Seminal Fluid PDF - Forensic Biology

14 Identification of Semen 14.1 Biological Characteristics A typical ejaculation releases 2–5 mL of semen, which contains seminal fluid and sperm cells (spermatozoa). A normal sperm count ranges from 107 to 108 spermatozoa per milliliter of semen. The spermatozo...

14 Identification of Semen 14.1 Biological Characteristics A typical ejaculation releases 2–5 mL of semen, which contains seminal fluid and sperm cells (spermatozoa). A normal sperm count ranges from 107 to 108 spermatozoa per milliliter of semen. The spermatozoa are formed from spermatogonia in the seminiferous tubules of the tes- tes. This process of generating spermatozoa is referred to as spermatogenesis (Figure 14.1). The spermatozoa are then transported and stored in the tubular network of the epididymis where they undergo functional maturation (spermatogenesis and maturation take approximately 3 months). The epididymis joins the ductus deferens, which transports matured sperm from the epididymis to the ejaculatory duct. From there, spermatozoa follow the ejaculatory ducts into the prostatic urethra where they are joined with secretions from the prostate. Figure 14.2 illustrates the anatomy of the male reproductive system. Seminal fluid is a complex mixture of glandular secretions. A typical sample of seminal fluid contains the combined secretions of several accessory glands. Seminal vesicle fluid accounts for approximately 60% of the ejaculate. Various proteins secreted by the seminal vesicles play a role in the coagulation of the ejaculate. Additionally, seminal vesicle fluid contains flavin, which causes semen to fluoresce under ultraviolet light, often utilized when searching for semen-stain evidence. Prostatic fluid secretions account for approximately 30% of the ejaculate. The components of this fluid are complex as well. This portion of semen contains high concentrations of acid phos- phatase (AP) and prostate-specific antigen (PSA). Both are useful markers for the identification of semen in forensic laboratories. The epididymis and the bulbourethral secretions each account for approximately 5% of the ejaculate. A vasectomy is the surgical removal of a bilateral segment of the ductus deferens. The sur- gery prevents spermatozoa from reaching the distal portions of the male reproductive tract. However, a vasectomized male can still produce ejaculate that contains only seminal vesicle fluid, prostatic fluid, and bulbourethral fluid. The condition by which males have abnormally low sperm counts is known as oligospermia. Azoospermia is a condition that causes males to produce no spermatozoa. However, the secretion of seminal fluid is not affected in males who have these conditions. DNA derived from epithelial cells can be isolated from the seminal fluids of these individuals. 14.1.1 Spermatozoa A human spermatozoon has three morphologically distinct structures: the head, the middle piece, and the tail (Figure 14.3). The head contains a nucleus with densely packed chromo- somes. At the tip of the head is the acrosomal cap, which is a membranous compartment 257 Forensic Biology, Second Edition Figure 14.1 Spermatogenesis. In the seminiferous tubules (left) of the testes, spermatogonia (located at peripheral area of the seminiferous tubules) are differentiated to spermatids (located at the center of the seminiferous tubules). The spermatids are eventually differentiated to matured spermatozoa (right). (© Richard C. Li.) Bladder Ejaculatory duct Prostatic Seminal vesicle gland Prostatic Ductus urethra Bulbourethral deferens gland Vasectomy Epididymis Seminiferous tubule External Testis urethral orifice Figure 14.2 Male reproductive system and accessory glands (unilateral view). (© Richard C. Li.) containing enzymes essential for fertilization. The head is attached to the middle piece through a short neck where the mitochondria that provide the energy for moving the tail are located. The tail or flagellum is responsible for spermatozoon motility. In contrast to other cell types, a mature spermatozoon lacks various intracellular organelles such as an endo- plasmic reticulum, Golgi apparatus, lysosomes, and peroxisomes. In a normal male, at least 60% of spermatozoa have normal morphology, so morphological abnormalities can often be observed. 