Method for detecting enzymatic activity using particle agglutination

A method is disclosed for determining enzymatic activity in a liquid sample by particle agglutination or inhibition of particle agglutination.

TECHNICAL FIELD 
This invention relates to particle agglutination based diagnostic methods 
for detecting enzymatic activity in liquid test samples. 
BACKGROUND ART 
It is known that the reaction of an enzyme with its substrate generates a 
product, The enzyme is a true catalyst because it is not consumed in the 
reaction, but is free to generate repetitively more product. The rate at 
which the product is generated is referred to as the turnover number, a 
number that varies for different enzymes. In practice, enzymatic activity 
is most conveniently detected by monitoring a spectrophotometric 
absorbance of the substrate or product that varies as a result of 
enzymatic turnover. It is known that some naturally occurring substrates 
and/or their corresponding products do not possess readily utilizable 
absorbance peaks, making the spectrophotometric detection of enzymatic 
activity difficult. In some cases a synthetic substrate can be designed 
for the detection of enzymatic activity. 
Synthetic substrates can be designed to be chromogenic or fluorogenic, 
i.e., when catalyzed by the enzyme, an optically detectable change in the 
substrate and/or product is produced. The most significant limitation to 
the use of synthetic substrates to detect enzymatic activity is that such 
substrates cannot always be prepared for the desired enzyme. Detailed 
knowledge is required of the catalytic properties of each specific enzyme 
in order to properly design a useful synthetic substrate. For example, 
synthetic chromogenic substrates for thrombin or Factor X.sub.a have been 
designed for the detection of thrombin or Factor X.sub.a activity. These 
synthetic substrates replace the natural substrate, fibrinogen, in 
chromogenically based assays for these enzymes. 
Immunoassays for the detection of analytes are known. The so-called 
heterogeneous immunoassays can involve an incubation of an antibody with 
the analyte in the presence of a labeled antigen of substantially 
identical immunological properties vis-a-vis the antibody as the analyte. 
The amount of labeled antigen bound to the antibody or free in solution is 
determined after an appropriate separation procedure, as a measure of the 
amount of analyte present. The first such assay was described by R. S. 
Yalow and S. A. Berson in 1959 (Nature 184:1648). This assay, called a 
radioimmunoassay, utilized a radionuclide as the label. The use of enzymes 
as labels to replace the radionuclide with the latter's self-evident 
storage, handling and safety problems, was described by van Weeman and 
Schuurs in 1971 (FEBS Letters, Volume 15, 232 (1971) and U.S. Pat. No. 
3,791,932, issued Feb. 12, 1974 on an application filed Jan. 27, 1972). 
The art has seen numerous variations on the basic theme of van Weemen and 
Schuurs. 
It is known that multiepitopic antigenic substances can agglutinate 
particles with multiple epitope receptors to produce agglomerates, e.g., 
influenza virus and sheep erythrocytes, respectively. These agglomerates 
can be detected using light scattering type measurements. 
Agglutination-inhibition assays are known for the detection of antigens 
and haptens in liquid test samples. Typically, the binding of a 
multivalent antibody to highly refractive particles coated with the 
antigen or hapten is inhibited in a competitive fashion by the antigen or 
hapten in the test sample. (See Craig et al., U.S. Pat. No. 4,401,765 
issued Aug. 30, 1983.) These assays are generally limited in sensitivity 
to antigens and haptens in the concentration range of 10.sup.-7 to 
10.sup.-9 M. 
A continuing problem in immunoassays utilizing enzymes as labels is the 
inability to detect analytes at concentrations below 10.sup.- 10 M in a 
relatively short period of time, typically thirty minutes or less, 
preferably ten minutes or less. High turnover number enzymes have been 
used as labels to maximize signal production. Such enzymes include 
.beta.-galactosidase, horseradish peroxidase, alkaline phosphatase, 
glucose oxidase, .beta.-lactamase, and urease. The use of chromogenic 
substrates with these enzymes can provide sensitivity to about 10.sup.-10 
M. The use of fluorogenic substrates could lead to even greater 
sensitivity. However, all biological samples contain fluorescent material, 
e.g., porphyrins, which can interfere with the fluorogenic substrate 
measurements. This disadvantage can be overcome with additional sample 
processing, specifically, separating the bound from enzyme-labeled 
complex. Interfering fluorescent materials can be removed during this 
separation, with the constraint that measurements of only the bound 
enzyme-labeled complex can be made. 
High sensitivity immunoassays using enzyme require an undesirble amount of 
time for the generation of detectable levels of enzymatically generated 
chromophore or fluorophore, i.e., roughly thirty to ninety minutes to 
achieve optimum sensitivity, even with high turnover number enzymes and 
synthetic substrates. 
There is a continuing need in the art for a method to detect low levels of 
enzymatic activity in a test sample in a short amount of time, typically 
ten minutes or less. There is also a need for a method to measure 
enzymatic activity when no readily measurable product results from either 
natural or synthetic substrates. 
DISCLOSURE OF THE INVENTION 
This need is met by the present invention which, in a first aspect is an 
agglutination based method for detecting an enzyme in a liquid test 
sample, comprising: 
(1) forming an agglutination system comprising: 
(i) highly refractive particles having a ligand disposed thereon, 
(ii) a binding partner specific for the ligand and capable of binding to at 
least two ligands, the binding partner and ligand being present at 
concentrations which permit agglutination, and 
(iii) a substrate for the enzyme to be detected, the enzyme, substrate, 
ligand and binding partner being such that a reaction between the enzyme 
and substrate produces a product which competes with the ligand for the 
binding partner; 
(2) contacting the test sample suspected of containing the enzyme with the 
agglutination system to produce the product which competes with the ligand 
for the binding partner; 
(3) measuring a physical property of the agglutination system, which 
property is a function of agglutination; and 
(4) relating the measurement to the amount of enzyme initially present in 
the test sample. 
In another aspect, the present invention is an agglutination based method 
for detecting an enzyme in a liquid test sample, comprising: 
(1) forming an agglutination system comprising: 
(i) highly refractive particles having coated thereon a substrate capable 
of being converted into a ligand upon reaction with the enzyme to be 
detected; and 
(ii) a binding partner capable of binding to at least two ligands, but 
incapable of binding to the substrate; 
(2) contacting the test sample suspected of containing the enzyme with the 
agglutination system to convert the substrate into the ligand; 
(3) measuring a physical property of the agglutination system which is a 
function of agglutination; and 
(4) relating the measurement to the amount of enzyme initially present in 
the test sample. 
