Journal of Immunoassay

ISSN: 0197-1522 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ljii19

Dot Blot Immunoassay for Detection of Human Semen D. V. Rao & V. K. Kash yap To cite this article: D. V. Rao & V. K. Kash yap (1992) Dot Blot Immunoassay for Detection of Human Semen, Journal of Immunoassay, 13:4, 537-544, DOI: 10.1080/15321819208019834 To link to this article: http://dx.doi.org/10.1080/15321819208019834

Published online: 23 Sep 2006.

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Date: 14 June 2016, At: 14:52

JOURNAL OF IMMUNOASSAY, 13(4), 537-544 (1992)

DOT BLOT IMMUNOASSAY FOR DETECTION OF HUMAN SEMEN

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D V

Rao

V

k

I(

Kashyap

Central Forensic Science Laboratory Bureau of Police Research & Development (M.H.A.) Government of India Ramanthapur, HYDERABAD 500 013.

-

I N D I A . A B S T R A C T A sensitive and specific dot blot immunoassay based on prostate specific antigen (PSA) antibodies and a radiolabelled Protein A detection system was developed for detection of trace amounts of human semen in stains. The method was found highly sensitive and detected semen stain extracts diluted 10,000 times. Semen stains u p to five years old were successfully detected by t h e method. The assay is found t o be highly suitable for semen detection i n forensic analysis.

(KEYWORDS : Dot blot immunoassay, Semen, Nitrocellulose, P30 antibodies). INTRODUCTION In

of

cases of

semen on t h e

Microscopic acid

alleged

genital

examination

phosphatase,

sexual assaults,

area or

demonstrating

the a p p r e l

and biochemical tests

choline o r

spermine

(1-5)

of

t h e presence

victims is important.

based on t h e presence of a s routinely

practiced

for

t h e detection of semen have limitations (6-7). Immunological detection of

methods

biological

have

substances

provided

better

methods

including semen components

been

reported

and specific,

(11-14).

Though

enzyme

immunoassays

stains

a r e simple

sensitivities may b e impaired by matrix effects (15).

537 Copyright 0 1992 by Marcel Dekker, Inc.

the

(8-10) and

many enzyme immunoassays for t h e detection of traces of semen its have

for

In

RAO AND KASHYAF

538

the

present

immunoassay

study.

we

have

prostatic

using

labelled Protein A

developed

an

specific antigen

for the

visualization

the nitrocellulose membrane.

of

ultrasensitive

dot

blot

and

IlZ5

(PSA) antibodies

immune complexes formed on

The specificity of PSA antibodies and the

sensitivity of the radiolabel makes the assay highly mitable for forensic investigation.

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MATmIALS A M ) METHODS Polyclonal prchased

antibodies

from Dakopatts,

Denmark.

human

PSA

raised

in

rabbit

were

Protein A was obtained from Sigma

USA and Nitrocellulose membrane was supplied by Advanced

Chemicals Co. Microdevices

against

Private

Limited,

Ambala,

I l Z 5 was obtained from

India.

Bhabha Atomic Research Centre, Bombay, India.

A l l other chemicals were

of analytical grade and procured locally. Semen and other body f l u i d s

: Semen from normal vasectomized

persons

and patients suffering from pathogenic and malignant diseases of the male genital

system,

volunteers.

vaginal

Bull,

smears,

buffalo,

saliva

and

urine

were

collected

from

goat, dog, horse, monkey and pig semen were

obtained from the Government Veterinary

Hospital,

Hyderabad.

From a l l

the body fluids, stains were prepared by pwring 0.05~11 of sample over sterile cotton cloth.

Human semen samples used i n the study were from

individuals aged from 1 5 to 70 years.

The stains of various body fluids

and semen were prepared from a minimum of five individuals. Stain Extraction

:

The stains were extracted i n 200 u l of phosphate

buffered saline, pH 7.2,

150 mmol/L (PBS) as explained elsewhere (16).

5.0111 of the extract was used i n the study. Radioiodination of

Protein A

:

Protein A was radioiodinated according

to the method of Franker and Speck (17).

Briefly, 0.5uci of radioactive

DOT BLOT IMMUNOASSAY

539

NaI was allowed to react with 50Oug of Protein A a t 4OoC in a test tube coated

with

Iodogen.

The reaction

m i n u t e s with occasional shaking.

was allowed

to

take

place for

15

The labelled Protein A was separated

from t h e free label using a l O m l column packed with Sephadex G-50. Dot

Blot

membrane

Immunoassay

Stain

:

and a i r dried.

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was

were

The non-specific

with 2% bovine serum albumin membrane

extracts

washed three

spotted

on

nitrocellulose

binding sites

were blocked

(BSA) i n PBS a t 37OC for one hour.

times

with

PBS containing

at

37OC for 4 hours.

antibodies

were

able

(satisfactorily).

The

and incubated with for

one hour

changes of dried

at

(This was to

bind

membrane

the

to

the

was

membrane

washed

in

t i m e s i n PBS at which

dilution

maximum

bound

three

Tween-20

0.005%

(PBST) and incubated with antibodies t o PSA diluted 1 : l O O O

The

PSA

antigen

changes of

autoradiography.

