(Exam 2) MLS 420 Immunoassay Methods part 1

immunology

The study of the body's immune system and its function and disorders.

serology

The study of serum and antigen and antibody interactions in the serum.

immuno

An immune response that causes the body to generate antibodies.

assay

A test.

immunoassay

This uses antibodies and antigens to detect an analyte.

serology

We use immunoassays because infectious diseases are quicker than growing organisms so we look for Antibodies through _____ rather than culturing the virus.

analyte

This is what we are trying to detect in the patient and the reaction is measurable.

antibody

Reagent ______ production for test kits use monoclonal versus polyclonal antibodies.

analyte interference

This is when something alters the concentration of the analyte being measured in the patient sample

analyte dependent

This is the interring substance reacts with the reagent antibody or antigen impacting the ability of analyte to react with the reagent.

high dose hook effect

This is when the analyte is in excess, mainly impacts the step of heterogenous sandwich assays, and the high concentration saturates the reagent capture and detecting antibody.

decrease, dilute

The high dose hooks effect has false ______ with the signal at high concentration. If this is suspected in the patient, we need to _____ the patient antigen.

biotin

This interfering substance is:

-Water soluble vitamin B7

-Impacts assays that are based on streptavidin and biotin

-Competitive assays lead to false positives

-Non-competitive assays lead to false negatives.

complement

This interfering substance is:

-can lead to false negatives/positives because binds to Fc portion of antibodies that block the analyte bind sites

-Heat to 56 C in water bath to inactivate

-Non-treponemal syphilis testing in RPR.

heterophile antibodies

This interfering substance is:

-endogenous antibody that forms in the body

-Form against one antigen but can react with similar antigens from different species

-Can react with assay reagent antibodies/antigens

-May lead to false elevated or falsely low results.

autoantibodies

This interference substance is:

-Causes false positives and negatives by binding to bridging or blocking binding sites on reagents or patient analyte

-PEG can be used to remove autoantibodies.

treatment with antibody therapy

This interfering substance is:

-Use of therapeutic antibodies results in human anti-mouse antibodies that are a subset of heterophil antibodies and can interfere with reagent antibodies

-False positive or negative concentrations

treatment with hormone-binding proteins

This interfering substance is:

-Alter concentrations of hormones binding up or removing the hormone in the specimen

-Causes false negatives

cross-reacting substance

This interfering substance is:

-substances of similar structure to the analyte of interest with analyte specificity concern and false positive concentrations

-Metbaolism of the analyte cyclosporine and cortisol that could cross-react with the assay.

analyte-independent

The interfering substance does not react with the reagents.

pre-analytical issues

This is when the wrong anticoagulant is used for collection or improper time of collection for hormones.

lipemia

This blocks binding sites, interfere with detection method, and lipoclear or fasting specimens need to be used.

hemolysis

This releases material/compounds from lysed RBCs that may bind to analyte or block binding sites on reagent and cross-reactivity of released compounds can interfere with detection methods and redraw the specimen.

zone of equivalence

This is the amount of multivalent antigen and antibody approximately equal and this is where we want the best results for greatest lattice formation and highest test sensitivity.

postzone

This is when the antigen is in excess.

prozone

This is when the antibody is in excess.

postzone

This is the insufficient amount of antibody to form cross-links, lack of cross linking results in minimal immune complex formation, there are false negatives of analyte being detected, and redraw patient in 1-2 weeks.

prozone

This is the insuffient amount of reagent antigen to form immune complexes with no cross linking of epitopes on antigens to form lattices, false negative test results, and need to serial dilute patient sample until equivalence is obtained.

cross-reactivity

When an antibody formed towards one antigen reacts with a different antigen than to which it was formed against, very easily broken because not lock and key.

law of mass action

Free reactants are in equilibrium with bound reactants, rate of forward reaction equals rate of backward reaction that has the high sensitivity and high affinity/avidity.

