Methods for detecting shigella bacteria or antibodies to shigella bacteria with an immunoassay

A method for detecting antibodies to Shigella bacteria in an animal or biological sample therefrom, comprising the steps of:

• a) contacting said biological sample with a Shigella bacterium or a fragment thereof having an enhanced antigenic property, wherein said Shigella bacterium is harvested from a culture of a Shigella species, wherein said culture is at about early log phase, between early log phase and stationary phase, or at about stationary phase grown in vitro under a set of conditions comprising:
• 1) a divalent cation chelator selected from the group consisting of about 1.0 to about 25 .mu.M of BAPTA/AM, about 0.5 to about 10 mM of EGTA, and about 1 to about 100 .mu.M of EGTA/AM;
• 2) at a temperature between about 30.degree. and about 42.degree. C.;
• and
• 3) in air or an microaerophilic condition, wherein the microaerophilic condition comprises i) about 5% to about 20% CO.sub.2 with about 80% to about 95% air;
• ii) about 5% to about 20% CO.sub.2 with about 80% to about 95% N.sub.2 ;
• or iii) about 5% to about 10% O.sub.2 with about 10% to about 20% CO.sub.2 with about 70% to about 85% N.sub.2 ;
• and
• b) detecting an antibody present in said biological sample binding to the Shigella bacterium or fragment thereof wherein said detecting is by means of an immunoassay.

A method for detecting Shigella bacteria in an animal or biological sample therefrom, comprising the steps of:

• a) contacting said biological sample with an antibody that binds the Shigella bacteria, or a fragment thereof, said antibody having been obtained using a Shigella bacterium having an enhanced antigenic property, wherein said Shigella bacterium having an enhanced antigenic property is harvested from a culture of a Shigella species, wherein said culture is at about early log phase, between early log phase and stationary phase, or at about stationary phase, grown in vitro under a combination of conditions comprising:
• 1) about 0.05% to about 3% bile or about 0.025% to about 0.6% of one or more bile acids or salts thereof;
• 2) at a temperature between about 30.degree. C. and about 42.degree. C.;
• 3) in air or a gas mixture, wherein the gas mixture comprises i) about 5% to about 20% CO.sub.2 with about 80% to about 95% air;
• ii) about 5% to about 20% CO.sub.2 with about 80% to about 95% N.sub.2 ;
• or iii) about 5% to about 10% O.sub.2 with about 10% to about 20% CO.sub.2 with about 70% to about 85% N.sub.2 ;
• and
• 4) a divalent cation chelator selected from the group consisting of 0 to about 100 .mu.M of BAPTA/AM, 0 to about 10 mM of EGTA, and 0 about 100 .mu.M of EGTA/AM, and
• b) detecting an antibody binding to the Shigella bacteria or fragment thereof present in said biological sample, wherein said detecting is by means of an immunoassay.

A method for detecting Shigella bacteria in an animal or biological sample therefrom, comprising the steps of:

• a) contacting said biological sample with an antibody that binds the Shigella bacteria, or a fragment thereof, said antibody having been obtained using a Shigella bacterium having an enhanced antigenic property, wherein said Shigella bacterium having an enhanced antigenic property is harvested from a culture of a Shigella species wherein said culture is at about early log phase, between early log phase and stationary phase, or at about stationary phase, grown in vitro under a set of conditions comprising:
• 1) a divalent cation chelator selected from the group consisting of about 1.0 to about 25 .mu.M of BAPTA/AM, about 0.5 to about 10 mM of EGTA, and about 1 to about 100 .mu.M of EGTA/AM;
• 2) at a temperature between about 30.degree. and about 42.degree. C.;
• and
• 3) in air or an microaerophilic condition, wherein the microaerophilic condition comprises i) about 5% to about 20% CO.sub.2 wits about 80% to about 95% air;
• ii) about 5% to about 20% CO.sub.2 with about 80% to about 95% N.sub.2 ;
• or iii) about 5% to about 10% O.sub.2 with about 10% to about 20% CO.sub.2 with about 70% to about 85% N.sub.2 ;
• and
• b) detecting an antibody binding to the Shigella bacteria or fragment thereof present in said biological sample wherein said detecting is by means of an immunoassay.

A diagnostic immunoassay kit for detecting production of antibodies to Shigella bacteria or for detecting Shigella bacteria, comprising:

• a) a Shigella bacterium or a fragment thereof or antibodies thereto, wherein said Shigella bacterium has an enhanced antigenic property, and is harvested from a culture of a Shigella species grown in vitro under a combination of conditions comprising:
• 1) about 0.05% to about 3% bile or about 0.025% to about 0.6% of one or more bile acids or salts thereof;
• 2) at a temperature between about 30.degree. C. and about 42.degree. C.;
• 3) in air or a gas mixture, wherein the gas mixture comprises i) about 5% to about 20% CO.sub.2 with about 80% to about 95% air;
• ii) about 5% to about 20% CO.sub.2 with about 80% to about 95% N.sub.2 ;
• or iii) about 5% to about 10% O.sub.2 with about 10% to about 20% CO.sub.2 with about 70% to about 85% N.sub.2 ;
• and
• 4) a divalent cation chelator selected from the group consisting of 0 to about 100 .mu.M of BAPTA/AM, 0 to about 10 mM of EGTA, and 0 about 100 .mu.M of EGTA/AM, and
• b) at least one kit component selected from the group consisting of microtiter plates, buffers to maintain pH and a conjugated antibody.

A diagnostic immunoassay kit for detecting production of antibodies to Shigella bacteria or for detecting Shigella bacteria, comprising:

• a) a Shigella bacterium or a fragment thereof or antibodies thereto, wherein said Shigella bacterium has an enhanced antigenic property, and is harvested from a culture of a Shigella species grown in vitro under a set of conditions comprising:
• 1) a divalent cation chelator selected from the group consisting of about 1.0 to about 25 .mu.M of BAPTA/AM, about 0.5 to about 10 mM of EGTA, and about 1 to about 100 .mu.M of EGTA/AM;
• 2) at a temperature between about 30.degree. and about 42.degree. C.;
• and
• 3) in air or an microaerophilic condition, wherein the microaerophilic condition comprises i) about 5% to about 20% CO.sub.2 with about 80% to about 95% air;
• ii) about 5% to about 20% CO.sub.2 with about 80% to about 95% N.sub.2 ;
• or iii) about 5% to about 10% O.sub.2 with about 10% to about 20% CO.sub.2 with about 70% to about 85% N.sub.2, and
• b) at least one kit component selected form the group consisting of microtiter plates, buffers to maintain pH and a conjugated antibody.