Diagnostic Testing

Various analytical approaches, including enzyme inhibition assays and immunoassays, have been described for Bik determination.

For enzyme inhibition assays, urine is the preferred specimen [4]. Interestingly, Bik can be measured by the inhibition of trypsin in urine but not in plasma. Urinary Bik analysis may also be performed by antibody staining, latex agglutination, and radioimmunoassay (RIA) [4]. Despite the analytical approach used, all Bik forms are measured together. The enzyme inhibition method involves adding known amounts of trypsin to the specimen and monitoring trypsin inhibition. Trypsin activity is assessed by detection of by-products from a cleavable substrate. Dipstick methods are available for the rapid detection of trypsin inhibitors in urine [15, 17-19].

Immunoassays for Bik, based on polyclonal antibodies (pAb), are affected by cross-reaction with Tamm-Horsfall protein (THP). This problem can lead to the generation of false positive results in cases of proteinuria [14]. In contrast, immunoassays that utilize plasma suffer from cross-reactivity to IaI [23]. The cross-reactivity with THP is due to complexed N-linked glycan, whereas cross-reactivity with IaI is due to bound Bik [14]. Cross-reaction with a-1-glycoprotein (AGP) also does not appear to be a significant factor in blood.

Monoclonal antibodies generated with purified Uri eliminated cross-reaction to THP and IaI in urine and plasma, respectively. Antibodies directed at the N-linked glycan allowed measurement of Bik in blood without IaI cross-reactivity. Antibodies directed at the peptide allowed measurement of urinary Bik without THP cross-reactivity. These antibodies do not cross-react with aprotinin. These antibodies allow estimating the IaI family in blood.

Despite its lack of specificity, Bik determination has been shown to correlate well (p < 0.01) with other indexes of inflammation, including C-reactive protein (CRP), WBC count, and erythrocyte sedimentation rate (ESR) [4]. The urine strip is an alternative to a blood CRP measurement. The Bik test was more predictive of upper respiratory and urinary tract infections as well as kidney diseases. Furthermore, it was more sensitive to bacterial and viral infection vs CRP. Because plasma proteins, that is CRP, are not cleared and circulate until hepatic metabolism, urinary Bik appears to be a better predictor of an abnormal WBC, ESR, and neutrophil degranulation.

Receiver operator curves (ROC) have demonstrated the superiority of urinary Bik vs CRP in predicting vascular inflammation, viral and bacterial infection. Bik determination by immunoassay is better able to separate patients with inflammation, that is fewer false positives and higher correlation to CRP and WBC, vs enzyme inhibition methods. Urinary IL-8 activity is also increased in acute and active inflammatory conditions and correlates positively with inflammatory markers.

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