An ELISA method for the identification of salivary amylase

An ELISA method for the detection of salivary amylase in dried stains using a monoclonal anti-human salivary amylase antibody was developed. Studies demonstrated the assay to be sensitive down to 0.0002 Sigma units and showed a linear response between absorbance and salivary amylase activity between 0.002 and 0.2 units. The assay showed no cross reactivity with either commercially purchased human pancreatic or bacterial amylase. Sample studies utilizing swabs from several human body fluids showed that 100% of all saliva containing swabs (sixteen of sixteen) and 13% of non-saliva human body fluid swabs (eight of sixty-three) showed a net absorbance with the method. Of these eight non-saliva swabs yielding a net absorbance, none exceeded a salivary amylase activity of 0.003 units. In contrast, only three of the sixteen saliva-containing swabs (swabs produced from saliva diluted 1:5, 1:6, and 1:10, respectively) showed an activity below 0.2 units. Of these swabs, the 1:100 dilution showed the lowest activity (0.048). This value is still more than ten times that of the non-saliva containing swab with the highest activity.     

Amylase levels in semen and saliva stains

Amylase levels were determined for 148 semen samples and 20 saliva samples as well as for their corresponding stains. The effect of aging on the detectability of amylase activity in these stains was also investigated. The Phadebas amylase test was used for the quantitative assay of amylase. High levels of amylase in fluid saliva resulted in high levels being detected in saliva stains. Lower levels present in most seminal fluids produce little or no detectable amounts of amylase in stains. Interpretations are made as to the possible sources of amylase activity found in stains from laboratory casework based on both the amylase concentration and the elapsed time between collection and analysis. The evidential value of the presence or absence of amylase activity in casework stains is also discussed.     

7-[125I]iodoclonazepam: purification by high-performance liquid chromatography for use in a very sensitive benzodiazepine radioimmunoassay of broad specificity

The purification of 7-[125I]iodoclonazepam by high-performance liquid chromatography (HPLC) for use in a very sensitive benzodiazepine radioimmunoassay is described. A silica column is used with a non-aqueous eluent and sequential ultra-violet and gamma-ray detection. A commercially available antiserum is used at a dilution of 1:1000. Blood samples are diluted 10-fold with buffer before analysis and only 25 microliters of diluted sample are required per assay tube. Benzodiazepines, but not the radiolabel, appear to be bound by blood proteins in competition with the antiserum and so, if undiluted blood is assayed, erroneously low results are obtained. The minimal sample requirement and the high sensitivity of the assay described here largely avoid this problem while maintaining acceptable detection limits. For diazepam, the detection limit is 2.5 ng/ml in blood or urine (after correction for the initial 10-fold dilution) and therapeutic or sub-therapeutic levels of many other benzodiazepines can be detected. In practice, the assay is reliable, simple to perform and extremely economical.     

The rapid determination of the ABO group from body fluids (or stains) by dot enzyme-linked immunosorbent assay (dot-ELISA) using enzyme-labeled monoclonal antibodies

Using ABH enzyme-labeled monoclonal antibodies, the authors could rapidly detect the ABO group from body fluids and body fluid stains by the dot enzyme-linked immunosorbent assay (dot-ELISA). In this test, the antigen was immobilized on nitrocellulose paper; the entire piece of paper was coated with an appropriate dilution of enzyme-labeled McAb directly against the antigen of interest; and, finally, 3,3'-diaminobenzidine (DAB) substrate solution was added. The site of a positive reaction is clearly visible as a brown spot. We analyzed 521 samples and got satisfactory results. We also analyzed 99 practical case samples by this method and achieved the same results as those obtained by other researchers using other methods. This method is accurate, simple, direct, rapid, and sensitive; it also produces easily observed results, requires no equipment, and can be completed in 30 min. The test proved to be clearly more sensitive for the detection of the ABO blood group in secretor saliva than the conventional hemagglutination inhibition test. Also saliva diluted 10(-4) to 10(-5) and the ABO group of nonsecretor saliva and urine could be easily detected by this method.     

Comparison of Catalytic and Immunological Amylase Tests for Identifying of Saliva from Degraded Samples

The stability of salivary α‐amylase is a critical factor in both catalytic and immunological method‐based forensic saliva identification. This study aimed to assess the sensitivity of catalytic and immunological tests on degraded saliva samples. Degraded saliva stains were prepared by microbial decomposition using humid soil. Salivary α‐amylase activity was catalytically detected both qualitatively and quantitatively using the Phadebas^® amylase test. As immunological methods, we conducted qualitative and quantitative tests using the RSID?‐saliva test and ELISA, respectively. Salivary α‐amylase activity of degraded samples (incubated at 37°C for 12 h) was significantly lower than that of controls in the quantitative tests. All the degraded samples obtained by the humid soil produced negative results in the Phadebas^® tests, but showed positive results in the RSID?‐saliva test and ELISA. These results suggest that immunological tests are effective for testing degraded saliva samples that have lost their enzymatic activity.     

