A method for the detection or quantitation of a water-borne parasite, such as Cryptosporidia. The detection or quantitation is accomplished by an electrochemiluminescence assay comprising the steps of filtering water to obtain a sludge thought to contain the parasite or a fragment thereof; extracting a sample of said sludge in a extraction medium to form antigenic derivatives of said parasite; forming an assay mixture comprising a sample of said extracted sludge and an antibody specific to said antigenic derivative; incubating said assay mixture to permit binding of said antibody and said antigenic derivative; and conducting an electrochemiluminescence assay for the bound complex of antibody and antigenic derivative, thereby detecting or quantitating the Cryptosporidia in said water.

BACKGROUND 
Cryptosporidium and CryDtosporidiosis 
Cryptosporidium is a genus of protozoan parasite commonly found in the 
gastrointestinal tract of vertebrates. There are eight named species of 
Cryptosporidium, C. parvum is infectious for 79 species of mammals, 
including humans, causing acute gastroenteritis. Unlike most parasites, C. 
parvum lacks host specificity among mammals and is able to cross- infect 
multiple host species. 
Cryptosporidiosium is transmitted as an oocyst via the fecal-oral route. 
Contaminated water, close human-to-human contact and contact with the 
excrement of infected livestock, zoo animals or domestic animals can lead 
to transmission of the parasite. When outside a host, the exogenous phase, 
Cryptosporidium exists as a sporulated oocyst. The oocyst consists of four 
sporozoites within a tough, two layered wall. The oocyst wall has defined 
inner and outer layers and a suture at one end. During excystation, the 
suture dissolves providing an opening through which the sporozoites leave 
the oocyst. When ingested by a suitable host, excysted sporozoites, the 
living matter, parasitize the cells of the gastrointestinal or respiratory 
tract. After reproduction, resporulation into oocysts occurs. Oocysts in 
the gastrointestinal tract are excreted with the fecal matter while those 
in the respiratory tract exit the body in respiratory and nasal 
secretions. 
Cases of infection by Cryptosporidium are commonly encountered in both 
developed and underdeveloped countries. The slightly higher prevalence of 
Cryptosporidiosis in the lesser developed countries can be attributed to 
poor sanitation, malnutrition, contaminated drinking water and close 
contact with infected persons and animals. 
The most common clinical sign associated with Cryptosporidiosis is 
diarrhea, which can be severe and result in weight loss and dehydration. 
Other common clinical symptoms include abdominal cramps, fever, nausea, 
vomiting, headache, fatigue, myalgia and inappetence. Infections with 
Cryptosporidium are generally in the small intestine, but have also 
occurred in the lungs, esophagus, stomach, and other organs. The clinical 
symptoms associated with the parasitic infection depend on the affected 
organ. 
The range of symptoms and the severity of the illness can vary greatly from 
one individual to another and can become life threatening. The symptoms of 
acute enteritis generally last one to two weeks in individuals who are 
otherwise immunologically healthy. Cryptosporidiosis represents a 
heightened threat in AIDS patients, malnourished persons, individuals with 
inherited immune deficiencies, and person receiving immunosuppressive 
drugs. 
All infections with Cryptosporidium are initiated by ingestion or 
inhalation of the oocyst. Because the parasite is transmitted in the form 
of an oocyst, oocysts have evolved to survive in harsh environmental 
conditions and are unusually resistant to natural stresses and chemical 
disinfectants. In addition, the presence of an exogenous oocyst 
encapsulating the protozoan parasite makes the parasite much more 
resistant to conventional water treatment processes. Measures to prevent 
or limit the spread of infection concentrate on eliminating or reducing 
infectious oocysts in the environment. For humans, disinfection procedures 
are sought to minimize person-to-person transmission and to deal 
effectively with contamination of water supplies. 
The fairly recent occurrence of large water-borne outbreaks has focused 
attention on the importance of understanding their transmission through 
the environment. Surface waters may be polluted naturally by infected 
animal excrement. Many waste disposal practices may lead to contaminated 
water courses and streams. Fecal contamination of waterways has recently 
led to massive outbreaks of C. parvum infection. Water polluted by these 
practices may then lead to the contamination of drinking water supplies or 
of food crops during irrigation. 
Chemical Composition and Decomposition of Cryptosporidium 
The protein, carbohydrate, and lipid composition of C. parvum is diverse 
and complex. Many of the components are antigenic and therefore function 
as immune response targets. Glycoproteins ranging from &lt;14 to 7200 kDA 
from disrupted oocytes, purified oocyst shells and purified sporozoites 
have been identified by SDS-PAGE gel electrophoresis. Many of these 
oocyte-derived proteins are glycosylated. Specific carbohydrate moieties 
have been identified. It has been determined that sporozoite glycoproteins 
with terminal N-acetyl-D-glycosamine residues may function in attachment 
of the parasite or somehow assist in invasion. These carbohydrates are 
expressed on the oocyte surface and are useful in immunological detection 
methods. sporozoites of Cryptosporidium can spontaneously excyst through a 
suture at one pole of the oocyst when warmed to about 37.degree. for 
approximately 90 minutes. This renders mechanical methods for oocyst wall 
disruption unnecessary to accomplish. 
