immunoassy techniques, etc. ch. 8 serology. for review: antigen: a substance which, when put into a...

IMMUNOASSY TECHNIQUES, ETC. Ch. 8 Serology


For Review:

Antigen: A substance which, when put into a body, stimulate the body to produce antibodies against it.

Antibodies: A protein in blood serum that attacks a specific antigen. It can either destroy it or neutralize it.

Agglutination: Clumping of RBCs due to antibodies attacking antigens


Q: Why do red blood cells agglutinate?

A: The antibodies for each antigen of blood type are able to bind at TWO binding sites to the antigen (sometimes called marker molecules). This enables the antibody to bond in two places and thus bind numerous RBCs together.

(See illustrations)


Q: Why do red blood cells agglutinate?


This agglutination can cause clotting which can be fatal in a transfusion.

Remember:

Blood type ____ has ________ antibody



Remember:

Blood type _A__ has _anti-B_ antibody



Remember:

Blood type _A__ has _anti-B_ antibodyBlood type ____ has ________ antibody



Remember:

Blood type _A__ has _anti-B_ antibodyBlood type _B__ has _anti-A_ antibody



Remember:

Blood type _A__ has _anti-B_ antibodyBlood type _B__ has _anti-A_ antibodyBlood type ____ has ________ antibody



Remember:

Blood type _A__ has _anti-B_ antibodyBlood type _B__ has _anti-A_ antibodyBlood type _O__ has _BOTH_ antibodies



Remember:

Blood type _A__ has _anti-B_ antibodyBlood type _B__ has _anti-A_ antibodyBlood type _O__ has _BOTH_ antibodiesBlood type ____ has ________ antibody



Remember:

Blood type _A__ has _anti-B_ antibodyBlood type _B__ has _anti-A_ antibodyBlood type _O__ has _BOTH_ antibodiesBlood type _AB_ has _NEITHER_ antibody


Antibodies can be artificially created to attack certain antigens (e.g. illegal drugs).

These antibodies are often used in drug testing.


The technique for producing these antibodies is called _________________.


The technique for producing these antibodies is called __immunoassy_____.



Step 1: Antigen (e.g. the illegal drug) is attached to a carrier protein molecule.




WHY?




WHY?

(We’ll come back to that question)



Step 2: The antigen (attached to its carrier protein) is injected into an animal (e.g. a rabbit).



Step 2: The antigen (attached to its carrier protein) is injected into an animal (e.g. a rabbit).

Days or weeks typically elapse while the body of the animal develops an antibody



Step 3: Spleen cells are extracted from the rabbit.



Step 4: Fuse the (normal) spleen cells with cancer cells.




WHY?




WHY?

(We’ll get back to that later as well)



Step 5: Let them grow in a petri dish. They will produce as many antibodies as you want.



Back to our questions:




Q: (from step 1) Why do we attach the antigen to a carrier protein before injecting it into the animal?




Q: (from step 1) Why do we attach the antigen to a carrier protein before injecting it into the animal?

A: The usually antigen is too small. (the larger molecule elicits a more potent immuno-response)




Q: (from step 4) Why did we fuse the normal spleen cells from the rabbit with cancer cells?





A: The answer is in the definition of the type of cells these produce called hybridoma cells.





A: hybridoma cells: Cells that are produced when normal cells are fused with myeloma or lymphoma (cancer) cells TO PRODUCE CHARACTERISTICS OF EACH.





A: Think about what a HYBRID (…e.g. a hybrid car…)





A: Therefore hybridoma cells have the benefits of both:






a) Normal cells capacity to produce the specific antibody







b) Cancer cells ability to reproduce quickly.







b) Cancer cells ability to reproduce quickly.So TOGETHER….



(This immunoassay technique is illustrated on pages 290 and 292 of the Saferstein text)



Also this immunoassay technique is a good one because it produces ______________ antibodies.



Also this immunoassay technique is a good one because it produces __monoclonal___ antibodies.



Q: What is the difference between monoclonal antibodies and polyclonal antibodies?




A: Polyclonal antibodies bond to MANY sites on a particular antigen




A: Polyclonal antibodies bond to MANY sites on a particular antigen Monoclonal antibodies bond to only ONE site on a particular antigen




A: Polyclonal antibodies bond to MANY sites on a particular antigen Monoclonal antibodies bond to only ONE site on a particular antigen

(See the hand-drawn illustrations)



Q: Why are monoclonal antibodies more desired than polyclonal antibodies?




A: Because monoclonal antibodies are more specific to the antigen in question.





I.e. polyclonal antibodies can often bond to antigens other than the one you want them to.





I.e. polyclonal antibodies can often bond to antigens other than the one you want them to.BOTTOM LINE: More false positive drug tests when using polyclonal antibodies as one of the reagents.



BOTTOM LINE: More false positive drug tests when using polyclonal antibodies as one of the reagents.

NOTE: ALL immunoassay techniques will produce antibodies that will give false positives (even the monoclonal antibodies). The key is to minimize it.