258  14.1 Biological Characteristics Acrosome Nucleus Mitochondria Head Midpiece Tail Neck Figure 14.3 Structure of spermatozoon. (© Richard C. Li.) 14.1.2 Acid Phosphatase Acid phosphatase (AP) consists of a group of phosphatases with optimal activity in an acidic pH environment. The greatest forensic importance of AP is that the prostate-derived AP contributes most of the AP activity present in semen. AP levels in semen are not affected by vasectomies. AP isoenzymes are also found in other tissues (Section 16.2). The half-life of AP activity at 37°C is 6 months. However, the half-life is decreased if a sample is stored in a wet environment. AP activity can be detected from dry seminal stains stored at –20°C up to 1 year. Low levels of prostatic AP are present in the sera of healthy males. Elevated levels of prostatic AP found in serum are useful in diagnosing and monitoring prostate car- cinoma. Many AP tests utilized in clinical testing may be used to identify semen for forensic applications. 14.1.3 Prostate-Specific Antigen Prostate-specific antigen (PSA) is a major protein present in seminal fluid at concentrations of 0.5–2.0 mg/mL. PSA is produced in the prostate epithelium and secreted into the semen. PSA can also be found in the paraurethral glands, perianal glands, apocrine sweat glands, and mam- mary glands. Thus small quantities can be detected in urine, fecal material, sweat, and milk. PSA can also be found at much lower levels in the bloodstream. An elevated plasma PSA is present in prostate cancer patients, and it is widely used as a screening test for this disease. PSA is also elevated in cases of benign prostatic hyperplasia and prostatitis. The synthesis of PSA is stimulated by androgen, a steroid hormone. PSA is a protein that has a molecular weight of 30 kDa and is thus also known as P30. It is responsible for hydrolyzing semenogelin (Sg), which mediates gel formation in semen (Section 14.1.4). PSA is a member of the tissue kallikrein (serine protease) family and is encoded by the KLK3 locus located on chromosome 19. In addition to PSA, other tissue kallikreins encoded by KLK2 and KLK4 loci are expressed in the prostate. The half-life for PSA in a dried semen stain is about 3 years at room temperature. The half-life is greatly reduced when a sample is stored in wet conditions. 14.1.4 Seminal Vesicle–Specific Antigen Human seminal vesicle–specific antigen (SVSA) includes two major types, semenogelin I (SgI) and semenogelin II (SgII), and constitutes the major seminal vesicle–secreted protein in semen. On ejaculation, SVSA forms a coagulum that is liquefied after a few minutes due to the degradation of SVSA by PSA. In humans, both SgI and SgII are present in a number 259 Forensic Biology, Second Edition of tissues of the male reproductive system, including the seminal vesicles, ductus deferens, prostate, and epididymis. They are also present in several other tissues such as skeletal muscle, kidney, colon, and trachea. They have also been found in the sera of lung cancer patients. The use of Sg as a marker for semen identification instead of PSA presents certain advantages. The concentration of Sg in seminal fluid is much higher than that of PSA, and this is beneficial for the sensitivity of detection. Sg is present in seminal fluid and absent in urine, milk, and sweat, where PSA can be found. Although Sg compounds are present in skeletal muscle, kidney, and colon, this is not a great concern because these tissue samples are not routinely collected for semen detection in sexual assault cases. 