Pretreatment of C. parvum oocytes with sodium hypochlorite (a "bleach") 
results in separation of the inner and outer oocyst walls. That is, while 
it is not necessary to pretreat oocysts within a reducing agent when 
excysting C. parvum, a slight increase in the rapidity of excystation 
occurs when bleach treated oocysts are incubated in PBS containing 0.01M 
cysteine HCL during the excystation process. Oocysts that have not been 
pretreated with bleach excyst somewhat when they are warmed to 
approximately 37.degree. C. The use of trypsin and bile salts, or bile 
salts alone, can increase or speed excystation of unbleached oocysts. 
Prior Art Assay Methods for Crytospyridium 
Immunological techniques have been used to detect C. parvum in 
environmental specimens. The availability of monoclonal antibodies for 
specific antigens of Cryptosporidium facilitated development of these 
methods. 
Immunofluorescence assays (IFA) are the most common assays used to detect 
Cryptosporidium oocytes in specimens and to detect the presence of a 
specific antibody. These methods employ fluorescent dyes which are 
combined with antibodies to make them fluoresce when exposed to 
ultraviolet light. In a typical IFA assay, water is filtered through a 
polypropylene cartridge filter or a flat, membrane filter. Both filters 
yield filtrates that are then subjected to purification before analysis by 
microscopy. The filtrate is removed from the filter and then centrifuged. 
Extraneous debris is removed by flotation over a sucrose solution. The 
supernatant is labeled with a fluorescein conjugated antibody against 
Cryptosporidium and examined by epifluorescence microscopy. 
Some commercial immunofluorescent assays and reagents used to detect 
Cryptosporidial oocytes include: (1) HydroFluor Combo, an 
immunofluorescent assay system based on an oocyst-specific monoclonal 
antibody (IgM, OW3) (2) Detect IF Cryptosporidium, an immunofluorescent 
assay system based on an oocyte-specific monoclonal antibody (IgM, C1), 
and (3) Crypto IF Kit, an immunofluorescent assay system based on an 
oocyst-specific monoclonal antibody. 
The disadvantages of immunofluorescence assays include their low recovery 
efficiency, long processing times, the need for highly trained analysts, 
high cost, the inability to discriminate viable or virulent strains and 
cross-reactivity of the probes with similar size and shaped algae. In 
addition, IFA detection often involves the time consuming and skill 
intensive step of looking at water sludge under a microscope for oocysts 
that have been labeled with a fluorescent antibody. It is also often 
difficult to distinguish oocysts from debris bound non-specifically by the 
antibodies. The procedure is expensive and often takes days to complete. 
Enzyme-linked immunosorbent assays (ELISA) using oocyte-reactive monoclonal 
antibodies is also used to detect Cryptosporidium in contaminated water 
samples. Two basic ELISA tests have been used in the past for detecting 
Cryptosporidium antigen in samples: (1) the double antibody sandwich 
technique for the detection of antigens, and (2) the enzyme-linked 
indirect immunosorbent assay for the detection of antibodies. 
In the double antibody sandwich method, antiserum is adsorbed to a well. 
Test antigen is added and, if complementary, binds to the antibody. An 
enzyme-linked antibody specific for the test antigen then binds to the 
antigen, forming a sandwich. The enzyme's substrate is then added, and the 
reaction produces a visible color change. In the indirect immunosorbent 
assay, an antigen is adsorbed to a well. Test antiserum is then added, 
with complementary antibody binding to the antigen. Enzyme-linked 
anti-human gamma globulin is then added. It binds to the bound antibody. 
The enzyme's substrate is then added, producing a visible color change. A 
difficulty encountered in enzyme-based assays is the deactivation of the 
enzyme by components of the assay mixture. A further difficulty is 
encountered in the wash step where strong forces overcome the 
antibody-antigen interaction. This leads to loss of assay precision. 
Detection assays based upon polymerase chain reactions (PCR) have also been 
used to detect oocysts in clinical or environmental samples. Several DNA 
and RNA regions of C. parvum have been sequenced and have been reported to 
be assay targets for parasite detection. 
Flow cytometry is another method used to detect parasitic contamination of 
water samples. Flow cytometry techniques can quantify whole oocysts but 
involves much preparation, and time and require extremely expensive 
equipment. 
Numerous problems are associated with prior art methods of detecting 
Cryptosporidium in water and environmental samples. In addition to those 
mentioned and the general lack of precise, recitable assays, prior art 
techniques generally require that samples be transferred to a laboratory 
or to another remote location for the conduct of the assay. Prior art 
techniques lack the requisite reliability, speed and sensitivity to 
accurately detect Cryptosporidium in contaminated water samples. 
