Saliva as a source of anti-Toxoplasma gondii

IgG for enzyme immunoassay in human

samples

B. F. C. Sampaio1, M. S. Macre2, L. R. Meireles1 and

H. F. Andrade Jr1,3

1) Laborat�orio de Protozoologia, Instituto de Medicina Tropical de

S~ao Paulo, 2) Instituto de Ciencias Biom�edicas and 3) Faculdade de

Medicina, Universidade de S~ao Paulo, S~ao Paulo, Brazil

Abstract

Toxoplasmosis, a highly prevalent disease, is mainly diagnosed by

serology. Incidence studies could be feasible in children, but ethical

concerns restrict blood sampling in this group. Saliva contains

small amounts of crevicular fluid IgG. Dot-ELISA and a protein A

IgG capture immunoassay were standardized for anti-Toxoplasma

gondii IgG in paired saliva and serum samples from 20 adult

volunteers. A frequency of toxoplasmosis of 19% (95% CI 12–28)

was found in 100 saliva samples from university graduates using

both assays. Toxoplasmosis immunoassays using saliva IgG are a

promising tool for the investigation of the epidemiology of this

disease in children and other vulnerable groups.

Keywords: Dot-ELISA, ELISA, IgG, immunoassay, protein A,

saliva, toxoplasma, toxoplasmosis

Original Submission: 30 July 2012; Revised Submission: 29

May 2013; Accepted: 31 May 2013

Editor: E. Bottieau

Article published online: 13 June 2013

Clin Microbiol Infect 2014; 20: O72–O74

10.1111/1469-0691.12295

Corresponding author: H. F. Andrade Jr, Laborat�orio de

Protozoologia, Instituto de Medicina Tropical de S~ao Paulo, Av.

Dr. E.C.Aguiar, 470 05403-000 S~ao Paulo, Brazil

E-mail: hfandrad@usp.br

Toxoplasmosis, a worldwide zoonosis, presents few or no

symptoms in most patients, despite occasional severe

disease [1]. Detection of specific antibodies in blood or

serum is the main tool for diagnosis. Saliva has been used

for monitoring drugs and hormones [2] and also for

diagnosis of viral [3], yeast [4] or parasitic [5–7] infectious

diseases. Saliva contains secretory immunoglobulin A, from

salivary gland plasma cells, but also IgG and IgM exuded in

the gingival crevicular fluid [8]. Saliva is easy and comfortable

to collect for the patient, especially children, and also easy to

process due to the absence of clotting and complement [9]. We

standardized Dot-ELISA and a protein A IgG capture immuno-

assay for use in purified saliva samples, followed by testing 100

university students’ saliva to determine the prevalence of the

infection in this group.

Materials and Methods

Young adult volunteers supplied 20 paired serum and saliva

samples and serology was validated with both commercial

assays and in-house comparative ELISA [6]. After this, saliva

samples from 100 university asymptomatic students (aged 18–

21 years) were tested using both assays. Those students were

unaware of their previous toxoplasmosis serology. All samples

were collected after informed consent, and the Ethics

Committee of the Medical College and the University of S~ao

Paulo (No. 031/11) approved this project. Each volunteer

provided 5–10 mL of saliva after mouth washing. The saliva

was ice chilled and cleared and supernatants were added at a

1:1 volume of chilled ethanol (�13°C). After 1 h in an ice bath,

globulins were pelleted and dissolved in one-tenth of the

original volume of pH 7.4 sodium borate buffer.

Immunoassays

Dot-ELISA. Dot-ELISA was prepared as previously described

[2]. Each strip contains 25 lg of soluble Toxoplasma gondii

antigen or Protein A paired dots. Strips were incubated with

serum or saliva samples for 1 h at room temperature under

agitation. After washings, anti-human IgG peroxidase conjugate

was added for 1 h and the reaction was revealed with 3,3 –

diaminobenzidine and H2O2 with digital imaging. At least one

dot must be stained, and if both dots were stained this was

considered a positive IgG detection.

For IgG capture ELISA, recombinant Staphylococcal protein

A was added to the solid phase for capture saliva IgG, followed

by binding of biotinylated tachyzoite saline antigen, which was

quantitated by avidin peroxidase. The reaction was revealed

using two chromogenic peroxidase substrates, such as OPD

and TMB, with appropriate controls in each plate.