14.2 Analytical Techniques for Identifying Semen The location of semen stains is usually carried out through visual examination. Particularly, the application of alternate light sources (ALSs) can facilitate searches for semen stains. The presumptive identification of semen is largely based on the detection of the presence of prostatic AP activity in a sample. However, most presumptive assays cannot completely distinguish pros- tatic AP from nonprostatic AP. Confirmatory assays for the identification of semen are available, including the microscopic examination of spermatozoa, the identification of PSA and SVSA, and the RNA-based assay. 14.2.1 Presumptive Assays 14.2.1.1 Lighting Techniques for Visual Examination of Semen Stains Lighting techniques can be used to aid in searching for semen stains. A dried semen stain fluo- resces under certain light sources such as ALSs or argon lasers. ALSs are most commonly uti- lized for the visual examination of semen stains (Chapter 1; Figures 14.4 and 14.5). Excitation wavelengths between 450 and 495 nm can be used, allowing for the visualization of fluorescence with orange goggles. However, this approach is not specific for semen. Other bodily fluid stains, such as saliva and urine stains, can also fluoresce with less intensity. Additionally, the intensity of the fluorescence can be affected by different colors of substrates, and the material, such as clothing, where semen stains have been deposited. Figure 14.4 A tabletop ALS device (left) for the detection of semen stains (right). (© Richard C. Li.) 260  14.2 Analytical Techniques for Identifying Semen (a) (b) Figure 14.5 Examining a garment for semen stains. (a) A potential semen stain (labeled) is found on a garment, and (b) the stain fluoresces when irradiated with an ALS device. (© Richard C. Li.) 14.2.1.2 Acid Phosphatase Techniques 14.2.1.2.1 Colorimetric Assays Colorimetric assays can be used for the presumptive identification of semen. The AP contained in semen can hydrolyze a variety of phosphate esters. It catalyzes the removal of the phosphate group from a substrate (Figure 14.6). Subsequently, an insoluble colored precipitate at sites of acid phosphatase activity is formed with a stabilized diazonium salt (usually in the form of zinc double salts). O O Acid P R phosphatase P OH HO O + R OH HO O Phosphate monoester Phosphate Alcohol Figure 14.6 Reaction catalyzed by acid phosphatases (EC 3.1.3.2). The optimal pH of the reaction is usually under pH 7. 261 Forensic Biology, Second Edition However, interference during a test by nonprostatic AP isoenzymes (multiple forms of AP), such as contamination by AP commonly present in vaginal secretions (Chapter 16), can create problems in specimens collected from victims. Thus, it is desirable to be able to increase the specificity of the assay for prostatic AP. One solution is the application of substrates that are hydrolyzed rapidly by the prostatic enzyme and at a slower rate by the other forms of AP iso- enzymes. For example, α-naphthylphosphate and thymolphthalein monophosphate are more specific to prostatic AP than phenyl phosphate and 4-nitrophenyl phosphate (Figure 14.7). The most common method for forensic applications is the use of α-naphthyl phosphate as a sub- strate. In the presence of AP, α-naphthylphosphate is hydrolyzed to phosphate and α-naphthol. Subsequently, the Fast Blue B, a stabilized diazonium salt, is added to carry out an azo coupling reaction, producing a purple azo dye (Figures 14.8 through 14.10). Prostatic AP is water soluble. Thus, a moistened cotton swab or piece of filter paper can be used to transfer a small amount of sample from a stain by briefly pressing onto the questioned stain area. The α-naphthylphosphate reagent is added to the swab or filter paper followed by the addition of Fast Blue B reagent. If a purple coloration develops within 1 min, the test is consid- ered a positive indication for semen. Color that develops after more than 1 min may arise from the activity of nonprostatic AP. Additionally, the prostatic enzyme is strongly inhibited by