sandwich Enzyme linked immunosorbent assay for Determination of ABH Blood type of semen stains stored under various conditions using anti-p84 monoclonal antibody as a marker of blood group substance in semen

ABSTRACT

Semen samples were obtained from 42 volunteer donors. The ABO and Lewis blood type of each semen donor were determined by conventional hemaglutination test. ABO blood type of each semen sample was determined by performing a Sandwich enzyme-linked immunosorbent assay [ELISA] in which p84 [which is a protein with ABO antigenic activity isolated from human seminal plasma] is captured with an anti-p84 monoclonal antibody. It was found that the concentration of p84 in semen was independent of the secretion status. The status can be determined as non-secretor status. The status can be determined as non-secretor when p84 but not the individual blood group substance [BGS] activity was detected. To determine the stability of BGS activity on p84, dried semen stains were prepared on 3 filter papers by blotting 100 micro L of semen on each one and allowing them to dry. After that, the first, second and third stains were kept at 2 °C, 20 °C and 25 °C respectively after dividing each one into 4 equal parts where part one, two, three and four of each stain were stored at its corresponding temperature for one hour, one month, 10 months and 36 months respectively. Each part was then cut into squares of different sizes and subjected to the Sandwich ELISA to detect the P84 activity of dried semen stain. After one hour at 20 °C and 25 °C, 62.29% and 34.42% of the original BGS activity was recovered. However after 36 months at 2 °C, 78.68% of p84 activity still remained. So, it could be hypothesized that the temperature affects the detection of the BGS activity on p84 in dried semen stain rather than the duration of storage. BGS activity o P84 could be detected from the pieces of 0.5 x 0.5 cm[2] even after storage for up to 36 months under various environmental conditions. It could be concluded that this Sandwich ELISA offers a sensitive and specific method for determining the ABO blood type of semen samples obtained from sexual assault victims and of dried semen stains kept under various environmental conditions