DETAILED DESCRIPTION OF THE INVENTION 
In an inhibition mode, the present invention makes use of an agglutination 
system which comprises highly refractive particles having a bindable 
substance, i.e., a ligand, on their surfaces. A binding partner 
specifically reactive with the ligand is reacted with the particles. The 
binding partner is multivalent, i.e., can bind with at least two ligands. 
The binding partner is used at a concentration which provides substantial 
agglutination of the highly refractive particles. Agglutination occurs 
because the multivalent binding partner can act as a cross-linking agent 
to link two particles together. The agglutination system also contains a 
substrate for the enzyme to be detected. The substrate, enzyme and ligand 
must be chosen to form an operational trio. Specifically, given the enzyme 
to be detected, the ligand must be chosen to have substantially identical 
properties vis-a-vis the binding partner as the product of the 
enzyme-substrate reaction. This condition assures that the product of the 
enzyme-substrate reaction will compete with the ligand for the binding 
partner, thus reducing the amount of binding partner available to 
participate in the agglutination reaction. In operation, the test sample 
is contacted with the agglutination system and the amount of subsequent 
agglutination inhibition determined in comparison with an enzyme-free 
control. In one aspect, the enzyme can be free in solution and the amount 
of enzyme can be determined by either direct agglutination or 
agglutination inhibition. Alternatively, the enzyme can be a label on an 
antibody molecule, antigen or hapten molecule, nucleic acid probe, etc., 
in which case the inhibition of agglutination is a direct measure of the 
substance on which it acts as a label. In one example of the latter case, 
the sample containing the analyte is contacted with a conjugate of the 
enzyme and a binding partner reactive with the analyte. The conjugate can 
be prepared prior to contact with the sample by convalent bonding between 
the enzyme and the substance on which it acts as a label. This conjugate 
is prepared by known methods that provide attachment of enzyme to the 
binding partner either directly or through an appropriate spacer arm, and 
which preserve both enzyme activity and the ability of the binding partner 
to bind its corresponding analyte. It is also possible to prepare 
appropriate conjugate by other means including the specific binding of 
avidin-labelled enzyme and biotin-labelled binding partner in instances 
when direct covalent attachment of enzyme to antibody is undesirable. This 
avidin/biotin labeling allows for preparation of conjugate formed by 
incubation of labeled enzyme and analyte binding partner prior to contact 
with the sample or during simultaneous contact with the sample. 
Alternatively, the conjugate of enzyme and binding partner can be formed 
in a sequential manner by adding sample, labeled binding partner, and 
labeled enzyme in any desired order. In all cases, the total number of 
binding sites provided by the binding partner is in molar excess relative 
to the analyte. A fraction of the conjugate binds to the analyte, and the 
remaining fraction remains free. Either the bound or the free conjugate is 
contacted with the agglutination system to inhibit agglutination. 
An experiment was carried out to demonstrate visually the unexpected 
improvement in enzyme activity detection when using the assay of this 
invention. Example 2 describes the visual detection of 
.beta.-galactosidase activity in a 3-minute interval using the 
agglutination inhibition mode of the invention. Using 
o-nitrophenyl-.beta.-D-galactopyranoside (ONPG) as the substrate, the 
yellow colored o-nitrophenolate anion product can be visually detected in 
3 minutes with .beta.-galactosidase concentrations down to 
2.times.10.sup.-2 IU/mL. However, when .beta.-galactosidase at 
2.times.10.sup.-3 IU/mL was used, no color was observable visually after 3 
minutes. When the agglutiation inhibition assay was used to detect the 
o-nitrophenol product, 2.times.10.sup.-3, 2.times.10.sup.-4 and 
2.times.10.sup.-5 IU/mL of .beta.-galactosidase showed visually 
non-turbid, partially turbid and fully turbid reaction mixtures, 
respectively. This shows that the assay of this invention allows visual 
detection down to between 2.times.10.sup.-4 and 2.times.10.sup.-5 IU/mL of 
.beta.-galactosidase in only 3 minutes. This is an approximately 100-fold 
improvement in the visual detection limit of this enzyme over that 
previously achievable with the prior art method. 
In a direct agglutination mode, the highly refractive particles have 
disposed on their surfaces the substrate for the enzyme, rather than the 
ligand. When the particles are reacted with the enzyme, the substrate is 
converted into the ligand. The binding partner is then able to cross-link 
separate particles to provide agglutination. Again, the enzyme can exist 
free in the test sample, in which case the agglutination phenomenon is a 
direct measure of the enzyme , or the enzyme can be the label on an 
antibody, antigen or hapten, nucleic acid probe, etc., in which case the 
agglutination phenomenon is an indirect measure of the substance on which 
it acts as a label. 
Enzymes which can be detected using the method of the present invention 
include, but are not limited to, .beta.-galactosidase, alkaline 
phosphatase,horseradish peroxidase, glucose oxidase, urease and 
.beta.-lactamase. When used as an indirect measure of a substance in a 
biological sample, such test samples can include blood, cerebrospinal 
fluid, serum, plasma, sputum, urine, nasal washings, genital and throat 
swabs, and other biological samples. 
Suitable highly refractive particles can be made from, for example, 
agarose, polydextran, polyacrylamide and polymeric latexes. Particle shape 
is not critical, although spherical particles are preferred because they 
are easiest to prepare and provide maximum lattice density in the 
agglutinated state. Particle size is somewhat critical. Preferred diameter 
for spherical particles is from about 30 nm to 100 nm for the 
agglutination inhibition mode. The most preferred particle is that 
described in U.S. Pat. No. 4,401,765, issued to Craig et al. on Aug. 30, 
1983 on an application filed Oct. 28, 1981. The disclosure of this patent 
is incorporated herein by reference. These particles have a highly 
refractive spherical polymer core preferably made of polyvinylnaphthalene 
and polystyrene. The core has disposed on its surface a reactive shell to 
which antigens, haptens, etc., can be covalently coupled. A convenient way 
to control particle size of the polymer particles is first to prepare a 
seed emulsion whose size can be controlled by the amount of surfactant 
used. After preparation of the seed emulsion, additional monomer and 
surfactant can be added at a controlled rate to increase the size of the 
particles in the seed emulsion. 
The outer shell polymer of the polymer particle can be prepared from a wide 
range of ethylenically unsaturated monomers having functional groups 
capable of reacting with compounds of biological interest. Optionally, the 
outer shell can also contain other ethylenically unsaturated monomers. The 
attachment of the shell polymer to the core can be accomplished by graft 
polymerization of the functional monomer to the residual ethylenically 
unsaturated groups in the core polymer or the functional monomer can be 
polymerized around the core to produce a contiguous shell. Preferred 
monomers include those containing an epoxy group such as glycidyl 
methacrylate, glycidyl acrylate, vinyl glycidyl ether, and methallyl 
glycidyl ether. Other functional groups include carboxyl, hydroxyl, amino, 
and aldehyde. 
