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ORIGINAL ARTICLE |
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| Year : 2011 | Volume
: 29
| Issue : 2 | Page : 106-112 |
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The relationship between salivary IgA levels and dental caries in children
E Ranadheer1, Ullal Anand Nayak2, N Venugopal Reddy3, V Arun Prasad Rao3
1 Department of Pedodontics & Preventive Dentistry, Karnavati School of Dentistry, Gandhinagar, Gujarat, India
2 Department of Pedodontics & Preventive Dentistry, Mahatma Gandhi dental college, Jaipur, Rajasthan, India
3 Department of Pedodontics & Preventive Dentistry, Rajah Muthiah dental college and hospital, Annamalai University, Tamilnadu, India
| Date of Web Publication | 9-Sep-2011 |
Correspondence Address:
E Ranadheer
Department of Pedodontics & Preventive Dentistry, Karnavati School of Dentistry, 907/A, Uvarsad, Dist: Gandhinagar, Gujarat - 382 422
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/0970-4388.84681
 Abstract | | |
Purpose: The aim of the study was to find the relationship between salivary IgA (s-IgA) levels and dental caries in children. Materials and Methods: A total of 40 children in the age group of 8 to 12 years were selected and divided into two groups. Group I with DMFT score 0 and Group II with DMFT score ≥3. The whole unstimulated s-IgA levels were estimated using ELISA method. Results: Whole s-IgA levels were significantly higher in group II with DMFT score ≥3 as compared with group I with DMFT score 0. Conclusions: There was an increase in s-IgA levels in caries-active mouth to give protection mechanism against dental caries and the Streptococcus mutans which are active in caries-active mouth. The s-IgA antibodies can play an important role in control of dental caries.
Keywords: ELISA, dental caries, DMFT, salivary IgA
How to cite this article:
Ranadheer E, Nayak UA, Reddy N V, Rao V A. The relationship between salivary IgA levels and dental caries in children. J Indian Soc Pedod Prev Dent 2011;29:106-12 |
How to cite this URL:
Ranadheer E, Nayak UA, Reddy N V, Rao V A. The relationship between salivary IgA levels and dental caries in children. J Indian Soc Pedod Prev Dent [serial online] 2011 [cited 2021 Jan 21];29:106-12. Available from: https://www.jisppd.com/text.asp?2011/29/2/106/84681 |
| Introduction | |  |
Dental caries is a multifactorial disease and one of the major contributing factors is saliva. Salivary components, its flow, viscosity, buffering capacity, etc., play a major role in the prevention, initiation, and progression of the disease. It helps in the prevention of the caries by its antibacterial effect. [1]
Secretory IgA is the main immunoglobulin in secretions, including saliva. It is the first line of defense of the host against pathogens which invade mucosal surfaces. Salivary IgA (s-IgA) antibodies could help oral immunity by preventing microbial adherence, neutralizing enzymes, toxins, and viruses; or by acting in synergy with other factors such as lysozyme and lactoferrin. [2]
Studies have also demonstrated a lower incidence of caries as a result of a high s-IgA concentration [3] and increase in s-IgA levels with increase in caries activity [4],[5] and some authors found no correlation between dental caries and IgA levels. [6],[7],[8],[9]
The aim of this study was to assess the levels of s-IgA in the unstimulated whole salivary samples of caries-free and caries-active children and to correlate the role of s-IgA in prevention of dental caries.
| Materials and Methods | | |
Study population
The study was designed and carried out in the department of Pedodontics and Preventive dentistry in collaboration with Department of Biochemistry. A total of 40 children were randomly selected from Chidambaram school children in the age group of 8 to 12 years. Children who had suffered from upper respiratory tract infection in the past one week were excluded from the study due to development of IgA and lysozyme. They were divided into two groups. Group I comprises of caries-free children with DMFT - 0 and group II comprises of caries-active children with DMFT ≥3. Using WHO 1997 criteria, their dental caries status was assessed. The selection criteria for the study samples were as follows:
- Children aged 8 to 12 years
- Children who had suffered from upper respiratory tract infection in the past one week were excluded from the study due to development of IgA and lysozyme.
- The children in the group II, the DMFT score had to be ≥3
Saliva collection
Unstimulated whole salivary samples were collected in the wide-mouthed sterile test tubes [Figure 1]. Subjects were informed in advance not to eat or drink (except for water) one hour before saliva collection to minimize possible food debris and stimulation of saliva. The samples were frozen and kept at -70°C until analyzed [Figure 2]. The samples were then thawed and centrifuged for 15 minutes at 12 000 g at 4°C to remove the mucin and debris and the supernatant was examined by ELISA. [10] The s-IgA concentration was evaluated by ELISA (Bethyl Laboratories, Inc. Human IgA ELISA Quantitation kit: Catalog No- E 80-102; Size- 1000 wells; Lot No- E 80-102-9; U.S.A.) [11] [Figure 3]. | Figure 3: Human IgA ELISA Quantitation Kit (Catalog No. E80-102), Bethyl Laboratories Inc. U.S.A
Click here to view |
The ELISA protocol was performed as described in Appendix-1. The s-IgA concentration was evaluated by ELISA. The ELISA protocol was performed in the Department of Biochemistry.