The detection of infectious C. parvum oocysts in water and other 
environmental samples is essential to detecting and treating contaminated 
water supplies. It is crucial, therefore, that specific, rapid and highly 
sensitive assays be developed to detect the presence of the parasite 
accurately and reliably. The known methods of enzyme immunoassays and 
immunofluorescence do not fulfill these requirements. The source, 
viability and pathogenicity of oocysts found in water or other 
environmental samples cannot be reliably determined using prior art 
methods. There is a need for routine epidemiological surveillance and 
environmental monitoring that can be conducted on site to provide early 
detection of the parasite. 
OBJECTS OF THE INVENTION 
A primary object of this invention is to provide a fast, sensitive, and 
precise assay for the detection or quantitation of oocysts of 
Cryptosporidium in environmental samples. 
It is another object of this invention to provide a fast, sensitive, and 
precise assay procedure for the detection or quantitation of sporozoites 
of Cryptosporidium in an environmental sample. 
It is a further object of this invention to provide a fast, sensitive, and 
precise assay procedure for the contemporaneous detection and quantitation 
of oocysts and sporozoites of Cryptosporidium in an environmental sample. 
Still a further object of the invention is to provide rapid quantitative 
assays for C. parva in decentralized settings, i.e., at water treatment 
plants, which are not labor-intensive and which employ reliable assay 
instruments. 
SUMMARY OF THE INVENTION 
The problems inherent in the prior art assays are solved in the 
electrochemiluminescence (ECL) based assays of the invention. These assays 
can detect Cryptosporidium in water sources. These ECL assays are rapid 
assays for the detection or quantitation of C. parva in decentralized 
settings, i.e., the water is assayed at the treatment plant. The simple 
quantitative assays are a major improvement over the prior art methods. 
They are precise and sensitive and are performed in a few hours without 
extensive labor. 
The assays can detect or quantify whole oocysts or fragments of oocysts 
using a method that is reliable, accurate and inexpensive, and can be 
performed rapidly using simple equipment. 
This invention is broadly in a method for the detection or quantitation of 
Cryptosporidium in water by an electrochemiluminescence assay comprising 
the steps of: 
a) filtering water to obtain a sludge containing oocysts of Cryptosporidia; 
b) extracting a sample of the sludge in an extraction medium to form at 
least one antigenic derivative of the oocysts; 
c) forming an assay mixture comprising a sample of the extracted sludge and 
an antibody specific to the antigenic derivative; 
d) incubating the assay mixture to permit binding of the antibody and the 
antigenic derivative; and 
e) conducting an electrochemiluminescence assay for the bound complex of 
antibody and antigenic derivative and thereby detecting or quantitating 
Cryptosporidia in the filtered water. 
The assay mixture contains a sample of extracted sludge, magnetic particles 
labeled with a first antibody specific to a first epitope of the antigenic 
fragment, a labeled probe comprising a second antibody specific to a 
second epitope of the antigenic derivative, and an ECL assay reagent. The 
second epitope may be the same as or different from the first epitope. 
The method of the invention can also be used for the detection or 
quantitation of sporozoite antigens of Cryptosporidia and to assays in 
which the Cryptosporidia oocysts' outer wall and sporozoites contained in 
the oocysts are assayed contemporaneously. 
The invention also relates to a kit for conducting the 
electrochemiluminescence assays for Cryptosporidia in water which includes 
an extraction medium for the extraction of antigenic derivations in oocyst 
walls and/or sporozoites from organic sludge obtained from water 
filtration and an antibody specific to an antigenic derivative in the 
extracted sludge. Kits for the conduct of an electrochemiluminescence 
assay for Cryptosporidium in water may also include components of an 
electrochemiluminescence assay, including magnetic particles labeled with 
an antibody specific to an epitope of an antigenic derivative of extracted 
Cryptosporidia oocysts, ECL label probes comprising an antibody specific 
to an epitope of the antigenic derivative, and assay reagents. 
The methods and assays for detecting Cryptosporidium in environmental 
samples offer distinct advantages over prior art detection methods. The 
assay can be conducted on the raw sludge, at the site of filtration, i.e. 
in a decentralized setting, so that there is no need to transport the 
specimens to another location for analysis. The results obtained using 
this invention are precise, accurate, rapid and overcome the problems 
inherent in prior art procedures. 
DETAILED DESCRIPTION OF THE INVENTION 
The invention is in an electrochemiluminescence assay for the detection of 
Cryptosporidia in sludge obtained from a raw water filter. The presence 
and amount of both oocysts and/or sporozoites can be determined in these 
assays. Thus, the assay can detect if the water is presently contaminated 
with living parasites or if only empty oocysts remain in the water sample. 
