Antigen-Antibody Interactions

Question 1

What is a hapten?

Answer 1

Small organic molecule that becomes part of an antigenic determinant only when coupled to a carrier molecule

Question 2

What is Ko?

Answer 2

The average intrinsic association constant, based on the ratio of [Ab-H] / [Ab]*[H]. Higher number = tighter binding

Question 3

What does heterogeneity of antibody refer to?

Answer 3

Number of binding sites which contribute to the total affinity.

Question 4

What is valence?

Answer 4

Number of sites on an Ig which interact with Ag. 10 for IgM, 2 or 4 for IgA. 2 for IgG.

Question 5

What is meant by specificity?

Answer 5

Antibodies are highly specific and preferentially react to the stimulus or Ag which induced them.

Question 6

How do antibodies influence solubility?

Answer 6

When Ab reacts with Ag, the complex precipitates out

Question 7

What is the equivalence point in a quantitative precipitin reaction?

Answer 7

Point at which antibody = antigen numbers, and there is no free antibody or antigen in the supernatant. Each antibody reacts with two antigens, and each antigen reacts with two different antibodies.

Question 8

Why does the highest weight of precipitant in the quantitative precipitin reaction happen when there is slightly more antigen than antibody?

Answer 8

Assuming the antigen is multivalent, only one end of an antibody needs to react with it to precipitate it out. So antibodies should be able to precipitate out more than one particle of antigen since they are divalent, assuming there are not many cases of multiple different antibodies binding the same antigen complex (i.e. albumin).

Question 9

What is an agar diffusion experiment?

Answer 9

Way to visualize the quantitative preciptin reaction in agar. Putting two wells of antigen and 1 well of antibodies, and there will be a line of equivalence where the average meetingpoint is.

Question 10

What is identity vs non-identity?

Answer 10

If the two antigen wells are the same identity as the antibody, the line will curve

If they are non-identity, there will be straight lines around the wells which will not interact in any way

Question 11

What is responsible for the partial identity interaction?

Assume Ag1 = 12, Ag2 = 23, and antibodies in well are anti-1 and anti12

Answer 11

If one of the antigens is 12, and the other one is 23, and antibodies placed in wells are anti-12 and anti 1, a very thick line will be between the ab and 12. However, a less thick line will be between ab and 23, since anti 1 will not react with 23. The excess anti-1 will continue the heavy line between 12 as a more like light, and will thus point towards the antigen of missing or only partial identity.

Question 12

How does Radial Immunodiffusion work?

Answer 12

Agar is filled with anti-gammachain antibodies. Standard curve is made with IgG antigen discs sitting on Agar diffusing out and reacting with agar making a ring. By plotting the log [Ag] vs ring diameter, a standard curve can be made to compare your own serum reaction on the agar to determine your IgG concentration.

Question 13

What are the antigenic determinants on A and B blood groups? What is the backbone?

Answer 13

A = PS backbone + galNAc
B = PS backbone + galactose
conjugated onto H substane via glycosyl transferases

Backbound is a glycoprotein called “H substance”

Question 14

What isoantibodies will be in circulation for blood types O and AB? What class are they and what do they cause if you have a donor mismatch?

Answer 14

O = anti-A and anti-B
AB = no isoantibodies (would be self-reactive)

IgM class, cause RBC agglutination

Question 15

What blood type is universal donor vs recipient?

Answer 15

O = universal donor (blood has no antigens)
AB = universal recipient (no isoantibodies to react with any blood coming in)

Question 16

How do you humanize mouse monoclonal antibodies?

Answer 16

Insert the gene for the human Fc region into the mouse Ab tumor gene which codes the correct Fv region.

Question 17

How does flow cytometry work?

Answer 17

Can quantify the number of cells in suspension and actually sort them by having a fluorescently tagged Ab directed towards a particular cell population (i.e. CD4+ or CD8+).

Question 18

How does a radioimmunoassay work?

Answer 18

Count the amount of drug / hormone by linking an antibody to radioactive iodine and putting it through a gammacounter. I.e. drug or hormone like vasopressin. Mainly used to detect if something is there at all.

Question 19

How do you measure antibody levels in serum via ELISA?

Answer 19

Put Ag at the bottom of a tube. Throw the unknown serum into the bottom of the tube, and then wash. Ab’s which bound the Ag will stick. Put in Anti-Ig which is enzyme-linked into the well, and wash it again. Now place substrate for that antibody and it will be converted, having a certain optic density. You can compare this to a standard curve for how much of the original antibody would be put in vs what the OD would be to determine the amount of Ab in the serum.

Question 20

How does a Western blot detect antibodies to specific microbial antigens? Use HIV components as an example?

Answer 20

Disrupt the HIV virus and run it in a gel, proteins will separate based on size. Take nitrocellulose paper and blot it on the gel to transfer protein. Put patient’s serum on nitrocellulose, then wash it. Add enzyme linked or radioactive anti-human-Ig, and scan for where human antibodies had bound to specific viral proteins. You can tell which HIV virus protein it stuck to based on how large the protein fragments were.