Average duplicate readings from each standard, control, and sample were taken. Intergroup comparison of various values was done with Scheffe test. Pearson correlation test was employed for correlation analysis. A standard curve was created by reducing the data using computer software capable of generating a four-parameter logistic (4-PL) curve-fit. The software used was SOFTMAX.
| Results | | |
The s-IgA levels in salivary samples of group I and II are shown in [Table 1] and [Table 2]. A total of 40 children were included in the study of the age group 8 to 12 years, 20 children in each group. Group I was with DMFT score - 0 and group II was with DMFT ≥3. [Table 1] shows the s-IgA levels in Group I (DMFT score - 0) and [Table 2] shows the s-IgA levels in Group II (DMFT score ≥3). The whole s-IgA levels were comparatively higher in children with DMFT ≥3 (Group II) with the mean level 11.760 mg/dl ± 1.8 and in children with DMFT score - 0, they were comparatively at lower level with the mean 7.585 mg/dl ± 2.48 [Table 3]. The result of the study showed that children with DMFT ≥3 had higher levels of whole s-IgA antibody when compared with children with DMFT score 0. Average duplicate readings from each standard, control, and sample were taken. Intergroup comparison of various values was done with Scheffe test. Pearson correlation test was employed for correlation analysis. A standard curve was created by reducing the data using computer software capable of generating a 4-PL curve-fit. The software used was SOFTMAX. | Table 1: Salivary IgA levels in unstimulated saliva of children with DMFT score - 0
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 | Table 2: Salivary IgA levels in unstimulated saliva of children with DMFT score ≥3
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 | Table 3: Comparison of salivary IgA levels in group with DMFT score = 0 (Group I) and group with DMFT ≥3 (Group II)
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| Discussion | | |
It is generally accepted that S. mutans is the primary causative agent of dental caries in human beings. [12],[13] Secretory IgA inhibits the attachment and adherence of oral bacteria to the epithelial and tooth surfaces. S-IgA neutralizes viruses, bacterial exotoxins, and enzymes that contribute to disease process. It also inhibits the attachment and adherence of oral bacteria to the epithelial and tooth surfaces. [7],[14] But, the relationship between s-IgA and dental caries has not been well established. Hence, the present study was aimed at assessing the relationship of salivary immunoglobulin-A with dental caries.
Saliva was collected by the method suggested by Dawes [15] as it was easy to obtain the child's cooperation. In this method, the child was asked to pool the saliva in the floor of the oral cavity and asked to spit intermittently. Unstimulated whole salivary samples were collected in the wide-mouthed sterile test tubes. Subjects were informed in advance not to eat or drink (except for water) one hour before saliva collection to minimize possible food debris and stimulation of saliva. The samples were frozen and kept at -70°C until analyzed. The samples were then thawed and centrifuged for 15 minutes at 12 000 g at 4°C to remove the mucin and debris and the supernatant was examined by ELISA. [10] The s-IgA concentration was evaluated by ELISA (Bethyl Laboratories, Inc. Human IgA ELISA Quantitation kit: Catalog No- E 80-102; Size- 1000 wells; Lot No- E 80-102-9; U.S.A.). [11]
Our results demonstrated that the mean levels of s-IgA are significantly higher in children with DMFT score ≥3 than those with DMFT score - 0. The increased levels of s-IgA in caries-active children might be a defensive mechanism to the greater number of S. mutans in whole saliva of caries-active children.
Many similar studies were conducted in the past and contradictory results were obtained. Studies conducted by Camling and Kohler, [7] Cogulu et al., [16] Parkash et al., [17] and Doifode and Damle [18] reported an increase in the levels of s-IgA with decrease in caries activity.
Jafarzadeh et al. [19] reported that s-IgA levels increased with age up to 60 years and then slightly decreased in subjects aged 61 to 70 years.
However, studies conducted by Koga et al., [6] Camling and Kohler, [7] and Shifa et al. [9] found no correlation between dental caries and s-IgA levels.
Our study results are in agreement with studies conducted by Defarias and Bezerra [4] and Bruno et al., [5] who found an increase in s-IgA levels with increase in caries activity. Our values were significantly higher in caries-active children.
| Conclusions | | |
There was an increase in s-IgA levels in caries-active mouth to give protection mechanism against dental caries and the S. mutans which are active in caries-active mouth. But, different authors have different views regarding the relation between the s-IgA and dental caries in children. The s-IgA antibodies can play an important role in control of dental caries. Very limited literature is available on the relationship between s-IgA levels and dental caries in children. Furthermore, long-term studies should be carried out in the field of s-IgA and dental caries with different DMFT scores.