Particles having diameters greater than 100 nm, preferably in the range of 
1000 nm to 100,000 nm can be prepared for visual agglutination tests 
according to methods previously described. These larger diameter particles 
are readily seen during agglutination reactions due to changes in their 
agglutination state. The direct agglutination mode is preferred for visual 
detection of particle aggregation because direct agglutination is easier 
to detect than agglutination inhibition. 
In the inhibition mode, the particles having the desired composition are 
prepared with appropriate ligands disposed on their surfaces. The ligand 
can be physically adsorbed or covalently attached (directly or through a 
so-called spacer arm). Methods for covalently attaching compounds to 
derivatized surfaces are well known. [Kiefer, Immunological Methods, 
Lefkovits & Perris, eds., New York: Academic Press, 1979, 137]In the 
direct agglutination mode, the particles have the substrate disposed on 
their surfaces. Methods of covalently attaching substrate are analogous to 
those for attaching ligand. 
To practice the agglutination inhibition mode of the present invention, the 
enzyme substrate reaction must produce a product which competes with the 
ligand for the binding partner. Additionally, the substrate must not be 
substantially reactive with the binding partner prior to enzymatic 
cleavage. For example, in the case of .beta.-galactosidase, a suitable 
substrate is ortho-nitrophenyl-.beta.-galactopyranoside (ONPG), and a 
suitable binding agent is an anti-ortho-nitrophenol or an 
anti-nitrohydroxybenzoic acid (NHB) antibody. When .beta.-galactosidase 
reacts with ortho-nitrophenyl-.beta.-galactopyranoside, the bond between 
the ortho-nitrophenyl group and the .beta.-galactopyranoside moiety is 
cleaved, yielding a free ortho-nitrophenol molecule. The antibody is 
reactive with the ortho-nitrophenol molecule, but not with 
ortho-nitrophenyl-.beta.-galactopyranoside. It is believed that the 
galactopyranoside group sterically prevents the binding of the 
antiortho-nitrophenol antibody with the ortho-nitrophenyl substituent 
(ONP). However, enzymatic cleavage of substrate produces a product which 
is capable of binding with the antibody. The required functional 
relationship between enzyme, substrate, ligand, and binding partner is 
further illustrated by the data in Table 1 which shows a comparison of 
agglutination rates provided by substrate and product. 
The data were generated by comparing the ability of ONPG and 3-nitro- 
4-hydroxybenzoic acid (NHB), an ONP analogue, to inhibit the agglutination 
of NHB-coated particles by an anti-NHB antibody. The data show that 
concentrations of NHB approximately three orders of magnitude below those 
of ortho-nitrophenylgalactopyranoside produce equivalent inhibition. NHB 
is the functional equivalent to the product resulting from hydrolysis of 
ONPG by .beta.-galactosidase. Therefore, there is no significant 
inhibition of agglutination absent the enzymatic cleavage of substrate. 
TABLE 1 
______________________________________ 
Agglutination Inhibition of 
NHB-bearing Particles by 
Enzyme Substrate (ONPG) and Product (NHB) 
ONPG or NHB Agglutination Rate 
Concentration (mA/minute; 405 nm) 
(Mole/liter) NHB ONPG 
______________________________________ 
0 .sup. 353 387 
10.sup.-9 331 405 
10.sup.-8 243 411 
10.sup.-7 102 408 
10.sup.-6 18 384 
10.sup.-5 0 360 
10.sup.-4 0 178 
10.sup.-3 0 24 
______________________________________ 
It will be appreciated by those skilled in the art that to demonstrate a 
maximally sensitive quantitative assay system with any operational trio of 
substrate, enzyme, and ligand, certain requirements must be met. The 
substrate must be in molar excess relative to the anticipated 
concentration of enzyme. Typically, substrate concentration is on the 
order of 5 to 10 K.sub.m (Michaelis constant). This requirement generally 
insures that first-order kinetics are observed with respect to substrate, 
and that reaction rate is not limited by insufficient subsrate 
concentration. When the enzyme of interest occurs in the assay as a 
reagent, the determination of appropriate substrate level is relatively 
straight-forward because the enzyme will occur in the system over an 
expected concentration range. When the enzyme of interest is the assay 
analyte and is also rare or difficult to isolate, a more empirical 
approximation of appropriate substrate level may be required to maintain 
favorable kinetics for a quantitative assay. The constraints in the 
relationship of enzyme and substrate levels just discussed are not as 
critical for qualitative results where the presence or absence of enzyme 
may be of interest. 
Another important consideration is the degree of cross-reactivity of 
binding partner with substrate. The selected level of substrate that 
insures first-order kinetics in quantitative and semi-quantitative 
systems, also insures that substrate will occur in much greater 
concentration than product being produced by enzyme activity. The law of 
mass action would suggest binding of binding partner to substrate if 
significant cross-reactivity existed. This would result in significant 
background agglutination in direct agglutination systems, and unacceptable 
levels of non-specific inhibition of agglutination in inhibition systems. 
Therefore, to achieve maximal assay sensitivity, binding partner is 
selected with little or no cross-reactivity with substrate when at least a 
2 to 3 order of magnitude concentration difference exists between 
substrate and product. This requirement provides that in direct 
agglutination systems, very low levels of product can be detected, and in 
inhibition systems, very low levels of product compete effectively with 
particle-bound ligand for binding partner combining sites. In both 
systems, a noticeable change in particle agglutiation state can be 
observed with very low levels of product only when binding partner 
cross-reactivity is minimized. Under quantitative conditions for most 
enzymes, substrate concentrations will range from 10.sup.-3 to 10.sup.-6 
M, and product will range from approximately 10.sup.-6 to 10.sup.-10 M. 
In the direct agglutination mode which requires the substrate to be 
disposed on the surface of the highly refractive particle, it is important 
that the cleavable bond not be sterically hindered from the enzyme by the 
particle itself. The use of a so-called spacer arm can provide sufficient 
distance between the cleavable bond and the particle surface to prevent 
hindrance. 
Preferred binding agents are antibody molecules. Antibodies are known to be 
highly specific for their respective antigens or haptens. In addition, 
antibodies are easily prepared and provide the multi-valency required for 
agglutination. Both monoclonal and polyclonal antibodies can be used in 
the present invention. 
