Enzyme-linked immunosorbent modified with

PAMAM dendrimers or modified PAMAM dendrimers (with oxiamine or sulfhydryl surface functionalities) exclusive of one another. These antisera recognize den-drimers in ELISA (enzyme-linked immunosorbent assays), dot blots and Western blots. The immunogenicity of dendrimer-protein conjugates has implications for therapeutic use of dendrimers as vaccines and we anticipate that antidendrimer antibodies will have applications in patterning and assembling nanostructures containing dendrimers. 

Eig. 5. Several endpoint detection methods were compared for the detection of immuno-polymerase chain reaction (IPCR) amplificate from a direct IPCR (Fig. 3A) of mouse-IgG. Although all IPCR/DNA-detection combinations were able to improve the detection limit of a comparable enzyme-linked immunosorbent assays (ELISA) of approximately 10 amol IgG in a 30-fL sample volume, several differences were observed in actual detection limit, and the linearity of the concentration/signal ratio dependent on the DNA quantification was applied. Best results were obtained for PCR-ELISA (see also Fig. 6) in combination with fluorescence- or chemiluminescence-generating substrates (b, c). With photometric substrates (d) or gel electrophoresis and subsequent spot densitometry (a), a 10-fold decrease in sensitivity was observed. In addition to the more sigmoid curve in gel electrophoresis, an enhanced overall error of 20% compared to 13% in PCR-ELISA was observed for two independent assays. The simple addition of a double-strand sensitive intercalation marker to the PCR-amplificate and measurement in a fluorescence spectrometer further decreased sensitivity (e) and appears therefore to be unsuited for IPCR amplificate quantification. (Figure modified according to references 37 and 65.)... 

Approximately 2.5-5 X 10 cells were seeded into 24-well plates and allowed to adhere overnight. Dulbecco s modified Eagle s media (phenol red free) with supplements were added to the cells (Mitrovic et ah, 1994). The cells received no treatment, received interferon-y (IFN-y) (500 U/ml) and interleukin-1/8 (IL-1/3) (300 U/ml), or were pretreated with pentoxifylline (PTX) (1 mg/ml for 24 hr) before stimulation with IFN-y and IL-lj3. After 4-7 days of stimulation, cell supernatants were removed and stored at —40°C. Samples were thawed, and soluble TNF-Rl and TNF-R2 levels were assayed using enzyme-linked immunosorbent assay kits [human soluble tumor necrosis factor receptor type 1 (sTNF-Rl) and sTNF-R2 Quantikine kits, R D Systems, Minneapolis, MN]. For the sTNF-Rl kit the minimum detectable dose is 1 pg/ral. For the TNF-R2 kit the minimum detectable dose is 0.5 pg/ml. PTX was provided courtesy of Hoechst-Roussel Pharmaceuticals (Somerville, NJ). Values are expressed as means SD. 

Another technique, the enzyme-linked immunosorbent assay (ELISA), combines the specificity of antibodies with the sensitivity of an enzyme assay. The ELISA can be performed in a variety of combinations that involve either a specific antibody or the total cellular protein immobilized on a solid support, such as the wells of a plastic microplate. In one version of the method, the sandwich ELISA, the primary antibody is bound to the wells. When a mixture of proteins is added, the protein of interest binds to the antibody, and other proteins are washed away. A second labeled antibody, specific to a different epitope on the protein, is added, and the amoruit of signal is proportional to the amoimt of the particular protein in the sample. The method can be modified to detect specific antibodies in a mixture by using their antigen as the immobilized bait. ELlSAs also have the advantage of being able to be performed in 96-weU plates so many samples can be analyzed in one experiment. 

Experiment I. In a time-course experiment, mucosal PGE production and phospholipid fatty acid profile were assessed at d 0,4,8,12, and 16 of dietary treatment in formula-fed and naturally reared piglets (n = 5 piglets per time per dietary treatment). Mucosal cells were scraped from proximal ends of the small intestine and frozen at -80°C for later lipid analysis. Lipids were extracted by a modified Folch procedure (15). Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were separated by thin-layer chromatography (16), and fatty acids in each phospholipid fraction were analyzed by gas chromatography. For eicosanoid measures, fresh mucosal tissue was incubated in Kreb s Ringer bicarbonate buffer as described previously (17). PGE2 was extracted from the incubation media with ethyl acetate and quantified using a competitive enzyme-linked immunosorbent assay (Cayman Chemical, Ann Arbor, MI). 

Albumin activated with glutaraldehyde has been coupled with catalase and the product used in a thermometric enzyme-linked immunosorbent assay. Folic acid has been used to modify albumin in a carbodi-imide-mediated reaction. The folate-derivative was then coupled to agarose and used for affinity chromatography. Albumin alkylated with l-[ C]iodoacetamide has been used in the development of a new method for preparing affinity chromatography matrices. ... 

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