An affinity chromatography-gel filtration device for preparing thyroid microsomal antigen.
On the basis of conventional differential centrifugation for preparing crude thyroid microsomal antigen (TMAg), we have employed Sepharose 4B gel filtration and affinity chromatography separately to study the elution pattern in terms of absorbance and antigenic activity. The result indicates that thyroglobulin (TG) exists in two forms in crude TMAg, i.e., 'free TG' and 'membrane-bound TG'. TMAg is present in two forms in the eluate: (1) the TM fragment or TMAg polymer, which is produced at a higher rate and has greater antigenic activity, but which is less pure; (2) soluble TMAg, which is produced at a lower rate and has less antigenic activity, but which is more pure. We have developed an affinity chromatography-gel filtration (AC-GF) device which is a combination of affinity chromatography and a Sepharose 4B column. Sephadex G-50 is placed between the rubber stopper and Sepharose 4B in the GF column to ensure intactness of the entire system. With such a device, the AC removes the contaminated TG from TM homogenate, and allows the latter to pass directly from AC to GF for rechromatography. This device extracts the full advantages of both methods and each compensates for any deficiency of the other. Using this one-step procedure, one has the greatest chance of removing TG and obtaining TM fragments of TMAg polymers of higher antigenic activity, as well as separating small amounts of more purified soluble TMAg. Thus, the newly developed method meets the need of large quantities of TMAg for practical application, and at the same time the more purified preparations can be used for analytical purposes.
Wang
L
L
Department of Immunology, Tianjin Medical College, China.
Zheng
W F
WF
D001324:Autoantigens / Q000302:isolation & purification*; D002498:Centrifugation; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008861:Microsomes / Q000276:immunology; D013056:Spectrophotometry, Ultraviolet; D013961:Thyroid Gland / Q000276:immunology*
D001324:Autoantigens