Sludge collected from a filtrate of raw water is treated directly to 
release antigens in soluble form from the parasites for detection. 
Living and dead parasites can be detected from the filtrate sample. The 
assay therefore provides qualitative or quantitative information regarding 
previous contamination of the water (no viable parasites) as well as 
regarding present contamination (infective, sporozoite-containing 
parasites). 
The method generally comprises the steps of: 
a) filtering the water to obtain a sludge containing oocysts of 
Cryptosporidia; 
b) extracting a sample of the sludge in an extraction medium to form 
derivatives of oocysts including at least one antigenic derivative; 
c) forming an assay mixture comprising a sample of the extracted sludge and 
an antibody specific to the antigenic derivative; 
d) incubating the assay mixture to permit binding of the antibody and the 
antigenic derivative; and 
e) conducting an electrochemiluminescence assay for the bound complex of 
antibody and antigenic derivative and thereby detecting or quantitating 
the Cryptosporidia. 
Glycosylated proteins in the walls of the oocysts and sporozoite serve as 
antigenic derivatives for these assays. The protein, carbohydrate, and 
lipid composition of C. parvum is diverse and complex. Many of these are 
antigenic and thus function as immune-response targets. Identification and 
characterization of C. parvum antigens has been facilitated by protocols 
for purifying oocysts and sporozoites. Many protein/glycoprotein molecules 
from disrupted oocysts, purified oocyst shells and purified sporozoites, 
ranging from &lt;14 to 7200 KDA have been identified in protein-stained 
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. 
Monoclonal antibodies specific to these glycosylated proteins in the wall 
of the oocyst and the sporozoite can be used in these immunoassays. 
The mAb OW3 identifies C. parvum sporozoite cDNA that encodes a putative 
670 amino acid sequence with significant homology to both hsp70 and a 78 
kDa glucose regulation protein. This mAb recognizes a &gt;200-kD C. parvum 
oocyst wall antigen on western blots. Although this molecule may be a 
component of the oocyst wall, the Gly-Gly-Met-Pro repeat of C. parvum 
hsp70 should be highly immunogenic and may simply have an antigenic 
feature in common with COWP-190. The mAb OW-IGO, developed to detect 
antigenic moieties within the outer wall of C. parvum oocysts, also labels 
the fibrillar material in the parasitophorous vacuole of developing 
macrogametes, microgametocytes, and sporulating oocysts. On western blots, 
the antibody recognizes major bands at 250 and 40 kDa and additional 
smaller bands. Most western blot activity is abolished by periodate 
treatment, again suggesting carbohydrate on the outer wall. 
The sludge collected from a filtrate of raw water is treated to release the 
antigens from the parasites so that they can be detected by immunoassays. 
The extraction and solubilization of the antigens requires an acid 
treatment of the parasites in the presence of bile salts at approximately 
40.degree. C. This treatment activates protease enzymes that help to break 
down the oocysts walls. The sample is then treated with a base such as 
tris to provide a pH between 5.5 and 8. The sample is then treated with 
disruptive agents such as ionic or non-ionic detergents and/or urea or 
formamide incubated at elevated temperatures in buffered solutions. The 
sample may then be treated with reducing agents such as mercapto-ethanol 
or dithiolthreitol. Excess bile salts or detergent-reagents can be removed 
or sequestered by adding different non-ionic detergents or BSA or other 
animal serum albumin or immunoglobulin to the extract, leaving the target 
antigens in solution and in a state ready to bind with specific 
complementary antibodies. Excess chaotropic agents can be removed by 
dialysis or enzymatic means or chromatography, or their effects by 
dilution. Excess reducing agents may be removed by treatment with: oxygen 
(or air) saturated buffer, diamide, iodine solution (0.05 N in water), 
tert-butyl hydroperoxide, ferricyanide, hydrogen peroxide, iodoacetomide, 
N-methyl maleimide, 2-(2-pyridyl dithio) ethanol. 
The assays of the invention employ procedures for treating the parasite 
oocytes with bile salts at elevated temperatures, in vitro, to induce 
excystation and to release antigens from both the oocysts and the 
sporozoites. These antigens can then be detected and quantitated in an 
immunoassay. 
The antigens in solution in the treated sludge suspension, consisting of 
antigens released from the oocyst membranes and the sporozoites, if any, 
are incubated with antibodies specific for the antigenic derivatives, 
e.g., antibody OW3 which is specific for an oocyst wall antigen of &gt;20okD. 
Antibody OW3 recognizes linear repetitive epitopes characterized from 
Western blot experiments. Linear epitopes are not destroyed after 
detergent treatment and therefore remain active with antibodies following 
solubilization of the sludge. Once the antigens are in solution, the ECL 
immunoassay can be conducted. 
