Zeitschrift for

Z Rechtsmed (1984) 93:117-121 Rechtsmedizin

© Springer-Verlag 1984

A New Method in Criminology:

Use of ELISA to Detect AFP on Different Materials
with Monoclonal Anti- -fetoprotein

E. Kurucz 1, F. K4sa 1, l~. Monostori 2, and I. Ando 2

1 Dept. of Forensic Medicine, University of Szeged, Kossuth Lajos sgt. 40, H-6724 Szeged,
Hungary
2 Institute of Genetics, Biologic Research Center, Hungarian Academy of Sciences,
H-6701 Szeged, Hungary

Summary. A new method has been introduced to distinguish normal adult

serum stains from fetal or newborn serum and amniotic fluid stains with
E L I S A in cases of criminal abortion and infanticide. The method is based on
the sensitive detection of a-fetoprotein (AFP) by a two-site enzyme immuno-
assay (EIA) following its elution with high efficiency from different mate-
rials (e.g., cotton, paper, synthetic fabric, or glass) by phosphate-buffered
0.5 M NaC1 solution
Key words: Monoclonal antibody - E L I S A technique - Fetal umbilical cord
blood-stain - Amniotic fluid stain

Zusammenfassung: Es wurde eine Methode erarbeitet, um in Ffillen von

Abtreibung bzw. Kindest6tung (T6tung des Neugeborenen) zwischen den
vom Neugeborenen herrtihrenden biologischen Spuren (Blut- und Frucht-
wasserflecke) und den von anderen Individuen stammenden Blutflecken
unterscheiden zu k6nnen. Von der Oberflfiche der verschiedenen Spuren-
trfiger (Baumwollstoff, Papier, Kunstfaser und Glas) wurden die einge-
trockneten Flecke mit 0,5 M NaC1 enthaltendem Phosphatpuffer eluiert und
dann aus den Eluaten das Alpha-Fetoprotein mit monoklonalen Anti-
k6rpern mit Hilfe der ELISA-Technik nachgewiesen.
Schliisselw6rter: Monoklonale Antik6rper - ELISA-Technik - Nabelblut-
flecke - Fruchtwasserflecke

In special cases of homicide, i.e., criminal abortion and infanticide, the demon-
stration of findings relating to the fetus or infant at the site of the criminal act is
of great importance in medicolegal laboratory investigations. If the origin of a
blood stain is unknown, the distinction between fetal umbilical cord serum or
amniotic fluid and adult serum would provide and additional possibility for
evidence in medicolegal practice.
Offprint requests to: F. K6sa, M.D., Associate Professor (address see above)
118 E. Kurucz et al.

A F P is a glycoprotein that has been detected at very low concentration

(10ng/ml) in normal adult serum and in higher concentrations [1, 4, 5, 8] in
maternal and fetal sera and amniotic fluid. It is synthesized by the yolk sac and
fetal liver. After birth it decreases gradually, reaching the level of normal
healthy adults with in 1 month [2, 6, 9]. In standard clinical practice measure-
ment of the A F P level in maternal serum and in amniotic fluid is used for the
detection of neural tube defects [3, 11].
Fetal and adult blood stains may be distinguished via the detection of AFP.
To detect A F P from dried and eluted samples on different trace supports (e.g.,
cotton, paper, synthetic fabric or glass), we have used a sandwich E L I S A tech-
nique [2, 10].

Reagents

The two monoclonal antibodies used for E L I S A were described by Monostori

et al. [7]. Anti-AFP-1 antibody was used to coat the plate. Anti-AFP-33 anti-
body was conjugated with horseradish peroxidase according to Wilson and
Nakane [12]. The A F P standard was obtained from D A K O .