dextrorotatory tartrate ions. Thus, these inhibitors, particularly tartrate, allow a distinction to be made between prostatic AP and other AP isoenzymes. Prostate and vaginal acid phosphatase can also be distinguished by using gel electrophoresis (Chapter 16). O O H 3C O O HO P OH O P OH O H3C OH CH(CH3)2 CH(CH3)2 (a) (b) OH O O O P OH NO2 O P OH (c) OH (d) OH O HO O O O P OH (e) CH3 Figure 14.7 Chemical structures of acid phosphatase substrates. (a) α-Naphthyl phosphate, (b) thymolphthalein monophosphate, (c) phenyl phosphate, (d) 4-nitrophenyl phosphate, and (e) MUP. 262  14.2 Analytical Techniques for Identifying Semen O HO P OH O α-Naphthyl phosphate AP OH α-Naphthol – 2Cl H3CO + + N N N N OCH3 Fast Blue B salt OH H3CO OH N N N N OCH3 Azo dye Figure 14.8 A colorimetric acid-phosphatase assay. In this assay, α-naphthylphosphate is hydrolyzed by acid phosphatase to phosphate and α-naphthol. The α-naphthol is subsequently converted into a purple azo dye with a diazonium salt such as Fast Blue B salt. AP, acid phosphatase. (a) (b) (c) Figure 14.9 AP colorimetric assay. (a) A small amount of sample from the stain is transferred using a moistened cotton swab. (b) The substrate reagent is applied, followed by adding Fast Blue B reagent. (c) Purple coloration indicates a positive reaction. (© Richard C. Li.) 263 Forensic Biology, Second Edition Figure 14.10 Photo of a colorimetric acid-phosphatase assay using α-naphthylphosphate as a substrate. (© Richard C. Li.) HO HO O O HO O O O OH HO HO P AP P + O O CH3 CH3 MUP MU Figure 14.11 The principle of MUP assay for detecting acid phosphatase activity. In the presence of acid phosphatase, 4-methylumbelliferone phosphate (MUP) is hydrolyzed, forming phosphate and 4-methylumbelliferone (MU), which fluoresces. AP, acid phosphatase. 14.2.1.2.2 Fluorometric Assays Fluorometric methods are more sensitive than the colorimetric detection of AP and are used for semen stain mapping. AP catalyzes the removal of the phosphate residue on a 4-methylumbel- liferone phosphate (MUP) substrate (Figure 14.11), a reaction that generates fluorescence under ultraviolet light. A piece of moistened filter paper, marked for proper orientation and identifica- tion, is used for transferring the prostatic AP. The evidence to be tested, a garment for example, is covered by the filter paper. Gloved hands are used to press the filter paper onto the stained area, ensuring that the evidence is in close contact with the paper. The filter paper is lifted from the evidence and examined in a dark room using long-wave ultraviolet light to detect any back- ground fluorescence, which is then marked on the paper. The paper can then be sprayed with MUP reagent in a fume hood. The AP reaction on the paper can be visualized immediately. Areas where semen is present can be visualized as fluorescent areas on the filter paper (Figure 14.12). 14.2.2 Confirmatory Assays 14.2.2.1 Microscopic Examination of Spermatozoa The cells from a questioned stain on an absorbent material can be transferred to a microscope slide by extracting a small portion of a stain with water, followed by gentle vortexing. The sus- pension is then transferred to a slide and evaporated at room temperature or fixed with low heat. Alternatively, it can be transferred by dampening the stain with water and rubbing or rolling it onto a microscope slide. 264  14.2 Analytical Techniques for Identifying Semen (a) (b) (c) Background fluorescence Semen stains (d) (e) (f ) Figure 14.12 Fluorometric assay of acid phosphatase for locating semen stains. Evidence item (a) is closely covered by a piece of moistened filter paper (b) to allow transfer of a small amount of stain. The orientation of the paper is marked (c). The paper is lifted. The background fluorescence is marked under UV light (d). The filter paper is treated with MUP (e). The presence of fluorescence under ultraviolet light indicates semen stains (f). (© Richard C. Li.) Microscopic identification of spermatozoa provides