ECL assay techniques provide a sensitive and precise measurement of the 
presence and concentration of an analyte of interest. In such techniques, 
electrochemiluminescence is triggered by a voltage impressed on the 
working electrode of an ECL cell at a particular time and in a particular 
manner. The luminescence produced by the ECL label is measured and 
indicates the presence or quantity of the analyte. 
Electrochemiluminescence assays can be performed with or without the need 
for a separation step during the assay procedure and at maximum signal 
modulations for different concentrations of analyte, so that precise and 
sensitive measurements over a broad range of concentration of analyte can 
be made. Nonseparation assays include those which employ microparticulate 
matter suspended in the assay sample to bind one or more of the binding 
components of the assay. 
For a fuller description of ECL techniques, reference is made to PCT 
published application U.S. Ser. No. 85/01253 (WO86/02734), PCT published 
application number U.S. Ser. No. 87/00987 (WO 87/06706), and PCT published 
application U.S. Ser. No. 88/03947 (WO89/04302). These publications and 
those referred to below are incorporated by reference. While it had been 
expected in the art that luminescence from electrochemiluminescent 
moieties would be absorbed, scattered, or otherwise suffer interference 
from the microparticulate matter, U.S. application Ser. No. 539,389 now 
abanoned, continued by U.S. application Ser. No. 08/413,336 (PCT published 
application U.S. Ser. No. 89/04919(WO90/05301)) teaches sensitive, 
specific binding assay methods based on a luminescent phenomenon wherein 
inert microparticulate matter is specifically bound to one of the binding 
reactants of the assay system. The assays of that application may be 
performed in a heterogeneous (one or more separation steps) assay format 
and may be used most advantageously in a homogeneous (nonseparation) assay 
format. 
The signal from labeled species can be enhanced by concentrating them 
before subjecting them to a measurement step. U.S. application Ser. No. 
08/255,824 now U.S. Pat. No. 5,705,402 PCT published application U.S. Ser. 
No. 92/00982 (WO92/14138) relates to a method of particle based 
electrochemiluminescence measurement wherein particles are brought into 
close contact with the electrode using a magnet to impose a magnetic field 
which collects the particles at the electrode. 
Particles useful in electrochemiluminescence assays advantageously have a 
diameter of 0.01 to 200 .mu.m and a surface component capable of binding, 
directly or directly, to the analyte. The particles are suspended in the 
ECL system, and are advantageously magnetically responsive. 
The assay requires an electrolyte. Generally, the electrolyte is in the 
liquid phase, for instance as a solution of a salt in water. The 
electrolyte is, in certain embodiments of the invention, a buffered system 
such as an aqueous solution of sodium phosphate/sodium chloride or an 
aqueous solution of sodium phosphate/sodium fluoride. 
As described in PCT published application U.S. Ser. No. 89/04859 
(WO90/05296), it is desirable to include a reductant, typically an amine 
or amine moiety (of a larger molecule) which can be oxidized and 
spontaneously decomposed to convert it into a highly reducing species, 
which in turn facilitates the electrochemiluminescent phenomenon. A wide 
range of amines and corresponding amine moieties can be utilized in 
practicing the present invention. Amines (and corresponding moieties 
derived therefrom) which are advantageously utilized in the present 
invention, including aliphatic amines, such as primary, secondary and 
tertiary alkyl amines, the alkyl groups of each having from one to three 
carbon atoms, as well as substituted aliphatic amines. Tripropyl amine is 
especially preferred. 
As described in PCT published application U.S. Ser. No. 89/04915 (WO 
90/05302) the assays of the invention are desirably carried out in the 
presence of an enhancer utilized in an amount sufficient so that in its 
presence the desired increase in emission of electromagnetic radiation, 
typically a compound of the formula 
##STR1## 
wherein R is hydrogen or C.sub.n H.sub.2n+1, R' is C.sub.n H.sub.2n, X is 
0 to 70, and n is from 1 to 20. 
The apparatus for carrying out the assays of the invention includes a 
working electrode or other triggering surface to apply an electric 
potential to trigger the reactions which cause the particle-linked ECL 
moiety to electrochemiluminesce and magnets capable of collecting the 
particle-linked ECL moiety at the electrode. Further details of apparatus 
for carrying out the ECL assays of the invention are disclosed in 
published PCT applications U.S. Ser. No. 89/04854 (WO 90/05411) and U.S. 
Ser. No. 90/01370 (WO 90/11511). 
The assays of the invention can be performed in any of the conventional 
formats, i.e., direct, indirect, forward, reverse or competitive formats. 
Sandwich immunoassays, as are well known in the field of diagnostics in 
general and in ECL detection specifically, are preferred. Such assays 
involve an analyte (antigen) which is bound by two antibodies: a "primary" 
or capture antibody which is bound to a solid surface by, for example, 
being labeled with biotin, and a "secondary" or label antibody which is 
labeled with an electrochemiluminescent species such as 
Ru(bpy).sub.3.sup.2+. Hence, in such an assay, a streptavidin-coated solid 
support is bound to a biotinylated primary antibody. The primary antibody 
is bound to the analyte (the antigen, if present), which antigen is bound 
to the Ru(bpy).sub.3.sup.2+ - labeled secondary antibody. 