A rapid method to detect dried saliva stains swabbed from human skin using fluorescence spectroscopy

Saliva on skin is important in forensic trace evidence. If areas where saliva is present can be outlined, this may lead to DNA analysis and identification. This study describes a rapid and non-destructive method to detect dried saliva on the surface of the skin by fluorescence spectroscopy. Eighty-two volunteers deposited samples of their own saliva on the skin of their ventral forearm. A control sample of water was deposited at three different sites on the contralateral arm. Saliva and water control were then allowed to air-dry. Swab samples were taken from dried saliva and control sites and were dissolved in 0.1M KCl solution. Emission spectra were obtained from the solution and were characterized by a principal maximum at 345-355nm with excitation at 282nm. The fluorescence emission intensity was greater than background readings obtained from the control swab site in 80 of 82 volunteers (approximately 97.6%). The fluorescence profile of saliva samples were similar to those obtained from aqueous samples of pure amylase and tryptophan, an endogenous fluorophore in alpha-amylase. The presence of an emission peak at 345-355nm with excitation at 282nm could provide a strong presumptive indication of saliva deposition.     

Detection of human seminal γ-glutamyl transpeptidase in stains using sandwich ELISA

A sensitive and specific sandwich ELISA for human seminal γ-glutamyl transpeptidase (γ-GTP) was developed using a combination of monoclonal antibodies, SG1 and SG3, which we produced. For semen identification in forensic samples, we modified the assay so as to be more sensitive and to establish efficient extracting conditions. After testing the extracting abilities of several detergents, CHAPS and deoxy-BIGCHAP were chosen as the solubilizer. Polystyrene beads coated with SG1 were incubated with samples extracted by the detergents, and further with biotinylated SG3, followed by peroxidase-labeled streptavidin. γ-GTP was detected only in seminal samples. The sensitivity of this assay was 0.01 ng/ml of seminal γ-GTP equivalent to 10⁷ times diluted semen, which was ten times as compared with the previous plate assay. No significant seminal γ-GTP was detected in other biological stains such as blood, saliva and vaginal smear. The extract of a 500 fold diluted seminal stain, 8 months old, showed the detection limit. Seminal γ-GTP was detectable even in 14-year-old stains.     

Evaluation of amylase testing as a tool for saliva screening of crime scene trace swabs

Amylase testing has been used as a presumptive test for crime scene saliva for over three decades, mainly to locate saliva stains on surfaces. We have developed a saliva screening application for crime scene trace swabs, utilising an amylase sensitive paper (Phadebas^® Forensic Press test). Positive results were obtained for all tested dried saliva stains (0.5–32 μL) with high or intermediate amylase activity (840 and 290 kU/L). Results were typically obtained within 5 min, and all samples that produced DNA profiles were positive. However, salivary amylase activities, as well as DNA concentrations, vary significantly between individuals. We show that there is no correlation between amylase activity and amount of DNA in fresh saliva. Even so, a positive amylase result indicates presence of saliva, and thereby presence of DNA. Amylase testing may be useful for screening in investigations where the number of DNA analyses is limited due to cost, e.g., in volume crime.     

Developmental Validation of RSID™-Saliva: A Lateral Flow Immunochromatographic Strip Test for the Forensic Detection of Saliva

Abstract:  Current methods for forensic identification of saliva generally assay for the enzymatic activity of α-amylase, an enzyme long associated with human saliva. Here, we describe the R apid S tain ID entification (RSID™-Saliva), a lateral flow immunochromatographic strip test that uses two antisalivary amylase monoclonal antibodies to detect the presence of salivary amylase, rather than the activity of the enzyme. We demonstrate that RSID™-Saliva is accurate, reproducible, and highly sensitive for human saliva; RSID™-Saliva detects less than 1 μL of saliva. The sensitivity of RSID^TM-Saliva allows investigators to sample a fraction of a questioned stain while retaining the majority for DNA-STR analysis. We demonstrate that RSID™-Saliva identifies saliva from a variety of materials (e.g., cans, bottles, envelopes, and cigarette-butts) and it does not cross-react with blood, semen, urine, or vaginal fluid. RSID™-Saliva is a useful forensic test for determining which evidentiary items contain saliva and thus may yield a DNA profile.     