the proof of a seminal stain. Histological staining can facilitate microscopic examination. The most common staining technique is the Christmas tree stain (Figure 14.13). The red component known as Nuclear Fast Red (NFR) is a dye used for staining the nuclei of spermatozoa in the presence of aluminum ions. The green component, picroindigocarmine (PIC), stains the neck and tail portions of the sperm. The acro- somal cap and the nucleus stain pink-red, and the sperm tails and the midpiece stain blue-green. Epithelial cells, if present in the sample, appear blue-green and have red nuclei. Additionally, fluorescent detection utilizing SPERM HY-LITER Fluorescent Staining Kit can facilitate the identification of spermatozoa. Laser capture microdissection (LCM) has been shown to be an effective technique for sepa- rating spermatozoa from nonsperm cells (i.e., epithelial cells from the victim) on a glass slide (Figure 14.14). This technique involves using a thin layer of a thermosensitive polymer that is placed on the surface of an LCM cap. Once spermatozoa are identified on the slide under a microscope, a polymer-containing LCM cap is placed over the spermatozoa on the slide. An infrared laser melts the polymer and causes it to adhere only to the targeted spermatozoa. The spermatozoa are then lifted off the slide. This allows spermatozoa to be separated and placed into snap-cap tubes for forensic DNA analysis. 14.2.2.2 Identification of Prostate-Specific Antigen Over the years, a number of methods have been utilized to detect PSA: immunodiffusion, immunoelectrophoresis, enzyme-linked immunosorbent assay (ELISA), and immunochro- matographic assays. ELISA and immunochromatographic assays have been found to be the most sensitive methods (Chapter 11). 14.2.2.2.1 Immunochromatographic Assays Commercially produced immunochromatographic kits such as the PSA-check-1 (VED-LAB, Alencon), Seratec® PSA Semiquant (Seratec Diagnostica, Göttingen), and One Step ABAcard 265 Forensic Biology, Second Edition (a) (b) Figure 14.13 Human spermatozoa stained with Christmas tree stain. (a) Staining spermatozoa on a microscope slide and (b) stained spermatozoa. (© Richard C. Li.) Figure 14.14 A microscopic device for fluorescent detection utilizing SPERM HY-LITER Fluorescent Staining Kit (left) and a laser-capture microdissection device for separating sperm cells from other types of cells (right). (© Richard C. Li.) PSA® (Abacus Diagnostics, California) are available. These devices utilize antihuman PSA anti- bodies. In the ABAcard PSA® assay, a labeled monoclonal antihuman PSA antibody is con- tained in a sample well, a polyclonal antihuman PSA antibody is immobilized on a test zone of a nitrocellulose membrane, and an antiglobulin that recognizes the antibody is immobilized on a control zone (Figures 14.15 and 14.16). 266  14.2 Analytical Techniques for Identifying Semen -S-S- -S-S- - - -S -S -S -S -S -S -S -S - - Anti-PSA Ab Bond anti-PSA Ab - -S -S -S-S- -S -S - -S-S- - -S -S -S -S - PSA - - -S -S -S-S- -S-S- -S -S -S -S -S -S - - -S-S- - -S -S -S -S - Unbond anti-PSA Ab (a) (b) (c) Test zone (d) Control zone Figure 14.15 Immunochromatographic assays for identification of PSA in semen. (a) In a sample well, PSA in a semen sample is mixed with labeled anti-PSA Ab. (b) The PSA binds to the labeled anti-PSA Ab to form a labeled Ab–PSA complex. (c) At the test zone, the labeled complex binds to an immobilized anti-PSA Ab to form a labeled Ab–PSA–Ab sandwich. (d) At the control zone, the labeled anti-PSA Ab binds to an immobilized antiglobulin and is captured at the control zone. Ab and PSA represent antibody and prostate-specific antigen, respectively. (© Richard C. Li.) Figure 14.16 Semen identification using an immunochromatic device (ABAcard PSA). The negative (left) and positive (right) results are shown. The “C” band indicates that the test is valid. The “T” band indicates the presence of human blood. The sample well is labeled as “S”. (© Richard C. Li.) 