Antisera or other body fluids such as ascites containing the anti-ligand 
antibody can be used directly in the agglutination system or can be 
purified to provide an immunoglobulin fraction, an IgG fraction, or an 
affinity purified IgG fraction, all of which can also be used as the 
binding partner in the agglutination system. It is also possible to use an 
IgM fraction. Fragments of IgG can also be used, e.g., F(ab').sub.2. 
Given the choice of a particular enzyme to be detected, the type of 
cleavable bond can be ascertained, e.g., for .beta.-galactosidase, the 
bond is a .beta.(1-4) ether linkage between .beta.-galactopyranoside and 
another sugar (glucose or fructose) or a suitable substitute such as ONP. 
A natural substrate containing the bond can be identified or a synthetic 
substrate containing the bond can be devised. One can then determine the 
chemical identity of the product(s) resulting from the reaction of the 
enzyme and substrate, which in turn provides the identity of suitable 
binding partner and ligand that must be chosen to form an operative 
system. Specifically, the reaction of enzyme with substrate must produce a 
product which can compete with the ligand for the binding partner. 
Examples of operative systems of enzyme, substrate, ligand and binding 
partner are shown in the table below: 
TABLE 2 
__________________________________________________________________________ 
Enzyme Substrate Ligand Binding Agent 
__________________________________________________________________________ 
.beta.-galactosidase 
ONP--.beta.-galactopyranoside 
Ortho-nitrophenol 
Anti-ortho-nitrophenol 
Alkaline phosphatase 
-p-nitrophenyl-phosphate 
para-nitrophenol 
Anti-para-nitrophenol 
Tyrosine-amino-transferase 
tyrosine -p-hydroxyphenyl- 
Anti- -p-hydroxy- 
pyruvic acid 
phenylpyruvic acid 
.gamma.-oxoglutarate 
glutamic acid 
Anti-glutamic acid 
Tyrosine decarboxylase 
tyrosine tyramine Anti-tyramine 
Biotinyl-CoA--Synthetase 
biotin-CoA--ATP 
biotin-C--S--CoA 
Anti-thioacetyl-CoA 
.beta.-galactosidase 
galacto-pyranosyl derivatives 
thyroxine Thyroxine 
of aromatic hydroxyl Binding 
group of thyroxine Globulin 
Acetylcholine-sterase 
biotinylcholine 
biotin avidin 
__________________________________________________________________________ 
Preferred systems are: 
(1) .beta.-galactosidase, ONP-.beta.-galactopyranoside, ortho-nitrophenol 
and anti-ortho-nitrophenol antibody; and 
(2) alkaline phosphatase, p-nitrophenyl-phosphate, paranitrophenol and 
anti-para-nitrophenol antibody. 
As can be seen from the table above, the substrate must provide at least 
one bond which can be cleaved by the enzyme to yield at least one product 
which is reactive with the binding partner. One of the products will be 
chosen to serve as the ligand. In general, the ligand will be a hapten, 
i.e., a small molecule which can be bound by an anti-hapten antibody, but 
which cannot directly elicit an immune response. The ligand can be coupled 
by known methods to a suitable high molecular weight carrier to form an 
appropriate immunogen. The immunogen can be injected into an 
immunocompetent animal to elicit an anti-hapten immune response. The 
immunized animal may then provide suitable antiserum after an appropriate 
immunization schedule, or suitable immunosensitized cells for use in a 
recognized monoclonal antibody producing process such as that of Kohler 
and Milstein [Nature, 256:495, 1975]. 
Multivalent binding agents other than antibodies may be useable in the 
present invention, provided that for a given enzyme to be detected, a 
substrate can be designed to provide a cleavage product which can function 
as a ligand for the biasing agent. For example, thyroxine binding globulin 
(TBG) is known to bind thyroxine. If one desires to detect 
.beta.-galactosidase, a substrate comprising a covalent conjugate of 
thyroxine and galactopyranoside may be designed so that the reaction of 
the enzyme and substrate yields thyroxine which can be bound by TBG. It is 
believed that an ether linkage formed from the hydroxyl group of thyroxine 
and the hydroxyl group of the C.sub. 1 carbon of galactopyranoside will 
provide a suitable substrate. Other multivalent binding agents which may 
prove to be useful in the invention include C-reactive protein, avidin, 
and amyloid A protein. 
In either the direct agglutination or agglutination inhibition modes, the 
physical properties of the system, especially optical properties, will 
change with time. Examples of these detectable physical properties include 
absorbance at a given wavelength of incident light and the intensity of 
monochromatic light scattered at a selected angle relative to the incident 
beam. In addition, the size distribution of aggregated, highly refractive 
particles can be determined. 
Absorbance at a given wavelength can be measured using a standard 
spectrophotometer. Light scattering can be measured using a nephelometer. 
Size distribution of aggregarted, high refractive index particles can be 
measured using an optically-based or electrically-based particle counter. 
The preferred method is the measurement of absorbance at a selected 
wavelength chosen on the basis of high turbidimetric signal and low 
interference by additional components of the test system. For example, 
when the system of this invention comprises .beta.-galactosidase, 
ONP-.beta.-galactopyranoside, ortho-nitrophenol, and anti-nitrophenol 
antibody, the substrate has a significant absorbance maximum at 340 nm. 
Therefore, a source of interference would be created by the substrate if 
the reaction system were monitored at 340 nm, which is the optical 
wavelength for measuring the turbidimetric signal. As a result, the 
turbidimetric signal is monitored at 405 nm to diminish the absorbance 
contribution from the substrate. 
The invention is illustrated by the following nonlimiting examples.

EXAMPLE 1 
Spectrophotometric Measurement of .beta.-galactosidase Activity 
The following example illustrates the ability of the method provided by the 
present invention to measure as little as 2.5.times.10.sup.-4 units per mL 
of .beta.-galactosidase within four minutes. In the example, the highly 
refractive particle used was a polystyrene core with an intermediate shell 
of polyvinylnaphthalene and an outer shell of polyglycidyl methacrylate. 
The binding agent was an antibody prepared against the hapten 
3-nitro-4-hydroxybenzoic acid. The antibody cross-reacted significantly 
with ortho-nitrophenol. The ligand was 3-nitro-4-hydroxybenzoic acid, 
which was coupled covalently through the carboxyl group to the outer shell 
of polyglycidyl methacrylate on the highly refractive particles. The 
substrate for .beta.-galactosidase was 
ortho-nitrophenyl-.beta.-D-galactopyranoside. The method was performed in 
the agglutination inhibition mode. The rate of agglutination was 
determined by measuring the absorbance at 405 nm at two predetermined 
times. 