Results

Dot-ELISA

Dot-ELISA results demonstrated consistency between

the serum and saliva samples, without false-negative or

ª2013 The Authors

Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases

RESEARCH NOTE PARASITOLOGY

false-positive samples, as shown in Fig. 1(a). We also

performed a double-dot design Dot-ELISA protein A confir-

matory test on the samples, with one dot spot with protein A

for total IgG detection and a T. gondii antigen second spot for

specific IgG detection in order to avoid the absence of IgG in

saliva samples. IgG was detected in all of the saliva samples,

including the negative and positive control samples, as shown

in Fig. 1(b). We tested 100 university student saliva samples in

this assay, and stained spots are shown in Fig. 1(c), which

demonstrate that all of the saliva samples contained IgG, as

shown in the upper SPA-stained dots, and 19 were clearly

positive for IgG and anti-T. gondii antibodies. The data

demonstrated a frequency of 19% (12.1–28.3 95% confidence

interval). These preliminary data were not confirmed by

blood sampling.

Protein A IgG capture ELISA

We performed the protein A IgG capture ELISA using two

peroxidase substrates with different specificities for

anti-T. gondii IgG detection. Data from 100 university student

saliva samples are shown in Fig. 2. As can be seen, all positive

or negative samples exhibited the same reactivity as shown in

Dot-ELISA, with better discrimination using TMB as peroxi-

dase substrate, which is more sensitive for peroxidase

detection.

Discussion

Our preliminary data demonstrated that the Dot-ELISA

immunoassay detected IgG antibodies against T. gondii in the

(a)

(b)

(c)

FIG. 1. Dot-ELISA results for anti-T. gondii

IgG detection in human saliva. (a) Results of

the volunteers’ paired saliva and serum samples

tested in quadruplicate. Dots contain T. gondii

soluble antigen. Upper lanes, 1/200 serum;

lower lane, 19 reconstituted saliva. Arrows

indicate specific IgG-positive samples. (b) Dou-

ble dot design: upper dot was adsorbed with

Staphylococcal IgG-binding protein A and lower

dot was adsorbed with T. gondii soluble antigen

and reacted with 19 reconstituted saliva from

the volunteers used in A. (c) Dot design as

described in B using samples from university

graduation students, as described in the Meth-

ods section. SPA, protein A dots; TGA, T. gondii

soluble antigen dots.

FIG. 2. Protein A IgG capture ELISA assay of 100 samples of human

saliva using T. gondii biotinylated antigen and revealed using OPD or

TMB. Dot-ELISA positive (+) = closed symbols; Dot-ELISA negative (�)

= open symbols; TMB (circles) and OPD (squares). The y-axis was

divided at the threshold of the 95% confidence interval cut-off of the

negative samples. The mean and the SEM for each sample are shown.

ª2013 The Authors

Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20, O72–O74

CMI Research Note O73

saliva with the same efficiency as in the serum in our small

sample, which is the material most commonly used for this

purpose. We found no correlation of the reactivity in saliva

with its protein content or total IgG content, and both assays

were well controlled for avoiding false-negative results for the

absence of IgG. We confirmed the detection efficiency using

samples of saliva obtained from a large number of young adults

at our university, using either Dot-ELISA or a protein A

capture ELISA. We used this population due to their availability

for expanding saliva testing without attempting to detect IgG

avidity or any other acute infection predictor. Our Dot-ELISA

results are similar to those described in previous studies [10],

in which anti-rubella IgG antibodies were detected in saliva,

and the pioneering study in which anti-T. gondii antibodies

were detected in saliva [6,11].

Saliva could be an extremely important antibody source for

specific groups of patients, such as children, who are often

protected from conventional invasive blood collection proce-

dures. Saliva has also demonstrated positive results in groups

of adults in patient adherence studies; it increases the

confidence and perseverance of individuals undergoing treat-

ment because there is no need to use invasive needles during

consultations, which helps avoid absenteeism [12]. Our data

using Protein A as IgG capture molecule give very good results,

The absence or low levels of IgG in the saliva could be

responsible for the low efficiency of a number of Dot-ELISA

assays reported elsewhere, which did not include careful

controls [13]. Many studies support the idea that in older

volunteers, the rate of serological positivity for toxoplasmosis

is greater [14]. Our results demonstrated a 19% frequency of

serological positivity, which is lower than that demonstrated in

other Brazilian studies; for example, a 50% frequency was

observed in teenagers in low-income neighbourhoods in the

same city [6], and a >70% frequency was observed in adults in

the Brazilian Amazon [15]. We attributed the low frequency

observed in our study to the high-income origin of our

volunteers or university students. The Dot-ELISA and protein

A capture ELISA for the detection of IgG anti-T. gondii in saliva

are promising tools because they use a non-invasive method of

collection. In addition, the technique is highly sensitive and

highly specific and can be performed routinely and easily in the

laboratory at a low cost. The assay also demonstrates potential

for the development of epidemiological studies or quick tests

using small amounts of saliva.

