Antibodies adsorbed

Appropriate controls should always be run with any immunocytochemical procedure. Controls may include omitting the primary antibody, substituting pre-immune serum, normal serum, or normal IgG for the primary antibody, adsorbing the primary antibody against the antigen, or immunostaining with an unrelated antibody. 

Immunoassays can be accomplished in systems where an unknown antigen (rather than antibody) competes with labeled antigen for specific antibody adsorbed onto the surface of a solid phase (such as the lower inner wall of a plastic tube). This method can be used effectively for measuring serum antibody levels (Clinical Assays Division of Travenol Laboratories, 620 Memorial Drive, Cambridge, Massachusetts, 02139) the more highly purified the labeled antigen, the less background noise there is likely to be in the assay system. 

For in vitro labeling, 0.2 n-Ci pH]farnesyl pyrophosphate (Du Pont -New England Nuclear) and 2 ng of the CaaX-tagged protein is added to 20 il of reticulocyte lysate (Promega) as source of farnesyl transferase. The mixture is adjusted to contain 5 mM MgC. To carry out the full modification, 1 jxl of dog pancreatic microsomes (Promega), must also be added. The mixture is then incubated for 30 min at 37 °C. Labeled proteins can be recovered from the reaction mixture by immunoprecipitation with an appropriate antibody adsorbed to protein A-Sepharose, and analyzed by SDS-PAGE and fluorography (Wiedlocha etal., 1992). 

Both phases are then diluted to 1 ml with PBS (0 "C), followed by immunoprecipitation with antibodies adsorbed to protein A-Sepharose. The precipitated material is analyzed with SDS-PAGE and tiuorography. 

Limit of Detection. Detection limits of approximately 10p,g/mL (lOmg/L) have been attained with routine nephelometric and turbidimetric methods this limit has been lowered by 10 to 100 fold using antibodies adsorbed onto latex particles. RID methods are able to detect down to a minimum of 10 to 20p.g/mL the detection limits have been lowered even further by using special low-level plates (Calbiochem-Behring Corp., La Jolla, Cahf.). Methods using labeled antibodies (e.g., RIA, EIA, chemiluminescence) are very sensitive and can measure levels as low as nanograms per milliliter level. 

Butler JE, Ni L, Nessler R, Joshi KS, Suter M, Rosenberg B, Chang J, Brown WR, Cantarero LA. The physical and functional behavior of capture antibodies adsorbed on polystyrene. J Iimnunol Methods 1992 150 77-90. 

JH Peterman, PJ Tarcha, VP Chu, JE Butler. The immunochemistry of sandwich ELISAs. IV. A comparison of the antigen capture capacity of antibodies adsorbed or covalently attached to polystyrene. J Immunol Meth 111 271, 1988. 

An additional control has been performed for antibody. It is known that the clone 4 and clone 5 bind to the Mb molecule simultaneously (27). If antibody is adsorbed to the PDMS surface initially, introduced into the solution, some of the clone 4 antibody adsorbed to the PDMS surface will remain active and will bind Mb from solution. The bound Mb is thus surface-confined without interacting with the PDMS surface and in an orientation that the clone 5... 

Antibody adsorbed on immunoadsorbent was eluted with 5 M guanidine HCl, and after dialysis precipitin curves were performed with anti-BSA. 

Multiplex detection using UPT was performed by Corstjens et al. [36] for the simultaneous detection of human antibodies against human immunodeficiency virus, Myobacterium tuberculosis and hepatitis C virus by using different antibodies adsorbed on adjacent test lines and one type of UCNPs. Hampl et al. [38] used two different types of particles (a thulium oxysulfide phosphor with blue emission and an erbium oxysulfide phosphor with green emission, both after being excited at 980 nm) for simultaneous detection of mouse IgG and ovalbumin. 

The question as to why, in the case of bovine albumin films, the amount of adsorbed antibodies increases with the number of underlying monolayers is of importance. First, it seems probable that the antibody molecules are piled up on top of each other in the thick layers of antibodies adsorbed on four double layers of bovine albumin. If this is true, the same process might take place in other cases. It has often been reported that undiluted immune sera gave much thicker layers of specifically adsorbed material than diluted sera. Bateman, Calkins and Chambers (19) found increments in thickness of 200 A and 60 A with undiluted and diluted serum, respectively. These variations have usually been assumed to result from a different orientation of the adsorbed antibody molecule which in the case of the rabbit antibody has approximately the dimensions of 40 X 270 A. Great variations in the thickness of adsorbed layers of antibodies have also been observed by electron microscopy. For instance, Anderson and Stanley (24) reported an adsorbed thickness of 225 A of rabbit antibodies on the surface of tobacco mosaic virus molecules but they also have observed much smaller increments. 

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