Legal Medicine xxx (2015) xxx–xxx

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Legal Medicine
journal homepage: www.elsevier.com/locate/legalmed

Brief Communication

Detection of proline-rich proteins for the identification of saliva

by enzyme-linked immunosorbent assay
Akihisa Igoh a,⇑, Sho Tomotake a, Yusuke Doi a,b
a
Forensic Science Laboratory of Okayama Prefectural Police H.Q., 1-3-2 Tondacho, Kita-ku, Okayama 700-0816, Japan
b
Department of Legal Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science, 2-5-1 Shikatacho, Kita-ku, Okayama 700-8558, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Saliva is one of the most common body fluids found at a crime scene. Therefore, identifying saliva is
Received 5 October 2014 important in forensic science. However, the current protein marker assays used to identify saliva are
Received in revised form 28 December 2014 not sufficiently specific. Although proline-rich proteins (PRPs) are highly specific for saliva, their forensic
Accepted 30 December 2014
potential has not yet been investigated. In this study, we developed enzyme-linked immunosorbent
Available online xxxx
assays (ELISAs) to detect acidic salivary PRP HaeIII subfamily 1/2 (PRH1/2) and basic salivary PRP 2
(PRB2). The specificity, sensitivity, and efficiency of the ELISAs for PRH1/2 and PRB2 were compared with
Keywords:
those of the ELISA for statherin (STATH), a known protein marker for saliva. The levels of PRH1/2 were
Enzyme-linked immunosorbent assay
Saliva identification
significantly higher in saliva and saliva stains than in other body fluids (nasal secretions, urine, semen,
Proline-rich proteins vaginal fluid, blood, and sweat). PRB2 and STATH were detected in both nasal secretions and saliva.
PRP HaeIII subfamily 1/2 The PRH1/2 ELISA showed sensitivity similar to that of STATH ELISA. The detection rate of PRH1/2 ELISA
Salivary PRP 2 was almost similar to that of STATH ELISA, followed by the ELISA for PRB2. The PRH1/2 ELISA had higher
Statherin specificity for saliva than STATH ELISA. Therefore, the PRH1/2 ELISA has potential as a method to identify
saliva for forensic investigation.
Ó 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction protein markers is important to develop more convenient and

cost-effective methods.
The identification of body fluids is important in forensic inves- Statherin (STATH) is a known protein maker used to identify
tigations, to help determine the events that took place and to inter- saliva [4]. However, the protein is also detected in nasal secretions
pret the DNA results. Saliva is often found at crime scenes, for [14]. Although STATH has higher specificity for saliva than a-amy-
example, on cigarette butts, on the rim of drink cans, or on the skin lase, more specific protein markers are needed. Proline-rich pro-
of victims in sexual assaults. teins (PRPs) are coded by a multigene family of seven genes.
Methods to detect a-amylase are commonly used to identify Differential RNA splicing and proteolytic cleavage after secretion
saliva [1,2]. However, a-amylase is also present in other body result in more than 20 different PRPs [15,16]. PRPs are classified
fluids including semen, vaginal fluid, blood, sweat, and urine into acidic, basic, and glycosylated groups. Acidic PRPs are involved
[3,4]. Recently, methods for detecting histatin 3 (HTN3) mRNA in typical oral processes such as mineral homeostasis and neutra-
[5] and bacterial DNA [6] have been reported. Simple, quick, and lization of toxic substances in the diet and are present only in sal-
cost-effective methods are indispensable for forensic investigation, iva. Basic PRPs are present in saliva, nasal secretions, and bronchial
because of the increasing numbers of samples requiring analysis. mucus and may have a more general protective function. Although
Forensic institutes routinely use protein-based methods to acidic PRPs are specifically expressed in salivary glands [17,18], to
identify body fluids. For example, hemoglobin and prostate specific date, the proteins have not been investigated to identify saliva in
antigens are used as protein markers for blood and semen, respec- forensic science.
tively [7,8]. Moreover, several immunochromatographic assay kits Enzyme-linked immunosorbent assays (ELISAs) have been
are commercially available. These kits enable simple and quick developed to identify body fluids such as semen, urine, and saliva
identification of some body fluids [9–13], but investigating novel [3,4,19–21]. These assays can be used to analyze crude samples
that have not been subjected to processing and purification. In
the present study, we used ELISAs to evaluate the expression of
⇑ Corresponding author. Tel./fax: +81 86 221 8592. acidic salivary proline-rich protein HaeIII subfamily 1/2 (PRH1/2)
E-mail address: op-qphif108@pref.okayama.jp (A. Igoh). and basic salivary proline-rich protein 2 (PRB2) in various body