Appendix -1
Human IgA ELISA Quantitation Kit (Catalog No. (E80-102)
Bethyl Laboratories Inc. U.S.A [Figure 3]
Conditions
Storage: 2 - 8°C
Range of detection: 500 - 7.8 ng/ml
Shelf life: 1 year from date of receipt
Kit Materials
- Coating antibody
Goat anti-Human IgA-affinity purified
Concentration: 1 mg/ml
Catalog No: A80-102A
Amount: 1 ml
Working dilution: 1/100 - Calibrator
Human Reference Serum
Catalog No: RS10-110
Amount: 0.1 ml
Working range: 500 - 7.8 ng/ml - HRP Detection Antibody
Goat anti-Human IgA-HRP conjugate
Concentration: 1 mg/ml
Catalog No: A80-102P
Amount: 0.1 ml; Working Dilution: 1 : 10, 000 - 1 : 1,50,000
ELISA Starter Accessory Package, used with ELISA Quantitation Kits
Catalog No. E101
Kit Materials
- Microtiter Wells
Nunc MaxiSorp C bottom well Modules and Frames, #445101
10 - 96 well plates
5 - 8 well strips - Coating Buffer
Carbonate-bicarbonate buffer capsules, Sigma
Chemical #C3041 - Wash Solution
50 mM Tris buffered saline, pH 8.0, 0.05% Tween
20; Sigma Chemical# T9039 - Postcoat Solution
50 mM Tris buffered saline, pH 8.0, 1% BSA;
Sigma Chemical # T6789 - Sample/Conjugate Diluent
Postcoat Solution plus 10% Tween 20; Sigma
Chemical # P7949 - Enzyme Substrate
TMB Peroxidase Substrate and Peroxidase
Solution B; Kirkegaard and Perry
Buffer Preparations
- Coating Buffer
The contents of 1 capsule was dissolved in 100 ml of distilled water = 0.05M carbonate-bicarbonate, pH 9.6. The prepared coating buffer was stored at room temperature. - Wash Solution
Each packet of wash solution was dissolved in 1 litre of distilled water and stored at 2 - 30°C. - Postcoat Solution/Blocking Solution
The post coat solution was dissolved in 1litre of distilled water and stored at 2 - 8°C, - Sample/Conjugate Diluent
0.5 ml 10% Tween 20 was added to postcoat solution for use as sample and conjugate diluent and stored at 2 - 8°C. - Enzyme Substrate
Equal volumes of each reagent were mixed. The required quantity was prepared and stored at 2 - 8°C. - Notes
2M H2SO4 was added to stop the enzyme substrate reaction
Step-by-step method for human S-IgA quantitation
(All steps were performed at room temperature.)
Coat with Capture Antibody
- Standards, samples, blanks and /or controls were analyzed in duplicate
- 96 μl of capture antibody (A80-102A) was diluted to 9,600 μl Coating Buffer and coated on each well
- Coated plate was incubated for 60 minutes.
- After incubation, the capture antibody was aspirated from the solution from each well with the SKAN WASHER- 300 version - B Model- 12010 (Skatron Instruments, Norway. U.S.A) [Figure 4][Figure 5].
- Each well was washed with wash solution with SKAN WASHER- 300 version - B Model- 12010 (Skatron Instruments, Norway. U.S.A) as follows:
- Each well was filled with wash solution
- Wash solution was removed by aspiration
- This was repeated for 3 washes.
Blocking (Postcoat) solution
- 200 μl of Blocking (Postcoat) Solution was added to each well.
- Incubated for 30 minutes at RT.
- After incubation, the Blocking (Postcoat) Solution was removed from each well and washed three times as in step A1
Standards and Samples
- Standards were diluted in sample diluent according to the chart below
- Since our samples had got more s-IgA than the standards range given by the company, samples were diluted in sample Diluent.
- 1 μl of saliva samples were diluted to 100 μl in sample Diluent.
- 100 μl of samples were transferred to assigned wells [Figure 4]
- Incubated for 60 minutes at RT.
- After incubation, samples and standards were removed and each well was washed 5 times as in Step A1.
HRP Detection Antibody
- HRP Conjugate (A80-102P) was diluted in Conjugate Diluent. Dilution range was 1 : 10,000.
- 100 μl was transferred to each well.
- Incubated for 60 minutes.
- After incubation, HRP Conjugate was removed and each well was washed 5 times as in Step A1.
Enzyme Substrate Reaction [Figure 6].
- Substrate solution was prepared according to the manufacturer's recommendation.
- 9.6 ml of TMB Peroxidase substrate was mixed with 9.6 ml of Peroxidase solution in equal volumes
- 100 μl of substrate solution was transferred to each well.
- Incubated for 15 minutes at room temperature.
- To stop the TMB reaction, 100 μl of 2M H 2 SO4 was added to each well [Figure 7].
- Using a Microtiter plate reader, VERSAMAX, MOLECULAR DEVICES CALFIORNIA (U.S.A), the plate was read at the wavelength of 450 nm that is appropriate for the TMB Substrate solution used [Figure 8].
 | Figure 6: Antigen and Antibody reaction after adding TMB substrate (Blue color)
Click here to view |
Calculation of Results
- Average duplicate readings from each standard, control, and sample were taken.
- A standard curve was created by reducing the data using computer software capable of generating a four-parameter logistic (4-PL) curve-fit.
- The software used was SOFTMAX.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3]
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