A. Synthesis of NHB:Protein Conjugates 
Keyhole limpet hemocyanin (KLH) (750 mg, Calbiochem-Behring) was dissolved 
in 10 mL of 0.05M sodium bicarbonate buffer (pH 9.0) and dialyzed 
exhaustively with the bicarbonate buffer. An analog of ONP, 
3-nitro-4-hydroxybenzoic acid (NHB), (64 mg), was dissolved in 5 mL of 
dimethyl formamide and cooled to 4.degree. C. To this solution was added 
81 mg of 1-ethyl3-(3-dimethylaminopropyl) carbodiimide and 43 mg of 
N-hydroxysuccinimide. The solution was stirred at 4.degree. C. for 18 
hours. The dialyzed KLH solution was then added to the dimethyl formamide 
solution, the pH was adjusted to 8.5 with 0.2N sodium hydroxide, and the 
solution was incubated at 4.degree. C. for 8 hours. The reaction mixture 
was then dialyzed with deionized water to remove unreacted materials and 
the conjugate was lyophilized prior to storage at 4.degree. C. 
A separate conjugate using bovine serum albumin (BSA) as the carrier 
protein was prepared in an identical procedure using 500 mg of BSA. 
B. Polyclonal Anti-NHB Antibodies 
Three rabbits (New Zealand White, female) were injected subcutaneously with 
0.25 mg of the NHB:KLH conjugate (synthesized in part A above) emulsified 
in 1.0 mL of complete Freund's adjuvant. Three booster injections were 
given at approximately 21-day intervals as described above using 
incomplete Freund's adjuvant. Prior to each booster injection, the rabbits 
were bled and the serum tested for o-nitrophenol cross-reactive antibodies 
by a particle-enhanced turbidimetric inhibition immunoassay as described 
in U.S. Pat. No. 4,401,765, using the polymer particle reagent described 
in part D below. 
C. Preparation of Polyvinylnaphthalene/Polyglycidyl 
Methacrylate Core/Shell Polymer Particles 
A polystyrene emulsion was prepared at room temperature (20.degree. C.) in 
a 4L Erlenmeyer flask. The following ingredients were added sequentially: 
400 g of Dupanol WAQE [a Du Pont grade of a 30% sodium dodecyl sulfate 
(SDS) solution]was added to 2.5 L of deionized water, followed by 50 mL of 
styrene, 20 g sodium metabisulfite, 10 g of potassium persulfate 
(dissolved in 200 mL of water) and 125 mL of a ferrous sulfate solution 
(0.6 g of ferrous sulfate heptahydrate, and 0.25 g of sulfuric acid 
dissolved in 500 mL of nitrogen purged deionized water). The emulsion 
mixture was stirred and blanketed with nitrogen. After 10 minutes, 25 g of 
Aerosol OT-100 (American Cyanamid Co., Wayne, N.J.) dissolved in 375 mL of 
styrene was added at a rate of 30 mL/min. The mixture was stirred 
overnight. A sample of the emulsion, diluted 1:100, had an optical density 
of 0.171 at 340 nm. 
Polyvinylnaphthalene was prepared in a 250 mL round-bottom flask equipped 
with a magnetic stirrer and a reflux condenser. The round-bottom flask was 
placed in a boiling water bath. To provide a polyvinylnaphthalene 
intermediate shell on polystyrene core particles, 1.8 mL of the 
polystyrene emulsion, prepared above, was added to 98 mL of water, and 
heated to 95.degree. C. This mixture was then added to 11.7 g of 
2-vinylnaphthalene (Aldrich Chemical Co., Milwaukee, WI., purified by 
sublimation and chromatography on basic alumina in a dichloromethane 
solution), 300 mg of sodium bicarbonate and 90 mg of potassium persulfate. 
As soon as the 2-vinylnaphthalene had melted, 4 mL of 10% sodiumdodecyl 
sulfate was fed into the mixture at a rate of 0.3 mL/min. One hour after 
the beginning of the SDS feed, the polymerization was complete. The 
optical density of the product measured at 340 nm was 0.155 after diluting 
1:5000 in water. The average particle diameter was determined to be 74 nm 
by electron microscopy. The conversion of monomer to polymer was found to 
be 99.6% by gas chromatographic determination of the residual monomer. 
An outer shell of polyglycidyl methacrylate was formed on the 
polyvinylnaphthalene/polystyrene particles. Polymerization of glycidyl 
methacrylate was carried out in the same apparatus as above. One hundred 
four mL of the polyvinylnaphthalene/polystyrene particles was heated in a 
boiling water bath. One hundred milligrams of potassium persulfate and 
2.06 mL of glycidyl methacrylate (Aldrich Chemical Co.) were added. After 
20 minutes, the mixture was cooled. The conversion of monomeric glycidyl 
methacrylate to polymer aws found to be 99.0% by determination of the 
unreacted monomer concentration. The optical density of the product 
measured at 340 nm was 0.154 after diluting 1:5000 in water. 
D. Attachment of NHB to Polymer Particles 
An analog of o-nitrophenol, 3-nitro- 4-hydroxy benzoic acid (NHB, 144 mg) 
and 1-ethyl- 3-(3-dimethylaminopropyl) carbodiimide (156 mg) were 
dissolved in 5 mL of dimethylsulfoxide and allowed to sit at room 
temperature for 2 hours. In a second 5 mL volume of dimethylsulfoxide was 
dissolved 420 mg of 2,2'-oxybis (ethylamine) dihydrochloride. The two 
solutions were then combined and allowed to incubate at room temperature 
for approximately 72 hours. 
The NHB conjugate solution (0.333 mL), prepared as described above, and 
0.06 mL of a 10% w/v GAFAC.RTM. RE- 610:water solution (GAFAC.RTM. RE- 610 
is an anionic surfactant from GAF Corp., NY) were diluted in 4.4 mL of 5 
mM sodium phosphate buffer (pH 8.0). The pH of the solution was adjusted 
to pH 10.0-10.1 and 1.2 mL of core/shell polymer particles (part C above) 
were added to the reaction. The mixture was heated to 70.degree. C. for 3 
hours and allowed to cool to room temperature. The particle reagent was 
diluted with 15 mM sodium phosphate buffer (pH 7.0) containing 0.1% 
GAFAC.RTM.. The particles were centrifuged for 90 minutes at 20,000 rpm to 
form a pellet, and the supernatant was discarded. The particles were 
resuspended in the phosphate/GAFAC.RTM. solution and recentrifuged. The 
centrifuge and resuspension process was repeated a total of four times to 
remove unreacted NHB conjugate. After the final wash, the particle reagent 
was brought to a total volume of 10 mL in the 15 mM sodium phosphate 
buffer (pH 7.0) containing 0.1% GAFAC.RTM.. 