http://dx.doi.org/10.1016/j.legalmed.2014.12.011
1344-6223/Ó 2015 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Igoh A et al. Detection of proline-rich proteins for the identification of saliva by enzyme-linked immunosorbent assay.
Leg Med (2015), http://dx.doi.org/10.1016/j.legalmed.2014.12.011
2 A. Igoh et al. / Legal Medicine xxx (2015) xxx–xxx

fluids (nasal secretions, saliva, urine, semen, vaginal fluids, blood, Horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG
and sweat), and to determine whether these proteins could be used was purchased from Sigma Aldrich (St. Louise, MO, USA), and
as markers for the forensic identification of saliva. HRP-conjugated goat anti-rabbit IgG was purchased from KPL
(Gaithersburg, MD, USA).
2. Materials and methods Anti-PRH1/2 and anti-PRB2 were diluted (1:1000) with 0.05%
Tween-20 in phosphate buffered saline (PBST), and anti-STATH
2.1. Sample collection and treatments was diluted (1:500) with PBST. The HRP-conjugated mouse anti-
goat IgG and HRP-conjugated goat anti-rabbit IgG were diluted
2.1.1. Sample collection (1:1000) with PBST for PRH1/2 and PRB2 ELISA, respectively. The
All procedures involving human volunteers were approved by HRP-conjugated mouse anti-goat IgG was diluted (1:5000) with
the Ethical Committee of Human Genome and Genomic Analysis PBST for STATH ELISA.
from the Japanese Association of Forensic Science and Technology.
Samples were collected from consenting adults. Nasal secretions 2.2.2. Sample preparation
(n = 5), saliva (n = 20), semen (n = 5), blood (n = 5), vaginal fluids Vaginal fluid swabs were cut into 5 mm  5 mm squares and
(n = 10), urine (n = 5), and sweat (n = 5) were collected from volun- the sample was extracted with 100 ll of 0.05 M bicarbonate buffer
teers aged 26 to 57 years. Blood samples were collected from the (BCB; pH 9.6). Body fluids and vaginal fluid extracts were diluted
brachial vein into Venoject II tubes (TERUMO, Tokyo, Japan). Vagi- with BCB (1:100 to 1:6400). Filter papers with sweat samples were
nal fluids were self-collected by the volunteers by swabbing the cut into 10 mm  10 mm squares and extracted with 250 ll of BCB
vaginal wall with sterile cotton swabs, regardless of the menstrual by pipetting. The extracts were diluted with BCB (1:2–1:64).
cycle. Sweat samples were collected from the facial region and Vaginal fluid stain samples were prepared by extracting with
arms after exercise, using filter paper strips. Saliva, nasal secre- 100 ll of BCB by pipetting. The extracted vaginal fluid samples
tions, and other body fluids were collected in sterile plastic tubes. (5 ll) were diluted (1:100) with BCB on ice. Other body fluid stains
With parental consent, saliva samples were also collected from were extracted with 250 ll of BCB by pipetting on ice. The sample
twelve children aged between 2 months and 12 years. Collected extracts (250 ll) were centrifuged at 7900g for 3 min at 4 °C. The
body fluids were stored at 80 °C until required for further supernatants were diluted with BCB (1:2–1:64).
analysis.
2.2.3. ELISA
2.1.2. Stain preparation Diluted samples (50 ll per well) were added to 96-well multi-
Body fluid stains were prepared as follows. Five microliters of tier plates (SUMILON MS7296F; Sumitomo Bakelite, Tokyo, Japan)
saliva were spotted onto filter papers (10 mm  10 mm squares) and incubated at 37 °C for 1 h. Each well was blocked with 200 ll
