Forensic St5enc.eIntenational, 55 (1992) 161- 172 Elsevier Scientific Publishers Ireland Ltd.

161

A SENSITIVE SANDWICH ENZYME IMMUNOASSAY FOR y-SEMINOPROTEIN AND ITS APPLICATION TO SEX DISCRIMINATION OF BLOOD AND BLOODSTAINS

N. YUKAWAa, T. KOHNOb, E. ISHIKAWAb and K. TAKAHAMA8 aDepartment of Legal Medicine and bDepartment of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki 889-16 (Japan)

(Received1April 7th 1992) (Accepted June 8th, 1992)

A sensitive sandwich enzyme immunoassay for r-seminoprotein (~30, prostate-specific antigen) is described for sex discrimination of blood and bloodstains. A polystyrene ball coated with rabbit antiy-seminoprotein IgG was incubated with 7-seminoprotein and, after washing, with affinity-purified rabbit anti-y-seminoprotein Fab’-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as hydrogen donor. The detection limit of y-seminoprotein was 0.15 pg per assay. Blood levels of y-seminoprotein, measured using 1 - 10 gl of blood, were at least 3.3-fold higher in male adults than in female adults. The ratio of y-seminoprotein in terms of pg to hemoglobin in terms of mg was significantly higher in male adults than in female adults. Thus, the measurement of y-seminoprotein or both y-seminoprotein and hemoglobin was useful for the discrimination of blood and bloodstains of male and female adults, although with some limitations. Keg words: y-Seminoprotein; tion: Bloodstain

~30; Prostate-specific

antigen; Enzyme immunoassay; Sex discrimina-

Introduction On the basis of electrophoretic studies of human seminal plasma, a glycoprotein with a molecular mass of -23 000 Da was purified from human seminal plasma and called y-seminoprotein [l]. Later, a semen-specific protein purified from human seminal plasma was designated p30 [2] and a prostate-specific antigen was purified from prostatic tissues [3]. y-Seminoprotein and the prostatespecific antigen have been found to be identical to each other from their amino acid sequences [4 - 61 and appear to be identical to p30 [‘7]. y-Seminoprotein is present at readily detectable levels in semen and blood of male adults but at very low levels, if any, in blood, secretions and tissues of female subjects [8- 111. Therefore, the usefulness of the detection of y-Correspolzdence to: Keiichi Takahama, Professor of Legal Medicine, Department Medical College of Miyasaki, Kiyotake, Miyazaki 889-16, Japan. 0379-0738/92/$05.00

0 1992 Elsevier Scientific Publishers Printed and Published in Ireland

Ireland Ltd.

of Legal Medicine,

162

seminoprotein in forensic tests has been suggested and various methods for this purpose have been developed. These methods include competitive enzyme immunoassay [12] and sandwich enzyme immunoassay [13 - 161. The detection limits of y-seminoprotein by these methods are 10 - 3200 pg per assay and 0.2 - 8 pg/l of semen [13- 161 and these methods are apparently useful for demonstrating the presence of semen in various materials. However, y-seminoprotein in blood has not been measured for forensic purposes and a higher sensitivity may be useful for the detection of y-seminoprotein in materials containing blood. This paper describes a sensitive sandwich enzyme immunoassay for yseminoprotein and its usefulness tested for sex discrimination of blood and bloodstains. Materials and Methods Buffers

The regularly used buffers were 10 mmol/l sodium phosphate buffer, pH 7.0, containing 1 g/l bovine serum albumin (fraction V, Armour Pharmaceutical Co., Kankakee, IL, USA) (buffer A) and 10 mmol/l sodium phosphate buffer, pH 7.0, containing 0.1mol/l NaCl (buffer B). y-Seminqwotein standard

Lyophilized y-seminoprotein included in a commercial kit for enzyme immunoassay (y-Sm Chugai R, Chugai Pharmaceuticals Co., Tokyo, Japan) was reconstituted to a concentration of 200 pg/l according to the instructions of the manufacturer and was used as standard. Antibody

