Differentiation between human and chimpanzee in bloodstains by enzyme-linked immunosorbent assay (ELISA) using antihuman serum

An enzyme-linked immunosorbent assay (ELISA) for species identification of human bloodstains using two commercially available antisera against human serum is described. Human bloodstains were to be distinguished from those of chimpanzees and other animals using raw antisera, and the differentiation between human and chimpanzee became more evident when those antisera were absorbed with a small amount of chimpanzee plasma. Human bloodstains could clearly be identified by the present method even after 4 weeks of aging at room temperature.     

Lewis typing of human bloodstains by enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b)

The Lewis blood grouping of human dried bloodstains could be determined by an enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b) antibodies with an avidin-biotin complex (ABC). The bloodstains aged 1 year were used as samples, and approximately 1 mg of the stains was enough to type each Lewis antigen reliably by this method. The Lewis substances of 106 individual stains were correctly typed regardless of their ABO blood group system.     

Gm typing by enzyme-linked immunosorbent assay (ELISA)

A solid-phase ELISA for Gm typing is described. A mixture of anti-Gm serum (or monoclonal anti-Gm antibody) and test serum was incubated in microtiter wells coated with IgG or its fragments of appropriate Gm type. After washing of the wells, the bound antibody was detected with peroxidase-labeled second antibody. The Glm(3), G3m(16), and G3m(21) antigens could be identified by this technique. Since some of the human anti-Gm sera and anti-Rh0 sera required for the conventional hemagglutination-inhibition method are hard to obtain, the ELISA system using anti-Gm antibodies and no anti-Rh0 sera may serve as an alternative to the conventional method.     

Identification of fetal bloodstains by enzyme-linked immunosorbent assay for human alpha-fetoprotein

A rapid and highly sensitive enzyme-linked immunosorbent assay (ELISA) for determination of human alpha-fetoprotein (AFP) using commercially available reagents was devised and applied to identification of fetal bloodstains. When experimentally prepared bloodstains, 1 by 2 mm in area, were submitted to analysis, only fetal bloodstains showed positive reactions in the present ELISA. The reactions did not change significantly when these bloodstains were stored at room temperature for one week. The present ELISA seems to be suitable for forensic science practice.     

An enzyme-linked immunosorbent assay (ELISA) for prostate-specific antigen.

A sandwich ELISA for human prostate-specific antigen (PSA) is described. Optimal assay conditions, resulting in a sensitive assay with a low background, are presented. The method uses a hyperimmune antiserum produced in the New Zealand white rabbit, against human semen PSA. The IgG fraction of the antiserum was conjugated with horseradish peroxidase and used in the sandwich ELISA method. The anti-PSA IgG showed no cross reactions with saliva, normal blood, female urine, vaginal fluid, or menstrual blood. On occasions, a blood sample showed a non-specific cross-reaction, which was detected by non-immune rabbit IgG. This reaction could be caused by rheumatoid factors, as indicated by experiments with a series of known IgG and IgM rheumatoid antibodies, although other heterophilic antibodies could not be eliminated. The recovery of PSA added to blood plasma, saliva and vaginal fluid was affected by three factors; (a) protein concentration (dilution) of body fluid; (b) nature of the protein; and (c) amount of PSA added.     

Lewis typing of human saliva stains by enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b) antibodies

The Lewis blood grouping of human saliva stains could be detected by an enzyme-linked immunosorbent assay (ELISA) using anti-Le(a) and anti-Le(b) monoclonal antibodies with an avidin-biotin complex (ABC). The saliva stains (1.0 by 1.0 cm in size) were used as samples and not only could the Lewis substances of 57 individual stains be correctly typed by this method, but also it was clarified that there are several different secretion patterns of amounts of Le(a) and Le(b) substances in 3 individual Lewis types.     

An examination of a contaminated seminal stain using absorption-elution and enzyme-linked immunosorbent assay (ELISA)

A semen stain, apparently contaminated with a detergent cleanser, was received for examination. The contamination interfered with the normal biochemical reactions of such stains. Treatment of the sample enabled ABO groups to be determined.     

