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ORIGINAL ARTICLE
Year : 2011  |  Volume : 29  |  Issue : 2  |  Page : 106-112
 

The relationship between salivary IgA levels and dental caries in children


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 Publication9-Sep-2011

Correspondence Address:
E Ranadheer
Department of Pedodontics & Preventive Dentistry, Karnavati School of Dentistry, 907/A, Uvarsad, Dist: Gandhinagar, Gujarat - 382 422
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-4388.84681

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   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 2019 Aug 18];29:106-12. Available from: http://www.jisppd.com/text.asp?2011/29/2/106/84681



   Introduction Top


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 Top


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 1: Collection of saliva

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Figure 2: Salivary samples stored at -70°C (Cryo scientific Inc)

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Figure 3: Human IgA ELISA Quantitation Kit (Catalog No. E80-102), Bethyl Laboratories Inc. U.S.A

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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 Top


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 Top


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 Top


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

  1. Coating antibody

    Goat anti-Human IgA-affinity purified

    Concentration: 1 mg/ml

    Catalog No: A80-102A

    Amount: 1 ml

    Working dilution: 1/100
  2. Calibrator

    Human Reference Serum

    Catalog No: RS10-110

    Amount: 0.1 ml

    Working range: 500 - 7.8 ng/ml
  3. 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

  1. Microtiter Wells

    Nunc MaxiSorp C bottom well Modules and Frames, #445101

    10 - 96 well plates

    5 - 8 well strips
  2. Coating Buffer

    Carbonate-bicarbonate buffer capsules, Sigma

    Chemical #C3041
  3. Wash Solution

    50 mM Tris buffered saline, pH 8.0, 0.05% Tween

    20; Sigma Chemical# T9039
  4. Postcoat Solution

    50 mM Tris buffered saline, pH 8.0, 1% BSA;

    Sigma Chemical # T6789
  5. Sample/Conjugate Diluent

    Postcoat Solution plus 10% Tween 20; Sigma

    Chemical # P7949
  6. 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.
Figure 4: Adding saliva samples to the wells coated with IgA antibody

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Figure 5: Washing the wells with SKAN WASHER-300 version

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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)

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Figure 7: H2SO4 added to stop the TMB reaction (Yellow color)

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Figure 8: Microtiter plate reader VERSAMAX, MOLECULAR DEVICES (U.S.A)

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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.


 
   References Top

1.Whelton H. The anatomy and physiology of salivary glands. In: Edgar WM, O'Mullane DM, editors. Saliva and oral health, 2 nd ed. London: British Dental Association; 1996. p. 2  Back to cited text no. 1
    
2.Marcotte H, Lavoie MC. Oral Microbial Ecology and the Role of Salivary Immunoglobulin A. Microbiol Mol Biol Rev 1998;62:71-109.  Back to cited text no. 2
[PUBMED]  [FULLTEXT]  
3.Bratthall D, Serinirach R, Hamberg K, Widerstrom L. Immunoglobulin A reaction to oral streptococci in saliva of subjects with different combinations of caries and levels of mutans streptococci. Oral Microbiol Immunol 1997;12:212-8.  Back to cited text no. 3
    
4.Defarias DG, Bezerra AC. Salivary antibodies, amylase and protein from children with early childhood caries. Clin Oral Investig 2003;7:154-7.  Back to cited text no. 4
    
5.Bruno B, Pezzini A, Menegazzi M. Salivary levels of immunoglobulin and dental caries in children. Boll Soc Ital Biol Sper 1985;61:381-6  Back to cited text no. 5
    
6.Koga-Ito CY, Martins CA, Balducci I, Jorge AO. Correlation among mutans streptococci counts, dental caries, and IgA to Streptococcus mutans in saliva. Bras Oral Res 2004;18:350-5  Back to cited text no. 6
    
7.Camling E, Kohler B. Infection with the bacterium Streptococcus mutans and salivary IgA antibodies in mothers and their children. Arch Oral Biol 1987;32:817-23.  Back to cited text no. 7
    
8.Everhart DL, Rothenberg K, Carter WHJr, Klapper B. The determination of antibody to streptococcus mutans serotypes in saliva for children ages three to seven years. J Dent Res 1978;57:631-5  Back to cited text no. 8
    
9.ShifaS, MuthuMS, AmarlalD, RatnaPrabhuV. Quantiative assessment of IgA levels in the unstimulated whole saliva of caries-free and caries-active children. J Indian Soc Pedod Prev Dent 2008;26:158-6.  Back to cited text no. 9
    
10.Bass-Agholme M, Dahllöf G, Modéer T, Engström PE, Engström GN. Periodontal condition and salivary immunoglobulin in individual with Down's syndrome. J Periodontol 1998;69:1119-23.  Back to cited text no. 10
    
11.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.  Back to cited text no. 11
    
12.Kohler B, Andreen I,Jonsson B. The earlier the colonization by Mutans streptococci the higher the caries prevalence at 4 years of age. Oral Microbial Immunol 1988;3:14-7.  Back to cited text no. 12
    
13.Tenovuo J,Aalternen AS. Antibody responses to Mutans streptococci in children. Proc Finn Dent Soc 1991;87:449-61.  Back to cited text no. 13
    
14.Michalek SM, Childers NK. Development and outlook for a caries vaccine. Crit Rev Oral Biol Med 1990;1:37-54.  Back to cited text no. 14
[PUBMED]  [FULLTEXT]  
15.Dawes C. Factors influencing salivary flow rate and composition. Edgar WM, O'Mullane DM, editors. Saliva and oral health. 2nd ed. London: British Dental Association; 1996.p.27  Back to cited text no. 15
    
16.Cogulu D, Sabah E, Kutukculer N, Ozkinay F. Evaluation of the relationship between caries indices and salivary secretory IgA, salivary pH, buffering capacity and flow rate in children with Down's syndrome. Arch Oral Biol 2006;51:23-8.  Back to cited text no. 16
[PUBMED]  [FULLTEXT]  
17.Parkash H, Sharma A, Banerjee U, Sidhu SS, Sundaram KR. Humoral immune response to mutans streptococci associated with dental caries. Natl Med J India 1994;7:263-6.  Back to cited text no. 17
[PUBMED]    
18.Doifode D, DamleSG. Comparison of salivary IgA levels in caries free and caries active children. Int JClin Dent Sci 2011;2:10-4   Back to cited text no. 18
    
19.Jafarzadeh A, Sadeghi M, Karam GA, Vazirinejad R. Salivary IgA and IgE levels in healthy subjects. Braz Oral Res 2010;24:21-7.  Back to cited text no. 19
[PUBMED]  [FULLTEXT]  


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


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