Rabbit anti-prostate-specific antigen (y-seminoprotein) IgG was obtained from DAKO-immunoglobulins a/s, Copenhagen, Denmark. F(ab’)z was prepared by digestion of IgG with pepsin, and Fab ’ was prepared by reduction of F(ab ‘)z with 2-mercaptoethylamine [17]. The amount of IgG and its fragments was calculated from the absorbance at 280 nm [17]. Biotinyl nonspecifti goat IgG-y-seminoprotein conjugate Puriftiation of y-senvinoprotein. r-Seminoprotein was purified from pooled

seminal plasma by column chromatography using CM-sepharose CL-6B (Pharmacia Fine Chemicals AB, Uppsala, Sweden), DEAE(diethylaminoethyl)sepharose fast flow (Pharmacia Fine Chemicals AB), Con A sepharose (Pharmacia Fine Chemicals AB) and Ultrogel AcA 54 (IBF biotechnics, Villeneuve-laGarenne, France) according to the method of Lilja [18]. The amount of yseminoprotein was measured using a commercial protein assay kit (Bio-Rad Laboratories, Richmond, CA, USA) based on the method of Bradford [19]. Bovine serum albumin was used as standard. Mercaptoswcinylated r-sem.hwprotein. y-Seminoprotein was mercaptosuccinylated using S-acetylmercaptosuccinic anhydride (Nacalai Tesque, Inc.,

163

Kyoto, Japan) [17]. The average number of thiol groups introduced per yseminoprotein molecule was 2.3 [17]. 6-MaleimidokanoyLrwnspe&fk goat IgG. Nonspecific goat IgG was prepared from nonspecific goat serum by fractionation with NacS04 followed by passage through a column of DEAE-cellulose [17]. Maleimide groups were introduced into nonspecific goat IgG using N-succinimidyl-6-maleimidohexanoate (Dojindo Laboratories, Kumamoto, Japan) [17,20]. The average number of maleimide groups introduced per IgG molecule was 19 [17]. N-Biotinyl-2mercaptoethy2amine. An aliquot (0.2 ml) of 44 mmol/l biotin-Nhydroxysuccinimide (Zymed Laboratories, Inc., San Francisco, CA, USA) was incubated with 2.0 ml of 4.4 mmol/l2-mercaptoethylamine (Nacalai Tesque, Inc.) in 0.1 mol/l sodium phosphate buffer, pH 7.0, containing 5 mmolfl EDTA at 30°C for 30 min. After incubation, 0.1 ml of 1 mol/l Tris-HCl buffer, pH 7.0, was added to the reaction mixture to eliminate remaining biotin-N-hydroxysuccinimide [21]. 6-Makimidohexanoyl-biotinyl-nonspecific goat IgG. 6-Maleimidohexanoyl-nonspecific goat IgG (13.6 mg) in 3.6 ml of 0.1 mol/l sodium phosphate buffer, pH 6.0, was incubated with an aliquot (0.24 ml) of the N-biotinyl-2-mercaptoethylamine solution at 30°C for 30 min and the mixture was subjected to gel filtration on a column (1.0 x 30 cm) of sephadex G-25 (Pharmacia Fine Chemicals AB) using the same buffer [21]. The average number of biotin residues introduced per IgG molecule was 8.0, which was calculated from the decrease in the number of maleimide groups [17]. Biotinyl wnspecifti goat IgG-y-semhoprotein con&gate. 6-Maleimidohexanoyl-biotinyl-nonspecific goat IgG (0.60 mg, 4 nmol) in 0.29 ml of 0.1 mol/l sodium phosphate buffer, pH 6.0, was incubated with mercaptosuccinylated y-seminoprotein (0.59 mg, 20 nmol) in 0.91 ml of the same buffer containing 5 mmol/l EDTA at 4°C for 16 h. After incubation, the reaction mixture was incubated with 120 ~1of 0.1 mol/l2-mercaptoethylamine in 0.1 mol/l sodium phosphate buffer, pH 7.0, containing 5 mmol/l EDTA at 30°C for 15 min and subjected to gel filtration on a column (1.5 x 45 cm) of Ultrogel AcA 22 (IBF biotechnics) using 0.1 mol/l sodium phosphate buffer, pH 7.0. The average number of y-seminoprotein molecules conjugated per IgG molecule was 1.3, which was calculated from the decrease in the number of maleimide groups [17]. Proteiwsepharose 4B

Biotinyl nonspecific goat IgGy-seminoprotein conjugate (0.13 mg) and strept&din (3.0mg, Bethesda Research Laboratories, Life Technologies, Inc., MD, USA) we:re coupled to 13 mg and 1 g, respectively, of CNBr-activated sepharose 4B (Pharmacia Fine Chemicals AB) according to the instructions of the manufacturer. The amount of biotinyl nonspecific goat IgG-y-seminoprotein conjugate was measured using the commercial protein assay kit (Bio-Rad Laboratories) based on the method of Bradford [19]. Bovine serum albumin was used as standard. The amount of streptavidin was calculated from the absorbance at 282 nm [221.

