The specificity of the binding is due to specific chemical constitution of each antibody. The antigenic determinant or epitope is recognized by the paratope of the antibody, situated at the variable region of the polypeptide chain. The variable region in turn has hyper-variable regions which are unique amino acid sequences in each antibody. Antigens are bound to antibodies through weak and noncovalent interactions such as electrostatic interactions, hydrogen bonds, Van der Waals forces, and hydrophobic interactions,In an antibody, the Fab (fragment, antigen-binding) region is formed from the amino-terminal end of both the light and heavy chains of the immunoglobulin polypeptide. This region called V (variable) domain is composed of amino acid sequences that define each type of antibody and their binding affinity to an antigen. The combined sequence of variable light chain (VL) and variable heavy chain (VH) creates three hypervariable regions (HV1, HV2, and HV3). In VL these are roughly from residues 28 to 35, from 49 to 59, and from 92 to 103, respectively. HV3 is the most variable part. Thus these regions are the paratope, the binding site of antigen.

the attachment may help

These are all excellent questions, Mr. Shey, that only an inquisitive student would dare to ask.  There are probably different explanations for each phenomenon.

1) Why do we need to block in immunological assays (WB, ELISA..etc)? 

When an antibody source is serum, there may be a million different antibody specificities in 10 microliters.  By chance, some percentage of these antibodies will have physicochemical properties that include capacity to interact with (usually) hydrophobic surfaces.  This is not a physiologically meaningful binding; it’s really an artifact of the system, which may even be mediated by the Fc region of or perhaps carbohydrate on the antibody molecule.  Blocking is an attempt to saturate such potential binding sites on the solid phase surface (or possibly to other macromolecules derived from the sample that bound to the solid phase) so that such artifactual binding does not contribute to assay background.

2)  Why do we sometimes have pre-immune sera reacting to antigens?

There could be several explanations for this. One reason could be a reflection of the same phenomenon as in the previous description --  just the coincidental presence of some irrelevant immunoglobulin that  has affinity for the test antigen perhaps through its Fc region or even a polyspecific antibody  in which a portion of its Fab has some affinity for a region of the test antigen.  This may or may not have immunological significance.  The more interesting explanation is that the preimmune repertoire includes a population of antibodies that have meaningful affinity for the antigen and, as B cell Ig receptors, will  ultimately mediate enhanced Ab response when immunogen is introduced.   This could actually reflect prior exposure to an antigen similar to the immunogen.

3)  Why do we have cross-reactivity?

Here you’re probably imaging that there could be some overlap in the epitopic structure of the test antigen with that of another molecule.  Often, however, it is difficult to provide a simple structural explanation for such phenomena, so invoking unknown secondary or tertiary structural similarities between the two antigens may be imagined.   There are also well-documented cases in which a “cross-reacting” antigen is dissimilar from the cognate antigen, but its capacity to bind to the same (monoclonal) antibody can be explained by the relatively large size of the paratope (see Dr. Abed’s description of Ab structure) and the possibility that epitopes on the cognate antigen and the “X-reacting” antigen are binding to different or partially overlapping regions on the antibody.

But overall, Robert, non-specific binding in the world of antibodies is a reflection of the huge heterogeneity in the circulating immune repertoire, its high concentration, and the reality that there may be many structural ways for molecules to interact with each other. Use of blocking reagents, adding detergents to the reaction, using dilute antibody preps and short incubation times are all attempts to minimize what are usually considered (sometimes incorrectly) assay artifacts.

Dr. Rubin, that was amazing how you have described your answers. It felt like listening to a class lecture.

Dr. Rubin 

A possible good informative answer to the question.

Appreciable !