tion-inhibition, neutralization, and complementfixation tests were performed on sera of rabbits inoculated with Venezuelan equine encephalomyelitis (VEE) virus in combination with Freund's adjuvants and in Hank's salt solution. This study, indicated that the complete adjuvants (i.e., with mycobacteria) considerably increased the antibody response to VEE virus. MIycobacterium butyricum (M. smegmatis) appeared to be more effective than MI. tuberculosis H37Ra. In the absence of mycobacteria, the response was much less pronounced. Paper electrophoretic
1. The control of precision and accuracy in a clinical chemistry laboratory can be achieved only by knowledgeable and careful use of "concurrent standardization" together with the use of control sera in routine analysis. That is, within each series of analyses, both reference standards and control sera should be included as the only means by which the ultimate in reliability can be achieved.
2. The use of standard curves or calibration factors can be tolerated only when the pure reference standard is difficult to obtain, prepare, or maintain. In such cases, however, the curves or factors should be rechecked frequently and a control serum analyzed concurrently with the unknown.
3. The manufacturer or supplier should refrain from advertising his product as simultaneously a standard and a control. Secondly, he should ascertain the shelf-life of his product under the most rigorous conditions likely to be encountered and indicate this information on the label of the individual bottle or on the container of that lot of control sera. Finally, he should make every effort to reduce the cost of his product in order to make it practical for the clinical chemistry laboratory to establish and maintain an adequate control system.
Summary
The effect of pH, time, temperature, and buffer systems on solutions of fluorescein isothiocyanate and its protein conjugates was examined fluorometrically. The fluorescence of the isothiocyanate and of the protein conjugate was maximal at about pH 8.7 and pH 10.7, respectively. The stability of their fluorescence above pH 7 was affected adversely by increases in pH or temperature. The protein conjugate, however, showed maximum stability at pH 10.5 and above. The type of buffer (carbonate, phosphate, borate, Tris and barbiturate) did not affect the fluorescence of the free dye significantly. On the other hand, increasing the molarity of the buffer caused a decrease in stability of fluorescence of the free dye but did not seriously affect the fluorescence of the conjugate.
The absorbance of fluorescein isothiocyanate and its conjugates increased with increasing pH. In 1 N NaOH, the effect of pH variation on absorbance was minimized.
By increasing the pH or temperature of the reaction mixture during conjugation, fluorescein isothiocyanate reacted more readily with the protein. Conditions may be selected to obtain the desired degree of label with short conjugation periods. Conjugation of a bovine anti-Brucella abortus globulin sample for 30 min at pH 9.45 and room temperature was as effective as conjugating at pH 8.75 for 18 hr at 5°C. No apparent loss of biologic activity as the result of conjugation was observed.
The binding of fluorescein isothiocyanate to protein resulted in a loss of fluorescence efficiency, the extent of loss being dependent upon the pH of measurement.
Existing procedures in fluorescent antibody studies should be reevaluated on the basis of the variables discussed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.