用ELISA检测精液及精斑中精子抗原的研究

本实验应用单克隆抗人精子抗体和酶标记羊抗人精子抗体,采用ELISA方法确定精子抗原成份的存在。对10份新鲜精液,15份精斑进行了验测,其结果阳性率为100%。新鲜精液(精子数约10,000万个/ml)稀释100万倍,精斑浸出液稀释50万倍,均可出现阳性。对唾液斑、尿斑、乳汁斑、阴道斑、汗斑及输精管结扎的精液均为阴性。实验结果表明,本法检验精子抗原具有灵敏度高,特异性好的优点。     

Comparison of modern techniques for saliva screening

Saliva stains present a unique challenge in the forensic setting, often challenging the analyst to weigh the value of presumptive indication of the fluid versus the potential for DNA analysis to yield identification information. There are many situations in which determining the presence of a body fluid is probative and further corroborates DNA evidence. That said, even a minute portion of sample consumed by a screening test could mean the difference between a full, partial, or null profile obtained through DNA analysis. The basis of presumptive testing or screening of saliva has historically been based on the presence of amylase, a component found in relatively high concentrations in human saliva versus other body fluids and substances. Though the current available methods for the screening of saliva in a forensic application have grown in number, the popularity of these methods seemingly has not. This study attempts to identify a specific and sensitive saliva screening test by comparing three modern techniques--the recently released SALIgAE, Phadebas, and starch-iodine mini-centrifuge test--on the basis of sensitivity, specificity, mixtures, and simulated casework samples while also considering sample consumption. The Phadebas method for presumptive saliva testing detected dilutions of neat saliva down to 1:200 versus considerably less sensitive results with SALIgAE and the starch-iodine mini-centrifuge test. Utilizing a screening test with a high degree of sensitivity, such as Phadebas, allows an analyst to gain a maximum amount of information in the form of body fluid indication and DNA results because of the consumption of a small portion of sample.     

Applicability of two commercially available kits for forensic identification of saliva stains

The RSID-saliva test and the SALIgAE-saliva test are two recently developed forensic saliva detection kits. In this study, we compared the sensitivity and the specificity of the two test kits with the Phadebas amylase test by analyzing amylases from various sources including human, animals, plants, and micro-organism. The data demonstrate that the RSID-saliva test and the SALIgAE-saliva test offer higher sensitivity and specificity for the detection of saliva than the Phadebas amylase test. The detection limits of the RSID-saliva test, the SALIgAE-saliva test, and the Phadebas amylase test equate to 10, 4, and 1000 nL, respectively for human saliva. The RSID-saliva test and the SALIgAE-saliva test were further evaluated by analyzing semen, vaginal secretion, breast milk, blood, urine, sweat, and feces. The results of the two tests are in good agreement. The two tests reacted with urine, breast milk, and feces, but not with semen, vaginal secretion, blood, and sweat.     

Identification of saliva stains by determination of the specific activity of amylase.

The specific activity (enzyme activity/protein concentration) of amylase was determined for the identification of saliva stains. The specific activity of amylase in saliva stains rapidly decreased during the first hour but, from 1 to 28 days, this decrease was much less when the stains were kept at room temperature. Stains of various human biological materials, breast milk, nasal secretion, meconium and vaginal secretion showed comparatively high amylase activity, but the saliva stains could be differentiated by their high specific activity of amylase, over 2 I.U./mg. When saliva stains were contaminated with blood or vaginal secretions at various ratios, the specific activity of amylase decreased with increase in the ratio of contaminant, especially when the contaminant was blood. However, the specific activity of amylase was still higher than 2 I.U./mg even after one fifth volume of blood was added or after five volumes of the extract of the stains of vaginal secretions were added.     

Radioimmunoassay detection limits for 19-OH F1 alpha/F2 alpha prostaglandin in normal, infertile and vasectomized semen stains. Analysis of saliva, sweat and urine for possible non-specific or matrix effects

The sensitivity of a simple radioimmunoassay (RIA) for the detection of 19-OH prostaglandin F1 alpha/F2 alpha (PG F) has been evaluated on a number of semen samples from vasectomized, infertile and fertile donors. The specificity of the test has been examined by testing saliva, sweat and urine from a number of male and female donors. The assay technique could readily detect the PG in semen stains prepared from 0.2 microliter of semen from normal, infertile and vasectomized donors. The detection limit of the assay system, based on the observed displacement, was calculated to be approximately 0.05 microliter semen. The assay could be conducted over a pH range of 7.5-10.5 even after the PG has been heated to 100 degrees C. The 19-OH series of PG were absent from sweat, saliva and female urine using the normal assay protocol; volumes in excess of 100 microliters of some urines particularly from women in labour and those with acute urinary tract infection showed some displacement. Low levels of PG were detected in 50% of the male urine analysed. However, urine samples from men who had engaged in recent sexual activity contained relatively high concentrations of PG which could be readily detected in 10 microliters of urine. These results emphasise the potential of these compounds as specific and sensitive markers for the presence of human semen.     

