Immunology & Serology - Lecture - 7 - Antigen-Antibody Reactions - Part 3 - Complete

IgM

(Differences between IgM and IgG in Lattice Formation)

What is that strong agglutinin that looks like a star?

IgM

(Differences between IgM and IgG in Lattice Formation)

Manifests a pentameric structure so it looks like a star.

IgM, 35 nm

(Differences between IgM and IgG in Lattice Formation)

It is a strong agglutinin because of its diameter about _____.

IgG

(Differences between IgM and IgG in Lattice Formation)

It is small in size

IgG

(Differences between IgM and IgG in Lattice Formation)

Its flexibility is restricted or limited at the hinge region

IgG

(Differences between IgM and IgG in Lattice Formation)

It may largely prohibit multivalent binding, thus it cannot bridge the distance between particles (e.g., from one RBC to another RBC).

Controlling the surface charge

It is a way of enhancing lattice formation by decreasing the buffer's ionic strength through the use of low ionic strength saline (LISS)

Neutralizing the surface charge

It is a way of enhancing lattice formation by adding 5-30% albumin. This permits erythrocytes to come into contact easily and approach each other more closely

Increasing viscosity

It is a way of enhancing lattice formation by adding polyethylene glycol (PEG) or dextran. These agents reduce the water of hydration around cells and allow them to come into a closer proximity for Ab to join together.

Using enzymes

It is a way of enhancing lattice formation by using bromelain (from pineapple), trypsin (from the stomach of pancreas of pigs), and ficin (from figs). These enzymes reduce the RBC surface charge through chemical group cleavage and decreasing hydration

Agitating centrifuging

It is a way of enhancing lattice formation by providing physical means to increase cell-cell contact and thus accentuate agglutination

Altering the temperature or pH

It is a way of enhancing lattice formation by adjusting the temperature to where immunoglobulins agglutinate best

5-30% Albumin

What is added for the enhancement of lattice formation by neutralizing the surface charge? Which permits erythrocytes to come into contact easily and approach each other more closely.

Polyethylene glycol or Dextran

What is added for the enhancement of lattice formation by increasing viscosity?

These agents reduce the water of hydration around cells and allow them to come into a closer proximity for Ab to join together.

Bromelain

What pineapple enzyme can be added for the enhancement of lattice formation by using enzymes?

Papain

What papaya enzyme can be added for the enhancement of lattice formation by using enzymes?

Trypsin

What enzyme from pig stomach or pancreas can be added for the enhancement of lattice formation by using enzymes?

Ficin

What fig enzyme can be added for the enhancement of lattice formation by using enzymes?

30-37°C

When adjusting the temperature or pH to enhance lattice formation, at what temperature do IgG class antibodies best react?

4-27

When adjusting the temperature or pH to enhance lattice formation, at what temperature do IgM class antibodies best react?

Room temperature

When adjusting the temperature or pH to enhance lattice formation, at what temperature do naturally occurring antibodies against the ABO blood groups (IgM Class) best react?

20-25°C

What temperature is room temperature?

37°C

When adjusting the temperature or pH to enhance lattice formation, at what temperature do antibodies to other human groups (IgG class) best react?

pH 6.5-7.5

When adjusting the temperature or pH to enhance lattice formation, at what pH is optimal antigen-antibody reaction achieved?

Direct Agglutination

This type of agglutination reaction can only take place when antigens

Direct Agglutination

The best example for this type of agglutination reaction is the usage of bacterial antigens used to test for the presence of unknown patient antbodies

Direct Agglutination

What type of agglutination reaction are the Widal test and Hemagglutination test?

Widal test

This direction agglutination test is a rapid screening test for typhoid fever, which uses the following antigens:

-O antigen (Somatic)

H antigen (Flagellar)

Fourfold increase in antibody titer over time

What is considered a significant result in the Widal test?

Hemagglutination

It is a direction agglutination test that involves erythrocytes

Hemagglutination

The best example for this type of direction agglutination test is the ABO blood group typing of human RBCs

IgM type antisera

What is used in hemagglutination test to determine the presence of absence of the A and B antigens since there are only two antigens under the ABO blood group?

