Detecting water-borne parasites using electrochemiluminescence

A method for the detection or quantitation of Cryptosporidia in a sample comprising:

• (a) extracting the sample in an extraction medium;
• (b) solubilizing, in said extraction medium, an antigen of Cryptosporidia oocysts and/or sporozoites;
• (c) forming an assay mixture comprising,
• (i) said solubilized antigen and
• (ii) an antibody specific to said antigen;
• (d) incubating said assay mixture under conditions sufficient to permit binding of said antibody and said antigen thereby forming an antibody-antigen complex;
• and
• (e) determining the presence of said antibody-antigen complex, thereby detecting or quantitating Cryptosporidia in the sample.

A method for the detection or quantitation of Cryptosporidia in a water sample comprising:

• (a) filtering said water sample to obtain a retentate comprising Cryptosporidia present in the water sample
• (b) extracting the retentate in an extraction medium;
• (c) solubilizing, in said extraction medium, antigens of oocysts of said Cryptosporidia, including at least one first antigen from the outer wall of said oocysts and at least one second antigen of a sporozoite contained within said oocyst;
• (d) forming a first assay mixture comprising,
• (i) a first portion of said solubilized antigens, and
• (ii) a first antibody specific to said first antigen;
• (e) forming a second assay mixture comprising,
• (i) a second portion of said solubilized antigens, and
• (ii) a second antibody specific to said second antigen;
• (f) incubating said first assay mixture under conditions sufficient to permit binding of said first antibody and said first antigen forming a first antibody-antigen complex;
• (g) incubating said second assay mixture under conditions sufficient to permit binding of said second antibody and said second antigen thereby forming a second antibody-antigen complex;
• (h) determining the presence of said first antibody-antigen complex;.and
• (i) determining the presence of said second antibody-antigen complex, thereby detecting or quantitating both the oocysts and sporozoites in the sample.

A method for the detection or quantitation of Cryptosporidia in a sample comprising:

• (a) extracting the sample in an extraction medium;
• (b) solubilizing, in said extraction medium, antigens of oocysts of said Cryptosporidia, including at least one first antigen from the outer wall of said oocysts and at least one second antigen of a sporozoite contained within said oocyst;
• (c) forming a first assay mixture comprising,
• (i) a first portion of said solubilized antigens, and
• (ii) a first antibody specific to said first antigen;
• (d) forming a second assay mixture comprising,
• (i) a second portion of said solubilized antigens, and
• (ii) a second antibody specific to said second antigen;
• (e) incubating said first assay mixture under conditions sufficient to permit binding of said first antibody and said first antigen forming a first antibody-antigen complex;
• (f) incubating said second assay mixture under conditions sufficient to permit binding of said second antibody and said second antigen thereby forming a second antibody-antigen complex;
• (g) determining the presence of said first antibody-antigen complex;
• and
• (h) determining the presence of said second antibody-antigen complex, thereby detecting or quantitating both the oocysts and sporozoites in the sample.

A method for the detection or quantitation of Cryptosporidia in a water sample comprising:

• (a) filtering said water sample to obtain a retentate comprising oocysts and/or sporozoites of Cryptosporidia present in the water sample;
• (b) extracting said retentate in an extraction medium
• (c) solubilizing, in said extraction medium, an antigen of said oocysts and/or sporozoites;
• (d) forming an assay mixture comprising,
• (i) said solubilized antigen,
• (ii) a capture antibody specific to said antigen,
• (iii) a label probe comprising a label antibody specific to said antigen and an electrochemiluminescent moiety, and
• (iv) a magnetic particle linked to or capable of capturing said capture antibody;
• (e) incubating said assay mixture under conditions sufficient to form a complex comprising said magnetic particle, said capture antibody, said antigen and said label probe;
• and
• (f) conducting an electrochemiluminescence assay for said complex, thereby detecting or quantitating Cryptosporidia in said water sample.--

A method for the detection or quantitation of Cryptosporidia in a sample comprising:

• (a) extracting said sample in an extraction medium;
• (b) solubilizing, in said extraction medium, an antigen of said oocysts and/or sporozoites;
• (c) forming an assay mixture comprising,
• (i) said solubilized antigen,
• (ii) a capture antibody specific to said antigen,
• (iii) a label probe comprising a label antibody specific to said antigen and an electrochemiluminescent moiety, and
• (iv) a magnetic particle linked to or capable of capturing said capture antibody;
• (d) incubating said assay mixture under conditions sufficient to form a complex comprising said magnetic particle, said capture antibody, said antigen and said label probe;
• and
• (e) conducting an electrochemiluminescence assay for said complex, thereby detecting or quantitating Cryptosporidia in said sample.

