What is an antibody?
Antibodies are host proteins produced in response to the presence of foreign bodies, infectious organisms and their toxic products. They are synthesized primarily by plasma cells and circulate throughout the blood and lymph where they bind specifically to foreign molecules (antigens) and speed its removal from the host.

What are antibodies used for?
The interaction of an antibody with an antigen forms the basis of all immunochemical techniques. Antibodies are produced to identify specific antigens. Their affinity to specific antigens aid in learning more about peptides and proteins. Some applications include:

- Quantification: Radioimmunoassay and enzyme immunoassay.
- Localization: Immunostaining via avidin/streptavidin.
- Neutralization of peptides and other antigens.
- Precipitation of peptides and other antigens.

How are they produced?
Antibodies are produced by plasma cells. The host's response to an antigenic presence can be characterized as two responses: the primary response and the secondary response. The primary response is a relatively weak response to the initial antigenic exposure. A series of specialized events during the primary response prepare the host for subsequent exposure to the same antigen. When the antigen is reintroduced, the secondary response is relatively more rapid and intense. To fully optimize antibody production, the peptide/antigen must be prepared in a way to induce the strongest and most appropriate response. Since most peptides are too small to induce an adequate response, the peptides are normally conjugated to protein carriers (such as KLH, thyroglobulins, BSA, etc.). This larger complex boosts the response. Antibodies harvesting usually takes place within six months.

How to determine neutralization amounts for peptide/antibodies?
To determine the amount of peptide needed to neutralize 1 ml of neat, undiluted antiserum: 1ml of antiserum contains 10mg of IgG. Among the 10mg total IgG, only 1% (under estimate) is against the specific antigen, therefore: (10mg)(1%)= 0.1mg or 100 mg of specific IgG against the antigen.

Molecular weight of IgG=150,000 daltons, therefore, 100mg of IgG/150,000= 0.00066mmol or 0.66nmol 0.66nmol of antibody can neutralize twice the amount of peptide or 1.32nmol of specific peptide. Weight of peptide needed to neutralize = (1.32nmol)(M.W. of peptid)=X ng of peptide needed.

To determine the amount of antibody needed to neutralize 1mg of peptide: 1mg of peptide/MW of peptide=X mmol (X times 1000nmol)1000 nmol of peptide needs 1000/2 nmol of specific IgG to be neutralized 1000/2 nmol of IgG= (1000/2 nmol)(MW of IgG)=Y ng of specific IgG. Since Y ng is the minimum needed, we recommend an overdose amount to be determined by end user.

Will an antibody cross-react with a different species not listed in the catalog?
An antibody may cross react with peptide of a different species. Our antibodies are raised against a specific synthetic peptide sequence and if the sequence of another species is identical or highly similar to the sequence of our synthetic peptide, the possibility of recognition is high.
For peptide sequences, please check our website, Bachem catalog, or call your nearest Bachem sales office. In the case of unestablished cross reactivity of a sequence from an untested species, we cannot guarantee recognition by the antibody. However, we do offer cross reactivity testing upon request.

What is the shelf life?
All the RIA kits have an expiration date of 6 weeks after preparation which is the expiration date of the tracer (¹²⁵I-peptide). The other components such as RIA buffer, goat anti-rabbit IgG (GARGG), normal rabbit serum (NRS), antibody, and standard are stable for 6 months from the day of receipt when stored at 2-8°C. Once rehydrated, there is no guarantee on any of these components. Two weeks is the expected cutoff point for viability after rehydration.
All the EIA kits have an expiration date of 4 months from shipping date. Lyophilized antisera are guaranteed for 1 year from shipping date at -20°C.

What is the dynamic range/standard curve?
For RIA kits, we have three ranges: 0.1-64pg/tube, 1-128 pg/tube, and 10-1280 pg/tube. For EIA kits, we have two ranges: 0-10 ng/ml and 0-25 ng/ml. For your specific kit, please check your datasheet.

What is the assay time?
For RIA and RIA kits, extraction-free - 3 days (2 overnight incubation) (not including extraction procedure). For EIA kit - 4.5 hours (not including extraction procedure). For EIA kits, extraction-free - overnight due to incubation time. For staining kits - 3.5 hours to 2 days.

What type of sample can be measured but does not require extraction?
Sample extraction is strongly recommended for all RIA and EIA kits. For the extraction-free kits, if the peptide level in the sample to be measured is lower than the sensitivity of the kit, the extraction procedure may still be necessary to concentrate the sample.

Can components from different lots be combined?
Each lot is specifically formulated to give optimal sensitivity. Therefore, we do not recommend combining components with different lot numbers. In addition, we cannot guarantee the performance of our products when used outside of our protocol.

Can the diluted antisera for RIA or the antibody in RIA or EIA kits be used for staining?
The diluted antisera for RIA and the antibody for RIA or EIA kits are too diluted for staining purpose. They should not be used for staining.

What is the dilution for immunohistochemistry antisera?
For immunofluorescence staining, the suggested starting dilution is 1:200. For PAP or ABC method, it is 1:500 to 1:1000.