reversible

In the law of mass action, the Ag-AB reaction are _____ due to non-covalent bonds.

screening test

Presumptive test with high sensitivity, rule in or out possibility of disease, and does not diagnose disease.

confirmatory tests

Test only those that were positive using the screening test, high specificity, and diagnosis of disease/condition.

monoclonal antibody

Highly specific for a single epitope on a multivalent antigen made from a single B cell clone and are all identical.

monoclonal

_____ antibodies reduce false positive due to specificity and lack the ability to crosslink multivalent antigens.

polyclonal antibody

Different multivalent antibodies to multiple epitopes on a multivalent antigen

polyclonal

_____ antibody decreased specificity and more cross-reactivity, increased diagnostic sensitivity, ability to cross-link multivalent antigen, injected in rabbits or mice.

affinity

Attractive force between one Fab piece of an antibody and a single epitope on an antigen

avidity

The sum of forces binding multivalent antibody to multivalent antigen with the number of Fab pieces and number of identical epitopes—how tightly they bond together

ionic strength

Charge on cells, antibodies, and antigens and most are negatively charged

shielding/zeta potential

This is when antigens and antibodies repel each other due to their charge and this can prevent immune complex formation if not taken into account.

normal saline, LISS

For shielding/zeta potential, use solutions that neutralize shielding like ______ or ______.

pH

This affects the overall change of the reaction and extremes of this can cause conformation changes and a neutral _____ is optimum.

reaction time

This is the rate of Ag-Ab reactions to form immune complexes.

22 C

What is the temperature for IgM antibody binding?

37 C

What is the temperature for IgG antibody binding?

heterogenous

In ______ assays there is a separation step.

homogenous

In _____ assays there is no separation step.

heterogenous

This removes unbound labeled reagent and unbound patient sample from the assay.

adsorption, precipitation, solid phase

What are the three separation methods for heterogenous assays?

adsorption

This heterogenous assay separation method is with charcoal coated with dextran or resin traps with unbound labeled reagent or using the magnetic particles in the Ag-Ab complexes absorbed.

precipitation

This heterogenous assay separation method is when Ag-ab complexes are precipitated out of solution through acidification.

solid phase

This heterogenous assay separation method is a known antigen or antibody is attached to a solid surface and the patient analyte binds to the reagent becoming immobilized.

homogenous

This is no removal of the unbound labeled reagent and unbound patient sample from the assay.

unlabeled

This assay is the visual assessment of reaction with no tag on reagent antigen or antibody that requires a high concentration of the Ag and Ab, like staph latex, tube ABO.

direct

The ____ unlabeled assay is known antibody detects patient antigen and is involved in the initial ag-ab complex.

indirect

The _____ unlabeled assay is reagent antibody not involved in the initial ag-ab complex formation.

labeled

This is not visual rely on detecting the ta on the reagent antigen or antibody when it binds to the patient analyte, more sensitive than the unlabeled assays, and detects smaller concentration of the analyte in the patient sample.

enzyme, chemiluminescent, fluorescent

What are the threee labeled assays?

enzyme

What type of labeled tests are these:

-Horseradish peroxidase

-Alkaline phosphatase

-Beta-D-galactosidase

-G6P dehydrogenase.

chemiluminsescent

What type of labeled tests are these:

-Luminol

-acridinium esters

-Ruthenium oxalate

-Nitrophenyl oxalates

fluorescent

What type of labeled tests are these:

-Fluorescein

-Rhodamine

-Phycoerythrin

-Europium

competitive

This type of assay is when there is patientt sample and labeled reagent antigen added in the same step.

indirectly/inversely

Competitive assays are ______ proportional to analyte quantity because the less of the bound labeled reagent antigen detected the more antigen present in patient sample.

non-competitive

This assay has high levels of sensitivity and specificity that utilizes two different reagent antibodies of one labeled and one unlabeled and patient sample is added in a separate step that labeled.