164

Rabbit anti-y-seminoprotein Fob ’-per&e

conhate An&y-seminoprotein Fab’ was conjugated to horseradish peroxidase (EC 1.11.1.7) (grade I, lyophilized RZ = 3.0, Boehringer Mannheim GmbH, Mannheim, Germany) using N-succinimidyl-6-maleimidohexanoate [20]. The amount of the conjugate was calculated from peroxidase activity [17].

Affinity-purification of Fab ‘-peroxidase coqiqate

Anti-y-seminoprotein Fab’-peroxidase conjugate (3.4 mg) in 0.21 ml of buffer A containing 0.1 mol/l NaCl was applied to a column (3 x 3 mm) of biotinyl nonspecific goat IgG-y-seminoprotein-sepharose 4B using the same buffer at a flow rate of 0.6 ml/h. After washing the column with the same buffer, the conjugate bound to the column was eluted with 0.5 ml of 3.2 mmol/l HCl, pH 2.5, at a flow rate of 6.0 ml/h and the eluates (0.5 ml) were immediately neutralized by addition of 55 ~1 of 1 mol/l sodium phosphate buffer, pH 7.0, containing 10 g/l bovine serum albumin. The amount of affinity-purified conjugate was 154 j.4g. The affinity-purified conjugate (145 pg) was passed through a column (3 x 12 mm) of streptavidin-sepharose 4B using buffer A containing 0.1 molil NaCl at a flow rate of 0.3 ml/h and subsequently subjected to gel filtration on a column (1.5 x 100 cm) of Ultrogel AcA 44 (IBF Biotechnics) using buffer B containing 0.1 g/l bovine serum albumin [23,24]. Anti-y-seminoprotein IgG-coated polystyrene balls

Polystyrene balls (3.2 mm in diameter, Immuno Chemical Inc., Okayama, Japan) were coated with rabbit anti?-seminoprotein IgG (0.1 g/l) by physical adsorption [25]. Blood samples

Blood was collected into syringes with heparin from ten healthy males aged 23-34 years and ten healthy females aged 22-30 years. Bloodstain on filter paper

Blood (30 ~1) placed on filter paper (phenylketonuria-filter paper for massscreening, Toyo Roshi Co. Ltd., Tokyo, Japan) was maintained at room temperature for 7 days and stored at 4°C until use.

filter paper discs with bloodstain Filter paper discs with bloodstain of 3 mm in diameter were punched out. The volume of blood per disc was estimated to be 2.7 ~1[26]. Further discs (n = 2 - 8) with bloodstain were immersed in 0.31 ml of buffer A containing 0.5 mol/l NaCl and 1 g/l NaN3 and allowed to stand at 20°C for 3 h.

Extraction of y-seminoprotein jknn

Sandwich enzyme immunoassay fory-semiruqwokin An anti-y-seminoprotein IgG-coated polystyrene ball was incubated with yseminoprotein standards, blood or bloodstain extracts in a total volume of 0.15 ml at 20°C for 3 h with continuous shaking and at 4°C for 16 h without shaking.