E. .beta.-galactosidase Activity (Prior Art) 
All assays were performed at 37.degree. C. on the aca.RTM. discrete 
clinical analyzer (Du Pont Co., Wilmington, DE.). A stock solution of the 
enzyme .beta.-galactosidase (Sigma Chemical Co., St. Louis, MO.) was 
prepared at a concentration of 4.5.times.10.sup.3 units/mL based on the 
activity as determined by the manufacturer. Serial dilutions of the stock 
.beta.-galactosidase solution were prepared over the range 
4.5.times.10.sup.3 -4.5.times.10.sup.-3 units/mL. A 0.05 mL sample of each 
.beta.-galactosidase dilution was injected automatically into an aca.RTM. 
analytical test pack followed by 4.95 mL of a 0.15M phosphate buffer (pH 
7.8) containing 3% polyethylene glycol 8000, 0.1% GAFAC.RTM., and 2 mM 
magnesium chloride. The contents of the pack were then heated to 
37.degree. C., and the enzymatic reaction was initiated by the addition of 
o-nitrophenyl-.beta.-D-galactopyranoside (ONPG) to a final concentration 
of 0.1 mM. Enzymatic activity was determined by measuring the difference 
in the absorbance at 405 nm (rate of change) 29 seconds and 46 seconds 
after the ONPG addition. 
F. .beta.-galactosidase Activity Using the Agglutination 
Inhibition Mode of the Present Invention 
The .beta.-galactosidase serial dilutions prepared in part E above also 
were used for these experiments. A 0.05 mL sample of each 
.beta.-galactosidase dilution was automatically injected into an aca.RTM. 
analytical test pack followed by 4.95 mL of a 0.15M phosphate buffer (pH 
7.8) containing 3% polyethylene glycol 8000, 0.1% GAFAC.RTM., and 2 mM 
magnesium chloride. The contents of the pack were heated to 37.degree. C., 
and the reagents in the first four pack dimples were released. The 
reaction mixture at this point consisted of 0.1 mM ONPG and 0.016% solids 
of the NHB particle reagent synthesized in part D above. The turbidimetric 
inhibition reaction was initiated 3.5 minutes later by the addition of 
0.008 mL of rabbit anti-NHB antiserum prepared in part B above. The 
increase in turbidity due to particle aggregation was measured as the 
difference in the absorbance at 405 nm (rate of change) 29 seconds and 46 
seconds after antibody addition. Table 3 shows the data for 
.beta.-galactosidase activity comparing the prior art method and the 
method of this invention. 
The data show that the prior art method had a sensitivity of between 0.25 
and 2.5 units/mL, while the method provided by the present invention had a 
sensitivity of about 2.5.times.10.sup.-4 units/mL, which corresponds to 
almost a thousand-fold increase. 
TABLE 3 
______________________________________ 
.beta.-Galactosidase Activity Measurements 
.beta.-Galactosidase 
Activity in Assay 
Prior Art Present Invention 
Units/mL (mA/min at 405 nm) 
(mA/min at 405 nm) 
______________________________________ 
25 647 nd 
2.5 53 nd 
0.25 2 12 
0.025 0 56 
0.0025 0 151 
0.00025 0 184 
0 0 186 
______________________________________ 
nd = not determined 
EXAMPLE 2 
Measurement of .beta.-galactosidase Activity 
This example illustrates the ability of the method provided by the present 
invention to detect visually as little as 2.times.10.sup.-4 IU/mL of 
.beta.-galactosidase within three minutes. This example utilized a highly 
refractive polystyrene core-polyglycidyl methacrylate shell particle with 
covalently attached 3-nitro-4-hydroxybenzoic acid ligand, antibody 
cross-reactive with o-nitrophenol, and 
o-nitrophenyl-.beta.D-galactopyranoside substrate for .beta.-galactosidase 
to carry out the assay in the agglutination inhibition mode. 
A. Preparation of Polyglycidyl Methacrylate Core/Shell Polymer Particles 
A polystyrene emulsion was prepared at room temperature (20.degree. C.) in 
a 5-L round-bottomed flask. The following ingredients were added 
sequentially: 1750 mL of deionized water, 400 g of Dupanol WAQE, 50 mL of 
styrene filtered through an aluminum oxide column, 10 g of sodium 
meta-bisulfite (dissolved in 250 mL of deionized water), 10 g of potassium 
persulfate (dissolved in 200 mL of deionized water) and 125 mL of a 
ferrous sulfate solution (0.6 g of ferrous sulfate heptahydrate, and 0.136 
mL of concentrated sulfuric acid dissolved in 300 mL of nitrogen purged 
deionized water). The mixture was stirred and blanketed with nitrogen. 
After 10 minutes, 25 g of Aerosol OT-100 (American Cyanamid Co., Wayne, 
NJ) dissolved in 375 mL of styrene was added at a rate of 14 mL/min. The 
mixture was stirred overnight, filtered and stored. 
To prepare polystyrene particles, the following ingredients were added 
sequentially to a 500-mL Erlenmeyer flask: 160 mL of deionized water, 480 
mg of sodium bicarbonate, 160 mg of potassium persulfate, 12.8 mL of 
polystyrene emulsion (prepared above) and 40 mL of styrene. The flask was 
placed in a boiling water bath and after temperature equilibration, 17.6 
mL of Dupanol was added at a flow rate of 1 mL/min and allowed to react 
for one hour. An outer shell of polyglycidyl methacrylate was formed on 
the polystyrene particles by adding 6.4 mL of glycidyl methacrylate, 0.48 
mL of ethyleneglycol dimethacrylate and 160 mg of potassium persulfate to 
the flask. The reaction was continued at 100.degree. C. for eight minutes 
and was then quenched to room temperature, filtered and stored. 
B. Attachment of an o-Nitrophenol analog to Polymer Particles 
An analog of o-nitrophenol, 3-nitro-4-hydroxybenzoic acid (NHB 67.45 mg), 
and 103.7 mg of disuccinimidyl carbonate were dissolved in 7.80 mL of 
dimethylsulfoxide. Then 0.10 mL of triethylamine was added and the 
solution was stirred for 1 hour. While stirring, 95.7 mg of polyether 
polyamine (PEPA, U.S. Patent 4,581,337) was dissolved in 1.30 mL of 
dimethylsulfoxide, the two solutions were combined and allowed to incubate 
at room temperature for approximately 12 hours. 
