Comparison of three PCR assays for the evaluation of interferon-beta biological activity in patients with multiple sclerosis.
BACKGROUND
The gene expression of the myxovirus-resistant protein A (MxA) gene is a sensitive measure of the biological response of therapeutically applied interferon-beta (IFNbeta) and of its reduced bioavailability due to inhibiting factors such as IFNbeta-induced neutralizing antibodies (NAbs).


METHODS
We compared three methods for MxA mRNA quantification in 826 peripheral blood mononuclear cell (PBMC) samples obtained from patients with multiple sclerosis (MS). MxA mRNA measurements were performed using quantitative-competitive (qc)-PCR, real time-PCR, and the new semi-quantitative (sq)-PCR assay (MxA IBRIDOGEN).


RESULTS
According to the treatment status (untreated samples versus NAb-negative treated samples), real time-PCR gave the highest specificity (93%). Slightly lower specificities were obtained with qc-PCR and sq-PCR (both 91%). qc-PCR showed the highest sensitivity (97%) compared with both real time-PCR (94%) and sq-PCR (95%). A positive correlation was found between qc-PCR and real time-PCR measurements (rspearman=0.776; p<0.0001), which also showed 90% agreement based on a statistically calculated threshold. Likewise, sq-PCR evaluations showed 84% and 79% agreement with qc-PCR and real time-PCR measurements, respectively. In addition, we showed a concordance of 89% between three sq-PCR kits.


CONCLUSIONS
All three methods displayed high specificity for MxA gene expression analysis, allowing the detection of patients in whom IFNbeta did not have any biological action. qc-PCR and real time-PCR are both useful during clinical trials demanding quantitative data of biological activity, whereas sq-PCR could prove useful for routine screening purposes because it is easy to perform and can be done in not specialized laboratories.
Gilli
Francesca
F
Regional Multiple Sclerosis Center (CReSM) & Clinical Neurobiology, Orbassano, S. Luigi Gonzaga Hospital, Torino, Italy. sclerosi.multipla@sanluigi.piemonte.it
Marnetto
Fabiana
F
Stefanuto
Guglielmo
G
Rinaldi
Valentina
V
Farinazzo
Federica
F
Malucchi
Simona
S
Capobianco
Marco
M
Caldano
Marzia
M
Sala
Arianna
A
Bertolotto
Antonio
A
D000906:Antibodies / Q000097:blood; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D016899:Interferon-beta / Q000627:therapeutic use*; D009103:Multiple Sclerosis / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D064547:Myxovirus Resistance Proteins; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D016896:Treatment Outcome
D000906:Antibodies; C579071:MX1 protein, human; D064547:Myxovirus Resistance Proteins; D012333:RNA, Messenger; D016899:Interferon-beta; D019204:GTP-Binding Proteins