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Host Organism:lama glama X
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T4 antibody
triclocarban (1-(4-Chlorophenyl)-3-(3,4-dichlorophenyl)urea) chemical
(Gonzalez Lab; Facultad de Química; Universidad de la República; Uruguay Cat# INQ-T4, RRID:AB_2721894)
monoclonal antibody
Anal Chem, 83, 7213-7220. "Two adult male llamas (Lama glama) #807 and #856 from the Montevideo municipal zoo were immunized according to animal welfare regulations, intramuscularly with 600 μg of the TCC-Thy conjugate in Freund Incomplete Adjuvant at days 0, followed by 4 booster injections every 3 weeks. One month after the last booster 150 mL of blood was collected in double blood collection bags with anticoagulant. Additionally, 10 mL was collected in plastic tubes to obtain the serum fraction.
...we proceeded with the construction of a VHH library on the premise that a careful selection process would allow us to isolate high affinity binders.
Total RNA was extracted from 108 peripheral blood lymphocytes from llama 807 and retrotranscribed to cDNA, amplified and clone in pComb3X as described.. The phage displayed VH-VHH library, which had a size of 3.4 × 107 clones, was panned against TCC-BSA. Initial panning experiments using acidic elution proved to be unsuccessful because, despite the high rate of TCC-BSA-positive and BSA-negative phage clones, their binding was hardly inhibited by soluble TCC. On the basis of this result, we devised a new panning strategy by using decreasing concentration of the free hapten in the successive rounds of panning, and selected clones from the third round by differential screening on plates coated with limiting amounts of TCC-BSA in the presence or absence of serial dilutions of TCC in the 0–1000 ng/mL range. Clones showing the best different in the absence or presence of 20 ng/ml of TCC were selected.
DNA sequencing revealed that five different clones were selected, all corresponding to sdAbs and not to conventional antibodies. All contain the hallmark substitutions in FR 2, which stabilize the structure of single domain antibodies and are characteristic of VHHs. These are residues F, E, R and F/G at positions 42, 49, 50 and 52, respectively (figure S-2, supporting information). Moreover, as frequently found in these antibodies, the variable domains possess long CDR3 regions (15–22 amino acids). Based on the classification of the VHH heavy-chain regions proposed by Harmsen et al. 20, clones T4-10 appear to belong to subfamily 1, while the VHH domain of clone T11 shows two additional cysteine residues, at positions 55 in FR2 and inside the CDR3, which are characteristic of subfamily 3. Except for the significant similarity between clones T9 and T4, all other clones present important differences in their CDRs. Interestingly, this observation suggests that they derived from independent B cell clones and recognized TCC in a different way."
T4
lama glama
Gonzalez Lab; Facultad de Química; Universidad de la República; Uruguay
INQ-T4
T9 antibody
triclocarban (1-(4-Chlorophenyl)-3-(3,4-dichlorophenyl)urea) chemical
(Gonzalez Lab; Facultad de Química; Universidad de la República; Uruguay Cat# INQ-T9, RRID:AB_2721896)
monoclonal antibody
Anal Chem, 83, 7213-7220. "Two adult male llamas (Lama glama) #807 and #856 from the Montevideo municipal zoo were immunized according to animal welfare regulations, intramuscularly with 600 μg of the TCC-Thy conjugate in Freund Incomplete Adjuvant at days 0, followed by 4 booster injections every 3 weeks. One month after the last booster 150 mL of blood was collected in double blood collection bags with anticoagulant. Additionally, 10 mL was collected in plastic tubes to obtain the serum fraction.
...we proceeded with the construction of a VHH library on the premise that a careful selection process would allow us to isolate high affinity binders.
Total RNA was extracted from 108 peripheral blood lymphocytes from llama 807 and retrotranscribed to cDNA, amplified and clone in pComb3X as described.. The phage displayed VH-VHH library, which had a size of 3.4 × 107 clones, was panned against TCC-BSA. Initial panning experiments using acidic elution proved to be unsuccessful because, despite the high rate of TCC-BSA-positive and BSA-negative phage clones, their binding was hardly inhibited by soluble TCC. On the basis of this result, we devised a new panning strategy by using decreasing concentration of the free hapten in the successive rounds of panning, and selected clones from the third round by differential screening on plates coated with limiting amounts of TCC-BSA in the presence or absence of serial dilutions of TCC in the 0–1000 ng/mL range. Clones showing the best different in the absence or presence of 20 ng/ml of TCC were selected.