In the invention, a monoclonal antibody specific to a first epitope of an 
antigen of interest--OW3 in the case of an assay for oocyst antigens, E3E 
in the case of an assay for sporozoite antigens--is conjugated to biotin. 
A monoclonal antibody specific to a second epitope of the antigen of 
interest (which may be the same as the first epitope) is conjugated to an 
ECL label ("TAG"). The extracted sludge and a solution of the biotinylated 
primary antibody are combined and incubated. The TAG-labelled antibody is 
then added and incubated to permit binding of TAG-labelled antibody and 
analyte. Following this incubation, a suspension of streptavidin coated 
beads is added to combine with the biotin on the primary antibody. 
The oocyte antigen immune complex is detected using an analyzer such as the 
ORIGEN.RTM. Analyzer. ORIGEN.RTM. Analyzers and reagents are available 
from Igen, Inc., Gaithersburg, Md. The immune complex that is quantitated 
in this procedure consists of the streptavidin coated paramagnetic beads 
bound by biotin to the complex of the antibody, OW3, which is bound to the 
OW3 specific antigen, which in turn is bound to another OW3 antibody, 
conjugated to the electrochemiluminescent moiety that produces the light 
in the analyzer. The magnetic beads and bound sandwich are brought to the 
surface of the electrode by a magnet associated with the flow cell. A 
voltage is applied to the electrode. Light produced by the ECL moiety is 
detected and quantitated by a photomultiplier. 
The solubilized material generated in the treatment of the sludge collected 
from the water filter includes all of the antigenic derivatives from C. 
parvum in the filtered water. Thus, a single preparation of solubilized 
material can be used to assay for both the Cryptosporidium oocyst and the 
sporozoites that it contains. It is important to quantitate both the 
number of viable sporozoites in the water, because they constitute the 
human-infective form of the parasite, as well as the oocysts from which 
they may have been liberated. The latter measurement may represent a 
present concentration of viable oocysts or a concentration of inviable 
oocysts, revealing a history of the contamination. 
Monoclonal antibody, 3E3, is specific for the highly immunogenic 27kD 
sporozoite surface antigen (p27). The 3E3 monoclonal antibody is 
conjugated to biotin and purified from the reaction mixture. A polyclonal 
antiserum prepared to the same highly immunogenic 27 kD sporozoite surface 
antigen (p27) is conjugated to a TAG and is then purified from the 
reaction mixture. A solution of the biotinylated antibody (in an assay 
diluent) is added to the treated sludge and incubated for about 1 hour at 
about 37.degree. C. The solution of the TAG-labelled antiserum (in assay 
diluent) is then added to the mixture and incubated under the same 
conditions. Following this incubation, a suspension of streptavidin coated 
beads are added and incubated for about 30 minutes at room temperature. 
The sporozoite antigen immune complex is detected using an Analyzer (such 
as the ORIGEN Analyzer) as described above. 
The detection or quantitation assays for one or both of the oocyst or 
sporozoite antigens can be performed essentially contemporaneously, i.e., 
one after the other. A significant advantage over the prior art methods is 
that one can determine if the water sample is actually infected with live 
sporozoites or whether there are merely non-infectious empty oocytes 
present. In addition the sludge collected on the water filter can be 
analyzed directly without extensive pretreatment. 
The method and assay of the invention are described further with reference 
to the following examples.

EXAMPLES 
Example 1 
WATER SAMPLE COLLECTION 
100 to 1000 liters of potable/clean water is filtered through a cuno-type 
filter. The filter membrane along with the sludge filtrate is cut free and 
removed from the holder. The membrane sheet is placed in a plastic 
container and 1 liter of a 0.1% Tween 80.TM. detergent solution is added 
to the container. The filter is agitated for 20 minutes and the sludge 
suspension is decanted into a centrifuge bottle. A second 1 liter of 
detergent solution is added to the container and the filter is agitated 
again for 20 minutes. The second liter is then decanted into a centrifuge 
bottle and the 2 liters of sludge filtrate suspension is centrifuged at 
7,280 g for 12 minutes. The bottles are removed from the centrifuge and 
the supernatant liquid is carefully aspirated off to leave a pellet in 
approximately 20 to 30 mls liquid in the bottles. The remaining liquid is 
vigorously shaken to resuspend the pellet and transferred to a 50 ml 
centrifuge tube. The bottle is rinsed with a 0.1% Tween.TM. 80 solution 
and added to a 50 ml centrifuge tube using .apprxeq.5 ml distilled water, 
if necessary, to balance the centrifuge tubes. The 50ml tubes are 
centrifuged at 2190 g for 10 minutes, and the supernatant liquid is 
decanted. The 2 pellets are pooled and resuspended. The final volume of 
the concentrate is measured in microliters with a pipetman. 
