Chapter 5: Serology Techniques
Section 5.1 only
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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 Li
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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
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Method
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▪
▪
▪
▪
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
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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
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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 purplecolored dye
α-naphthyl acid phosphate monosodium salt
sodium phosphate + naphthol
Acid phosphatase
napthol + Brentamine
Purple azo dye
Coupling reaction
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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
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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
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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
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amylose
amylopectin
Forensic Biology by Richard Li
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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 blueblack
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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
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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
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Immunochromatographic ELISAs
 Rapid and simple test
 Used as screening test in the field for seminal and
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saliva stains and for species identification
High-dose effect
Highly sensitive (too sensitive?)
Specificity still under debate
We will perform these in lab
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Positive test
T
C
T
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Negative test
C
T
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