Material and Methods

Preparation and processing of samples: 50, 100, 500, and 1,000 gl fetal umbilical cord serum
and blood, amniotic fluid, normal adult serum and whole blood were applied to different solid
supports, such as cotton, paper, synthetic fabric, and glass. The samples were eluted for I h at
room temperature with 1 ml phosphate-buffered 0.5 M NaC1 at pH 7.2. The protein concen-
trations of the eluates were measured at 280nm with a Spectromom 195 spectrophotometer.
The extinction coefficient was E I ~ g/rnl 1.65. Sandwich ELISA is based on the method of
Uotila et al. [10]. Briefly, the wells of polystyrene microtiter plates (DYNATECH) were coat-
ed with 100 ~tl anti-AFP-1 monoclonal antibody solution (2.5 gg/ml) diluted with carbonate
buffer of pH 9.6. The plates were incubated at 4°C overnight. After incubation, the wells were
washed three times with phosphate-buffered saline of pH 7.2 containing 0.05% Tween 20
(PBS-Tween). One hundred microliters standard AFP solutions or eluate samples were added
to the wells. The standards and sample dilutions were made in PBS-Tween buffer containing
0.5% bovine serum albumin (Sigma). After three washing steps and the addition of 100gl
anti-AFP-33 HRPO, a dilution of 1:2,500 was incubated in each well at 37°C for 1 h. After
three washing steps, 200 gl substrate solution (20 mg o-phenylenediamine and 10 ~t130% H202
in 60 ml phosphate-citric acid buffer of pH 6.0) was added to each well. After a 30-min incuba-
tion at 37°C, the reaction was stopped with 50 gl 4 N H2SO4,and the absorbance was meas-
ured at 492 nm with a Gilford spectrophotometer.

Results

The dried samples could be stored at room temperature for at least 1 week
without change. The concentrations of the recovered proteins were measured
spectrophotometrically at 280 nm.
Figure 1 shows the amount of total protein in fetal umbilical cord serum
(Fig. la), amniotic fluid (Fig. l b ) and adult serum (Fig. lc), and in their eluates
from cotton, paper, synthetic fabric, and glass supports. Of the total protein
A New Method in Criminology 119

[mg] [ xlO~Jg] ./
/

Q /,/
100 /
/ 1000- //
/, /
/" ,
<. //
/
500
./ o z

u_ ///z • .,¢
z ~"
z
"7
87P~10
8 100"
z
5 s0-
(23
eY
I3. 13.
-3

op.-
e
~t¢ i
so 16o s6o 1~ E;~i
APPLIEDA.OUNT OF FOET~_ uM~uaL APPLIED AMOUNT OF AMNIOTICFLUID
CORD SERUM THE AMOUNT OF TOTAL PROTEIN CONTENT
--- IN ORIGINAL SAMPLES
THE AMOUNT OF TOTAL PROTEIN CONTENT
- - [N ELUATES FROM
--- IN OR@NAL SAMPLES
SOLID SUPPORTS • COTTON x SYNTHETIC FABRIC
- - IN ELUATES FROM
°PAPER ~' GLASS
SOLID SUPPORTS • COTTON * SYNTHETIC FABRIC
o PAPER t OLASS

[mg]

100 /
/
/"

<
///I /
/
/

o
z 10,

o
E Fig. la--e. The total protein content in
fetal umbilical cord serum, in amniotic
fluid and adult serum, and in their
-g/ i eluates from different solid supports
so i~o s~o i~ EpTj
APPLIED AMOUNT OF NORMAL ADULT SERUM
c
THE AMOUNT OF TOTAL PROTEIN CONT[NT
--- IN ORIGINAL SAMPLES
- - IN ELUATES FROM
SOLID SUPPORTS "COTTON " SYNTHETIC FABRIC
oPAPER OOLASS

content 30%-90% were eluted from the fetal and adult blood stains and amnio-
tic fluid stains dried on cotton, paper, synthetic fabric, and glass supports. To
calculate the unabsorbed and eluted AFP from the samples, AFP standards
were used in the range of 1-100 ng/ml. Figure 2 shows the calibration curve for
these AFP standards. The AFP contents of the original and eluted samples of
fetal umbilical cord serum and amniotic fluid are presented in Fig. 3a and b. The
 120 E. Kurucz et al.

