Test performance, CalproLab (ALP)

Note: All design verification studies were performed on faeces extract samples (diluted 1:100) or EDTA plasma samples (diluted 1:20), using the ELISA procedure described in the kit package insert.

Inter-assay precision, faeces extracts

Mean results from three different laboratories, each testing two different kit lots: six samples were tested a total of 10 times over five days:

Concentration (ng/mL)	Concentration (mg/kg)	%CV
6.4	32.1	13
34.1	171	6.0
73.7	368	4.8
117	583	6.1
243	1215	6.8
395	1977	7.9

Intra-assay precision, faeces extracts

Mean results from three different laboratories, each testing two kit lots: six samples were tested with 10 replicates in one run:

Concentration (ng/mL)	Concentration (mg/kg)	%CV
5.7	28.7	6.7
34.5	173	3.6
77.1	385	4.1
118	592	4.0
242	1210	4.2
393	1966	5.0

Intra-assay precision, EDTA plasma

Tested using one kit lot; six samples were diluted 1:20 and tested with 10 replicates in one run:

Concentration (ng/mL)	Concentration (x 20)	%CV
8.3	166	3.8
37	740	4.7
58	1160	2.9
68	1360	3.5
149	2980	3.2
183	3660	10

Recovery

Faeces: 85 – 105%; tested with faecal extract spiked with purified Calprotectin at five different levels.

Plasma: 81 – 105%; tested with plasma spiked with purified Calprotectin at five different levels.

Limit of Quantification:

5 ng/mL; tested with faeces extract, plasma and purified Calprotectin. Samples were analysed five times over five days. The mean CV for the different samples and determinations at this level was 12%.

Limit of Detection:

< 5 ng/mL; calculated as mean (Sample Dilution Buffer; n= 32) + 5x SD.

Agreement with Calpro Calprotectin ELISA CAL0100

Satisfactory correlation and agreement has been found between samples analysed in CAL0100 and in CALP0170:

Faeces extracts: R² = 0.792 (48 – 1250 mg/kg, n=118 samples), R² = 0.864 (48 – 500 mg/kg, n=85 samples)

Plasma: R² = 0.977 (260 – 6400 µg/L, n=16 samples)

Interference

No observed interference on the ELISA from commonly used pharmaceuticals: Prednisolon, Imurel, Salazopyrin and Ciprofloaxcin.

Extraction precision

Two faeces samples were extracted 10 times each, using the procedure described in section 7.1.1, and the extracts analysed in the ELISA. Mean results from three different laboratories and one kit lot: