Assay method for detecting the presence of bacteria

A method for detecting the presence of bacteria which comprises:

• a) combining a sample to be tested for the presence of a selected bacteria with a magnetic solid support having immobilized thereon first antibodies to said selected bacteria, to thereby capture cells of said selected bacteria from said sample, said antibodies being attached to said solid support through a protein A intermediate such that the Fab portions of said antibodies extend outward from the surface of said solid support, said magnetic solid support being nonporous magnetic beads having a diameter of from 50 nm to 1 .mu.m;
• b) exposing to a magnetic field said solid support having bound thereto said first antibodies and captured bacteria cells to thereby separate said magnetic solid support from said sample;
• c) culturing said magnetic solid support and captured bacteria cells on a solid medium to form colonies of said bacteria;
• d) contacting said colonies with a colony lift membrane whereby colony material from said colonies adheres to said membrane;
• e) contacting said membrane and said colony material adhered thereto with second antibodies specific for said selected bacteria, whereby said second antibodies bind to said colony material from said colonies of said selected bacteria;
• f) contacting said membrane with labeled antibodies specific for said second antibodies, whereby said labeled antibodies bind to said second antibodies bound to said colonies of said selected bacteria;
• and
• g) treating said membrane to provide evidence of the presence on said membrane of the label on said labeled antibody and colonies of said selected bacteria.

A method for determining the quantity of Listeria in a sample which comprises:

• a) combining a sample to be tested for the presence of Listeria with a magnetic solid support having immobilized thereon antibodies to said Listeria, to thereby capture cells of said Listeria from said sample, said magnetic solid support being non-porous magnetic beads having a diameter of from 50 nm to 1 .mu.m;
• b) exposing to a magnetic field said magnetic beads having bound thereto said first antibodies and captured Listeria cells to thereby separate said magnetic solid support from said sample;
• c) culturing the thus captured magnetic solid support and captured Listeria on a solid medium to form colonies of said Listeria;
• d) contacting said colonies with a colony lift membrane whereby some colony material adheres to said membrane;
• and
• e) treating said membrane to detect the presence on said membrane of material from said colonies of said
• Listeria and to provide a quantification of the amount of Listeria in said sample.

A method for determining the quantity of Listeria in a sample, said method being capable of detecting one colony forming unit per 25 ml of sample, said method comprises:

• a) combining a sample to be tested for the presence of Listeria with a magnetic solid support having immobilized thereon first antibodies to said Listeria, to thereby capture cells of Listeria from said sample, said antibodies being attached to said solid support through an antibody linking compound such that the Fab portions of said antibodies extend outward from the surface of said solid support, said magnetic solid supporter being nonporous magnetic beads having a diameter of from 50 nm to 1 .mu.m;
• b) exposing to a magnetic field said solid support having bound thereto said first antibodies and captured Listeria cells to thereby separate said magnetic solid support from said sample;
• c) culturing said magnetic solid support and captured bacteria cells on a solid medium to form colonies of said Listeria;
• d) contacting said colonies with a colony lift membrane whereby colony material from said colonies adheres to said membrane;
• e) contacting said membrane and said colony material adhered thereto with second antibodies specific for said Listeria, whereby said second antibodies bind to said colony material from said colonies of said Listeria;
• f) contacting said membrane with antibodies conjugated with a label and being specific for said second antibodies, whereby said labeled antibodies bind to said second antibodies bound to said colonies of said selected bacteria;
• g) treating said membrane to provide evidence of the presence on said membrane of said label and colonies of said Listeria;
• and
• h) determining the number of colonies on said membrane to provide a quantitative measure of the number of said Listeria in said sample.

A method for detecting the presence of bacteria in a sample which comprises:

• a) combining a sample to be tested for the presence of a selected bacteria with a magnetic solid support having immobilized thereon antibodies to said selected bacteria, to hereby capture cells of said selected bacteria from said sample, said magnetic solid support being non-porous magnetic beads having a diameter of from 50 nm to 1 .mu.m;
• b) exposing to a magnetic field said magnetic beads having bound thereto said antibodies and captured bacteria cells to thereby separate said magnetic solid support from said sample;
• c) culturing the thus captured magnetic solid support and captured bacteria cells on a solid medium to form colonies of said selected bacteria;
• d) contacting said colonies with a colony lift membrane whereby colony material adheres to said membrane;
• and
• e) treating said membrane to detect the presence on said membrane of material from said colonies of said selected bacteria.

A method for detecting the presence of bacteria in a sample, said method being capable of detecting one colony forming unit per 25 ml of sample, said method comprises:

• a) combining a sample to be tested for the presence of a selected bacteria with a magnetic solid support having immobilized thereon first antibodies to said selected bacteria, to thereby capture cells of said selected bacteria from said sample, said antibodies being attached to said solid support through an antibody linking compound such that the Fab portions of said antibodies extend outward from the surface of said solid support, said magnetic solid support being nonporous magnetic beads having a diameter of from 50 nm to 1 .mu.m;
• b) exposing to a magnetic field said solid support having bound thereto said first antibodies and captured bacteria cells to thereby separate said magnetic solid support from said sample;
• c) culturing said magnetic solid support and captured bacteria cells on a solid medium to form colonies of said selected bacteria;
• d) contacting said colonies with a colony lift membrane whereby colony material from said colonies adheres to said membrane;
• and
• e) treating said membrane to detect the presence on said membrane of material from said colonies of said selected bacteria.

A method for determining the quantity of Listeria in a sample which comprises:

• a) combining a sample to be tested for the presence of Listeria with a magnetic solid support having immobilized thereon antibodies to said Listeria, to thereby capture cells of said Listeria from said sample, said antibodies being attached to said solid support through an antibody linking compound such that the Fab portions of said antibodies extend outward from the surface of said solid support, said magnetic solid support being non-porous magnetic beads having a diameter of from 50 nm to 1 .mu.m;
• b) exposing to a magnetic field said magnetic beads having bound thereto said antibodies and captured Listeria cells to thereby separate said magnetic solid support from said sample;
• c) culturing said magnetic solid support and captured Listeria cells on a solid medium to form colonies of said Listeria;
• d) contacting said colonies with a colony lift membrane whereby some colony material adheres to said membrane;
• and
• e) treating said membrane to detect the presence on said membrane of material from said colonies of said Listeria and to provide a quantification of the amount of Listeria in said sample.