and air-dried at room temperature for 1 week (n = 20) or 1 year of Block Ace (Dainippon Sumitomo Pharma, Osaka, Japan) at 37 °C
(n = 8). Sterile cotton swabs with vaginal fluid samples (n = 10) for 1 h. The wells were then washed three times using 250 ll of
were cut into 5 mm  5 mm squares and air-dried at room tem- PBST per well and 50 ll of diluted anti-PRH1/2 or anti-PRB2 were
perature for 1 week. Filter papers with sweat samples (n = 5) were added to each well. The plates were incubated at 37 °C for 1 h. The
cut into 10 mm  10 mm squares and air-dried at room tempera- plates were then washed three times with 250 ll of PBST per well
ture for 1 week. Other body fluids stains (n = 5 for each fluid) were and incubated with 50 ll of diluted HRP-conjugated rabbit anti-
prepared as for saliva stains and air-dried at room temperature for goat IgG or HRP-conjugated goat anti-mouse IgG per well at
1 week. 37 °C for 1 h. The plates were then washed five times with 250 ll
of PBST per well. In PRH1/2 and PRB2 ELISA, 50 ll of TMB + Sub-
2.1.3. Mixed stains strate Chromogen (Dako Cytomation, CA, USA) were added to each
Mixed stains were prepared by spotting 5 ll of saliva and well and incubated at room temperature for 3 min. Color develop-
semen onto filter papers (10 mm  10 mm squares), and 20 ll of ment was stopped by the addition of 50 ll of 1 M H2SO4. Absor-
saliva were spotted onto sterile cotton swabs with vaginal fluid bance was measured at a wavelength of 450 nm using a
samples (3 mm  3 mm squares). Both mixed stains were air-dried Molecular Devices SPECTRA max PLUS 384 (Molecular Devices,
at room temperature for 1 week. CA, USA). In STATH ELISA, the procedure of color development
and the measurement of absorbance value were followed accord-
2.1.4. Simulated casework samples ing to the previous report [4]. Each absorbance value was normal-
Rolling papers from cigarette butts were cut into 10 mm  ized by subtracting the primary antibody blank absorbance value.
10 mm squares. The rims of drink bottles and cans were wiped
with sterile cotton swabs. The skin of volunteers’ arms was licked 2.3. Data analysis
and dried for 1 h, and the trace was wiped with sterile cotton
swabs. The swabs were cut into 3 mm  3 mm squares. The ELISA data for each dilution ratio were statistically analyzed
by one-way ANOVA with Scheffé’s multiple-comparison test.
2.2. Enzyme-linked immunosorbent assay
3. Results
2.2.1. Reagents
Goat polyclonal antibody against the near N-terminus of human 3.1. Specificity and sensitivity of ELISA for detecting adult body fluids
PRH1/2 (anti-PRH1/2), purified by affinity chromatography, was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The expression of PRH1/2 and PRB2 in body fluids and the
Rabbit polyclonal antibody against the C-terminus of human expression of STATH in nasal secretions and saliva were evaluated
PRB2 (anti-PRB2), purified by affinity chromatography, was pur- by performing ELISA. The PRH1/2 absorbance values of saliva sam-
chased from Abgent (San Diego, CA, USA). Goat polyclonal antibody ples at dilutions ranging from 1:100 to 1:200 were significantly
against the N-terminus of human STATH (anti-STATH; Santa Cruz, higher than those of other body fluids (nasals secretions, semen,
CA, USA), purified by affinity chromatography, was donated by Dr. vaginal fluid, urine, blood, and sweat; p < 0.05; Fig. 1A). Further-
Sakurada of the National Research Institute of Police Science. more, the PRH1/2 absorbance values of diluted saliva (1:6400)