PBST

10 u l of 1125 Protein A showing lOuci activity i n PBS Then t h e

37OC.

membrane

was

washed

with

several

PBST for one hour a t room temperature and a i r dried.

membrane

the

was

placed

in

a

polythene

bag

The autoradiography was done a t -2OOC

and

kept

The for

for 24 hours

with ORWO X-Ray film and K i r a n intensifying screens. Specificity Study vaginal

: Normal semen and semen from vasectomized

smears of human adult females who had abstained from sex for

1 5 days prior t o collection, semen of bull,

i n t h e study. Sensitivity 1:100,

persons,

buffalo,

saliva, urine,

goat, dog, horse,

or normal humans and

monkey and pig were included

The extract of 20 stains of each t y p e were tested.

Study

1:lOOO

blood,

:

Normal human

and 1 : l O O O O

in PBS.

semen extract 5 . 0 ~ 1 sample of

was diluted l : l O , each dilution was

spotted on nitrocellulose membrane and t h e assay was performed. Effect of Disease suffering

from

:

Semen samples w e r e also collected from patients

pathogenic

and

malignant

diseases

of

the

male

genital

RAO A N D KASHYAP

540

system t o find cut t h e suitability of the assay in detection of semen stains derived from such persons. from

prostate

cancer,

Semen samples from persons suffering

non-malignant

tumcurs,

gonorrhea

and

syphillis

were tested in the study and 10 samples for each case were included. Effect of storage

were stored at room temperature (25°C-400C)

cloth month,

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Normal human semen samples prepared on cotton

:

six

months,

for one week,

one year and five years and tested

applicability of t h e assay in detection of old stains.

t o ascertain

For each duration,

extracts from 50 different stains were tested in t h e study.

Semen stains

were cut and extracted in PBS a s explained above.

of size 2.0cm'

one

5.0~1

of t h e extract was assayed from each.

RESULTS A l l t h e human semen stains, irrespective of t h e i r fertility status.

i.e.

normal or vasectomized,

were successfully detected by the assay.

Other human body fluids and semen derived from non-human sources gave negative results. Semen

extracts

diluted

up

to

10,000

times

could

be

clearly

demonstrated by the assay. Semen

from

prostatic cancer,

40

patients

with

urogenital

prostatic benign hyperplasia,

a l l gave positive results.

disease,

10

each

of

gonorrhea and syphillis ,

Positive results w e r e obtained on every one

of t h e 50 samples of human semen stains stored up t o 5 years.

DISCUSSION The assays for detection of semen have not lost t h e i r relevance i n spite of t h e advent of DNA Profiling (18) which can provide infallible

information about t h e identity of t h e source of semen in an alleged rape

DOT BLOT IMMUNOASSAY case,

because

the

54 1

characterization

of

semen

is

as

essential

as

in

the

individualization. Biochemical

analysis

discovery of a 32kd semen

(19).

of

protein

Because

of

semen

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be a

Antibodies

highly with

the

synthesis

suitable marker high

has

resulted

(PSA) which is highly specific t o human

presence i n semen is independent of to

components

of

in

PSA

spermatozoa.

the

prostate,

its

PSA has been found

of human semen i n

forensic analysis.

to

characterized

specificity

PSA

have

been

and

several enzyme immunoassays for detection of PSA i n semen and its stains have been

reported

(20).

However,

fail to detect m i n u t e

they often

quantities of semen i n dried old stains.

The present assay successfully

detected semen stain extracts diluted 10,000 times.

As

proteins

interactions,

in

dot

strongly bind t o nitrocellulose through blot

assays

sufficient for t h e t e s t which

even

nanogram

quantities of

revealed

that

a

PSA

are

is more sensitive than micro ELISA ( 2 1 ) .

The antibodies used in t h e assay a r e of high avidity, study h a s

hydrophobic

1:lOOO

dilution

gives

and t h e titration

satisfactory

results.

This dilution has been used throughout the study. The detection of PSA i n vaginal washings of victims of rape after 48 hours is not possible,

whereas prostatic acid phosphatase (PAP) can

be detected a t t h i s time ( 2 3 ) , but t h e specificity and stability of PAP

i n urinogenital swabs of victims and i n semen stains is poor compared t o PSA

. Protein A binds t o a wide variety of immunoglobulins of different

species and its affinity to rabbit IgG is high.

A h a s led t o the u s e of radiolabelled protein

This property of Protein

A for t h e detection of

immune complexes, instead of enzyme labelled second antibody.

542

RAO AND KASHYAP Further,

the

radiolabel

is

uninfluenced

which can affect t h e enzyme labels in ELISA.

by

the

sample

matrix

The o t h e r merit of t h e

assay is its successful detection of semen stains a s old a s 5 years with remarkable

sensitivity.

The

assay

has

better

sensitivity

than

the

commercially available PSA immunoassay kits. Another

advantage

of

the

present

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original results for long periods, t h e dot blot format, processed.

assay

is t h e availability

unlike enzyme immunoassays

a large number of

(23).

of In

samples can be simultaneously

Antigen loaded membranes after blocking can be stored for

periods of up to five years without any loss of t h e sample for further analysis.

ACKNOWLEDGEMENTS The authors a r e thankful Hyderabad,

for providing

facilities for

Rao thanks BPR&D for providing acknowledge

the

skillful

t o Dr.

T R carrying

Baggi,

Director,

CFSL,

out t h e work.

D V

financial assistance.

secretarial

assistance

The authors also

rendered

by

Mr.

R

Madhusudhan.

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