Acknowledgements

We are grateful to Claudio C. J. Ekman for the sampling

procedures and to all the volunteers for kindly supplying the

samples of saliva or blood. H. F. Andrade Jr was a fellow of

CNPq. Barbara F. C. Sampaio was a fellow of CAPES, and a

portion of this work was used for her Master’s degree. This

work was supported in part by LIMHCFMUSP.

Transparency Declaration

The authors declare no conflicts of interest.

References

1. Leal FE, Cavazzana CL, de Andrade HF Jr, Galisteo AJ Jr, de Mendonc�aJS, Kallas EG. Toxoplasma gondii pneumonia in immunocompetent

subjects: case report and review. Clin Infect Dis 2007; 44: e62–e66.

2. Haeckel R. Procedures for saliva sampling. J Clin Chem Clin Biochem

1989; 27: 246–247.

3. Perry KR, Brown DW, Parry JV, Panday S, Pipkin C, Richards A.

Detection of measles, mumps, and rubella antibodies in saliva using

antibody capture radioimmunoassay. J Med Virol 1993; 40: 235–240.

4. Jeganathan S, Ufomata D, Hobkirk JA, Ivanyi L. Immunoglobulin A1 and

A2 subclass of salivary antibodies to Candida albicans in patients with

oral candidosis. Clin Exp Immunol 1987; 70: 316–321.

5. Feldman M, Plancarte A, Sandoval M, Wilson M, Flisser A. Comparison

of two assays (EIA and EITB) and two samples (saliva and serum) for

the diagnosis of neurocysticercosis. Trans R Soc Trop Med Hyg 1990; 84:

559–562.

6. Macre MS, Pires M, Meireles LR, Angel SO, Andrade HF Jr. Serology

using rROP2 antigen in the diagnostic of toxoplasmosis in pregnant

women. Rev Inst Med Trop Sao Paulo 2009; 51: 283–288.

7. Wang Z, Xue C, Lou W et al. Non-invasive immunodiagnosis of

Schistosomiasis japonica: the detection of specific antibodies in saliva.

Chin Med J (Engl) 2002; 115: 1460–1464.

8. Kaufman E, Lamster IB. The diagnostic applications of saliva–a review.

Crit Rev Oral Biol Med 2002; 13: 197–212. Review.

9. Wong DT. Salivary diagnostics: scientific and clinical frontiers. Adv Dent

Res 2011; 23: 350–352.

10. Morris M, Cohen B, Andrews N, Brown D. Stability of total and

rubella-specific IgG in oral fluid samples: the effect of time and

temperature. J Immunol Methods 2002; 266: 111–116.

11. Hajeer AH, Balfour AH, Mostratos A, Crosse B. Toxoplasma gondii:

detection of antibodies in human saliva and serum. Parasite Immunol

1994; 16: 43–50.

12. Stekler JD, Swenson PD, Coombs RW et al. HIV testing in a

high-incidence population: is antibody testing alone good enough? Clin

Infect Dis 2009; 49: 444–453.

13. Shukla N, Husain N, Agarwal GG, Husain M. Utility of cysticercus

fasciolaris antigen in Dot ELISA for the diagnosis of neurocysticercosis.

Indian J Med Sci 2008; 62: 222–227.

14. Zarkovic A, McMurray C, Deva N, Ghosh S, Whitley D, Guest S.

Seropositivity rates for Bartonella henselae, Toxocara canis and Toxo-

plasma gondii in New Zealand blood donors. Clin Experiment Ophthalmol

2007; 35: 131–134.

15. Cavalcante GT, Aguilar DM, Camargo LM et al. Seroprevalence of

Toxoplasma gondii antibodies in humans from rural Western Amazon,

Brazil. J Parasitol 2006; 92: 647–649.

ª2013 The Authors

Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20, O72–O74

O74 Clinical Microbiology and Infection, Volume 20 Number 1, January 2014 CMI