A method for the detection or quantitation of Cryptosporidia in a water sample comprising:

• (a) filtering said water sample to obtain a retentate comprising Cryptosporidia present in the water sample
• (b) extracting the retentate in an extraction medium;
• (c) solubilizing, in said extraction medium, antigens of oocysts of said Cryptosporidia, including at least one first antigen from the outer wall of said oocysts and at least one second antigen of a sporozoite contained within said oocyst;
• (d) forming a first assay mixture comprising,
• (i) a first portion of said solubilized antigens,
• (ii) a first capture antibody specific to said first antigen,
• (iii) a first label probe comprising a first label antibody specific to said first antigen and a first electrochemiluminescent moiety, and
• (iv) a first magnetic particle linked to or capable of capturing said first capture antibody;
• (e) forming a second assay mixture comprising,
• (i) a second portion of said solubilized antigens,
• (ii) a second capture antibody specific to said second antigen,
• (iii) a second label probe comprising a second label antibody specific to said second antigen and a second electrochemiluminescent moiety, and
• (iv) a second magnetic particle linked to or capable of capturing said second capture antibody;
• (f) incubating said first assay mixture under conditions sufficient to form a first complex comprising said first magnetic particle, said first capture antibody, said first antigen and said first label probe;
• (g) incubating said second assay mixture under conditions sufficient to form a second complex comprising said second magnetic particle, said second capture antibody, said second antigen and said second label probe;
• (h) determining the presence of said first complex;
• and
• (i) determining the presence of said second complex, thereby detecting or quantitating both the oocysts and sporozoites in the water sample.

A method for the detection or quantitation of Cryptosporidia in a sample comprising:

• (a) extracting the sample in an extraction medium;
• (b) solubilizing, in said extraction medium, antigens of oocysts of said Cryptosporidia, including at least one first antigen from the outer wall of said oocysts and at least one second antigen of a sporozoite contained within said oocyst;
• (c) forming a first assay mixture comprising,
• (i) a first portion of said solubilized antigens,
• (ii) a first capture antibody specific to said first antigen,
• (iii) a first label probe comprising a first label antibody specific to said first antigen and a first electrochemiluminescent moiety, and
• (iv) a first magnetic particle linked to or capable of capturing said first capture antibody;
• (d) forming a second assay mixture comprising,
• (i) a second portion of said solubilized antigens,
• (ii) a second capture antibody specific to said second antigen,
• (iii) a second label probe comprising a second label antibody specific to said second antigen and a second electrochemiluminescent moiety, and
• (iv) a second magnetic particle linked to or capable of capturing said second capture antibody;
• (e) incubating said first assay mixture under conditions sufficient to form a first complex comprising said first magnetic particle, said first capture antibody, said first antigen and said first label probe;
• (f) incubating said second assay mixture under conditions sufficient to form a second complex comprising said second magnetic particle, said second capture antibody, said second antigen and said second label probe;
• (g) determining the presence of said first complex;
• and
• (h) determining the presence of said second complex, thereby detecting or quantitating both the oocysts and sporozoites in the sample.

A composition of matter comprising:

• (a) a retentate formed by filtering a water sample;
• (b) an extraction medium comprising at least one surfactant in an amount effective to solubilize at least one antigen of Cryptosporidia oocyst;
• and
• (c) a complex comprising said antigen and an antibody specific for said antigen.

A kit for quantifying Cryptosporidia in a sample comprising in one or more containers:

• (a) an extraction medium comprising at least one bile salt or ionic detergent in an amount effective to solubilize at least one cell wall antigen from oocysts of Cryptosporidia;
• (b) a capture antibody specific for said antigen;
• (c) a label antibody specific for said antigen;
• and
• (d) a solid support linked to or capable of capturing said capture antibody.