Abstract
Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). Although conventional antibodies dominate current assay development, recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. We expressed VHHs from an immunized alpaca and developed a VHH-based immunoassay using 3-phenoxybenzoic acid (3-PBA), a major metabolite of pyrethroid insecticides as a model system. A phage VHH library was constructed, and seven VHH clones were selected by competitive binding with 3-PBA. The best immunoassay developed with one of these VHHs showed an IC 50 of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameters were further improved by using the phage borne VHH, IC 50 = 0.1 ng/mL and LOD = 0.01 ng/mL. Both assays showed a similar tolerance to methanol and dimethylsulfoxide up to 50% in assay buffer. The assay was highly specific to 3-PBA and its 4-hydroxylated derivative, 4-hydroxy 3-PBA, (150% cross reactivity) with negligible cross reactivity with other tested structural analogues, and the recovery from spiked urine sample ranged from 80 to 112%. In conclusion, a highly specific and sensitive VHH for 3-PBA was developed using sequences from immunized alpaca and phage display technology for antibody selection.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1165-1171 |
| Number of pages | 7 |
| Journal | Analytical Chemistry |
| Volume | 84 |
| Issue number | 2 |
| DOIs | |
| State | Published - Jan 17 2012 |
Fingerprint
ASJC Scopus subject areas
- Analytical Chemistry
Cite this
Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay. / Kim, Hee Joo; McCoy, Mark R.; Majkova, Zuzana; Dechant, Julie E; Gee, Shirley J.; Tabares-Da Rosa, Sofia; González-Sapienza, Gualberto G.; Hammock, Bruce D.
In: Analytical Chemistry, Vol. 84, No. 2, 17.01.2012, p. 1165-1171.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay
AU - Kim, Hee Joo
AU - McCoy, Mark R.
AU - Majkova, Zuzana
AU - Dechant, Julie E
AU - Gee, Shirley J.
AU - Tabares-Da Rosa, Sofia
AU - González-Sapienza, Gualberto G.
AU - Hammock, Bruce D.
PY - 2012/1/17
Y1 - 2012/1/17
N2 - Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). Although conventional antibodies dominate current assay development, recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. We expressed VHHs from an immunized alpaca and developed a VHH-based immunoassay using 3-phenoxybenzoic acid (3-PBA), a major metabolite of pyrethroid insecticides as a model system. A phage VHH library was constructed, and seven VHH clones were selected by competitive binding with 3-PBA. The best immunoassay developed with one of these VHHs showed an IC 50 of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameters were further improved by using the phage borne VHH, IC 50 = 0.1 ng/mL and LOD = 0.01 ng/mL. Both assays showed a similar tolerance to methanol and dimethylsulfoxide up to 50% in assay buffer. The assay was highly specific to 3-PBA and its 4-hydroxylated derivative, 4-hydroxy 3-PBA, (150% cross reactivity) with negligible cross reactivity with other tested structural analogues, and the recovery from spiked urine sample ranged from 80 to 112%. In conclusion, a highly specific and sensitive VHH for 3-PBA was developed using sequences from immunized alpaca and phage display technology for antibody selection.
AB - Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). Although conventional antibodies dominate current assay development, recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. We expressed VHHs from an immunized alpaca and developed a VHH-based immunoassay using 3-phenoxybenzoic acid (3-PBA), a major metabolite of pyrethroid insecticides as a model system. A phage VHH library was constructed, and seven VHH clones were selected by competitive binding with 3-PBA. The best immunoassay developed with one of these VHHs showed an IC 50 of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameters were further improved by using the phage borne VHH, IC 50 = 0.1 ng/mL and LOD = 0.01 ng/mL. Both assays showed a similar tolerance to methanol and dimethylsulfoxide up to 50% in assay buffer. The assay was highly specific to 3-PBA and its 4-hydroxylated derivative, 4-hydroxy 3-PBA, (150% cross reactivity) with negligible cross reactivity with other tested structural analogues, and the recovery from spiked urine sample ranged from 80 to 112%. In conclusion, a highly specific and sensitive VHH for 3-PBA was developed using sequences from immunized alpaca and phage display technology for antibody selection.
UR - http://www.scopus.com/inward/record.url?scp=84862926222&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84862926222&partnerID=8YFLogxK
U2 - 10.1021/ac2030255
DO - 10.1021/ac2030255
M3 - Article
C2 - 22148739
AN - SCOPUS:84862926222
VL - 84
SP - 1165
EP - 1171
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 2
ER -