267 Forensic Biology, Second Edition The assay is carried out by loading an extracted sample into the sample well. The antigen in the sample binds to the labeled antibody in the sample well to form an antigen–antibody complex. The complex then diffuses across the nitrocellulose membrane. At the test zone, the immobilized antihuman PSA antibody binds with the antigen–antibody complex to form an antibody–antigen–antibody sandwich. The ABAcard PSA® uses a pink dye that allows for the visualization of a positive test with a pink line at the test zone. In the control zone, unbound labeled antihuman PSA antibody binds to the immobilized antiglobulin. This antibody–anti- globulin complex at the control zone also results in a pink line. The test is considered valid only if the line in the control zone is observed. The presence of human PSA results in a pink line at both the test and control zones. The absence of human PSA produces a pink line in the control zone only. A positive result can appear within 1 min; a negative result is read after 10 min. However, the high-dose hook effect, an artifact that may cause false-negative results (Chapter 10), occurs when high quantities of seminal fluid are tested. 14.2.2.2.2 ELISA The ELISA method can be used to detect PSA with anti-PSA antibodies. The most common method used in forensic serology is antibody sandwich ELISA, in which an antibody–antigen– antibody sandwich complex is formed (Figure 14.17). The intensity of the signal can be detected and is proportional to the amount of bound antigen. The amount of PSA can also be quantified by comparing a standard with known concentrations. Although this method is specific and highly sensitive, it is time-consuming. Chapter 11 discusses the principle of ELISA in further detail. Labeled PSA Anti-PSA Ab antiglobulin -S-S- -S - -S (different epitope) -S -S - -S-S- -S-S- - - -S -S -S -S -S -S -S -S - - Anti-PSA Ab - - - - -S -S -S -S -S-S- -S-S- -S-S- -S-S- -S -S -S -S -S -S -S -S -S -S -S -S - - - - (a) (b) (c) (d) Figure 14.17 Use of ELISA for identification of PSA in semen. (a) Sample containing PSA is applied to polystyrene tubes in which anti-PSA Ab is immobilized. (b) The PSA binds to immobilized Ab to form a PSA–Ab complex. (c) A second anti-PSA Ab, specific for a different epitope of PSA, is added to form an Ab–PSA–Ab sandwich. (d) A labeled antiglobulin then binds to the Ab–PSA–Ab sandwich. The bound antiglobulin can be detected by various reporting schemes. Ab and PSA rep- resent antibody- and prostate-specific antigen, respectively. (© Richard C. Li.) 268  14.2 Analytical Techniques for Identifying Semen 14.2.2.3 Identification of Seminal Vesicle–Specific Antigen 14.2.2.3.1 Immunochromatographic Assays Commercially produced immunochromatographic kits include the RSID® -Semen test (Independent Forensics, Hillside, IL) and the Nanotrap Sg. In the RSID® -Semen assay, a labeled monoclonal anti-Sg antibody is contained in a sample well, and a second monoclonal anti-Sg antibody, to a different epitope of Sg, is immobilized on the test zone of the membrane. An anti- globulin that recognizes the antibody is immobilized on a control zone (Figure 14.18). The sample can be prepared by cutting a small portion of a stain or a swab and is extracted for 1–2 h in an extraction buffer (200–300 μL). Approximately 10% of the extract is removed and mixed with the running buffer. The assay is carried out by loading an extracted sample into the sample well. The antigen in the sample binds to the labeled anti-Sg antibody in the sample well to form a labeled antibody–antigen complex that then diffuses across the mem- brane. At the test zone, the solid-phase anti-Sg antibody binds with the labeled complex to form a labeled antibody–antigen–antibody sandwich. The antigen in the sample produces a pink line at the test zone. In the control zone, unbound labeled anti-Sg antibody binds to the solid-phase antiglobulin. This labeled antibody–antiglobulin complex at the