**2.5 Data collection**

Data were collected by taking venous puncture for measuring total serum IgE and IgG4 from subjects' blood samples and taking saliva for measuring lipopolysaccharide of *Porphyromonas gingivalis* concentrations [23]. Total serum IgE and IgG4 were assessed by direct-sandwich ELISA (R&D System Europe Ltd., Abingdon, UK) according to the manufacturer's protocol. Briefly, total serum IgE was detected by diluting plasma (1:200), transferring it to pre-coated plates, and adding the supplied conjugate. However, total serum IgG4 were detected using monoclonal antibody against IgG4, followed by additions of blocking solution, diluted plasma sample (1:100,000) or standards, and conjugate, with washing between the steps. Total serum IgE and IgG4 ELISAs were developed with the supplied TMB substrate and stop solutions. Total serum IgE and IgG4 concentrations were determined using assay-specific 7-point calibration curves generated with the


**35**

*Correlation between Salivary Lipopolysaccharide of Porphyromonas gingivalis with Circulatory…*

manufacturer-supplied standard. A value of 0.01 pg/ml was assigned for concen-

culture from salivary secretion, added to 49.5 ml of sterile saline [23]. From this suspension, two serial 1:100 dilutions were made, and 0.1 ml was plated onto plate count agar from the last dilution [23]. Following that, lipopolysaccharides of *Porphyromonas gingivalis* were extracted by commercially optimized assay coupled with chromogenic substrate from pellet. We measured the concentration of extracted lipopolysaccharide by Pierce LAL Chromogenic Endotoxin Quantitation Kit. All measurements were done in duplicate and values averaged

The obtained data were tabulated and analyzed using Statistical Package of Social Science (SPSS version 17, IBM, New York, USA). First, univariable linear regression procedures were conducted to examine associations between circulatory Ig-E and all determinants. In advance, dummy variables were created for all categorical determinants. Second, a multiple linear regression analysis with a stepwise exclusion method was conducted with all continuous and dummy variables. Determinants that seemed relevant for prediction of activation were kept in the

A total of 20 periodontally healthy children accepted to participate in this study; 8 were males and 12 were females. The average age was 10.52 ± 2.78 years (range,

Analysis of correlation between salivary LPS-Pg and Ig-E levels showed a strong

Analysis of correlation between salivary LPS-Pg and Ig-G4 levels showed a very

As shown in **Table 3**, univariate logistic regression analysis uses gender, age, height, weight, BMI, family size, insurance status, number of colony-forming unit (CFU) of Pg, level of salivary LPS-Pg, level of circulatory Ig-G4 as the independent variables, and circulatory Ig-E as the dependent variable. Data revealed that number of colony-forming units (CFU) of Pg, level of salivary LPS-Pg, and level of

= 0.695; *p* < 0.001, n = 20), and there was a positive correlation as

= 0.796; *p* < 0.001, n = 20), and there was a positive correla-

6–16 years). The patients' general characteristics are as shown in **Table 2**.

*gingivalis* from subjects' saliva [23]. We obtained 500 μL of bacterial

We used a simple sample-processing method for PCR to detect *Porphyromonas* 

*DOI: http://dx.doi.org/10.5772/intechopen.81113*

trations below the limit of detection [23].

model. Statistical significance was set at *p* < 0.05 [23].

**3.1 Correlation between salivary LPS-Pg and Ig-E levels**

**3.2 Correlation between salivary LPS-Pg and Ig-G4 levels**

**3.3 Results of univariate logistic regression analysis**

circulatory Ig-G4 were associated with circulatory Ig-E (*p* < 0.05).

for analysis [23].

**3. Results**

correlation (r2

shown in **Figure 1**.

strong correlation (r2

tion as shown in **Figure 2**.

**2.6 Statistical analysis**

#### **Table 1.**

*Eligibility criteria for study participants.*

*Correlation between Salivary Lipopolysaccharide of Porphyromonas gingivalis with Circulatory… DOI: http://dx.doi.org/10.5772/intechopen.81113*

manufacturer-supplied standard. A value of 0.01 pg/ml was assigned for concentrations below the limit of detection [23].

We used a simple sample-processing method for PCR to detect *Porphyromonas gingivalis* from subjects' saliva [23]. We obtained 500 μL of bacterial culture from salivary secretion, added to 49.5 ml of sterile saline [23]. From this suspension, two serial 1:100 dilutions were made, and 0.1 ml was plated onto plate count agar from the last dilution [23]. Following that, lipopolysaccharides of *Porphyromonas gingivalis* were extracted by commercially optimized assay coupled with chromogenic substrate from pellet. We measured the concentration of extracted lipopolysaccharide by Pierce LAL Chromogenic Endotoxin Quantitation Kit. All measurements were done in duplicate and values averaged for analysis [23].
