1. Chapter 5: Serology Techniques Section 5.1 only

2.  Forensic Serology = Detection and identification of bodily fluids  Enzymatic assays ▪ Blood: peroxidase in heme group of hemoglobin ▪ Semen: acid phosphatase ▪ Saliva: amylase  Antigen-antibody assays ▪ Primary binding assays ▪ Secondary binding assays Forensic Biology by Richard Li2

3.  Enzymatic assays  Kastle-Meyer test for blood ▪ Tests for presence of peroxidase activity ▪ Peroxidases break down hydrogen peroxide to water and oxygen free radicals (O - ) ▪ Oxygen free radicals are strong oxidants and strip hydrogens off the K-M reagent (oxidize it) ▪ The reduced form of K-M is colorless but the oxidized form is bright pink ▪ Not the same dark red color of blood 3

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5. 5  Method ▪ Moisten Q-tip swab in distilled water ▪ Lightly touch suspected blood stain with tip of Q-tip ▪ Add one drop K-M reagent ▪ Add one drop hydrogen peroxide ▪ Look for fast color change to bright pink

6. 6  Limitations  False positive reactions ▪ Any strong oxidizing agent (e.g. bleach) ▪ Will oxidize K-M reagent even in the absence of peroxidase ▪ Any substance with peroxidase activity ▪ Bacteria ▪ Plants  Not species-specific ▪ Reacts with blood from any animal  Sensitivity (too high?) ▪ Will detect blood in urine, saliva, and other body fluids if trace amounts are present

7.  Enzymatic assays  AP test for semen ▪ Tests for presence of acid phophatase activity ▪ AP liberates naphthol from alpha-naphthol and the naphthol then reacts with brentamine to form a purple- colored dye 7 sodium phosphate + naphtholα-naphthyl acid phosphate monosodium salt Acid phosphatase Coupling reaction Purple azo dyenapthol + Brentamine

8. 8  Method ▪ Spray a Whatman paper circle with distilled water ▪ Lay the paper down over the suspected semen stain ▪ Leave in contact with stain 30-60 seconds ▪ Remove paper circle from stain and spray with AP spot solution ▪ Look for a rapid color change to purple

9. 9  Limitations  False positive reactions ▪ Any substance with acid phophatase activity ▪ Bacteria ▪ Plants ▪ Vaginal fluid  Not species-specific ▪ Reacts with semen from any animal ▪ Not usually a big problem in forensic casework ▪ Animal sperm are morphologically distinct from primate sperm under the microscope

10.  Enzymatic assays  Amylase test for saliva ▪ Tests for presence of amylase enzyme ▪ Amylase is present in saliva and small intestine ▪ Salivary amylase = ptyalin ▪ Pancreatic amylase = amylopsin  Breaks down starch to simple sugars  Two types of starch: amylose and amylopectin ▪ Amylose changes color from clear to deep blue-black in the presence of iodine 10

11. Forensic Biology by Richard Li11 amylose amylopectin

12. 12  Method ▪ Spray a Whatman paper circle with solution of soluble starch ▪ Lay the paper down over the suspected saliva stain ▪ Leave in contact with stain for 20 minutes ▪ Incubate in a 37 deg moisture chamber for 1 hour, then dry ▪ Spray with iodine and look for a lack of color change to deep blue- black

13. 13  Limitations  False positive reactions ▪ Any substance with amylase activity ▪ Bacteria ▪ Plants ▪ Vomit  Not species-specific ▪ Reacts with saliva from any animal that produces it ▪ Not usually a big problem in forensic casework ▪ Cats and dogs do not produce amylase

14.  Enzyme-Linked Immunosorbent Assays (ELISA) is most common type used  Very sensitive  Colorimetric or fluorometric signal is proportional to the amount of bound antigen  Often performed in wells  Detected by color change  Two methods: ▪ Well ▪ Cassette 14

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16.  Immunochromatographic ELISAs  Rapid and simple test  Used as screening test in the field for seminal and saliva stains and for species identification  High-dose effect  Highly sensitive (too sensitive?)  Specificity still under debate  We will perform these in lab 16

17. 17 T T C Positive test

18. 18 T C Negative test