165

y8eminoprotein standards were diluted with buffer A containing 0.5 mol/l NaCl and 1 g/l NaNa to a final volume of 0.15 ml. Blood (1 - 10 ~1)was mixed with deionized water to a total volume of 75 ~1, followed by addition of 75 ~1 of 20 mmol/l sodium phosphate buffer, pH 7.0, containing 2 g/l bovine serum albumin, 1 mol/l NaCl and 2 g/l NaNa. The volume of bloodstain extracts used was 0.15 ml corresponding to 1- 4 ~1 of blood. After removal of the incubation mixture, the polystyrene ball was washed twice by addition and aspiration of 2 ml of buffer B and :incubated with 10 ng of affinity-purified anti?-seminoprotein Fab’peroxidase conjugate in 0.15 ml of buffer A containing 0.1 mol/l NaCl at 20°C for 3 h. The polystyrene ball was washed twice as described above and transferred to another clean test tube. Bound peroxidase activity was assayed at 30°C for 90 min using 3-(4-hydroxyphenyl)propionic acid as hydrogen donor [27]. Fluorescence intensity was measured relative to 0.2 mg/l quinine in 50 mmovl H&SO4with a spectrofluorophotometer (RF-510, Shimadzu Seisakusho, Ltd., Kyoto, Japan). In experiments for measuring y-seminoprotein in blood and bloodstain extracts, antir-seminoprotein IgC-coated polystyrene balls were incubated with blood or bloodstain extracts and processed as described above with and without addition of the enzyme conjugate. Bound peroxidase activity in the absence of the enzyme conjugate was subtracted from that in its presence to correct false values due to nonspecific binding of hemoglobin. Expression of the detection limit of r-ssmiwprottk The detection limit of y-seminoprotein by sandwich enzyme immunoassay was

taken as the minimal amount of y-seminoprotein that gave a bound peroxidase activity significantly in excess of that nonspecifically bound in the absence of yseminoprolein (background). The existence of a significant difference from the background was confirmed by the t-test (P < 0.001, n = 5). Gel filtration

of seminal plasma

Pooled seminal plasma (0.1 ml) was mixed with 0.4 ml of buffer B containing 0.1 g/l bovine serum albumin and 1 g/l NaNa and subjected to gel filtration on a column (1.O x 60 cm) of Ultrogel AcA 54 (IBF biotechnics) using the same buffer at a flow rate of 17 ml/h. The fraction volume was 0.67 ml. Fractions were diluted 30 OOO-foldwith buffer A containing 0.5 mol/l NaCl and 1 g/l NaN3 to a final volume of 0.15 ml and subjected to sandwich enzyme immunoassay. Sandwich trnxyme immunoassay

for hemoglobin A Hemoglobin A was measured by sandwich enzyme immunoassay using rabbit anti-hemoglobin A &C-coated polystyrene balls and affinity-purified rabbit antihemoglobin A Fab’-peroxidase conjugate, which had been absorbed with Japanese monkey Hb-sepharose 4B and dog Hb-sepharose 4B [28]. Results and

DiSCU88iOn

A polystyrene ball coated with rabbit anti-y-seminoprotein IgC was incubated

166

with y-seminoprotein and, after washing, with rabbit anti-y-seminoprotein Fab’horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as hydrogen donor. Detection limit of y-semirwprotein

The detection limit of y-seminoprotein was 0.90 pg per assay, when anti-yseminoprotein Fab ’peroxidase conjugate was used without affinity-purification. In order to improve the detection limit, the conjugate was affinity-purified by elution at pH 2.5 from a column of y-seminoprotein-biotinyl-nonspecific goat IgG-sepharose 4B. The use of the conjugate in the eluates improved the detection limit only 2-fold, since the nonspecific binding of the conjugate to anti-yseminoprotein &G-coated polystyrene balls was increased 2.5-fold by affinitypurification. This might have been due to the presence in the eluates of yseminoprotein-biotinyl-nonspecific goat IgG conjugate released from the column and probably complexed with ant+-seminoprotein Fab ’-peroxidase conjugate. For elimination of the complex, the affinity-purified conjugate was passed through a column of streptavidin-sepharose 4B and subjected to gel filtration on a column of Ultrogel AcA 44. As a result, the nonspecific binding of the affinitypurified conjugate was lowered 1.3-fold by streptavidin-sepharose 4B and further 2.3-fold by Ultrogel AcA 44. The final detection limit of y-seminoprotein was 0.15 pg (5.0 amol) per assay (Fig. l), which was at least 66-fold lower than that achieved by methods previously reported for forensic purposes [12 - 161. Specificity of the sandwich enzym-eimmunoassay