Persisting fetal microchimerism does not interfere with forensic Y-chromosome typing

Forensic Y-chromosome typing applies Y-chromosomal polymorphisms to the analysis of male/female mixed stains such as vaginal swabs in rape cases. The sensitivity of this approach exceeds that of cytological techniques combined with autosomal DNA typing. Y-chromosome typing is based on the assumption that Y-chromosomal DNA found in tissue or secretions of women must originate from a male individual, usually the perpetrator. Nevertheless, it was shown recently that fetal cells can migrate into the female body during pregnancy and can persist for decades ("persisting fetal microchimerism"). The body of a woman after a pregnancy with a male embryo can thus display a small fraction of fetal cells with Y-chromosomes. Using high sensitivity PCR protocols (reamplification with nested primers and up to 60 PCR cycles) fetal cells were previously identified in a number of maternal tissues including skin, blood, muscle and solid organs. It is, however, not clear at present, whether these cells can occur in vaginal secretions, and whether they are capable of producing false positive results in forensic Y-chromosome typing. To evaluate these questions, 66 blood samples of women with at least one son and nine vaginal swabs of women without sexual intercourse in the last 2 weeks were amplified for a stretch of the SRY gene. Eight thyroid gland tissues with already established male fetal microchimerism were used as positive control samples. Blood samples of 10 young girls without history of pregnancy were used as negative controls. Using a PCR with 10 ng of extracted DNA and 30 PCR cycles ("routine sensitivity assay") none of the samples yielded positive results. However, in a PCR with 200 ng of extracted DNA and 45 PCR cycles ("high sensibility assay"), 14% of the blood samples of mothers and 33% of the vaginal swabs amplified for SRY. Our results thus show that increasing the sensitivity of the PCR method and the amount of template DNA produce positive results while protocols used for routine Y-chromosomal typing with small amounts of DNA (approximately 10 ng of DNA) and with a limited number of PCR cycles (approximately 30) can clearly eliminate this peril.     

Assessing the background levels of body fluids on hands

Forensic scientists are often asked to assist the court by evaluating the significance of finding body fluids on the hands of an individual; however, there is an absence of published data regarding the background levels of body fluids on hands. Whilst the scientist can use casework experience to inform the courts on the significance of the results, it would be advantageous to have data which could assist with this interpretation. This study was designed to ascertain the background levels of blood, semen, saliva, hairs/fibres and staining/debris on hands in the general population by sampling from delegates attending a scientific conference.The findings suggest that approximately one third of the population would be expected to have hairs or fibres on their hands and that females are more likely to have visible staining on their hands than males. Presumptive tests for blood and semen yielded negative results in all samples; however, almost 2 % of the samples were found to contain a very low number of sperm heads. In contrast, the majority of samples tested positive for the presence of saliva using the presumptive Phadebas® amylase test. The data supports the caution applied by forensic practitioners when evaluating the presence of saliva detected using the presumptive Phadebas® amylase test based on the lack of specificity and indicates that the RSID™-Saliva test would be more suitable to use.     

Enzyme-linked immunosorbent assay for A and B water soluble blood group substances

The conditions affecting an enzyme-linked immunosorbent assay for salivary blood group substances were investigated. It was found that A, B, and O secretor saliva samples would each bind both anti-A and anti-B typing reagents. The conditions that affected the assay response were optimized for maximum sensitivity and to give the highest resolution possible between the result for an antiserum binding to homologous antigen and the response for heterologous antigen-antibody combinations. Monoclonal antibodies eliminated the heterologous binding indicating that this binding was due to a lack of specificity of the routine typing reagents. A sensitive assay using the monoclonal antibodies to distinguish between samples of A and B secretor saliva is described.     

The sensitivity and specificity of red-starch paper for the detection of saliva.

The detection of saliva in forensic casework is extremely important in many types of cases. This study describes a relatively sensitive method, based on a red dye bound to starch, for the detection of amylase. The sensitivity and specificity of the method has been examined by testing over 50 household products, various body fluids and five laboratory chemicals. This study demonstrated for the first time that positive results can be obtained from certain washing powders as well as other household products. As well as detecting amylase in saliva, positive Red-Starch results were obtained from faeces and urine. The method was found to be suitable for the detection of mixtures of saliva and semen in conjunction with the Brentamine test for the detection of acid phosphatase.