salivary iga

106 JOURNAL OF INDIAN SOCIETY OF PEDODONTICS AND PREVENTIVE DENTISTRY | Apr - Jun 2011 | Issue 2 | Vol 29 |

E. Ranadheer, Ullal Anand Nayak1, N. Venugopal Reddy2, V. Arun Prasad Rao3Department of Pedodontics & Preventive Dentistry, Karnavati School of Dentistry, Gandhinagar, Gujarat, 1Mahatma Gandhi dental college, Jaipur, Rajasthan, 2,3Rajah Muthiah dental college and hospital, Annamalai University, Tamilnadu, India.

Correspondence: Dr.E. Ranadheer, M.D.S., Department of Pedodontics & Preventive Dentistry, Karnavati School of Dentistry, 907/A, Uvarsad, Dist: Gandhinagar, Gujarat 382 422, India. E-mail: [email protected]

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 populationThe 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

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DOI: 10.4103/0970-4388.84681

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Original article

The relationship between salivary IgA levels and dental caries in children

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..

Key words

ELISA, dental caries, DMFT, salivary IgA

AzharSRectangle

Ranadheer, et al.: Salivary IgA and dental caries in children

107JOURNAL OF INDIAN SOCIETY OF PEDODONTICS AND PREVENTIVE DENTISTRY | Apr - Jun 2011 | Issue 2 | Vol 29 |

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 DMFT3.UsingWHO1997 criteria, their dentalcaries status was assessed. The selection criteria for the study samples were as follows: Childrenaged8to12years Childrenwhohadsufferedfromupperrespiratory

tract infection in the past one week were excluded from the study due to development of IgA and lysozyme.

ThechildreninthegroupII,theDMFTscorehadtobe3

Saliva collectionUnstimulated 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 -70C until analyzed [Figure 2]. The samples were then thawed and centrifuged for 15 minutes at 12 000 g at 4C 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].

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 Tables 1 and 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-0andgroupIIwaswithDMFT3.Table1shows the s-IgA levels in Group I (DMFT score 0) and Table 2 shows the s-IgA levels in Group II (DMFT score3).Thewholes-IgAlevelswerecomparativelyhigherinchildrenwithDMFT3(GroupII)withthemean 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 3hadhigherlevelsof wholes-IgAantibodywhen

Figure 1: Collection of saliva

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

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



Figure 5: Washing the wells with SKAN WASHER-300 versionFigure 4: Adding saliva samples to the wells coated with IgA antibody

Figure 6: Antigen and Antibody reaction after adding TMB substrate (Blue color)

Table 1: Salivary IgA levels in unstimulated saliva of children with DMFT score - 0

Sample no DMFT score Salivary IgA levels(mg/dl)

1 0 7.4

2 0 8.3

3 0 6.1

4 0 5.3

5 0 6.3

6 0 5.6

7 0 5.7

8 0 8.5

9 0 5.7

10 0 12.3

11 0 8.1

12 0 6.6

13 0 4.4

14 0 8.8

15 0 8.3

16 0 13

17 0 11.3

18 0 6.7

19 0 9.5

20 0 7.2

Mean 7.585

Std dev 2.48

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.

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 childs 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 -70C until analyzed. The samples were then thawed and centrifuged for 15 minutes at 12 000 g at 4C 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

3thanthosewithDMFTscore0.Theincreasedlevels 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

Table 2: Salivary IgA levels in unstimulated saliva of children with DMFT score 3

Sample No DMFT score Salivary IgA levels(mg/dl)

1 4 8.4

2 3 12.8

3 4 14

4 5 12.2

5 4 11.8

6 4 10.4

7 4 9.5

8 5 10.9

9 3 12.6

10 3 13.9

11 5 10.9

12 4 11.4

13 5 9.5

14 3 10.6

15 4 11.5

16 3 11.1

17 5 9.4

18 4 10.7

19 5 8.9

20 4 10.6

Mean 4.05 11.76

Std Dev 0.50 1.85

Table 3: Comparison of salivary IgA levels in group with DMFT score = 0 (Group I) and group with DMFT 3 (Group II)Group N Mean s-IgA levels (mg/dl) Std

deviationP- value

(sig)Group I 20 7.585 2.488 0.05

(P0.05)Group II 20 11.760 1.859Significant at P 0.05

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

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



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]ConditionsStorage: 2 8CRange of detection: 500 7.8 ng/mlShelf life: 1 year from date of receiptKit Materials1. Coating antibody Goat anti-Human IgA-affinity purified Concentration: 1 mg/ml Catalog No: A80-102A Amount: 1 ml Working dilution: 1/1002. Calibrator Human Reference Serum Catalog No: RS10-110 Amount: 0.1 ml Working range: 500 7.8 ng/ml3. 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 : 1,50,000ELISA Starter Accessory Package, used with ELISA Quantitation Kits Catalog No. E101Kit Materials1. Microtiter Wells

