Long description
In immunoelectrophoresis against fresh mouse serum, a single precipitin line is obtained in the beta-1 region representing native C3. Against serum containing partly activated C3, a precipitin line is obtained which extends from the beta-1 into the alpha-2 region, demonstrating a gradient. In old serum containing totally activated C3 a single precipitin line in the alpha-2 region is obtained. Antisera to C3c cab also react with the fragments C3b, C3bi and smaller fragments, since they all carry antigenic determinants of the C3c domain. The product does not react with any other proteins component of mouse serum or plasma. The biotin immunoconjugate to mouse C3c is used to determine the presence and pattern of C3 in tissue lesions using immunohistochemical staining techniques. Locally deposited immune complexes in tissue usually contain complement, pointing to activation of the classical pathway. Complement activation in vivo implies active disease and may contribute to the elicitation of the pathogenesis and he extent of tissue destruction. In ELISA and Western blotting to identify mouse C3c in serum or other body fluids. As a second step an avidin or streptavidin conjugate of the user’s choice has to be used. This immunoconjugate is not pre-diluted. The optimum working dilution of each conjugate should be established by titration before being used. Excess labelled antibody must be avoided because it may cause high unspecific background staining and interfere with the specific signal. Working dilutions for histochemical and cytochemical use are usually between 1:50 and 1:200; In ELISA and comparable nonprecipitating antibody-binding assays between 1:500 and 1:1,000.
Antibody come from
C3 is the most abundant complement protein in mouse serum. Its biological function strongly resembles that of C3 in man and other laboratory animal species. It has a central role in the activation system being common to both pathways. Activation of C3 is achieved by very specific limited proteolysis resulting in the release of a number of degradation fragments. The anaphylotoxin C3a promotes smooth muscle contraction and increases vascular permeability: the large C3b fragment is involved in binding to the complement activator and can be interact with specific receptors to allow efficient clearance of the activating cell or particle; degradation fragments of C3b (C3bi, C3c, C3dg C3d) are important in receptor binding and clearance mechanisms, in virus neutralization and possibly in the immune response. The antiserum is raised against C3c, which is the major fragment resulting from C3 cleavage by C3 convertase and factor I. It is composed of an intact beta chain bound to two fragments of the alpha chain. Consequently the antiserum reacts with both native and activated C3. It may also react with the fragments C3b, C3bi and C3dg, since they all carry antigenic epitopes of the C3c domain. C3c is isolated and purified from pooled normal mouse serum. Freund’s complete adjuvant is used in the first step of the immunization procedure.
Storage
The lyophilized conjugate is shipped at ambient temperature and may be stored at +4°C; prolonged storage at or below -20°C. It is reconstituted by adding 1 ml sterile distilled water, spun down to remove insoluble particles, divided into small aliquots, frozen and stored at or below -20°C. Prior to use, an aliquot is thawed slowly at ambient temperature, spun down again and used to prepare working dilutions by adding sterile phosphate buffered saline (PBS, pH 7.2). Repeated thawing and freezing should be avoided. Working dilutions should be stored at +4°C, not refrozen, and preferably used the same day. If a slight precipitation occurs upon storage, this should be removed by centrifugation. It will not affect the performance of the immunoconjugate. Lyophilized at +4° C--at least 10 years. Reconstituted at or below -20° C--3-5 years. Reconstituted at +4° C--7 days
Technique
anti mouse, Antibody, Mouse, anti, Goat, antibody to, anti mouse antibodies, antibodies against human proteins, antibodies for, antibody Conjugates, goats, mouses