control zone also results in a pink line. The presence of Sg generates a pink line at both the test and control zones. The absence of Sg results in a pink line in the control zone only. Results may be read after 10 min. Validation studies have revealed that the sensitivity of the RSID-Semen kit for detecting seminal fluid can be as low as a 5 × 104-fold dilution. Species specificity studies have shown no cross-reactivity with various animal species including ruminants and small mammals. Bodily fluid specificity studies have also shown that the assay is not responsive to human blood, saliva, -S-S- -S-S- - - -S -S -S -S -S -S -S -S - - Anti-Sg Ab Bond anti-Sg Ab - -S -S -S-S- -S -S - -S-S- - -S -S -S -S - Sg - - -S-S- -S-S- -S -S -S -S -S -S -S -S - - -S-S- - -S -S -S -S - Unbond anti-Sg Ab Test zone Control zone (a) (b) (c) (d) Figure 14.18 Immunochromatographic assays for identification of semenogelin (Sg) in semen. (a) In a sample well, Sg in a semen sample is mixed with labeled anti-Sg Ab. (b) Sg binds to the labeled anti-Sg Ab to form a labeled Ab–Sg complex. (c) At the test zone, the labeled complex binds to an immobilized anti-Sg Ab to form a labeled Ab–Sg–Ab sandwich. (d) At the control zone, the labeled anti-Sg Ab binds to an immobilized antiglobulin and is captured at the control zone. Ab represents antibody. (© Richard C. Li.) 269 Forensic Biology, Second Edition urine, sweat, fecal matter, milk, or vaginal secretions. The assay results are not affected by con- dom lubricants or spermicides such as nonoxynol-9 and menfegol. However, the high-dose hook effect occurs when more than 3 μL of seminal fluid is tested. 14.2.2.3.2 ELISA Identification of Sg for semen detection has also been carried out with ELISA. Anti-Sg antibod- ies are utilized. An antibody–antigen–antibody sandwich complex is formed (Figure 14.19). The intensity of the colorimetric or fluorometric signals can be detected spectrophotometrically and is proportional to the amount of bound antigen. The amount of Sg can be quantified by compar- ing a standard with known concentrations. 14.2.2.4 RNA-Based Assays RNA-based assays (Chapter 11) have been developed to identify semen. The assays are based on the expression of certain genes in certain cell or tissue types. Thus, the techniques used in the identification of semen are based on the detection of specific types of mRNA expressed exclusively in spermatozoa and in certain cells of male accessory glands. These assays uti- lize reverse transcriptase polymerase chain reaction (RT-PCR; see Chapter 7) methods to detect gene expression levels of mRNAs for semen identification. Table 14.1 lists the tissue- specific genes utilized for semen identification. Compared to conventional assays used for semen identification, the RNA-based assay has higher specificity and is amenable to automa- tion. However, one limitation is that the RNA is unstable due to degradation by endogenous ribonucleases. Labeled Sg Anti-Sg Ab antiglobulin (different epitope) -S-S- - -S -S -S -S - -S-S- -S-S- - -S -S - -S -S -S -S -S -S - - Anti-Sg Ab - - - - -S-S- -S-S- -S-S- -S-S- -S -S -S -S -S -S -S -S -S -S -S -S -S -S -S -S - - - - (a) (b) (c) (d) Figure 14.19 Use of ELISA for identification of semenogelin (Sg) in semen. (a) Sample containing Sg is applied to polystyrene tubes in which anti-Sg Ab is immobilized. (b) Sg binds to immobilized Ab to form a Sg–Ab complex. (c) A second anti-Sg Ab, specific for a different epitope of Sg, is added to form an Ab–Sg–Ab sandwich. (d) A labeled antiglobulin then binds to the Ab–Sg–Ab sandwich. The bound antiglobulin can be detected by various reporting schemes. Ab represents antibody. (© Richard C. Li.) 270  Bibliography Table 14.1 Application of RT-PCR for Semen Identification Gene Symbol Gene Product Description Further Reading KLK3 Kallikrein 3 Also called prostate- Gelmini et al. (2001) specific antigen (PSA) PRM1 Protamine 1 DNA-binding Steger et al. 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