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Design and selection of a camelid single-chain antibody yeast two-hybrid library produced de novo for the cap protein of porcine circovirus type 2 (PCV2).

Fu X, Gao X, He S, Huang D, Zhang P, Wang X, Zhang S, Dang R, Yin S, Du E, Yang Z - PLoS ONE (2013)

Bottom Line: Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences.Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells.All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi, China.

ABSTRACT
Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs) are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid (Y2H) library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×10(6) cfu/3 µg and 2×10(9) cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.

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Determination of the specific interaction between Cap and anti-Cap VHHs by indirect ELISA.A, The plate was coated with PCV2 viral lysis (10−6.4 TCID50, 1∶800 dilution). B, The plate was coated with purified Cap protein (0.5 µg/ml). The positive serum in A and B was the anti-Cap serum taken from a pig. Negative 1 and 2 in A represent the wells coated with BSA (negative control 1) or the lysis of PCV2 uninfected FPRC cells (negative control 2), respectively. Negative 1 and in B represent the wells coated with BSA (negative control 1) and 0.5 µg/ml purified MBP tag (negative control 2). The absorbance at 450 nm indicated the binding activity between selected VHHs and Cap. Data is plotted as the average of 3 wells, with error bars representing the standard deviation.
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pone-0056222-g007: Determination of the specific interaction between Cap and anti-Cap VHHs by indirect ELISA.A, The plate was coated with PCV2 viral lysis (10−6.4 TCID50, 1∶800 dilution). B, The plate was coated with purified Cap protein (0.5 µg/ml). The positive serum in A and B was the anti-Cap serum taken from a pig. Negative 1 and 2 in A represent the wells coated with BSA (negative control 1) or the lysis of PCV2 uninfected FPRC cells (negative control 2), respectively. Negative 1 and in B represent the wells coated with BSA (negative control 1) and 0.5 µg/ml purified MBP tag (negative control 2). The absorbance at 450 nm indicated the binding activity between selected VHHs and Cap. Data is plotted as the average of 3 wells, with error bars representing the standard deviation.

Mentions: Indirect ELISA was used to detect the specific interaction between VHH antibodies and PCV2 viral lysis or purified Cap protein. For plate coated with PCV2 viral lysis, the measurement of virus titer was 10−6.4 TCID50 and the best coated concentration of the virus was 1∶800 (data not shown). The ELISA results indicated that most of the selected VHHs interacted with PCV2 viral lysis, among which 7 of 9 VHH clones were significantly stronger than the commercial PCV2 positive serum control (OD450>1.50, P<0.05) (Figure 7A). The reaction in the wells coated with BSA (negative control 1) or the lysis of PCV2 uninfected FPRC (negative control 2) was very weak (OD450<0.10).


Design and selection of a camelid single-chain antibody yeast two-hybrid library produced de novo for the cap protein of porcine circovirus type 2 (PCV2).

Fu X, Gao X, He S, Huang D, Zhang P, Wang X, Zhang S, Dang R, Yin S, Du E, Yang Z - PLoS ONE (2013)

Determination of the specific interaction between Cap and anti-Cap VHHs by indirect ELISA.A, The plate was coated with PCV2 viral lysis (10−6.4 TCID50, 1∶800 dilution). B, The plate was coated with purified Cap protein (0.5 µg/ml). The positive serum in A and B was the anti-Cap serum taken from a pig. Negative 1 and 2 in A represent the wells coated with BSA (negative control 1) or the lysis of PCV2 uninfected FPRC cells (negative control 2), respectively. Negative 1 and in B represent the wells coated with BSA (negative control 1) and 0.5 µg/ml purified MBP tag (negative control 2). The absorbance at 450 nm indicated the binding activity between selected VHHs and Cap. Data is plotted as the average of 3 wells, with error bars representing the standard deviation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585807&req=5

pone-0056222-g007: Determination of the specific interaction between Cap and anti-Cap VHHs by indirect ELISA.A, The plate was coated with PCV2 viral lysis (10−6.4 TCID50, 1∶800 dilution). B, The plate was coated with purified Cap protein (0.5 µg/ml). The positive serum in A and B was the anti-Cap serum taken from a pig. Negative 1 and 2 in A represent the wells coated with BSA (negative control 1) or the lysis of PCV2 uninfected FPRC cells (negative control 2), respectively. Negative 1 and in B represent the wells coated with BSA (negative control 1) and 0.5 µg/ml purified MBP tag (negative control 2). The absorbance at 450 nm indicated the binding activity between selected VHHs and Cap. Data is plotted as the average of 3 wells, with error bars representing the standard deviation.
Mentions: Indirect ELISA was used to detect the specific interaction between VHH antibodies and PCV2 viral lysis or purified Cap protein. For plate coated with PCV2 viral lysis, the measurement of virus titer was 10−6.4 TCID50 and the best coated concentration of the virus was 1∶800 (data not shown). The ELISA results indicated that most of the selected VHHs interacted with PCV2 viral lysis, among which 7 of 9 VHH clones were significantly stronger than the commercial PCV2 positive serum control (OD450>1.50, P<0.05) (Figure 7A). The reaction in the wells coated with BSA (negative control 1) or the lysis of PCV2 uninfected FPRC (negative control 2) was very weak (OD450<0.10).

Bottom Line: Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences.Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells.All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi, China.

ABSTRACT
Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs) are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid (Y2H) library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×10(6) cfu/3 µg and 2×10(9) cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.

Show MeSH
Related in: MedlinePlus