~
1,2-
f
1,0

u.t

0,5
Fig. 2. Calibration curve to calculate
the unabsorbed and eluted AFP from
0,1¸
I i the samples with the ELISA method
10 100 [ ng/ml ]
CONCENTRATIONS OF STANDARD AFP

Ing],
/
40000 /
./ [ngl
/ //
30000. / /"
//
/ //
/ //
1/ 100- /"
//
o 10000- / //
/ //
r--, /
/ 1/ /
-J /

1/
/
/ F-
u
Z
N 10.
u_ 1000-
z Z
m

§ t--
Z
8
,£- 200-
.,¢

so ioo so0 1oooI,ul 5O IOO sOd 1ooo

a APPL,EDAMOUNT OF FOETAL UMBILICAL b APPLIED AMOUNT OF AMNIOTIC FLUID
CORD SERUM
---AFP CONTENT IN O~DINAL SAMPLES
---AFP CONTENT IN O~OINAL SAMPLES --AFP CONTENT IN ELUATES FROM
- - A F P CONTENT IN ELUATES FROM SOLID SUPPORTS "COTTON x SYNTHETrC FABRIC
SOLID SUPPORTS "[OTTON * SYNTNETI[ FABRIC
o PAPER ~OLASS
o PAPER oOLASS

Fig.3a, b. The AFP contents of the original and eluted samples of fetal umbilical cord serum
and amniotic fluid

e x t e n t of A F P r e c o v e r y f r o m the solid supports w a s 1 0 % - 4 0 % . Similar results

w e r e o b t a i n e d w h e n w h o l e b l o o d was u s e d instead of s e r u m (data not s h o w n ) .
T h e r e w a s no d e t e c t a b l e A F P in either the original or the eluted n o r m a l adult
s e r u m and w h o l e b l o o d .

Discussion

In m e d i c o l e g a l practice, cases of criminal abortion and infanticide often necessi-

tate the d e t e c t i o n of traces relating to the fetus or the infant at the site of the
criminal act and the distinction b e t w e e n fetal and adult blood-stains. W e h a v e
d e v e l o p e d a t e c h n i q u e for the differentiation of fetal and adult dried s e r u m and
A New Method in Criminology 121

amniotic fluid samples mimicking traces of a criminal event. T h e distinction is

b a s e d on the detection of A F P . This is p e r f o r m e d with two m o n o c l o n a l anti-
A F P antibodies, which react with two distinct antigenic determinants on the
molecule. T h e advantages of using m o n o c l o n a l antibodies are their strict speci-
ficity and high affinity, and the r e a d y availability of standard antibodies, these
properties c o m p l y with the r e q u i r e m e n t s of medicolegal investigations. T h e
b l o o d , serum and amniotic fluid samples were dried on different solid supports.
O f the total p r o t e i n c o n t e n t 3 0 % - 9 0 % and 1 0 % - 4 0 % of the total A F P were
eluted f r o m the solid supports.
T h e sensitivity of the applied sandwich E L I S A technique allows the quanti-
tative d e t e r m i n a t i o n of A F P f r o m as little as 50 ~tl fetal b l o o d and 100 gl amnio-
tic fluid on a support, but there is no detectable A F P in the stains of adult b l o o d
or serum.
Acknowledgment. We are grateful to M.D.J. Szab6, Hospital II, Szeged (Hungary) for col-
lecting the fetal umbilical cord blood and maternal blood samples.

Abbreviations
AFP, a-fetoprotein; EIA, enzyme immunoassay; ELISA, enzyme-linked immunosorbent
assay; PBS-Tween, phosphate-buffered saline containing 0.05% Tween 20; HRPO, horseradish
peroxidase; anti-AFP-33 HRPO, antibody conjugated with horseradish peroxidase

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Received March 5, 1984