directly

The non-competitive assay is _____ proportional to analyte quantity because the more labeled reagent detected then the more analyte present in patient sample.

precipitation curve

This is the precipitation and agglutination based immunoassay that has ag and ab based assays influences by the amount of antigen and antibody present.

lattice formation

This is when the binding of antigens and antibodies happens to form stable complexes.

agglutination

This is an unlabeled immunoassay that relies on affinity and avidity for complex formation that has lattice formation important in visualizing the immune complexes.

sensitization, lattice formation, enhancement

Agglutination unlabeled immunoassay has the three steps of what:

sensitization

In agglutination, there is the _____ step that has antigen and antibodies starting to bind.

lattice formation

In agglutination, there is _____ where multivalent antibodies bind to epitopes on different antigen crosslinking the antigens.

enhancement

In agglutination, there is _____ that is not always done but is done to improve the binding of antigen and antibodies by reducing the charges like using LISS.

direct, indirect, reverse passive, agglutination inhibition

What are the four types of agglutination that can cause cross-linking?

quantitative

This is when serial dilutions to find end point titer to determine concentration

qualitative

This is when the presence or absence of the antibody or positive/negative results.

active (direct)

This type of agglutination is when the antigen is a natural part of the cell’s surface, a combination of insoluble antigen a with soluble antibody, can be used to detect antibody or antigens, and used for infectious diseases or blood banking.

hemagglutination

Active/direct agglutination is in tube ABO Rh typing that is also called ______ because it relies on the naturally occurring antigens on the RBCs.

passive (indirect)

This agglutination method is when antigens attached to a carrier molecule (antigens do not naturally occur on cell surface), detecting antibody in patient sample, and if the specific antibodies are present, they will cross-link the carrier molecules that create visible clumps.

reverse passive (indirect)

This agglutination method is when antigens do not naturally occur on cell surface, antibody is bound to the carrier molecule, and this is to detect antigen in patient sample.

inhibition

This agglutination method when detecting the antigen, usually a hapten, in the patient, this is a competition between known antigen and unknown in patient

no agglutination

In agglutination tests, a positive reaction will show ______.

agglutination

In agglutination tests, a negative reaction will show _____.

precipitation

This method is an unlabeled assay method that depends on numbers of epitopes on an antigen and number of Fab regions on antibodies.

soluble antibody

In the precipitation method, _____ binds to soluble antigen forming an immune complex that precipitates.

large

Detection of precipitation occurs when the immune ag-ab complexes become so _____ they precipitate out of solution.

nephelometry, flocculation, immunofixation electrophoresis

What are the three precipitation methods?

quantitative

Nephlometry is a _____ procedure that is the optimal method of measuring precipitation.

nephelometry

This measures the amount of light scattered by the precipitated immune complexes that depends on the number and size of complexes.

angle

Nephelmoetry measures light scattered at a specific ____ that can be anywhere from 10-90 degrees.

wavelength, directions

When monochromatic (incident) light interacts with a particle in solution, the scattered light is the same ____ as the incident light and it is scattered in all _____.

increase (directly proportional)

As the amount of ag-ab complex formation increases in nephelometry, then the amount of light scattered will _____.

automated, lipemia/hemolysis

An advantage of nephelometry is that it is _____, simple, many applications, sensitive and quantitative. A disadvantage is that it is high cost, and interfering substances of _____.

flocculation

This is a specific type of precipitation that reaction occurs over a narrow range of antigen concentrations and is used to detect the antibodies in the patient sample.

antigen

Flocculation uses a soluble reagent ____ that consists of fine particles and precipitate only the fine particles.

microscope

Flocculation is only visible with a _____ unless there is a carrier molecule used and this is susceptible to changes in pH, temperature, concentration, and rotation time.

flocculation

What are these examples of: traditional non-treponemal test with cardiolipin as the fine soluble antigen used in two syphilis tests.