IgM can span the distance from one RBC to another RBC as compared to IgG which is smaller

Why is IgM type antisera used in hemagglutination reaction?

Passive agglutination

What type of agglutination reaction utilizes particles that are coated with antigens not normally found on their surface?

Indirect agglutination

Other name for Passive agglutination

Passive agglutination

What type of agglutination reaction uses latex, gelatin, erythrocytes, and silicates?

Latex, gelatin, erythrocytes, silicates

If these carrier particles are found on the antigen, the type of agglutination is passive/indirect.

7 um

Erythrocyte size in Passive agglutination

0.8 um or less

Latex particle size in Passive agglutination

Latex particles

Most immunoserology test kits which utilize agglutination use these particles because these particles provide advantage of uniformity, stability, and consistency.

Latex particles

These particles in Passive agglutination are used in place of RBCs

Red blood cells

This particle, used in Passive agglutination, has a tendency that cross-reactivity with heterophile antibodies can happen, especially in nonhuman cells

Latex

This particle, used in Passive agglutination, is inexpensive, relatively stable, and shows no cross-reactivity with other antibodies.

Rheumatoid factor, Antinuclear antibody in SLE, Antibodies to Group A Streptococcus antigens, Trichinella spiralis, Treponema pallidum, and viruses like Cytomegalovirus, Rubella, Varicella zoster virus, and HIV-1/HIV

Passive agglutination can be used for the detection of...

Reverse passive agglutination

In this type of agglutination reaction, rather an antigen, an antibody is attached to a carrier particle.

Reverse passive agglutination

Example of this type of agglutination reaction are latex particles and their antibodies are attached on their surfaces. The Fab regions are facing outward.

Must be reactive, Active sites must face outward

In Reverse passive agglutination, what are the prerequisites for the antibody attached to the carrier particle?

Fab

What antibody region in Reverse passive agglutination should be facing outward?

Reverse passive agglutination

This type of agglutination reaction is also used to measure therapeutic drugs, hormones, and plasma protein levels (haptoglobin and CRP).

Reverse passive agglutination

This type of agglutination reaction is used to detect soluble antigens in urine, spinal fluid, and serum.

A patient sample containing the suspected antigen is allowed to react with the antibody-coated latex particles

What is the principle of reverse passive agglutination?

Latex particles

In the laboratory, most of the carrier particles utilized come as _______ because they can provide a rapid reaction

Passive agglutination

In this type of agglutination reaction, antigen is attached to the carrier particle and agglutination occurs if a patient antibody is present.

Reverse passive agglutination

In this type of agglutination reaction, antibody is attached to the carrier particle, and agglutination occurs if patient antigen is present.

Agglutination inhibition

This type of agglutination reaction is based on competition between soluble antigens and particulate for limited antibody-combining sites

Lack of agglutination

Positive reaction in Agglutination inhibition

Agglutination inhibition

This type of agglutination reaction usually involves haptens that are complexed to proteins. The hapten-protein conjugate is then attached to a carrier particle

Agglutination inhibition

This type of agglutination reaction is a highly sensitive assay capable of detecting small quantities of antigen

Agglutination inhibition

This type of agglutination reaction is used for the detection of illicit drugs (drugs for abuse): cocaine and heroin.

Agglutination inhibition

In this type of agglutination reaction, reagent antibody is added to the patient sample then incubated. If patient Ag is present, antigen-antibody combination results.

Positive = No agglutination

Negative = Agglutination

Positive

In Agglutination inhibition, when antigen-coated latex particles are added, no agglutination occurs, which is a (Positive/Negative) test

Negative

In Agglutination inhibition, if no patient antigen is there, the reagent antibody combines with latex particles, and agglutination results which is a (Positive/Negative) test.

Hemagglutination inhibition

This type of agglutination reaction utilizes a similar principle to agglutination inhibition, except that the indicator particles are erythrocytes.

Hemagglutination inhibition

This type of agglutination reaction is used to detect Abs to viruses, such as rubella, mumps, measles, influenza, parainfluenza, HBV, herpesvirus, RSV, and adenovirus.

Erythrocytes

This reagent in Hemagglutination inhibition has naturally occurring viral receptors.