Km(3) identification by enzyme-linked immunosorbent assay (ELISA) as an internal control for Km(1) activity determined by inhibition in dried bloodstains

A stability study comparing the identification of kappa marker Km(1), using the classical inhibition of agglutination, and the identification of Km(3), using an automated enzyme-linked immunosorbent assay (ELISA) technique, was done. Preliminary tests were performed to establish the specificity and sensitivity of the methods. Based on the results, the quantities of stain required to detect each marker were determined. Blood samples from 24 staff donors of known phenotype were aged at room temperature and at 37 degrees C in the dried stain and liquid forms. In addition, 192 stains from cases 1 to 7 months old and 76 staff-donor stains from 1 1/2 to 10 years old were tested in dried stain form. The known sensitivity of the ELISA technique was exploited by deliverately testing a decreased quantity of antigen. As control stains were aged beyond the detectable limits of sensitivity, results consistently showed an almost simultaneous success or failure to detect Km(1) and Km(3). This indicates that the interpretive criteria established for ELISA are sufficiently demanding to eliminate the danger of reporting false Km(-1) results but true Km(3) results.     

Detection of Lea substance in saliva stains by enzyme-linked immunosorbent assay (ELISA) using anti-gum arabic serum

It is known that rabbit anti-gum arabic (GA) serum has cross-reactivity with Lea antigen, and that, by using this cross-reactive anti-Lea antibody, the presence of Lea antigen in red blood cells and saliva can be demonstrated with accuracy. We have devised a rapid and highly sensitive method for detecting Lea substance in human saliva by the enzyme-linked immunosorbent assay (ELISA) method using an anti-Lea antibody isolated from anti-GA serum by affinity chromatography on Synsorb Lea. The ELISA plate, coated with the specific anti-Lea antibody, adsorbed the Lea substance in saliva which was subsequently identified by adding enzyme labeled anti-Lea IgG in that order. The method could detect the Lea substance in Le(a+) saliva stains as small as 0.1 by 0.1 cm in size that had been stored at room temperature for three weeks and in Le(a+) saliva stains 0.7 by 0.7 cm in size that had been stored for ten years. This method seems to be useful for quantitative analyses of the Lea substance in various body fluids.     

An enzyme-linked immunosorbent assay (ELISA) for trinitrotoluene (TNT) residue on hands

An enzyme-linked immunosorbent assay (ELISA) developed for the detection of trinitrotoluene (TNT) in munitions wastewater has been adapted to the detection of TNT residue on hands following contact. Using the procedure developed, as little as 50 pg of TNT could be detected. Accounting for sample size and dilution, the 50 pg equates to 15 ng of TNT recovered from the hands. Following contact with TNT, amounts ranging from 53 ng to more than 1500 ng were recovered from hands. The monoclonal anti-TNT antibodies showed no cross-reactivity with several other explosives or common contaminants. These preliminary results indicate promise for the development of a simple-to-use, immunoassay-based field test kit for TNT and, ultimately, other explosives.     

Identification of human urinary stains by enzyme-linked immunosorbent assay for human uromucoid

An enzyme-linked immunosorbent assay (ELISA) of the sandwich type for identification of human urinary stains using commercially available anti-human uromucoid was developed. When experimentally prepared urinary stains of humans and animals, 2 by 2 cm in area, were subjected to analysis, human stains could be differentiated from animal ones except chimpanzee and Old World monkey ones. Stains of other human body fluids showed negative reactions. The reactions did not decrease when human urinary stains were stored at room temperature for three months. The present ELISA provides a useful presumptive test for urinary stains of human origin.     

An enzyme-linked immunosorbent assay (ELISA) for human semen identification based on a biotinylated monoclonal antibody to a seminal vesicle-specific antigen

Monoclonal antibody mouse antihuman semen-5 (MHS-5) (immunoglobulin G1 [IgG1]) was biotinylated using N-biotinyl-w-aminocaproic acid-N-hydroxysuccinimide ester. This monoclonal antibody-biotin conjugate recognized low molecular weight peptide bands between 10.5 and 20 kilodaltons on immunoblots of liquefied semen. Immunodominant peptides had molecular weights of 10.5, 11.5, and 13.5 kilodaltons. An enzyme-linked immunosorbent assay (ELISA) developed with the biotinylated-MAb and streptavidin peroxidase demonstrated sensitivity curves with lower limits of 10 ng of seminal fluid protein per microtiter well using 50 ng per well of monoclonal antibody-biotin conjugate. Cross-reactivity studies on a panel of human biological fluids and tissues demonstrated no cross-reactivity or false positives using the monoclonal antibody-biotin conjugate. The sensitivity of the monoclonal antibody-biotin ELISA was compared to ELISA based upon a polyclonal secondary antibody-peroxidase conjugate. These findings indicate that this ELISA assay, based on a biotinylated monoclonal antibody to a seminal vesicle-specific antigen, may be useful for semen identification.     

