Lec12 Immunology methods

Question 1

Polyclonality of immune response

Answer 1

• in immune response get polyclonal expansion of B cells that undergo affinity maturation –> polyclonal antibodies may reaction to different epitopes/antigens/etc
• can isolate antibody produced from single B cell with single specificity and use as clinical tool

Question 2

How to make monoclonal antibodies

Answer 2

• immunize with HBsAg
• several weeks later isolate spleen cells
• fuse spleen cels with tumor cell line so grow indefinitely/spontaneously [hybridoma]
• select individual cells that make antibody of interest [for HBsAg]
• clone selected hybridoma
• can be made with any antigen
• can be chemically conjugated to enzymes that permit them to be detected in other assay

Question 3

peyers patch components

Answer 3

• mostly B cells

- some T, dendritic, macrophages

Question 4

what is a b cell hybridoma?

Answer 4

• fuse B cell with tumor cell so that will endlessly divide and grow spontaneously

Question 5

Steps for using ELISA to quantify HBsAg in serum?

Answer 5

Enzyme Linked ImmunoSorbent Assay: testing for quantity of HBsAg in serum

1. add monoclonal antibody specific for HBsAg to well
2. add serum [that may contain HBsAg which will thus bind to antibody]
3. Wash so only stuck stuff stays
4. add second enzyme-linked monoclonal antibody to HbsAg [to different epitope]
5. Add substrate that changes color proportional to amount of enzyme bound
6. Measure optical density to quantify amount of HBsAg present

Question 6

Steps for using ELISA to quantify antibody to HBsAg in serum [anti-HBsAg]

Answer 6

1. add HBsAg to well
2. add serum [that may contain antigen to HBsAg which will thus bind to HBsAg]
3. Wash so only stuck stuff stays
4. add second enzyme-linked monoclonal antibody to HbsAg [to different epitope]
5. Add substrate that changes color proportional to amount of enzyme bound
6. Measure optical density to quantify amount of HBsAg present