Spontaneous agglutination

In Hemagglutination inhibition, if there is presence of the virus, what is the result?

This is because the virus particles link the RBCs together

Agglutination reaction is inhibited

In Hemagglutination inhibition, if there is presence of patient antibody, what is the result?

Coagglutination

This type of agglutination reaction utilizes bacteria as the inert particles to which Ab is attached.

Staphylococcus aureus

It is the most frequently used bacteria because it has a protein A on its outer surface, which naturally adsorbs the Fc portion of antibody molecules

Staphylococcus aureus

What is the most frequently used bacteria in coagglutination?

Upwards

What direction are the active sites in coaggulination facing?

Streptococci, Neisseria meningitidis, Neisseria gonorrhoeae, Vibrio cholerae O139, Haemophilus influenzae

Coagglutination reagents are utilized in the detection and identification of the following bacteria

Advantage

Advantage or Disadvantage of Coagglutination

They are more refractory to changes in ionic strength.

Disadvantage

Advantage or Disadvantage of Coagglutination

Coagglutination has a difficult to read reaction because bacteria are not colored.

Advantage

Advantage or Disadvantage of Coagglutination

Coagglutination is highly specific.

Disadvantage

Advantage or Disadvantage of Coagglutination

It is not sensitive for detecting small Ag quantities.

Advantage

Advantage or Disadvantage of Coagglutination

Coagglutination particles have greater stability than latex particles.

Staphylococcus aureus

In Coagglutination, this bacteria's particles nonspecifically bind the Fc portion of immunoglobulin molecules.

When reagent antibody is used, combination with soluble patient antigen produces visible agglutination reaction.

Coomb's test

Is a method that detects non-agglutinating antibody through coupling with a second antibody.

Anti-human globulin test

Other name for Coomb's test

Anti-human globulin

It is a key component of the Coomb's test

Green

What is the color of Anti-human globulin in the Coomb's test?

Animals, Hybridoma technology

What can be used to make Anti-human globulin?

Fc

In Antiglobulin-mediated agglutination, anti-human globulin reacts with what portion of the human antibody attached to erythrocytes?

Anti-human globulin

What reagent in Antiglobulin-mediated agglutination functions similarly to IgM?

Anti-human globulin

This reagent in Antiglobulin-mediated agglutination causes agglutination because it is able to bridge the distance between cells that IgG alone cannot do.

Proportional

In Antiglobulin-mediated agglutination, what is relationship of the strength of the reaction to the amount of antibody coating the RBCs?

Direct antiglobulin test

This type of Antiglobulin-mediated agglutination is used to show in vivo attachment of antibody or complement to an individual's erythrocytes.

Direct antiglobulin test

This type of Antiglobulin-mediated agglutination is direct because it erythrocytes are tested directly as they come from the body.

Direct antiglobulin test

In this type of Antiglobulin-mediated agglutination, if IgG or complement is present on RBCs, the AHG (Coomb's reagent) is able to bridge the gap between RBCs and cause a visible agglutination

Indirect antiglobulin test

This type of Antiglobulin-mediated agglutination is used to establish the presence of a particular antibody in a patient or it can be used to type patient RBCs for specific blood group antigens.

Indirect antiglobulin test

This type of Antiglobulin-mediated agglutination follows a 2-step process.

First

Which step in the Indirect antiglobulin test is when the antibody is allowed to be combined with washed RBCs at 37° C then carefully washed to remove any unbound or free antibody floating in the sample?

Second

Which step in the Indirect antiglobulin test involves the addition of anti-human globulin, then a visible reaction occurs where Ab has been specifically bound?

Indirect antiglobulin test

This type of Antiglobulin-mediated agglutination is most often used to check for the presence of clinically significant alloantibody in patient serum when performing compatibility testing for a blood transfusion

Failure to wash, Improper centrifugation, Failure to add test serum or AHG, Use of expired reagents or those that have not been properly stored

Sources of Error in Performing the Coomb's test

Rapid, Relatively sensitive, Sample does not need to be viable, Simple to perform, Require no expensive equipment, Extremely portable

Advantages of agglutination reactions

Extremely portable

What advantage of agglutination reactions is because the tests are conducted on cards, tubes, and microtiter plates?