The NHB conjugate solution, prepared as described above (8.2 mL) and 1.2 mL 
of a 10% w/v GAFAC.RTM. RE-610:water solution were diluted in 100 mL of 5 
mM sodium phosphate buffer (pH 8.0). The pH of the solution was adjusted 
to pH 10.0-10.1 and 14.3 mL of core/shell polymer particles prepared in 
step A was added to the solution. The mixture was heated at 70.degree. C. 
for 2 hours and then allowed to cool to room temperature. The particle 
reagent was diluted with 100 mL of 15 mM sodium phosphate buffer (pH 7.0) 
containing 0.1% GAFAC.RTM.. The particles were centrifuged for 2 hours at 
19,500 rpm to form a pellet and the supernatant was discarded. The 
particles were resuspended in the phosphate/GAFAC.RTM. solution and 
recentrifuged. The centrifugation and resuspension process was repeated a 
total of four times to remove unreacted NHB conjugate. After the final 
wash, the particle reagent was brought to a total volume of 100 mL in the 
phosphate/GAFAC.RTM. buffer. 
C. Monoclonal Anti-NHB Antibodies 
Synthesis of a 3-nitro-4-hydroxybenzoic acid/KLH conjugate used for the 
development of antibodies cross-reactive with o-nitrophenol is described 
in Example 1, Part A. 
1. Mouse Immunization 
Two BALB/C mice were injected intraperitoneally with 0.1 mg each of NHB-KLH 
emulsified in 0.25 mL of complete Freund's adjuvant (total volume of 0.5 
mL per mouse). Five booster injections were given at 21 day intervals (0.1 
mg of NHB-KLH in incomplete Freund's adjuvant). A sixth boost was given 49 
days after the fifth booster. The final (7th) boost was given 21 days 
after the sixth boost. Fusion was done four days after final boost. 
2. Fusion 
Spleens were removed aseptically, and a single cell suspension prepared by 
passing the spleens through a wire mesh. Spleen cells were fused with 3.5 
.times.10.sup.7 P3Ag8.653 murine myeloma cells (ratio of 4.5:1 spleen 
cells to myeloma cells). The fusion was done by the addition of 1.8 mL of 
a polyethylene glycol (PEG) solution (42% v/v PEG 3350, 45.2% media and 
7.5% dimethylsulfoxide) to the pellet of myeloma and spleen cells that had 
been washed in serum free media. The mixture was allowed to stand for one 
minute. One mL of serum free media was then added slowly over one minute. 
Forty mL of serum free media was then added over five minutes. Feeder 
cells (peritoneal exudate macrophages) in 40 mL of Iscove's medium 
containing HAT (hypoxanthine, aminopterin, and thymidine) and fetal calf 
serum was added. The total 80 mL was plated into twenty 96 well microtiter 
plates. Clones were detected after one week in culture (7% CO.sub.2 
incubator). 
3. Screening 
Supernatants were harvested four weeks after the fusion. They were screened 
for antibody activity using a particle enhanced turbidimetric immunoassay 
(U.S. Pat. No. 4,401,765). Supernatants (0.025 mL) were added to 
microtiter wells containing 0.075 mL of buffer (150 mM phosphate, 3% PEG 
8000, 0.1% GAFAC.RTM., 2 mM MgCl.sub.2) containing NHB particle reagent 
(0.05 mL particle reagent from Example 1, Part D, to 10 mL buffer) pH 7.8. 
The contents of the plate were incubated at 37.degree. C. for five minutes 
and then examined for agglutination of the particles. A total of nine 
positive cell lines were detected from 700 clones. These supernatants were 
then screened for their ability to bind free NHB-BSA by the addition of 
NHB-BSA to the particle reagent-buffer in the microtiter well before 
addition of the culture supernatants. All nine clones showed inhibition of 
agglutination (lack of turbidity) when this was done. 
4. Cloning 
The cell lines were cloned at semi-limiting dilution, approximately 1 cell 
per well. An aliquot of cells was also frozen in liquid nitrogen as a 
safeguard against loss. Feeder cells (peritoneal macrophages) were used to 
promote growth. When clones were large enough, culture supernatants were 
retested for antibody as described in Part C (3) above. 
The cells of interest were then selected for cloning at limiting dilution, 
using strict Poisson statistics. When sufficient numbers of cells were 
present in the wells, the supernatants were again tested for antibody. The 
reclones were then frozen for storage. 
5. Chain Composition 
The heavy chain composition of the antibodies produced by the following 
cell lines was determined by double diffusion in agar gel using specific 
reagents. 
______________________________________ 
Heavy Chain 
Cell Line Isotype 
______________________________________ 
2/1 .gamma..sub.3 
2/2 .gamma..sub.1 
2/3 .gamma..sub.1 
2/4 .gamma..sub.1 
2/5 .gamma..sub.1 
2/6 .gamma..sub.b 
2/7 .gamma..sub.1 
2/8 .gamma..sub.1 
______________________________________ 
D. .beta.-galactosidase Activity (Prior Art) 
A stock solution of the enzyme .beta.-galactosidase (Boehringer Mannheim 
Co., Indianapolis, IN) was prepared at a concentration of 1 mg/mL (200 
units/mL) based on the activity supplied by the manufacturer. Serial 
dilutions of the stock .beta.-galactosidase solution were prepared over 
the range of 2.times.10.sup.2 to 2.times.10.sup.-2 units/mL. An enzyme 
substrate solution of o-nitrophenyl.beta.D-galactopyranoside (ONPG) was 
prepared at a concentration of 1 mM ONPG, 1 mM magnesium chloride and 150 
mM phosphate buffer, pH 7.8. Ten microliters of each of the enzyme 
dilutions were mixed with 990 microliters of the substrate solution and 
allowed to react for 2 minutes at room temperature. These mixtures were 
immediately diluted 1/10 with phosphate buffer (150 mM, pH 7.8) and color 
formation observed (see Table 5). 
E. .beta.-galactosidase Activity Using the Agglutination Inhibition Mode of 
the Present Invention 
The .beta.-galactosidase reaction mixtures prepared in Part D (prior to 
final dilution) were used immediately for these experiments. The mixtures 
were diluted 1/10 by addig 3 parts of a solution containing 0.10 mL 
GAFAC.RTM., 0.185 mL phosphate buffer and 0.015 mL of ONB particle 
concentrate (from Part B), 5 parts of a 10% (w/v) polyethylene glycol 8000 
solution and 1 part of a 1:100 dilution of monoclonal anti-NHB ascites 
fluid (clone 2/2). The solutions were observed for the formation of 
particle agglutination (turbidity) after 3 minutes. The data show that the 
agglutiation assay is approximately 100-fold more sensitive for measuring 
.beta.-galactosidase activity in a visual mode. 
