Saliva samples were obtained on Radford University’s campus by waiting on paved pedestrian walkways near dormitories and dining halls. Test subjects were identified by observable behaviors of participating in smoking or not smoking.
Procedure:
Experimental Details:
Subjects were approached and asked to confirm their observed smoker/non-smoker status. Once status was confirmed, subjects verbally gave consent to participate after obtaining a consent form and being informed about the study. Subjects were given approximately 3 oz. water in a sterile paper cup in order to rinse the mouth of any food or residues from smoking. Following the rinse, the subjects waited 5 minutes without consuming any foods or liquids before giving the saliva sample. Each 1 mL saliva sample was collected in a 2.0-mL conical vial with twist-on top. If the test subject showed difficulty giving a sample, a sterile, plastic pipette was provided in order to alleviate any difficulty. Saliva samples were stored in a Styrofoam cooler filled with crushed ice during sample collection and stored in a laboratory freezer for long-term storage.
In order to determine any statistical significance between the average IgA levels of the test groups, a direct enzyme-linked immune assay (ELISA) test was performed. A direct ELISA test measures the level of IgA in saliva by creating a color change in the samples which signifies the concentration of IgA in each sample. This color change is measured using a spectrophotometer.
ELISA Analysis:
Procedure:
- Confirmation of smoker/non-smoker status
- Consent obtained verbally
- Subject rinsed mouth with water
- Five minute waiting period
- Saliva sample collected
- Sample then frozen
Experimental Details:
Subjects were approached and asked to confirm their observed smoker/non-smoker status. Once status was confirmed, subjects verbally gave consent to participate after obtaining a consent form and being informed about the study. Subjects were given approximately 3 oz. water in a sterile paper cup in order to rinse the mouth of any food or residues from smoking. Following the rinse, the subjects waited 5 minutes without consuming any foods or liquids before giving the saliva sample. Each 1 mL saliva sample was collected in a 2.0-mL conical vial with twist-on top. If the test subject showed difficulty giving a sample, a sterile, plastic pipette was provided in order to alleviate any difficulty. Saliva samples were stored in a Styrofoam cooler filled with crushed ice during sample collection and stored in a laboratory freezer for long-term storage.
In order to determine any statistical significance between the average IgA levels of the test groups, a direct enzyme-linked immune assay (ELISA) test was performed. A direct ELISA test measures the level of IgA in saliva by creating a color change in the samples which signifies the concentration of IgA in each sample. This color change is measured using a spectrophotometer.
ELISA Analysis:
- Thawed samples
- Diluted 1:2 with coating buffer in 96-well plate
- Incubated 4 hours at 37 degrees Celsius
- Added 100 micro-liters of blocking buffer, Incubated 5 minutes at room temperature, this was repeated once more
- Added 100 micro-liters of primary antibody, Incubated at 20 minutes at room temperature
- Washed twice with blocking buffer to remove excess antibody
- Add 100 micro-liters of TMB substrate, Incubated 20 minutes at room temperature
- Observed absorbance in spectrophotometer at 450 nanometers.