Question

What steps can we take in order to create a lateral flow qualitative system for detecting blood using monoclonal and polyclonal antibodies? i.e starting with a sandwich ELISA

Answer 1

The steps for lateral flow qualitative system for detecting blood using monoclonal and polyclonal antibodies are as follows:

1)Add the primary antibody to the wells of the microtiter plate.

2)The antibody gets attached to the plastic present at the bottom of the plate through hydrophobic interactions.

3)Wash the microtiter plate with a buffer to remove the unbound excess primary antibody from the microtiter plate.

4)Add a blocking protiein like albumin or protein casein to block the non specific sites on the antibody molecule.

5)Add a known amount of antigen to the primary antibody in the plate. This antigen will bind to the antibody binding site on the primary antibody layered in the microtiter plate.

6)Add the secondary polyclonal antibody that contains an attached enzyme.

7)Add the colorless substrate antigen called as chromogen which is converted by the attached enzyme to a colored product.

8)The amount and the intensity of color produced due to the action of the enzyme is directly proportional to the captured antigen.

9)In the lateral flow assay, take the pad with the slide and add the sample that should be analyzed. In this situation it is blood.

10)The pad with the slide contains a coating of the primary antibody against the blood antigen.

11)Add the blood sample containing the antigen on the slide in the lateral assay system.

12)The antigen in blood will bind to the primary monoclonal antibody coated on the slide plate first strip

13) Then, then the sample reaches the secondary polyclonal antibody coated with the gold particles leading to the formation of the antigen-perimary antibody-seconday gold coated antibodies complex. This helps in the detection of the antigen present in the blood. This antigen can be used for the diagnosis of flu, viral infection or bacterial infections.

14)The indication of a color change indicates that the antigen is present in the blood sample with the binding of the primary and gold coated secondary antibody. If the color is not formed in the test region then it means that the antigen against which the primary antibody is coated on the plate is not present in the blood sample. The antigen present in the blood is different.