foundational serology techniques

presumptive vs. confirmatory test characteristics

presumptive: sensitive, rapid, simple, but not specific. usually colorimetric as opposed to fluorimetric or luminescent. 

confirmatory: specific. ID with higher certainty. identification

what is ELISA? how does it work?

enzyme linked immunosorbent assay.

used to detect antigens or antibodies. not confirmatory nor presumptive but separate. colorimetric. results are read spectrophotometrically.

1. well is coated with a known “capture” antibody which will bond to the target antigen being searched for.

2. excess unbound capture antibodies are washed away.

3. unknown sample is added with unknown antigens. any which match the capture antibody will bind together.

4. excess sample w/ unbound antigens is washed away. the bound ones will stay.

5. detection antibody is added, labeled with an enzyme. this will also bind to the target antigen, but it is labelled so they can be found easily.

6. excess washed away 

7. substrate is added, which will become colored by the labeled enzyme (assuming the target product was present and the labelled antibodies bound, did not wash away. )

8. the colored product will be measured by spectrophotometry to determine how much of the target antigen is present. (based on a standard curve)

this is the sandwich method ***

what is an immunochromatographic assay? how does it work?

relies on reactions between antigen and antibody. test strip contains immobilized antibodies which are labeled with a dye. sample is applied and capillary action moves it across the strip. if the antigen matches the antibody, they will combine and the dye on the labeled antibodies will become visible. a control line is present — known antigens are present to ensure a reaction for the control.  these assays are usually presumptive   

what is the high dose hook effect?

refers to when there is too much of a questioned substance present, e.g antigens, for the amount of antibodies present — result is a false negative or underestimated amount of substance, ironically because of too much substance. too many antigens — system is saturated and antigens cannot make contact with the antibodies so they do not bind and the colored line does not appear.

which presumptive immunochromatographic assays are used for which potential substances?

blood: hematrace (hemoglobin), RSID-blood (glycophorin A)

semen: PSA, RSID-semen(semenogelin)

saliva: RSID-saliva(HSA)

what are immunodiffusion tests? how do they work?

passive method. antigen and antibody are added to respective wells. they are allowed to diffuse (move). if they come into contact with each other, they will bind and create a cluster (precipitation), indicating a positive result. there are 2 tupes, single vs. double diffusion.

single vs. double immunodiffusion w/ examples?

single immunodiffusion: antibody or antigen gradient is established. antibody or antigen is loaded into sample well and diffuses — just one diffuses — if matches, then precipitation occurs and forms a ring around well proportional to amount of antigen, which is compared for quantification. i.e radial diffusion , e.g. the ring test

double immunodiffusion: both ab and ag are allowed to diffuse together (two dimensions), added into an established gradient with both ag and ab. migrate towards each other. precipitate forms at interface. e.g. ouchterlony

single: only ag diffuses towards the ab.

double: both ag and ab diffuse towards each other

what is the ring test?

test tube. diffuses up or down. single diffusion.

what is the ouchterlony test?

double diffusion. bunch of wells on plate. diffuse towards each other through gradient (agar)

what is an immunoelectophoretic method? how does it work?

antigens are separated by electrophoresis. antibody is loaded parallel to separated antigens and incubated for double diffusion. precipitate = match.

what is agglutination? how is it used in forensic bio?

clumping caused by antibody-antigen interaction. it is indicative of a positive reaction.

false positives can be caused by?

strong reducing or oxidizing reagents, like bleach. they catalyze the reaction instead of the substance of interest. 

why do we use presumptive tests on scenes?

they are rapid and sensitive. good indicator if continuing examination would be useful or a waste of time.

what role does semen play in the acid phosphatase PSA presumptive test?

acid phosphatase will catalyze an acid-base reaction (phosphate group very negative), reaction = color change 

why does hydrogen peroxide need to be added to presumptive tests?

it oxidizes the reaction and allows for the color change to occur with the complex formed by a positive reaction

why shouldn’t you read results of tests after 10 mins?

it will oxidize eventually even if negative because of the oxygen in the air

what are the problems with interpreting colorimetric tests?

colors are subjective — important to compare to controls which are performed at the same time