Another great example of immunoassay is EMIT



EnzymeMultipliedImmunoassayTechnique



EMIT is widely used to detect drugs & drug metabolites in urine that are difficult to detect



(Use the illustration on page 291 of Saferstein text)

First: Put an antibody for the drug testing for into urine sample




Second: Place a labeled version of the drug in the urine sample.




Second: Place a labeled version of the drug in the urine sample.(B/c the drug is labelled it is easy to detect)




Second: Place a labeled version of the drug in the urine sample.(B/c the drug is labelled it is easy to detect)Competition for binding sites will occur between the labelled drug and the unlabeled drug




Third: Test for the labeled drug.




Third: Test for the labeled drug.If no labeled drug is present then the antibody was able to remove all of it b/c no unlabeled drug was present (no competition)




Third: Test for the labeled drug.If (YES) labeled drug IS present then the antibody was NOT able to remove all of it b/c of the competition between labelled and unlabeled drug.




E.G of EMIT: Using the antibody for THC-9 carboxylic acid (which is a metabolite of marijuan=-


FORENSIC COLLECTION OF SEMEN



After locating semen stain(s) testing to confirm its identity should take place.

This can include DNA testing



Test method 1:Acid Phosphatase Test: Acid Phosphatase is an enzyme present in many bodily fluids.


FORENSIC COLLECTION OF SEMENTest method 1:Acid Phosphatase Test: Acid Phosphatase is an enzyme present in many bodily fluids.

However, acid phosphatase is present in far greater amounts in seminal fluid (anywhere from 50-1000 times more concentrated in fluid from prostate gland) when compared to other bodily fluids.



Acid phosphatase reacts with certain reagents (e.g. Sodium napthylphosphate and certain dyes) to produce a purple color.




So the purple color indicates a positive test for semen.





Therefore, the acid phosphatase test is a(n) ________________ test for the presence of semen.





Therefore, the acid phosphatase test is a(n) ___presumptive____ test for the presence of semen.





Therefore, the acid phosphatase test is a(n) ___presumptive____ test for the presence of semen.https://www.youtube.com/watch?v=fdnOTMXiUW0

https://www.youtube.com/watch?v=fdnOTMXiUW0





Acid Phosphatase Test: Acid Phosphatase is an enzyme present in many bodily fluids.

A false positive can be given from:a. fruit & veggie juices b. vaginal fluid



A false positive can be given from:a. fruit & veggie juices b. vaginal fluid

However, the purple color will typically occur much sooner if it is from seminal fluid (1 min or less from semen; several min from other substances)



Test method 2: Look under microscope for presence of sperm.




Under 400x magnification if sperm are seen then (obviously) that is positive for semen.




Under 400x magnification if sperm are seen then (obviously) that is positive for semen.

However, there are some hurdles to seeing sperm:



Test method 2:


A) Fragile nature of sperm (easily destroyed)



Test method 2:



B) oligospermia: low sperm count



Test method 2:



B) oligospermia: low sperm count C) aspermia: The absence of sperm

(e.g. would be the case if the person had a vasectomy)



Test method 3:

PSA (Prostate Specific Antigen) (aka “p30”)



Test method 3:


This antigen is injected into a rabbit. Produces polyclonal antibodies (e.g. “anti-p30”)



Test method 3:


Place BOTH the antigen and antibodies intoThe two wells of a spot plate:



Test method 3:



FORENSIC COLLECTION OF SEMENTest method 3:


FORENSIC COLLECTION OF SEMENTest method 3:

Electrophoresis causes antigen and antibody to move towards each other.


FORENSIC COLLECTION OF SEMENTest method 3: Then a precipitant is formed in the middle (represented by dashes). This is a positive test for p30 (& thus for semen)


COLLECTION OF RAPE EVIDENCE



Absence of semen does not necessarily mean that no sexual assault occurred.



When a rape victim disrobes to submit evidence:

a) stand on a clean bed sheetb) also stand on clean paperWHY?




a) stand on a clean bed sheetb) also stand on clean paperWHY? A: to collect items of evidence that fall off during disrobing.




a) stand on a clean bed sheetb) also stand on clean paperWHY? A: to collect items of evidence that fall off during disrobing.c) shoes off before stepping on paper




Collect each disrobed item and bag separately (in paper bags) to avoid contamination




Items with possible seminal stains must be handled with great care




DNA evidence can be present in tiny amounts, even from the perspiration of the assailant.

Thus examiner must wear gloves




A physical exam must take place ASAP. A Rape Victim Collection Kit should be there as well. This includes everything from forms about the victims medical history to collection bags for evidence. (Pics on pp. 307-309 of textbook)



DNA testing

The increase in the sensitivity of DNA testing has lead to use of DNA analysis in:

a) Bite wounds on a victimb) Underwear of suspect



Vaginal contents are examined for evidence of sexual activity.



Vaginal contents are examined for evidence of sexual activity.

Non motile sperm can remain alive and viable for up to 3 days.



Buccal swabs will be collected for DNA analysis from:

a) The suspect of the sexual assaultb) Any with whom the victim may have had

consensual intercourse within past 72 hr.

immunoassy techniques, etc. ch. 8 serology. for review: antigen: a substance which, when put into a...

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