Antigen coated multiwell plates

Tests that can be carried out on 96-well plates coated vrith antigen either as protein or as cell monolayers are ideal for these initial screens, bound anti- 

IncnlMte hybridoma supernatants with antigen-coated plates or cell monolayers 

Detect bound antibodies with 125-lodine labelled or enzyme labelled secondaiy antibodies 

For a first screen we use antibodies to mouse or rat F(ab )2 since these reagents will detect all antibody classes and subclasses. With a successful fusion the wells will contain more than one hybridoma colony and in some cases a number of colonies will be present. To determine if one or more of the colonies is secreting specific antibody, colonies are picked under the microscope using a Pasteur pipette and plated in DMEM-20% FCS into individual wells of a 24-well plate. The cells are allowed to grow to near confluence before re-testing for the presence of specific antibody. Additional tests are performed at this time to confirm the specificity of the selected antibodies. 

Preparation of antigen coated plates for screening culture supernatants 

---

Assay using antigen coated PS or PVC coated multiwell plates... 

Assay using antigen coated PS or PVC coated multiwell plates 15 Assay using live or fixed adherent cells grown in multiwell plates 16 Ligand-binding assays for anti-receptor antibcxlies 17... 

Transfer 50-pL aliquots of the mixtures to the antibody or antigen-coated PVC multiwell plate so that each well contains 1-4 x 104 cpm of radiolabel. 

These are useful for mapping epitopes, determining whether or not two antibodies crossreact, and for comparing antibody affinities. They are the basis of quantitative assays (RIA) and use multiwell plates coated either with antigen or antibody, as described in Section 2.3. above. The principle is to either ... 

To determine antigen-binding and H202-degrading properties of the b-Ab/ SA/b-catalase conjugate, use the plastic wells of 24-well Multiwell cultural plates (Nunc) coated with an antigen (see Note 3). To control the specificity of binding, use albumin-coated wells. 

---