Detection of Antibody to M Protein of Measles Virus in Patients with Subacute Sclerosing Panencephalitis: A Comparative Study on Immunoprecipitation

Abstract

Consistent results have not been obtained yet on the presence of antibody to the M protein of measles virus in the sera of patients with subacute sclerosing panencephalitis (SSPE).We performed a comparative study on various immunoprecipitation systems which appeared in the literature and found that the difference in the composition of the solubilizing buffer produced a large variety of results on the immunoprecipitation.[35S] Methionine-labeled Vero cells infected with the Edmonston strain of measles virus were solubilized by 10 different buffers, and reacted with hyperimmune rabbit serum to whole virus, monospecific antisera to H, NP, and M proteins of the virus, normal adults' sera, and the sera from 16 SSPE patients .The immune complex was absorbed by protein A and both solubilization and precipitation rates were compared with each viral protein.Although viral proteins were solubilized by all buffers, the solubilization rate varied considerably . M protein was solubilized and was not coprecipitated nonspecifically with any of the other viral proteins.Purified protein A conjugated to Sepharose was preferable to Staphylococcus aureus for absorption of the immune complex since the latter absorbed both viral and host proteins nonspecifically. The precipitation rates of the viral proteins also varied according to the buffers.Better solubilization of the viral proteins seemed to reduce their rate of precipitation for which the presence of SDS may be responsible, and the presence of the protease inhibitors may also affect the results of immunoprecipitation.Detection of M protein in the immunoprecipitates was largely influenced by the kind of buffer used: some buffers could detect it clearly , but others could not defect it at all. Among the solubilizing buffers tested, Saleh's buffer (Virology 93: 369-376 (1979)), which contains 0.5% DOC and 0.5% Triton X-100, was most reliable for detection of the anti-M antibody in the rabbit serum, because it showed a high solubilization and high precipitation rates of viral proteins without nonspecific absorption by protein A or coprecipitation of M proteins with any of the other proteins.Using this buffer, we could definitely detect M proteins in the immunoprecipitates from the sera of all six healthy adults and 15 out of 16 patients with SSPE. It was found, however, that the amount of M proteins in SSPE patients was lower than that in healthy adults and varied considerably.

Humoral Immune Response in Dogs with Old Dog Encephalitis and Chronic Distemper Meningo-encephalitis

“…In conclusion, we have confirmed and extended the observations of Ohara et al (1985) that the methodology can influence the assessment of antibody responses to morbilliviruses by immunoprecipitation. The discrepancies between our results and those reported earlier (Hall et al, 1979b) in which the same sera and CSFs have been shown to precipitate all the antigens of CDV are not easily explained.…”

“…The discrepancies between our results and those reported earlier (Hall et al, 1979b) in which the same sera and CSFs have been shown to precipitate all the antigens of CDV are not easily explained. It is possible, assuming that RIPA buffer was used in the earlier study, that non-specific adsorption (Ohara et al, 1985) is the explanation for the different results. We have had the advantage of using monoclonal antibodies to better define the various bands observed in immunoprecipitations as representing CDV structural proteins.…”

“…It has recently been shown that the results obtained by immunoprecipitation tests are very dependent on the balance between the solubilization of the antigens and their ability to be precipitated in any specific buffer system. Ohara et al (1985), for example, demonstrated that the M protein of MV could be precipitated by sera and CSFs obtained from SSPE patients when some buffer systems were used, whereas in others no precipitation was observed. This led us to investigate precipitation in RIPA buffer ( Lamb et al, 1978) or Saleh buffer (Saleh et al, 1979).…”

“…This led us to investigate precipitation in RIPA buffer ( Lamb et al, 1978) or Saleh buffer (Saleh et al, 1979). The latter was declared most effective for precipitation of MV antigens with SSPE sera (Ohara et al, 1985). In each set of experiments we included SDS-PAGE analysis of the lysate in order to ascertain that all CDV antigens had been solubilized and were present in the lysate for precipitation.…”

Antibody and T cell responses to measles virus polypeptides and synthetic peptides

“…Norrby et al [35] further confirmed the presence of high levels of antibodies to M protein in atypical measles by both RIPA and CF tests. Ohara et al [36] pointed out that the level of M protein detected in RIPA depends on the buffer used. Variation due to the assay used may also explain why Hankins and Black [37] detected almost normal levels of anti-M antibodies in SSPE patients by Western blot.…”