DNA sequencing revealed that five different clones were selected, all corresponding to sdAbs and not to conventional antibodies. All contain the hallmark substitutions in FR 2, which stabilize the structure of single domain antibodies and are characteristic of VHHs. These are residues F, E, R and F/G at positions 42, 49, 50 and 52, respectively (figure S-2, supporting information). Moreover, as frequently found in these antibodies, the variable domains possess long CDR3 regions (15–22 amino acids). Based on the classification of the VHH heavy-chain regions proposed by Harmsen et al. 20, clones T4-10 appear to belong to subfamily 1, while the VHH domain of clone T11 shows two additional cysteine residues, at positions 55 in FR2 and inside the CDR3, which are characteristic of subfamily 3. Except for the significant similarity between clones T9 and T4, all other clones present important differences in their CDRs. Interestingly, this observation suggests that they derived from independent B cell clones and recognized TCC in a different way."
T9
lama glama
Gonzalez Lab; Facultad de Química; Universidad de la República; Uruguay
INQ-T9
T10 antibody
triclocarban (1-(4-Chlorophenyl)-3-(3,4-dichlorophenyl)urea) chemical
(Gonzalez Lab; Facultad de Química; Universidad de la República; Uruguay Cat# INQ-T10, RRID:AB_2721895)
monoclonal antibody
Anal Chem, 83, 7213-7220. "Two adult male llamas (Lama glama) #807 and #856 from the Montevideo municipal zoo were immunized according to animal welfare regulations, intramuscularly with 600 μg of the TCC-Thy conjugate in Freund Incomplete Adjuvant at days 0, followed by 4 booster injections every 3 weeks. One month after the last booster 150 mL of blood was collected in double blood collection bags with anticoagulant. Additionally, 10 mL was collected in plastic tubes to obtain the serum fraction.
...we proceeded with the construction of a VHH library on the premise that a careful selection process would allow us to isolate high affinity binders.
Total RNA was extracted from 108 peripheral blood lymphocytes from llama 807 and retrotranscribed to cDNA, amplified and clone in pComb3X as described.. The phage displayed VH-VHH library, which had a size of 3.4 × 107 clones, was panned against TCC-BSA. Initial panning experiments using acidic elution proved to be unsuccessful because, despite the high rate of TCC-BSA-positive and BSA-negative phage clones, their binding was hardly inhibited by soluble TCC. On the basis of this result, we devised a new panning strategy by using decreasing concentration of the free hapten in the successive rounds of panning, and selected clones from the third round by differential screening on plates coated with limiting amounts of TCC-BSA in the presence or absence of serial dilutions of TCC in the 0–1000 ng/mL range. Clones showing the best different in the absence or presence of 20 ng/ml of TCC were selected.
DNA sequencing revealed that five different clones were selected, all corresponding to sdAbs and not to conventional antibodies. All contain the hallmark substitutions in FR 2, which stabilize the structure of single domain antibodies and are characteristic of VHHs. These are residues F, E, R and F/G at positions 42, 49, 50 and 52, respectively (figure S-2, supporting information). Moreover, as frequently found in these antibodies, the variable domains possess long CDR3 regions (15–22 amino acids). Based on the classification of the VHH heavy-chain regions proposed by Harmsen et al. 20, clones T4-10 appear to belong to subfamily 1, while the VHH domain of clone T11 shows two additional cysteine residues, at positions 55 in FR2 and inside the CDR3, which are characteristic of subfamily 3. Except for the significant similarity between clones T9 and T4, all other clones present important differences in their CDRs. Interestingly, this observation suggests that they derived from independent B cell clones and recognized TCC in a different way."
T10
lama glama
Gonzalez Lab; Facultad de Química; Universidad de la República; Uruguay
INQ-T10