EIA procedures/Cryoglobulins/Strep testing

Double antibody sandwhich

If an antigen-antibody complex is present the enzyme labeled antibody will attach.

The amount of product is directly proportional to the concentration of patient antigen.

Indirect ELA

Detection of antibody

the antigen coated to a solid surface is detected in two stages or layers.

Another enzyme labeled antibody is added to the tube. If a protein (antibody) - antigen complex is present the labeled antibody will attach.

The amount of product is directly proportional to the concentration of protein (antibody)

Competitive binding

The sample antigen and the labeled antigen compete to bind to the available capture antibodies

Indirectly proportional

Non competitive binding

antibodies are immobilized and the patient's sample is added. Unbound components are washed away before a labeled antibody is added to bind the analyte. A final substrate is added to produce a color change that is measured

Directly proportional

EIA testing

Uses specific anti human IgG or IgM

No serial dilutions are needed

Enzymes:horseradish peroxidase, glucose oxidase, alkaline phosphatase

Rocky mountain spotted fever

Rickettsia ricketsii, deer tick, rash present on palms, soles, spares face

Treated with doxycycline

Marine typhus endemic thyphus

Rickettsia typhi, Tick, rash only present 50% of the time, spares the palms

Treated with tetracyclines, chlorophenicol

Pregnancy test EIA

Detects hCG in urine or blood

Mouse anti hCG labeled with colloidal gold

+ve = goat anti hCG binds mouse

-ve = goat anti mouse binds mouse (ref line)

HCG Human chorionic gonadotropin

Glycoprotein possessing two subunits

Alpha similar to FSH, LH, TSH

Beta unique for hCG

Produced by trophoblastic cells of placenta

Increased hCG

First trimester

Higher in multiple births

Predict pregnancy

Decreased hCG

Ectopic pregnancy, lower hCG than normal intrauterine pregnancy for the same gestational age

HCG false negative

Drug interference, bacteria, or low specigic weight

Borrelia burgdorferi

Spirochete, white tailed deer, ixodes tick, IgM

Rash:bullseye, Bells palsy, multiple organs

Chronic disseminated stage

HTLV Human T cell lymphotrophic viruses

Retrovirus, STD, blood, breast milk

HTLV I/ HTLV II

Hairy cell leukemia, T cells are target, causing T cell malignancy

P24 capsid also on HIV

Lyme disease false positive

Borrelia burgdorferi

False = other spirochetes, ANA, RA, infectious mono, rocky mountain spotted fever

Helicobacter pylori antibodies

Peptic ulcer

Organism weakens coating of stomach and duodenum, neutralize gastric acid

Urease/mucinase = damage lining

Urea breath test

Test CO2 level byproduct from bacteria urease

ACLA anti cardiolipin antibodies

Antibodies attach to platelet membranes = thrombosis, thrombocytopenia, recurrent fetal loss

Present in antiphospholipid syndrome

Reacts with negatively charge phospholipids, detects antibodies to cardiolipin and beta2 glycoprotein

ACLA false positive

Transient in syphilis temporary

Beta 2 glycoprotein

More specific than. ACLA for detection of cardiolipin antibodies

Celiac disease

Inflammation of small intestines = villous atrophy

Malabsorption of nutrients

Markers = Gliadin, tTG, endomycelial IgA

Gliadin

Breakdown product of gluten

Increase indicates failure of diet

IgA and IgG mucosal lining

tTG

Tissue transglutaminase

Enzyme responsible for cross linking proteins modifies gliadin

Endomycelial antigens forms a complex with gliadin = initiate immune response

IgA and IgG

Endomycelial IgA

Antibody to damage formed in the intestinal lining

IFA procedure using monkey esophagus

Cryglobulins

Abnormal proteins that precipitate when cooled and dissolve when heated, auto immune type reaction

Cryoglobulin procedure

Keep at 37C after collection

Spin and separate

Measure quantity at 4C remaining at room temp

Check for precipitation 24, 48, 72

Spun, resuspended in cold saline and electrophoresed = gamma region

Type I cryoglobulin

Monoclonal peak

IgG / IgM

Multiple myeloma, lymphomas, leukemias, Waldenstroms macroglobulinemia

Type II cryoglobulin

Mixed peaks, raised gamma region with monoclonal peak

Rheumatoid arthritis, Sjogrens syndrome

2 or more Ig, one is monoclonal

Type III cryoglobulin

Polyclonal, raised gamma region, no monoclonal peak

SLE, infections (hepatitis, IM, CMV)

Cryoglobulin symptoms

Raynauds phenomenom, vascular purpura, bleeding tendicies, cold induced urticaria

The temperature precipitation occurs is MORE important than the amount of precipitate

Group A streptococcus

Group A and Strep pyogenes

Throat and skin infection

Streptolysin O

DNAse B

Rheumatic fever

Can follow strep pharyngitis

Results in endocarditis

Acute glomerulonephritis

Cab follow pharyngitis or pyodermal infection

Edema, hematuria, elevated BUN, WBC, RBC and cast in urine

ASO

Neutralization procedure; antigen that possesses a biologic activity that ends on the addition of antibody which neutralizes the antigen, preventing the activity

Serum + SOR (streptolysin O reagent)

Sheep RBCs

Spin and read for hemolysis

Last tube with no hemolysis is end point = +ve

Streptolysin O

Inactive in oxidized state, lytic in reduced

Body produce antibody

<85 todd units

Preschool and adults

<170 todd units

School age

Rapid antigen test

Latex particles with Strep O

Look for agglutination = +ve