Please cite this article in press as: Igoh A et al. Detection of proline-rich proteins for the identification of saliva by enzyme-linked immunosorbent assay.
Leg Med (2015), http://dx.doi.org/10.1016/j.legalmed.2014.12.011
 A. Igoh et al. / Legal Medicine xxx (2015) xxx–xxx 3

Fig. 1. Specificity of ELISA for the detection of PRH1/2 (A), PRB2 (B), and STATH (C) in body fluids. Body fluids were diluted with bicarbonate buffer using dilutions ranging
from 1:100 to 1:6400. Absorbance values are presented as mean ± SD for saliva (n = 20), vaginal fluid (n = 10), or other body fluids (n = 5 for each fluid). The absorbance of
saliva in PRH1/2 ELISA was significantly higher than that of other body fluids (⁄p < 0.05) as indicated by one-way ANOVA with Scheffé’s multiple-comparison test.

were higher than the PRH1/2 absorbance values of other bodily cut-off absorbance value, and the AAV value at the dilution limit
fluids. The exclusion of absorbance values was fixed as 0.2 to avoid was 0.21.
non-specific absorbance from other body fluids and negative con-
trol sample. The values lower than 0.2 yielded positive results only 3.3. Specificity and sensitivity of ELISA for body fluid stains
for the saliva samples and all other body fluids tested negative
(Table 1). The data also revealed moderate inter-individual In the PRH1/2 ELISA performed on one-week-old stains of var-
variation in PRH1/2 expression. The detection limit of the PRH1/2 ious body fluids the absorbance of saliva was higher than that of
ELISA for saliva samples was determined to be 0.03 ll (50 ll at other body fluids (data not shown). Furthermore, the PRH1/2
1:1600 dilution) by the comparison of the average absorbance absorbance values of extracted saliva stains (1:16) were higher
(AAV) values in each dilution ratio with the cut-off absorbance than those of other bodily fluid stains. Of 20 saliva samples, 13
value, and the AAV value at the dilution limit was 0.26. tested positive for PRH1/2, and all other body fluid stains tested
ELISA detected both PRB2 and STATH in saliva and nasal secre- negative. PRH1/2 was also detectable in five of eight one-year-
tions (Fig. 1B and C). In the PRB2 ELISA, the exclusion of absorbance old saliva stains.
values was fixed as 0.2 to avoid non-specific absorbance from other
body fluids, except nasal secretions and saliva, and negative con- 3.4. Mixed stains and simulated casework samples
trol sample. The values lower than 0.2 yielded positive results for
the nasal secretions and saliva samples and all other body fluids The PRH1/2 ELISA was performed on mixed stains and simu-
tested negative (Table 1). In STATH ELISA, the cut-off absorbance lated casework samples to verify its usefulness for the forensic
value of 0.1 [4] yielded positive results for nasal secretions and sal- identification of saliva. The ELISA test successfully detected
iva samples (Table 1). PRH1/2 in the mixed stains, and on rolling papers from cigarette
butts, the rims of drink bottles and cans, and licked skin.

3.2. ELISA for detecting children’s saliva 4. Discussion

The detection of PRH1/2 and STATH in the saliva of children The aim of the present study was to assess the potential of PRPs
(including infants) was evaluated by ELISA. Five of twelve samples as protein markers and to develop a highly specific method for the
tested positive in both ELISAs. The limit of detection for PRH1/2 identification of saliva. The ELISA for PRH1/2 was highly specific
ELISA was determined to be 0.13 ll (50 ll at 1:400 dilution) by and sensitive for saliva, and might be useful to identify saliva in
the comparison of the AAV values in each dilution ratio with the a forensic setting.
The specificity, sensitivity, and detection rate of the PRH1/2 and
PRB2 ELISAs were compared with STATH ELISA for discriminating
saliva samples. STATH ELISA was only tested for nasal secretions
Table 1
and saliva, because previous studies have reported the specificity
Comparison of the specificity of PRH1/2, PRB2, and STATH ELISAs for saliva detection.
of the test for other body fluids [4,14]. The PRH1/2 ELISA had the
Body fluids Number Number of positive samples highest specificity and showed positive results only for saliva.
Detection Detection Detection The PRB2 and STATH ELISAs showed positive results for nasal
of of PRB2* of STATH* secretions and saliva samples. A previous study has shown that
PRH1/2*
PRB2, an example of the basic PRPs, is slightly expressed in nasal
Nasal secretions 5 0 3 2 secretions [18]. The current results agree with the findings of the
Saliva 20 16 12 17 earlier study. The PRH1/2 ELISA was as sensitive as STATH ELISA
Semen 5 0 0 –
Vaginal fluid 10 0 0 –
[4]. The detection rate of STATH ELISA for saliva in the present
Urine 5 0 0 – study almost agreed with previous results [4] and was almost simi-
Blood 5 0 0 – lar to that of the PRH1/2 ELISA (Table 1). In the semi-quantitative
Sweat 5 0 0 – PhadebasÒ amylase tube test, which is commonly used for saliva
*
Absorbance >0.2 for PRH1/2 and PRB2 (measured at 450 nm) or >0.1 for STATH investigation, the sensitivity is so high that approximately
(measured at 490 nm). A dilution of 1:100 was used for each sample. 0.00125 ll of saliva gives a positive result. However, the specificity

Please cite this article in press as: Igoh A et al. Detection of proline-rich proteins for the identification of saliva by enzyme-linked immunosorbent assay.
Leg Med (2015), http://dx.doi.org/10.1016/j.legalmed.2014.12.011
4 A. Igoh et al. / Legal Medicine xxx (2015) xxx–xxx

is so low that approximately 0.125 ll of blood, semen, sweat, and with other body fluids. Such samples are often found in sexual
urine also show positive results [4]. Therefore, saliva cannot be assault cases.
reliably identified using the PhadebasÒ amylase tube test. The
PRH1/2 ELISA might provide a viable alternative for saliva identifi- Acknowledgment
cation, because of the high specificity for saliva.
The PRH1/2 and STATH ELISA produced positive results for five We wish to thank Dr. Koichi Sakurada of the National Research
samples of children’s saliva. The detection rates in children’s saliva Institute of Police Science for providing the anti-STATH for ELISA.
were smaller than those in adults. This suggests that the expres-
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Please cite this article in press as: Igoh A et al. Detection of proline-rich proteins for the identification of saliva by enzyme-linked immunosorbent assay.
Leg Med (2015), http://dx.doi.org/10.1016/j.legalmed.2014.12.011