Pooled seminal plasma was fractionated by gel filtration on a column of Ultrogel AcA 54 and each fraction was subjected to the sandwich enzyme immunoassay. As shown in Fig. 2, a single and symmetric peak corresponding to a molecular weight of 30 000 was detected. This was consistent with the molecular weight of y-seminoprotein as determined by the amino acid and carbohydrate compositions (29 000) [6,29] and by gel filtration on a column of sephacryl S-200 (30 000) [7]. In order to further test the specificity, human renal/pancreatic kallikrein, to which y-seminoprotein shows 61% homology in amino acid sequences [30], was subjected to the sandwich enzyme immunoassay. With up to 3.0 ng per assay, no significant increase in bound peroxidase activity was observed. This indicated that the maximal blood concentration of human renal/pancreatic kallikrein not to interfere with the sandwich enzyme immunoassay for y-seminoprotein using up to 10 ~1of blood was 300 pg/l, which is at least 25-fold higher than its concentrations in healthy subjects (3.8 i 0.7 (SD) rg/l; range, 0.6-11.8 pg/l) [31]. Measurement of r-semkprotin

in blood

Hemoglobin in blood samples was nonspecifically adsorbed to anti-yseminoprotein IgG-coated polystyrene balls and was detected as peroxidase activity. When 1 ~1 and 10 ~1 of blood were subjected to the sandwich enzyme immunoassay, peroxidase activities due to nonspecifically adsorbed hemoglobin

16’7

y_Seminoprotein

Fig. 1. Dose-response

( pg per assay )

curve of r-seminoprotein

by sandwich enzyme immunoassay.

.+

2

3000 -

$ m E :: aG

2000

2

I! a m 5 9 s 5

12 Ii

‘Ooo

010

20

30 Elution Volume

40

50

( ml )

Fig. 2. y-Seminoprotein detected by sandwich enzyme immunoassay in pooled seminal plasma fractionated by gel f&ration on a column (1.0 x 60 cm) of Ultrogel AcA 54. Arrows 1- 4 indicate the elution vohunes of bovine serum albumin (MW: 66 200), horseradish peroxidase (Mw: 40 000), myoglobm (MW 17 800) and cytochrome C (MW 12 400), respectively.

168

were equivalent to y-seminoprotein levels of 0.00-0.18 cLgll (O.OO-0.18pg per assay) and 0.01-0.063 pg/l (O.lO-0.63 pg per assay), respectively. For correcting these false values, anti?-seminoprotein &G-coated polystyrene balls were processed with blood samples or bloodstain extracts in the same way as in the sandwich enzyme immunoassay, except that anti-y-seminoprotein Fab’-peroxidase conjugate was deleted, and bound peroxidase activity in the absence of the conjugate was subtracted from that obtained in the presence of the conjugate. Blood level of y-seminopotein

Blood levels of r-seminoprotein in ten healthy male subjects aged 23 - 34 years were 1.3 * 0.42 (SD) clgn (range, 0.50 - 1.8 rg/l), when 10 ~1of blood was used. The use of smaller volumes of blood (1 - 5 ~1)gave slightly (3 - 49%) higher values (1.5 f 0.47 (SD) pg/l; range, 0.54-2.4 clg/l) (Fig. 3). By contrast, blood levels of y-seminoprotein in ten healthy female subjects aged 22 - 30 years were 0.1 pgil or lower, when 5 - 10 ~1of blood was used. (The nature of the substance detected in females is unknown.) With smaller volumes

0 4

0.01

1



1



2



3

I 5

Volume of Blood Used per Assay

IO

( pl )

1

2

3

5

10

Volume of Blood Used per Assay ( pl )

Fig. 3. Level of r-seminoprotein in blood of ten healthy male subjects aged 23 - 34 years (0) and ten healthy female subjects aged 22 - 30 years (0). The detection limit of y-seminoprotein is indicated by a broken line. No y-seminoprotein was detected in some females using 1- 5 ~1 of blood and in one female even using 10 ~1 of blood.

Fig. 4. Ratio of y-seminoprotein in terms of pg to hemoglobin in terms of mg in blood for the healthy male (0) and female (0) subjects as shown in Fig. 3.