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.     

Detection and identification of HLA antigens in dried spleens by means of enzyme linked immunosorbent assay (ELISA)

HLA class-I and class-II antigens were detected in extracts of dry spleen tissue allowed to age from 1 to 17 months, using monomorphic anti HLA monoclonal antibodies and the ELISA technique. Using polymorphic HLA alloantisera samples of spleen tissue could be characterized and differentiated. The potential use of dried tissues and blood stains is discussed.     

Validity testing of commercial urine cocaine metabolite assays: III. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for detection of cocaine and cocaine metabolite

A validity assessment study was performed on the Genetic Diagnostic Enzyme Immunoassay test kit, a new enzyme-linked immunosorbent assay (GDC ELISA) for detection of cocaine and cocaine metabolite in urine. A set of 290 urine specimens, comprised of clinical cocaine urines collected from 5 male subjects who had received single doses of intravenous cocaine, drug-free urines spiked with cocaine, cocaine metabolites, cocaine isomers, and other drugs of abuse, were assayed by GDC ELISA. The results were compared with results by gas chromatography/mass spectrometry (GC/MS) assay for benzoylecgonine. Concordance was high between the GDC ELISA assay and GC/MS and with results reported earlier for other commercial assays. Detection times and specificity of the GDC ELISA antibody were most similar to those of the Abuscreen radioimmunoassay for cocaine metabolite. Overall, the assay produced no false negative or false positive results and appeared to be a reliable screening test for detection of cocaine and benzoylecgonine in human urine.     

Evaluation of an enzyme linked immunosorbent assay (ELISA) method for ABO and Lewis typing of body fluids in forensic samples.

An ELISA for the detection of the ABO group and secretor status of body fluids and stains other than blood is described, together with the validation procedures employed before its introduction into forensic casework. Criteria for the interpretation of results have been formulated for the method in use in this laboratory. The method was found to be reliable and to have a higher success rate than the haemagglutination techniques previously employed.     

A sandwich enzyme-linked immunosorbent assay for ABO blood typing of semen by using anti-p 84 monoclonal antibody as a marker of blood group substance in semen

A blood group substance (BGS), a protein with ABH antigenic activity, was isolated from human seminal plasma and designated as p 84 (Sato, 1995). We have developed a method for determining the ABO blood type of semen by performing a sandwich enzyme-linked immunosorbent assay (ELISA) in which p 84 is captured with an anti-p 84 monoclonal antibody, and evaluated the specificity and sensitivity of this method. Although BGS activity was detected in semen sensitively by this method, it was not detected in saliva, urine, breast milk, blood or vaginal secretions. Since the concentration of p 84 in semen was independent of the secretion status, the status can be determined as non-secretor when p 84 but not BGS activity was detected. To determine the stability of BGS activity on p 84, dried stains of semen on filter paper were kept at 4, 26, and 37 degrees C for 8 months, 2 years and 1 month, respectively, and their BGS activities were examined. After 8 months at 4 degrees C, over 60% of the original BGS activity was recovered from the stain. The activity could be detected even from a square as small as 0.25 by 0.25 cm. After 1 month at 37 degrees C and 2 years at 26 degrees C, 31 and 20% of the BGS activity, respectively, still remained. It could be detected from the pieces of 1.0 by 1.0 cm and 0.5 by 0.5 cm squares, kept for 1 month at 37 degrees C and 2 years at 26 degrees C, respectively. Finally, semen was mixed with saliva or blood at varying volumetric ratios and used for the sources of dried stains. The BGS activity of p 84 could be detected in the stains until the ratio between semen and saliva or blood reached 1:4. We conclude that this sandwich ELISA offers a more sensitive and specific method for determining the ABO blood type of semen samples obtained from sexual assault victims than existing methods, such as the conventional absorption-elution and classical hemagglutination-inhibition tests.     

Identification of male bloodstains by dot hybridization of human Y chromosome-specific deoxyribonucleic acid (DNA) probe

The sex determination of bloodstains was performed using a human Y chromosome-specific (DNA) fragment of 1.9-kb length as a hybridization probe. The DNA samples were taken from 1- and 4-week-old bloodstains of males and females, respectively. Strong signals with male DNA were observed by Y-probe, while faint signals with female DNA were detected. In addition, clear signals were observed in the extract samples from male bloodstains (16-week-old) on paper. Dot hybridization of the Y-probe would be widely applicable to studies on sex determination of medicolegal materials such as blood, bloodstains, teeth, and cadaverous parts.