarose, that is inert to the assay procedure, through a bridge 
that contains at least six carbons. 
In one preferred embodiment, where the assay is for HPL, the enzyme is 
beta-galactosidase; the conjugate is HPL coupled to the enzyme by MBS as 
in Ex. 1, in as close to a 12 to 1 molar ratio as is feasible; the 
pseudo-substrate is beta-D-galactosylamine, and the receptor is an 
anti-choriomammotropin antibody. To be complete, such a kit should also 
include enzyme substrate, and printed instructions for carrying out the 
assay, as outlined above and as described in detail in Ex. 1. In many 
cases, these instructions will include one or more standard curves. For 
this preferred embodiment, the preferred enzyme substrate is ONGP. 
For a kit to detect the presence of thyroxine, preferably the enzyme is 
beta-galactosidase, the conjugate consists of T-4 coupled to the enzyme as 
in Ex. 2, the pseudo-substrate is beta-D-galactosylamine, and the receptor 
is an anti-thyroxine antibody. The preferred enzyme substrate is again 
ONGP. 
The kit can be modified readily for use in the detection of general 
antibodies, such as, for example, IgG, IgM, IgE, or IgA. In this case the 
general antibody is the ligand, and the receptor is antibody to the 
general antibody, that is, an antibody to the particular immunoglobulin 
that is to be detected. 
Kit for Direct Assay Procedures 
In another embodiment, the kit may be designed for carrying out an assay 
procedure to detect the presence, if any, of a specific antibody in a 
medium suspected of containing it. 
Such a kit will include supplies of the following components, at 
concentrations and in amounts useful in carrying out the assay procedure: 
(1), a conjugate of an enzyme with the antigen or immunochemical equivalent 
thereof for the suspected specific antibody; 
(2), insoluble, immobilized pseudo-substrate for said enzyme, to which said 
enzyme normally binds, and 
(3), enzyme substrate that generates a color reaction when exposed to the 
enzyme or the enzyme moiety of the conjugate. 
In this case, the antibody is characterized by the ability, when bound to 
the antigen moiety of the antigen-enzyme conjugate, to inhibit the ability 
of the enzyme moiety of the conjugate to bind to the insoluble, 
immobilized pseudo-substrate. The reading of the assay result is made on 
liquid separated from the immobilized pseudo-substrate, preferably with an 
indicator-type of substrate for the enzyme, which may also be a part of 
the kit. Once again, ONGP is a preferred such substrate. 
Among the specific antibodies that may be detected in this way are those 
for rubella virus, hepatitis B surface antigen, and gonorrhea antigen. 
To be complete, this kit also must include printed instructions for 
carrying out the direct assay procedures, as outlined above and as 
described in detail in Ex. 4. These instructions preferably will include 
one or more standard curves. 
Conclusion 
The unknown typically is present in tremendous excess over that needed for 
a complete immunochemical reaction. Generally, the greater the excess, the 
more sensitive the assay. For a qualitative assay, it is possible for the 
conjugate to be in any relation to the unknown, preferably much less than 
is required for a complete immunochemical reaction. For a quantitative 
assay, the immunochemically reactive moiety of the conjugate must be 
present in at least an equal and preferably a greater amount than is 
needed for a complete immunochemical reaction with the receptor. The 
affinity gel is preferably always present to excess over that needed for 
completely reaction with the enzyme. 
In preparing the conjugate, the use of the proper molar ratio, and the 
purification of the conjugate by affinity chromatography as illustrated in 
Ex. 1, are very important to minimize the "noise" level of the assay. 
While the invention has been described in connection with specific 
embodiments thereof, it will be understood that it is capable of further 
modifications, and this application is intended to cover any variations, 
uses, or adaptations of the invention following, in general, the 
principles of the invention and including such departures from the present 
disclosure as come within known or customary practice within the art to 
which the invention pertains and as may be applied to the essential 
features hereinbefore set forth, and as fall within the scope of the 
appended claims.