Chapter 5.1: Serology Techniques
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Transcript Chapter 5.1: Serology Techniques
Chapter 5: Serology Techniques
Section 5.1 only
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
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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human semenogenlin
monoclonal goldlabeled murine anti
human semenogelin
antibody to epitope 1
monoclonal unlabeled
murine anti human
semenogelin antibody
to epitope 2
polyclonal unlabeled
goat anti murine
antiglobulin
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Positive RSID™
semen test
C
T
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Negative RSID™
semen test
C
T
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