Article Figures & Data
Figures
- FIGURE 1.
In vivo Ab production after the intranasal immunization with saline, HSA or sugar-conjugated HSA in BALB/c (A–D) and C57BL/6 (E and F) mice. Serum samples were taken after prime (▦), boost (□), and challenge (▪) as described in Materials and Methods. Ab of IgG (A and E), IgM, IgA, and IgG subclasses (B) specific for HSA were measured by indirect ELISA and estimated by endpoint titer. IgE titers specific for HSA (C and F) and the volume of serum total IgE (D) were determined by sandwich ELISA. Levels of HSA-specific IgE were presented as the absorbance at 450 nm from duplicate wells of 1/4 serum dilution. Results show the mean ± SEM of four individual serum per group. Data are representative of three (A–D) and two (E and F) separate experiments.
- FIGURE 2.
Analysis of Ag dose capable for the induction of Ab production. BALB/c mice were immunized intranasally with serial amounts of HSA (▪) or HSA-LNFPIII (•) described in Fig. 1. Amounts of Ag were determined by BCA protein assay. After 6th challenge, sera were collected, and HSA-specific IgG (A) and IgE (B) were analyzed. Results show the mean ± SEM of endpoint titer (A) or absorbance at 450 nm of 4× diluted serum (B) from four individual serum per group. Data are representative of two separate experiments.
- FIGURE 3.
In vivo Ab production after the intranasal immunization with saline, BSA or BSA-LNFPIII. Plasma samples were taken after prime (▦), boost (□), and challenge (▪) in an identical manner as described in Fig. 1. Ab of IgG (A) specific for BSA was measured by indirect ELISA and estimated by endpoint titer. IgE titers specific for BSA (B) were determined by sandwich ELISA and presented as the absorbance at 450 nm from duplicate wells of 1/4 serum dilution. Results show the mean ± SEM of four individual serum per group. Data are representative of two separate experiments.
- FIGURE 4.
A, Inhibition ELISA for HSA-LNFPIII-specific IgG using HSA (•) and free LNFPIII (□) as an competitor. Sera used in this study were from mice immunized and challenged with HSA-LNFPIII. Results are given as the average absorbance at 450 nm ± SEM of percent response from sera of four individual mice with inhibitors added divided by the response without inhibitors. B, Comparison of binding between HSA and Lewisx for IgE in murine sera intranasally immunized with HSA-LNFIII. Either biotinylated HSA or Lewisx was added as a detection reagent in captured IgE ELISA. Results show the mean ± SEM of four individual serum. Data are representative of two separate experiments.
- FIGURE 5.
Effect of free LNFPIII on HSA-specific IgG Ab production in response to immunization with HSA (▪) or HSA-LNFPIII (•). BALB/c mice were primed intranasally with HSA or HSA-LNFPIII (10 μg protein) simultaneously mixed with monovalent LNFPIII. Boost and challenge were performed as described in Materials and Methods, and free LNFPIII was given at same dose and 1/10, respectively. After 6 the challenge, sera were taken and endpoint titer of HSA-specific IgG Ab was determined described as above. Results show the mean endpoint titer ± SEM of four individual serum. Data are representative of two separate experiments.
- FIGURE 6.
Production of IL-4 (A), IL-5 (B), IL-10 (C), and IFN-γ (D) by nasal lymphocytes from BALB/C mice immunized intranasally with HSA, HSA-LNFPIII or saline. Nasal lymphocytes were isolated as described in Materials and Methods and restimulated in vitro with 10 μg/ml of HSA (□) or HSA-LNFPIII (▪) for 48 h. Cytokines were determined by ELISA. Results show the mean ± SEM of four different experiments. Detection limit was 6 pg/ml, 20 pg/ml, 40 pg/ml, and 20 pg/ml for IL-4, IL-5, IL-10, and IFN-γ, respectively.
- FIGURE 7.
Expression of CD80 (A–C) and CD86 (D–F) on B220+ nasal lymphocytes. Following 48 h in vitro recall stimulation with the same Ag as immunization; saline (A and D), HSA (B and E), or HSA-LNFIII (C and F), respectively, cell pellets were collected and stained by anti-CD80/CD86 and B220 mAbs coupled to FITC or PE, respectively, for flow cytometry analysis. Values given in the upper right quadrant indicate the mean percentage ± SEM of double-positive cells from four different experiments.
- FIGURE 8.
In vivo Ab production after i.p. (A and B) and s.c. (C and D) immunization with HSA, HSA adsorbed to alum, HSA-LNFPIII, or HSA-LNFPIII adsorbed to alum. Serum samples were taken 1 wk after the boost immunization as described in Materials and Methods. Levels of IgG (A and C) specific for HSA were determined by indirect ELISA and presented as endpoint titer. Levels of HSA-specific IgE (B and D) were determined by sandwich ELISA and presented as the absorbance at 450 nm from duplicate wells of 1/4 serum dilution. Results show the mean ± SEM of four individual serum per group. Data are representative of two separate experiments.
- FIGURE 9.
Priming effect of HSA-LNFPIII on Ab production following subsequent immunizaiton with HSA. A group of BALB/c mice was primed intranasally with HSA-LNFPIII and subsequently boosted and challenged with HSA alone in an identical manner as described in Materials and Methods. As a control, a group of mice was immunized with HSA alone throughout the experiment. Plasma samples were taken after prime (▦), boost (□), and challenge (▪). Titers of IgG (A) and IgE (B) specific for HSA were presented as the endpoint titer and the absorbance at 450 nm from duplicate wells of 1/4 serum dilution, respectively. Results show the mean ± SEM of four individual serum per group. Data are representative of two separate experiments.
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