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InSite® Semen Detection Kit

Abstract

This paper describes the development of the InSite® Test Kit.  This kit is comprised of two main components:  acid phosphatase (AP) test strips and prostate specific antigen (PSA) test strips, which work together to provide evidence of semen on garments and other items.  The included AP strips were found to detect semen down to a 1/2000 dilution, whereas comparative testing with two other acid phosphatase (AP) tests and a zinc test showed that their limit of detection was 1/150-1/300. The PSA strips detected semen to a 1/500,000 dilution, which was approximately the same as semenogelin test strips used in comparative testing.  Semen which was discharged onto undergarments was detectable by the AP tests up to 17 h, by the zinc test up to 17 h, and by the PSA and semenogelin tests up to 36 h after intercourse.   The AP test gave a more dramatic color change with small amounts of semen and therefore was chosen for inclusion in the kit over the zinc test.  The zinc test, on the other hand, was more specific and would be superior when testing directly with a vaginal swab.  In contrast with spermatozoa, which can be found in the vagina more than seven days after intercourse,  the marker proteins PSA and semenogelin became undetectable after 36 hours.  We attribute this difference to the acidic pH of the vagina, among other factors.

Introduction

Conservative statistics indicate that about 14% of women and 22% of men have had affairs sometime in their marriage [Ref. 1].  According to a recent study by the Centers for Disease Control, about 4% of both married men and women had more than one sexual partner in the previous twelve months [Ref. 2].   This figure rises to 15% in the case of unmarried couples cohabiting.  These data indicate that infidelity is a significant problem in the United States, and there exists a need to objectively test spouses for sexual activity.  For women, one such test is for the presence of semen.

When a man has sexual intercourse with a woman, semen is deposited into the woman's vagina.  Immediately after intercourse, most of the semen flows back out, but a some is retained in the vagina and slowly is discharged over a period of several days [Ref. 3].  Semen has over 900 identified proteins [Ref. 4] among which are semenogelin I and II (gel-forming proteins produced by the seminal vesicles), prostate-specific antigen (a protease which breaks down semenogelin), and acid phosphatase (which breaks down spermatozoa cell membranes) [Ref. 5]. These proteins can be identified by immunochromatographic assay, which forms the principle of the PSA test in the InSite kit.  Acid phosphatase can be detected by the classic test first described by Babson [Ref. 6], which forms the principle of the AP test in the InSite kit.  This test relies on the catalytic hydrolysis of 1-naphthyl phosphate to form 1-naphthol, which in turn reacts with an aryl diazonium salt, forming an intensely colored azo dyestuff.  In addition to proteins, semen also has unusually high concentrations of zinc (100-200 mg/L v. 1 mg/L in plasma) [Ref. 7].  Zinc acts to stabilize DNA inside spermatozoa and also may catalyze the gel-forming reaction between semenogelin I and II.  Semen may be detected by the modified zinc test of Hooft and van de Voorde [Ref. 8], which forms the principle of the zinc test developed during this research.

The semen flowing back out of a woman's vagina ("backflow") is deposited on her underwear or absorbent pad.  These items conveniently can be tested with the InSite kit.  The kit also can be used to test stains on other fabrics and surfaces.

There was some question as to how long after intercourse marker proteins like PSA and semenogelin could be detected, because of the acidic pH in the vagina, among other factors.  The predominant microorganism in the vagina is lactobacillus acidophilus, which produces lactic acid and hydrogen peroxide, creating a toxic environment for other bacteria and denaturing the three-dimensional structure of proteins, which structure is critical for their immunochromatographic detection.  The detection limits for these proteins were measured experimentally as described below.

Results and Discussion

Acid phosphatase test strips were prepared according to a modification of the procedure of Babson [Ref. 6].  Zinc test strips were prepared according to the method of Hooft and van de Voorde, using various filter papers as substrate.  PSA, semenogelin and two other AP tests were obtained commercially as described below.  In order to measure the relative sensitivity of these different tests, and their ability to detect semen on undergarments, comparative studies were performed with semen dilutions and with analysis of garments after intercourse.

Semen dilutions

The sensitivity of the zinc strips was tested by analyzing a series of dilute semen samples, and acid phosphatase tests were carried out simultaneously for comparison.  Semen was diluted with deionized water to levels of 1/10, 1/50, 1/100, 1/150, 1/200, 1/300, 1/500, 1/1,000 and 1/2,000 and tested with zinc strips and three acid phosphatase tests (prototype InSite®, CheckMate® and Phosphatesmo KM brands).  The zinc strips proved to be sensitive to a 1/150 dilution, the prototype InSite test had a detection limit of 1/100, the CheckMate® AP test was judged to have a detection limit of 1/150 and the Phosphatesmo KM test a detection limit of 1/300.  The AP tests were read after 15 seconds, but at high dilutions continued to slowly turn purple.  The results are shown in Fig. 1.  The zinc and InSite AP test strips in this experiment were prepared using Whatman Grade 1 filter paper.  The prototype InSite AP test (D) in this experiment turned out to be too weak, and the concentration of reagents in this strip later was increased.

The limit of detection of semen at a 1/150 dilution by the zinc spot test is generally consistent with the report of Hooft and van de Voorde, who reported a detection limit of 1/128 [Ref. 8].

The Phosphatesmo KM strips were clearly superior as a spot test for detecting semen in this experiment because of the dramatic color change, the small amount of enzyme needed for a reaction and the strip's overall design.  It was initially thought that, because of acidic conditions in the vagina, it might turn out that zinc would be the best test beyond a certain time frame, e.g. 12 hours.  This turned out not to be true, as shown below.  The Phosphatesmo KM strips were also somewhat expensive, at $5 each.

 

 

 

Control

 

Control

 

Control

 

Control

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B

C

D

 

 

 

1/10

 

1/10

 

1/10

 

1/10

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B

C

D

 

 

 

1/50

 

1/50

 

1/50

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B

C

 

 

 

1/100

 

1/100

 

1/100

 

1/100

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B

C

D

 

 

 

1/150

 

1/150

 

1/150

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B

C

 

 

 

1/200

 

1/200

 

1/200

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B

C

 

 

 

1/250

 

1/250

 

1/250

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1/300

 

1/300

 

1/300

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C

 

 

 

1/500

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1/1,000

A

 

 

 

1/2,000

 

1/2,000

A

B

 

 

 

0-1/2,000 Series

B

 

Figure 1.  Serial Dilutions of Semen and Analysis with Zinc Strips and Acid Phosphatase Tests. A=Phosphatesmo KM;  B=Zinc strips;  C=CheckMate® acid phosphatase test;  D=Early prototype InSite AP Test
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