Nunc MaxiSorp C bottom well Modules and Frames, #445101

10 - 96 well plates 5 - 8 well strips2. Coating Buffer Carbonate-bicarbonate buffer capsules, Sigma

Chemical #C30413. Wash Solution 50 mM Tris buffered saline, pH 8.0, 0.05% Tween

20; Sigma Chemical# T90394. Postcoat Solution 50 mM Tris buffered saline, pH 8.0, 1% BSA;

Sigma Chemical # T67895. Sample/Conjugate Diluent Postcoat Solution plus 10% Tween 20; Sigma

Chemical # P79496. Enzyme Substrate TMB Peroxidase Substrate and Peroxidase

Solution B; Kirkegaard and PerryBuffer Preparations CoatingBuffer 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.

WashSolution Each packet of wash solution was dissolved in 1

litre of distilled water and stored at 2 - 30C. PostcoatSolution/BlockingSolution The post coat solution was dissolved in 1litre of

distilled water and stored at 2 8C, Sample/ConjugateDiluent 0.5 ml 10% Tween 20 was added to postcoat

solution for use as sample and conjugate diluent and stored at 2 8C.

EnzymeSubstrate Equal volumes of each reagent were mixed. The

required quantity was prepared and stored at 2 8C.

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,blanksand/orcontrols

were analyzed in duplicate 96lof captureantibody(A80-102A)was

dilutedto9,600lCoatingBufferandcoatedon



Step ng/ml CalibratorRS10-110-3

Sample diluent

1 500 4 l 9.6 ml

2 250 1 ml from step 1 1 ml

3 125 1 ml from step 2 1 ml

4 62.5 1 ml from step 3 1 ml




each well Coatedplatewasincubatedfor60minutes. Afterincubation,thecaptureantibodywas

aspirated from the solution from each well with the SKAN WASHER- 300 version B Model- 12010 (Skatron Instruments, Norway. U.S.A) [Figure 5].

EachwellwaswashedwithwashsolutionwithSKAN WASHER- 300 version B Model- 12010 (Skatron Instruments, Norway. U.S.A) as follows:

Eachwellwasfilledwithwashsolution Washsolutionwasremovedbyaspiration Thiswasrepeatedfor3washes.Blocking (Postcoat) solution 200lof Blocking(Postcoat)Solutionwas

added to each well. Incubatedfor30minutesatRT. Afterincubation,theBlocking(Postcoat)

Solution was removed from each well and washed three times as in step A1

Standards and Samples Standardsweredilutedinsamplediluent

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.

1lof salivasamplesweredilutedto100linsample Diluent.

100lof samplesweretransferredtoassignedwells [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.

100lwastransferredtoeachwell. 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

100lof substratesolutionwastransferredtoeach well.

Incubated for 15 minutes at room temperature. TostoptheTMBreaction,100lof 2MH2SO4

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].

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

1. Whelton H. The anatomy and physiology of salivary glands. In: Edgar WM, O'Mullane DM, editors. Saliva and oral health, 2nd ed. London: British Dental Association; 1996. p. 2

2. Marcotte H, Lavoie MC. Oral Microbial Ecology and the Role of Salivary Immunoglobulin A. Microbiol Mol Biol Rev 1998;62:71-109.

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.

4. Defarias DG, Bezerra AC. Salivary antibodies, amylase and protein from children with early childhood caries. Clin Oral Investig 2003;7:154-7.

5. Bruno B, Pezzini A, Menegazzi M. Salivary levels of immunoglobulin and dental caries in children. Boll Soc Ital



13. Tenovuo J,Aalternen AS. Antibody responses to Mutans streptococci in children. Proc Finn Dent Soc 1991;87:449-61.

14. Michalek SM, Childers NK. Development and outlook for a caries vaccine. Crit Rev Oral Biol Med 1990;1:37-54.

15. Dawes C. Factors influencing salivary flow rate and composition. Edgar WM, OMullane DM, editors. Saliva and oral health. 2nd ed. London: British Dental Association; 1996.p.27

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.

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.

18. Doifode D, DamleSG. Comparison of salivary IgA levels in caries free and caries active children. Int JClin Dent Sci 2011;2:10-4

19. Jafarzadeh A, Sadeghi M, Karam GA, Vazirinejad R. Salivary IgA and IgE levels in healthy subjects. Braz Oral Res 2010;24:21-7.

Biol Sper 1985;61:381-66. 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

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.

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

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.

10. Bass-Agholme M, Dahllf G, Moder T, Engstrm PE, Engstrm GN. Periodontal condition and salivary immunoglobulin in individual with Downs syndrome. J Periodontol 1998;69: 1119-23.

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.

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.

How to cite this article: Ranadheer E, Nayak UA, Reddy NV, Rao VA. The relationship between salivary IgA levels and dental caries in children. J Indian Soc Pedod Prev Dent 2011;29:106-12.

Source of Support: Nil, Conflict of Interest: None declared.

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