Avian antibodies applied in particle enhanced turbidimetric immunoassay: Developement of serum/plasma calprotectin immunoassay and its clinical performance as a marker for bacterial infections

  • Datum:
  • Plats: Robergssalen, ingång 40, Akademiska Sjukhuset,, Uppsala
  • Doktorand: Nilsen, Tom
  • Om avhandlingen
  • Arrangör: Klinisk kemi
  • Kontaktperson: Nilsen, Tom
  • Disputation

Disputation

Calprotectin is a cytosolic protein in the granulocytes, consisting of S100A8 and S100A9. On the site of inflammation, the neutrophils release the cytosol as an inflammatory response. The circulating calprotectin concentration increases and can therefore be used as marker for neutrophil activation and inflammation.

To raise specific antibodies, it is crucial to immunize with pure calprotectin antigen. We purified calprotectin from human granulocytes by ion-exchange chromatography, dialysed it towards saline and concentrated it to required levels, suited for immunisation of the hens. The purified antigen solutions were assigned concentration values by the Biuret method and the purity was checked by SDS PAGE and size exclusion chromatography. The yield was approximately 2 mg purified antigens per unit of 450 ml blood.

A prototype calprotectin particle enhanced turbidimetric immunoassay was developed from the purified antigen and the affinity purified antibodies. The antigen was spiked into PBS to prepare calibrators and controls. The antibodies were coated to latex particles to prepare immunoparticles. The performance of the immunoassay was technically tested on a clinical chemistry analyser. LoQ, antigen excess, linearity, precision and calibration stability met the pre-set criteria.

In the production process of immunoparticles there are several factors affecting the performance of the assay. Investigating eight factors applying a Taguchi L12 screening, we experienced that conductivity and pH of conjugate buffer, coating grade and conductivity of dialysis buffer II affected the sensitivity and antigen excess the most.

The assay was used to measure clinical samples. Serum samples from elderly people aged 70+ were collected. Only patients with no infections were included to establish a reference interval for this patient group. The reference interval in serum was 0.3 mg/L to 2.5 mg/L for both genders. Furthermore, the plasma calprotectin immunoassay was tested clinically on critically ill patients to assess the ability of plasma calprotectin as an early marker for detection of bacterial infections. It showed promising results. Calprotectin was a better predictive marker for sepsis than procalcitonin and white blood cell count. Because some patients with an inflammation have low numbers of granulocytes, we examined the correlation between white blood cell count and the calprotectin levels in a group of patients with an inflammation. There was a weak correlation between the number of white blood cells and calprotectin concentration.