Example 2 
SOLUBILIZATION OF THE CRYPTOSPORIDIUM ANTIGENS 
50% of the volume of the resuspended concentrate derived in Example 1 is 
pippetted into a clean 50 ml centrifuge tube. A 10.times. concentrated 
stock solution of Hanks basic salt solution (HBSS) is adjusted to pH 2.5 
with HCl. A predetermined amount of the low pH HBSS 10.times. concentrate 
and predetermined amount of a 5% stock solution of bovine bile salts are 
added to the sludge suspension to give final concentrations of 0.5% bile 
salts and 1.times. HBSS. The sludge suspension is then incubated at 
40.degree. C. for 2 hours. After incubation, the solution is adjusted to 
pH 7 with tris base and a 3% stock solution of sodium dodecyl sulfate is 
added to give a final concentration of 0.3%. A predetermined amount of 200 
mM mercapto-ethanol or 100 mM dithiothreitol is added to give a final 
concentration of 20 mM mercapto-ethanol or 10 mM dithiothreitol. The 
suspension is mixed and incubated at 40.degree. C. for 1 hour. 
N-methylmaleimide is added to a final concentration of 20 mM. 5% stock 
solution of Triton X-100.TM. is added to give a final concentration of 
0.5% and bovine serum albumin is then added to the incubation mixture to a 
final concentration of 2% to sequester excess detergent and thereby 
improve immunoreactivity in the presence of the detergents. The solution 
is mixed and the final volume of the extraction mixture is measured in 
microliters with a pipetman. 
This extract is equivalent to 50% of the volume passed through the filter. 
The volume of the extract to be analyzed can be calculated as follows: 
Divide the extract volume in microliters by half the number of liters of 
water sampled. This will yield a volume ratio of microliters extract per 
liter of water sample. The water sample-equivalent to be analyzed should 
be at least 10% of the water volume passed over the filter. Thus 20% of 
the extract should be analyzed. The immunoassay volume to be analyzed 
should be between 20 and 50 microliters. 
Example 3 
PREATION OF OW3/ORIGEN TAG-NHS ESTER CONJUGATE 
0.5 mg of OW3 protein are dissolved in 500 .mu.l of PBS, pH 7.8, in a 
polypropylene vial. Ultra-filtration concentrating devices are used to 
achieve buffer exchange and concentration of the protein. ORIGEN TAG-NHS 
Ester stock solution is prepared by adding 50 .mu.l of DMSO to the 75 
.mu.g vial of TAG-NH3 Ester (Igen). The vial is gently twirled to wet the 
bottom and lower sides of the vial with the DMSO. This volume of DMSO 
dissolves the 75 .mu.g ORIGEN TAG-NHS Ester, resulting in 1.5 .mu.g/.mu.l 
stock solution. 
A 25:1 molar ratio of ORIGEN TAG-NHS Ester is added to the above OW3 
solution at 1 mg/ml. Based on molecular weights of 1057 and 750,000, 
respectively, 35 .mu.g/ml of the ORIGEN TAG-NHS Ester is added to the 
protein solution. The remaining ORIGEN TAG-NHS Ester is discarded. Vial 
contents are vortexed and incubated in darkness at room temperature for 60 
minutes. The reaction is stopped by adding 20 .mu.l of 2 M glycine and 
incubating in darkness at room temperature for an additional 10 minutes. 
Uncoupled ORIGEN TAG label is removed by loading the mixture onto a PD-10 
column (a prepacked Sephadex.TM. G-25 column manufactured by Pharmacia) 
previously equilibrated with PBS containing sodium azide or thimerosol. 
Due to the column's long separation time, it is covered with aluminum foil 
to shield the conjugate from the light. Two yellow bands formed as the 
separation of bound from free ORIGEN TAG proceeded. The labeled protein 
eluted first, followed by a second band corresponding to unconjugated 
ORIGEN TAG. Eight 0.5 ml fractions are collected after the sample volume 
entered the resin bed. 
Protein concentration in moles per liter was determined by using a standard 
protein assay (e.g., Bradford, Lowry, or a Pierce BCA protein Assay kit). 
An absorbance reading at an OD of 280 nm is avoided since TAG does alter 
the absorbance at this wavelength. The protein-containing fractions are 
collected and pooled to determine the final pooled protein concentration 
and molarity. The percent protein recovered ranged from 70-90%, depending 
on the separation technique employed. 
The absorbance of the TAG-OW3 at 455 nm is measured by using a 1 cm path 
cuvette. The absorbance value is divided by 13,700 to obtain the ORIGEN 
TAG concentration in moles per liter. 
The ORIGEN TAG:OW3 ratio is calculated by dividing the previously obtained 
value for ORIGEN TAG concentration by the previously obtained value for 
protein concentration. 
