Question

can u please help me with question 8 part d. And question 9

8. In ELISA, a. Why did you need to wash the wells after each step? b. Why do you need to assay positive and negative control samples as well as your own experimental samples? c. Why are enzymes used in this immunoassay? d. What causes hook effects in ELISA assays? And what are the consequences ? 9. In counting cells under the microscope what do you need and why? Think in materials and reagents

Answer 1

ELISA is Enzyme linked immunosorbent assay. ELISA is used to detect and measure concentration of antigen or antibody in sample.

Q.8 a) In the ELISA after every time we need to perform washing. Washing is done to remove any nonspecific background caused due to unbound antibody or protein or antigen. So washing will remove such nonspecific effects.

b) Positive and negative controls are used to check false positive results. Positive control will be one which will definitely give results we are expecting. Negative control is some buffer or water used in experiment. E.g if our experimental sample provide similar result like positive control and negative control doesnot give same result this means there are no false positive results.

If our experimental sample provide similar result like positive control and negative control also give sampe result that means there are false positive result some other than actual experimental. May occur due to nonspecific background in media or some other component.

d) hook effect is caused due to presence of excess amount of antibodies bound with excess amount of antigend. The consequence of this hook effect is that it will lead to false positive results.

Q.9 For cell counting requirements are

Reagents a)cell culture medium

b)Trypan blue solution

c) 70 percent ethanol

d) Trypsin

Materials

I)Hemocytometer

II) Microscope

III)flask

IV)Protective clothing and glooves

Cell culture medium is used to grow the desired cells.

Trypan blue dye is used for straining cells. Dead cells retain this dye. Live cells donot retain they have intact cell membrane.

70 percent ethanol used to clean Hemocytometer and cover slip before use.

Trypsin is used to make uniform cell suspension as cell are in clump form.

Hemocytometer is cell counting device which is specifically designed for cell counting.

Microscope used to see grid lines in Hemocytometer to perform counting number of cells.

Protective clothing and glooves are used to protect from stains during experiment. One major reason is Trypan blue. Because trypan blue is toxic and carcinogenic so it is required to protect your hands,eyes and other body parts.