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Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies.

Ulaeto DO, Hutchinson AP, Nicklin S - Monoclon Antib Immunodiagn Immunother (2015)

Bottom Line: Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex.Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA.The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites.

View Article: PubMed Central - PubMed

Affiliation: 1 Biomedical Sciences Department, Dstl Porton Down , Salisbury, United Kingdom .

ABSTRACT
Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites.

No MeSH data available.


Structure of RDX12 (A) and RDX14 (B). RDX and RDX derivatized haptens were synthesized as previously described.(7) Derivatized haptens were conjugated to carrier proteins as follows: 65 mg EDC-1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 75 mg SULFONHS 1-hydroxy-2-5-dioxo-3 pyrrolidine sulfonic acid monosodium salt were added to 16.51 mg of RDX hapten dissolved in 1 mL of 50% N,N-dimethyl formaldehyde (v/v) in water. The mixture was stirred at room temperature for 30 min. After mixing, 20 mg of BSA, KLH, or CGG, respectively, was dissolved in 2 mL water and added to the mixture, which was then stirred for a further 3 h at room temperature. Each conjugate was collected and dialyzed extensively against four 3 L changes of PBS. Animal studies were performed in accordance with the UK Scientific Procedures Act (Animals) 1986 and UK Codes of Practice for the Housing and Care of Animals Used in Scientific Procedures (1989).
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f1: Structure of RDX12 (A) and RDX14 (B). RDX and RDX derivatized haptens were synthesized as previously described.(7) Derivatized haptens were conjugated to carrier proteins as follows: 65 mg EDC-1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 75 mg SULFONHS 1-hydroxy-2-5-dioxo-3 pyrrolidine sulfonic acid monosodium salt were added to 16.51 mg of RDX hapten dissolved in 1 mL of 50% N,N-dimethyl formaldehyde (v/v) in water. The mixture was stirred at room temperature for 30 min. After mixing, 20 mg of BSA, KLH, or CGG, respectively, was dissolved in 2 mL water and added to the mixture, which was then stirred for a further 3 h at room temperature. Each conjugate was collected and dialyzed extensively against four 3 L changes of PBS. Animal studies were performed in accordance with the UK Scientific Procedures Act (Animals) 1986 and UK Codes of Practice for the Housing and Care of Animals Used in Scientific Procedures (1989).

Mentions: To avoid the above scenarios, two different derivatives of RDX were used in this project, RDX12, which has a relatively long derivative side chain, and RDX14, which has a very short derivative side chain (Fig. 1). RDX14 conjugates were used for immunizations of 5- to 8-week-old female BALB/c mice, and an RDX12 conjugate was used for screening of sera and hybridoma supernatants. The rationale for this is that only antibodies reactive with both derivatives will be selected, and the only features in common between the derivatives are those that are also shared by underivatized RDX. To further optimize the protocol, the derivatized haptens were conjugated to different carrier proteins, keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA), to ensure that carrier protein determinants were not part of the epitopes recognized by expanded antibodies. KLH-RDX14 conjugate emulsified in complete Freund's adjuvant (CFA) was used for all primary immunizations; but the mice were also simultaneously immunized with unconjugated BSA, emulsified in CFA, from a separate syringe at a separate site. Booster immunizations used a BSA-RDX14 conjugate emulsified in incomplete Freund's adjuvant. Hybridomas were generated by standard techniques using the X63-AG8 plasmacytoma fusion partner.(2,5)


Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies.

Ulaeto DO, Hutchinson AP, Nicklin S - Monoclon Antib Immunodiagn Immunother (2015)

Structure of RDX12 (A) and RDX14 (B). RDX and RDX derivatized haptens were synthesized as previously described.(7) Derivatized haptens were conjugated to carrier proteins as follows: 65 mg EDC-1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 75 mg SULFONHS 1-hydroxy-2-5-dioxo-3 pyrrolidine sulfonic acid monosodium salt were added to 16.51 mg of RDX hapten dissolved in 1 mL of 50% N,N-dimethyl formaldehyde (v/v) in water. The mixture was stirred at room temperature for 30 min. After mixing, 20 mg of BSA, KLH, or CGG, respectively, was dissolved in 2 mL water and added to the mixture, which was then stirred for a further 3 h at room temperature. Each conjugate was collected and dialyzed extensively against four 3 L changes of PBS. Animal studies were performed in accordance with the UK Scientific Procedures Act (Animals) 1986 and UK Codes of Practice for the Housing and Care of Animals Used in Scientific Procedures (1989).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4554551&req=5

f1: Structure of RDX12 (A) and RDX14 (B). RDX and RDX derivatized haptens were synthesized as previously described.(7) Derivatized haptens were conjugated to carrier proteins as follows: 65 mg EDC-1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 75 mg SULFONHS 1-hydroxy-2-5-dioxo-3 pyrrolidine sulfonic acid monosodium salt were added to 16.51 mg of RDX hapten dissolved in 1 mL of 50% N,N-dimethyl formaldehyde (v/v) in water. The mixture was stirred at room temperature for 30 min. After mixing, 20 mg of BSA, KLH, or CGG, respectively, was dissolved in 2 mL water and added to the mixture, which was then stirred for a further 3 h at room temperature. Each conjugate was collected and dialyzed extensively against four 3 L changes of PBS. Animal studies were performed in accordance with the UK Scientific Procedures Act (Animals) 1986 and UK Codes of Practice for the Housing and Care of Animals Used in Scientific Procedures (1989).
Mentions: To avoid the above scenarios, two different derivatives of RDX were used in this project, RDX12, which has a relatively long derivative side chain, and RDX14, which has a very short derivative side chain (Fig. 1). RDX14 conjugates were used for immunizations of 5- to 8-week-old female BALB/c mice, and an RDX12 conjugate was used for screening of sera and hybridoma supernatants. The rationale for this is that only antibodies reactive with both derivatives will be selected, and the only features in common between the derivatives are those that are also shared by underivatized RDX. To further optimize the protocol, the derivatized haptens were conjugated to different carrier proteins, keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA), to ensure that carrier protein determinants were not part of the epitopes recognized by expanded antibodies. KLH-RDX14 conjugate emulsified in complete Freund's adjuvant (CFA) was used for all primary immunizations; but the mice were also simultaneously immunized with unconjugated BSA, emulsified in CFA, from a separate syringe at a separate site. Booster immunizations used a BSA-RDX14 conjugate emulsified in incomplete Freund's adjuvant. Hybridomas were generated by standard techniques using the X63-AG8 plasmacytoma fusion partner.(2,5)

Bottom Line: Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex.Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA.The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites.

View Article: PubMed Central - PubMed

Affiliation: 1 Biomedical Sciences Department, Dstl Porton Down , Salisbury, United Kingdom .

ABSTRACT
Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites.

No MeSH data available.