169

of blood (1 - 3 pl), much higher (27 - 136%) values were observed, probably due to the fact that the amounts of y-seminoprotein measured were close to the detection limit (Fig. 3). However, these values were still at least 3.3-fold lower than thos#ein the male subjects. Ratio of y-semirwprotein

to hemoglobin in blood

The ratio of the amount of y-seminoprotein in terms of pg to the amount of hemoglobin in terms of mg was calculated for the same healthy subjects as described. above (Fig. 4). When the blood volumes used were 3 - 10 ~1,the ratios in the males and females were 8.8 * 3.1 (SD) (range, 2.8 - 14) and less than 0.80, respectively. When the blood volumes used were 1 - 2 ~1,the ratios in the males and females were 9.5 f 3.3 (SD) (range, 3.7- 15) and less than 1.1, respectively. Ratio of y-seminoprotein

to hemoglobin in bloodstains

Blood e#amplesof the same healthy subjects as described above were dried on filter papers and, 1 week after the preparation, 3-mm discs were punched out. Two, four and eight of the discs, corresponding to 5.4, 10.8 and 21.6 ~1of blood, respectively, were immersed in 0.81 ml of a buffer for extraction and an aliquot (0.15 ml) of the extracts corresponding to 1, 2 or 4.~1 of blood was subjected to the sandwich enzyme immunoassays for y-seminoprotein and hemoglobin. The recovery of y-seminoprotein from filter paper discs with bloodstain of male subjects was, 69% (range, 49 - 90%).

I

9

10 11

12

13

14

Aw

15

16 17

18 19

20

Adults

o-4

Fig. 5. Level of y-seminoprotein in serum of healthy male subjects aged 9 - 35 years (0) and healthy female subjects aged 22 - 31 years (0). Volumes of serum used per assay were 50 ~1 for female subjects, male subjects aged 9 - 10 years and some male subjects aged 11 - 12 years and 10 ~1 for the other subjects.

1

Female 4 6 6 4

21.6

8

0.30 f 0.052 < 0.20 0.28 * 0.081 < 0.098

< 0.40

10

5.4 10.8

2

4

2.7 f 0.95 2.8 zt 0.96 2.6 * 0.68

10 10 10

5.4 (1.5-4.6)

(0.14-0.38)

(0.25 - 0.37)

(1.2-4.8) (1.2-3.8)

Cl.7 1.0 ?? 0.20 co.78 1.1 * 0.37 co.43

8.1 zt 3.0 7.3 f 2.0

3.0 f 3.0

hfean f SD

Mean * SD

(0.50- 1.6)

(0.81- 1.3)

(3.5- 14) (3% 11)

(4.1- 14)

(Range)

to htmoglobin

o!&ected(pgfdisc) @awe)

Ratio of yseminoprotein

Amount of y-semiwprotein

10.8 21.6

of samples

correspanding to the number of discs used (4

2

Male

Number

Blood vo1um.e

4 8

Number of discs with bloooktuin for extraction

sex

RATIO OF y-SEMINOPROTEIN IN TERMS OF pg TO HEMOGLOBIN IN TERMS OF mg

TABLE

171

In the males and females, the ratios of y-seminoprotein in terms of pg to hemoglobin in terms of mg were 7.8 * 2.6 (SD) (range, 3.5 - 14) and less than 1.7, respectively (Table 1). The ratios in the males were at least 2-fold higher than those in the females. Discrimiwtion

of blood and bloodstains from mules and females

From the above results, the measurement of r-seminoprotein discriminated blood of male adults from that of female adults, and the ratio of y-seminoprotein to hemoglobin was useful for sex discrimination of bloodstains with no information on the blood volume. However, blood of male children may not be discriminated from that of female subjects. In order to clarify this point, y-seminoprotein in the serum of male subjects aged 9 - 20 years was measured (Fig. 5). The results indicated that blood of male subjects under 10 years of age and some male subjects aged 11 - 14 years could not, be discriminated from that of females. In addition, the presence of semen, prostatic tissues and/or severe anemia in materials’ to be examined may give misleading results. Interference by severe anemia may be overcome by measuring other proteins (e.g. serum albumin) as well as hemoglobin. Referenc:es 1

2

3 4

5 6

7 8 9

10

11

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A sensitive sandwich enzyme immunoassay for gamma-seminoprotein and its application to sex discrimination of blood and bloodstains.

A sensitive sandwich enzyme immunoassay for gamma-seminoprotein (p30, prostate-specific antigen) is described for sex discrimination of blood and bloo...
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