TABLE 4 
______________________________________ 
.beta.-Galactosidase Activity 
.beta.-Galactosidase Agglutination 
Activity Prior Art Inhibition Method 
______________________________________ 
.2 yellow non-turbid 
.02 light yellow 
non-turbid 
.002 colorless non-turbid 
.0002 colorless partially turbid 
.00002 colorless turbid 
______________________________________ 
EXAMPLE 3 
Direct Agglutination Assay for .beta.-Galactosidase Activity 
The following example illustrates the use of the direct agglutination 
method provided by the present invention for measuring enzyme activity. In 
the example, the highly refractive particles have a polystyrene core with 
an intermediate shell of polyvinylnaphthalene and an outer shell of 
polyglycidyl methacrylate. The binding agent would be an antibody prepared 
against the hapten 3-nitro-4-hydroxybenzoic acid. The substrate would be 
3-nitro-4-0-(.beta.-D-galactopyranosyl) benzoic acid which can be coupled 
covalently to the outer shell of the highly refractive particles. The 
enzyme .beta.-galactosidase would hydrolyze the galactopyranosyl 
derivative on the particle surface. Direct particle agglutination would 
then occur in the presence of the binding agent. The extent of 
agglutination could be observed visually to obtain a qualitative measure 
of enzyme activity or a change in the physical properties of the solution 
could be measured instrumentally to supply quantitative results. 
A. Synthesis of 3-Nitro-4-O-(.beta.-D-galactopyranosyl) benzoic acid 
A substrate for .beta.-galactosidase that is capable of attachment to 
highly refractive particles is required for the present example. The 
ligand, 3-nitro-4-O-(.beta.-D-galactopyranosyl) benzoic acid (NHB) would 
meet this requirement. An example of a procedure that could be adapted for 
the synthesis of this analog is provided by D. H. Leaback in an appendix 
to a paper by J. W. Woollen and P. G. Walker. [Clin. Chem. Acta. (1965) 
12, 647,658]. 
Acetobromogalactose (1 g, 2.44 mmole) and 3-nitro-4-hydroxybenzoic acid 
(0.40 g, 2.20 mmole) are dissolved in methanol (10 mL) and 1 N sodium 
hydroxide (2.2 mL) is added slowly with stirring. The mixture is left for 
16 hours at room temperature before the methanol is removed under reduced 
pressure to leave a syrup. The NGB material would then be deblocked and 
purified by procedures similar to those described in the above reference. 
B. Synthesis of Amine-Modified Polymer Particles 
The diamine 2,2-oxybis (ethylamine) dihydrochloride (420 mg) and 0.06 mL of 
a 10% w/v GAFAC.RTM./water solution can be added to 4.7 mL of 5 mM sodium 
phosphate buffer (pH 8.0). The pH of the solution is adjusted to pH 
10.0-10.1 and 1.2 mL of core/shell polymer particles (example 1, part C) 
are added to the mixture. The solution is heated to 70.degree. C. for 3 
hours and allowed to cool to room temperature. The modified particle is 
diluted with 15 mM sodium phosphate buffer (pH 7.0) containing 0.1% 
GAFAC.RTM.. The particles are centrifuged for 90 minutes at 18,000 rpm to 
form a pellet, and the supernatant is discarded. The particles are 
resuspended in the phosphate/GAFAC.RTM. solution and recentrifuged. The 
centrifugation and resuspension process is repeated a total of four times 
to remove unreacted diamine. After the final wash, the modified particles 
are brought to a total volume of 1 mL in the phosphate/GAFAC.RTM. buffer. 
C. Attachment of the NGB Ligand to Amine-Modified Polymer Particles 
The NGB substrate described in part A (9.1 mg, 0.026 mmole) is dissolved in 
0.17 mL dimethylsulfoxide containing 1-ethyl-3-(3-dimethyl aminopropyl) 
carbodiimide (5.2 mg, 0.027 mmole) and allowed to sit at room temperature 
for 2 hours. The NGB solution and 0.06 mL of a 10% w/v GARFAC.RTM./water 
solution would then be diluted in 2.0 mL of 5 mM sodium phosphate buffer 
(pH 7.0). The pH of the solution is adjusted to pH 7.0-7.1, and 1.0 mL of 
the amine-modified core/shell particles (part B above) is added to the 
reaction. The mixture is cooled to 4.degree. C. and incubated for 16 
hours. The particle reagent is diluted with 15 mM sodium phosphate buffer 
(pH 7.0) containing 0.1% GAFAC.RTM.. The particles are centrifuged for 90 
minutes at 18,000 rpm to form a pellet, and the supernatant is discarded. 
The particles are resuspended in the phosphate/GAFAC.RTM. solution and 
recentrifuged. The centrifugation and resuspension process is repeated a 
total of four times to remove unreacted NGB. After the final wash, the 
ligand (substrate) particles are brought to a total volume of 10 mL in the 
phosphate/GAFAC.RTM. buffer. 
D. .beta.-galactosidase Activity Using the Direct 
Agglutination Mode of the Present Invention 
The .beta.-galactosidase serial dilutions as prepared in example 1 (part E) 
would be appropriate for use in an assay. A convenient volume of each 
.beta.-galactosidase dilution is added to a mixture consisting of 0.15 M 
phosphate buffer (pH 7.8), 0.1% GAFAC.RTM., 1.46% w/v PEG 8000 and 0.02% 
solids w/v of the ligand particles (part C above). Reaction temperature 
must be equivalent for all assays and is selected as a matter of 
convenience, preferably between 25.degree. and 37.degree. C. The addition 
of .beta.-galactosidase to the assay mixture causes hydrolysis of the 
3-nitro-4-O-(.beta.-D-galactopyranosyl) benzoic acid on the particle 
surface to 3-nitro-4-hydroxy benzoic acid. Direct particle agglutination 
will then occur in the presence of an appropriate concentration of an 
antibody that binds the hydrolysis product on the particle but does not 
bind the unhydrolyzed ligand. The anti-NHB antibodies prepared in Examples 
1 and 2 above would be appropriate for use in these assays because they 
have been shown in the previous examples to cause agglutination of 
3-nitro-4-hydroxy benzoic acid coupled polymer particles. Direct particle 
agglutination can occur simultaneously with ligand hydrolysis, providing 
the anti-NHB antibodies are present prior to or simultaneous with the 
addition of .beta.-galactosidase. Alternatively, the agglutination 
reaction can be initiated at any convenient time after 
.beta.-galactosidase addition by delaying the addition of the anti-NHB 
antibodies. The extent of particle agglutination can be determined 
visually or instrumentally, depending on the requirements for qualitative 
or quantitative results.