Engineering topics

Rodkey, L. Scott

Publications and source records attributed to Rodkey, L. Scott.

6 records

NASA NTRS1988

Comparison of bioseparation methods for microgravity experiments

The efficiency of the 1-g version of the continuous-flow electrophoresis (CFE) system flown on Space Shuttle missions is compared with the efficiency of a commercial CFE for separating living cells (human kidney, liver, and pituitary-gland cells and T-lymphocytes). In addition, the CFE system and a reciprocal isoelectric focusing (RIEF) system are compared with respect to protein pyrification efficiency. Correlations were made among electrophoretic mobilities (EPMs), secretory functions of cells, and input sample concentrations. A significant reduction in mean and range EPM was observed when input sample concentrations exceeded a low threshohold. This effect was not observed in microgravity experiments conducted at sample concentrations three times greater than the threshold for the controls. Comparison of CFE and RIEF methods showed that there are apparent advantages for each method depending on the product. For example, RIEF purification of urokinase removed more protein impurities, but focused the enzyme at a pH different than the enzyme's known isoelectric point.

Morrison, Dennis R.↗

NASA NTRS1988

Ionic binding properties of carrier ampholytes

Radioactive ampholytes were synthesized with specific activity of 638 microCi/g. These were used in studies of ampholyte binding to target proteins under nonionic conditions. These radioactive ampholytes bound to target proteins but were dissociable in sodium chloride solutions with dissociation occurring in a concentration dependent way. The ampholytes could be dissociated from target molecules using excess unlabelled ampholytes synthesized in the laboratory as well as commercial ampholytes. Radioactive ampholytes were bound to target proteins with different isoelectric points, and the bound ampholytes were eluted and analyzed by recycling isoelectric focusing. The results showed that acidic proteins bound basic ampholytes and basic proteins bound acidic ampholytes.

Rodkey, L. Scott↗

NASA NTRS1988

Isoelectric focusing analysis of antibody clonotype changes occurring during immune responses using immobilized pH gradients

Serum was collected from rabbits at 2-day intervals following a single injection with tetanus toxoid or at weekly intervals following multiple injections with Micrococcus lysodeikticus cell walls. These sera were analyzed for the presence of individual clonotypes of specific antitetanus or antimicrococcal antibodies by isoelectric focusing in immobilized pH gradients with added carrier ampholytes followed by affinity immunoblotting. The affinity immunoblots obtained clearly defined both the rapid disappearance and late appearance of distinct subsets of antibody clonotypes during the response. These data demonstrate the application of affinity immunoblotting combined with immobilized pH gradients for detecting the subtle changes in specific antibody clonotype patterns which occur during an immune response.

Knisley, Keith A.↗

NASA NTRS1987

Isoelectric focusing-affinity immunoblot analysis of mouse monoclonal antibodies to the four human IgG subclasses

Isoelectric focusing (IEF)/affinity immunoblotting and enzyme-linked immunosorbent assay (ELISA) were used for parallel analysis of murine monoclonal antihuman IgG-subclass antisera (MoAbs). Coomassie Blue-stained protein bands in the pH region 5.5-8.0 were shown to be murine IgG by direct blotting onto nitrocellulose followed by detection with conjugated antimouse IgG. Use of IgG myeloma antigen-coated nitrocellulose in the IEF-affinity immunoblot allowed detection of the charge microheterogeneity of MoAbs. The MoAb group contained one to five major dense bands flanked by up to four minor fainter bands, all with pIs ranging from 6.1 to 7.8. Semiquantitative estimates of binding specificity in the IEF-affinity blot compared well with cross-reactivity data obtained from a quantitative ELISA.

Hamilton, Robert G.↗

NASA NTRS1987

Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

The quality control of murine hybridoma secretory products has been performed using two approaches for isoelectric focusing affinity immunoblot analysis: (1) a method in which antigen-coated nitrocellulose is placed on top of an acrylamide gel containing isoelectrically focused ascites to bind the antigen specific monoclonal antibody; and (2) a method in which focused ascite proteins were passively blotted onto nitrocellulose and specific monoclonal antibodies were detected with enzyme-conjugated antigen. Analysis by both methods of batches of ascites containing antihuman IgG antibodies that were produced by six hybridomas permitted effective monitoring of immunoreactive antibodies for pI microheterogeneity.

Hamilton, Robert G.↗

NASA NTRS1986

Affinity immunoblotting - High resolution isoelectric focusing analysis of antibody clonotype distribution

A sensitive and specific method is proposed for the analysis of specific antibody clonotype changes occurring during an immune response and for comparing multiple sera for antibody clonotype similarities. Polyclonal serum antibodies separated by isoelectric focusing (IEF) were analyzed by an affinity immunoblotting method using antigen-coated nitrocellulose membranes. Antibodies present on the surface of the acrylamide gels following IEF bind the antigen on the nitrocellulose when the coated nitrocellulose is laid over the gels. The technique has been used to analyze Ig clonotypes specific for five protein antigens and two carbohydrate antigens. Optimal antigen concentrations for coating the nitrocellulose membranes were found to range from 10-100 microgram/ml.

Knisley, Keith A.↗