LABELLING OF THE P27 ANTIGEN RECOGNIZING ANTIBODY 
Sporozoite (p27) antigen was prepared by immunopurification on a solid 
phase using 3E3 antibody from extracts prepared as described in Example 2, 
except that Cryptosporidium was obtained in highly concentrated form from 
infected calves. Rabbits were immunized following conventional methods. 
Antibodies were prepared using standard techniques. These antibodies were 
labelled following the protocol described in Example 3. 
The procedure followed is as described above for OW3, except that 17.6 
.mu.g (11.7 .mu.l of a 1.5 .mu.g/.mu.l solution prepared from the 75 .mu.g 
TAG product) of the ORIGEN TAG-NHS Ester is added to the protein solution. 
Example 4 
BIOTINYLATION OF OW3 ANTIGEN 
1.5 mg of OW3 antibody is dissolved in 1.5 ml of 150 mM potassium phosphate 
buffer, pH 7.8, containing 150 mM sodium chloride, and Aliquoted into 0.5 
ml fractions. 
1 mg of ORIGEN Biotin-LC-Sulfo-NHS Ester in 0.5 ml of sterile, distilled 
water is dissolved immediately prior to use resulting in a 2 mg/ml 
solution. To achieve a molar ratio of 10:1, 20:1 or 40:1 ORIGEN 
Biotin-LC-NHS ester to 0.5 mg of antibody in 0.5 ml of buffer, 9.3, 18.5, 
or 37 .mu.l, of the biotinylation reagent, respectively, are added to the 
antibody aliquots. The solution is incubated for 60 minutes at room 
temperature, and quenched by the addition of 20 .mu.l of 2 M glycine. 
Unreacted ORIGEN Biotin-LC-Sulfo-NHS Ester is removed by dialyzing the 
antibody solution against the desired final buffer of PBS containing 0.02% 
NaN.sub.3. Gel filtration on an appropriate size desalting column (such as 
a PD-10 disposable column from Pharmacia) or spin column, or use a 
microcentrator such as a Centricon-30 (Amicon) can also be employed to 
remove unreacted ORIGEN Biotin-LC-Sulfo-NHS Ester. 
The biotinylated antibody is stored at 4.degree. C. until ready for use. 
BIOTINYLATION OF 3E3 ANTIBODY 
This procedure is as described above for the OW3 antibody. 
Example 5 
ASSAY FOR OOCYST ANTIGENS 
A 30 microliter sample of treated sludge (Example 2) is pippetted into a 
12.times.70 mm test tube. 25 microliters of a 2 mg/ml solution of the 
biotinylated OW3 antibody (Example 4) in ORIGEN Assay Diluent (available 
from IGEN) is added to the tube, followed by the addition of 25 
microliters of 2 mg/ml solution of the TAG-labelled OW3 antibody in ORIGEN 
Assay Diluent (Example 3). The mixture is incubated at 37.degree. C. for 2 
hours, mixing periodically. Following incubation, 25 microliters of a 1 
mg/ml suspension of Dynal 2.8 micron streptavidin coated beads (equivalent 
to 25 micrograms) in ORIGEN Assay Diluent is added. The mixture is again 
incubated for 30 minutes at room temperature. The immunoassay incubation 
mixture is then diluted to 300 microliters with ORIGEN Assay Buffer. 
The tube is placed in the carousel of the ORIGEN Analyzer and the test 
cycle started. Using a peristaltic pump, the instrument draws an aliquot 
of the sample from the carousel tube and transports it to the 
electrochemical flow cell. The paramagnetic beads are collected at the 
electrode by a magnet associated with the flow cell. Thereafter a voltage 
is applied to the electrode. Light produced by the TAG is detected or 
quantitated by a photomultiplier. 
The immunoassay is calibrated by assaying for known quantities of 
Cryptosporidium oocysts counted by generally accepted procedures using a 
flow cytometer. The calibrator solution is prepared from freshly isolated 
oocyst from the stool of infected patients. 
ASSAY FOR SPOROZOITE ANTIGEN 
The assay procedure for the sporozoite antigen is as described above for 
the occyst antigen assay except using 3E3 antibody. 
The immunoassay is calibrated by assaying for known quantities of 
Cryptosporidium occysts containing live sporozoites. The calibrator 
solution is prepared from freshly isolated oocysts from the stool of 
infected patients and counted by generally accepted procedures using a 
flow cytometer. 
Example 6 
CONTEMPORANEOUS ASSAY FOR OOCYST AND SPOROZOITE ANTIGEN 
The ECL immunoassays of examples 7 and 8 can be performed contemporaneously 
(either simultaneously or in rapid succession). The concentrate derived in 
example 1 is solubilized as described in example 2. The solubilized 
product is divided into two equal portions. The portions are then assayed 
using the methods described in Examples 3 through 5.