Blood samples were collected for measurement of IL-
8 , TNF a, and ANCA into plain tubes with no anticoagulant
and for al-AT-E into potassium EDTA tubes. The samples wer e s e p a r a t e d by c e n t r i f u g a t i o n at 2 OOOg w i t h i n 30 m i n u t e s and s e r u m and p l a s m a s t o r e d at - 7 0 ° C u n t i l assayed.
8.2.4 Methods
(a) Serum IL-8 Immunoassay• 11-8 was measured by solid phase enzyme linked immunoassay (ELISA; Quantikine, R e s e a r c h and D i a g n o s t i c Systems, Minn e s o t a , USA). A monoclonal antibody specific for IL- 8 was coated onto the
microtitre plates provided. Standards with known amounts of IL- 8 and samples were pipetted, in duplicate, into the
wells, and any IL- 8 present bound by the immobilised
antibody. For detection, an enzyme linked polyclonal a n t i b o d y speci f i c for I L- 8 was applied. A s u b s t r a t e
solution containing tetramethylbenzidine and H2 O2 was
added, and the colour developed was proportional to the amount of IL- 8 bound in the initial step. A standard curve
Chapter 8; IL-8 and Neutrophil Activation
w a s d r a w n of o p t i c a l d e n s i t y (OD) a g a i n s t I L- 8
concentration (Fig.8.1). The lower limit of detection in this assay was 50 pg/ml. This was determined by adding 2 standard deviations to the mean optical density value of 5 zero standard replicates and calculating the corresponding concentration from the standard curve. The intra-assay c o e f f i c i e n t of v a r i a t i o n (CV) was 8 % (n=10) and the
interassay CV was 9.3% (n=5).
(b) Serum TNF a Immunoassay. TNF a was measured by solid phase ELISA (Quantikine). A monoclonal antibody specific for TNF a was coated onto the microtitre plates provided. Stan d a r d s with known a m o u n t s of TNF a and samples were pipetted, in duplicate, into the wells. For detection, an enzyme-linked polyclonal antibody specific for TNF a was applied. A substrate solution was added, as above, and a s t a n d a r d curve drawn of OD a g a i n s t the concentration of TNF a in the standard wells. The lower limit of detection for this assay was 10 pg/ml. The intra assay CV was 5.4% (n=10) and the inter-assay CV 4% (n=5).
(c) Human al - A T C o mplexed E l a s t a s e Immunoassay. P l a s m a a l - A T - E w as m e a s u r e d by a m o d i f i e d E L I S A as described in Chapter 7 {Section 7.2.3 (a)).
Chapter 8: IL-8 and Neutrophil Activation
(d) Immunoassay for ANCA against an Acid Extract of Neutrophil Cytoplasm. IgM and IgG ANCA were measured in
s e r u m by an E L I S A t e c h n i q u e u s i n g a c i d e x t r a c t e d neutrophil cytoplasm antigen prepared as described by Savage et al (274) . For each preparation, the optimal dilution for coating was determined using negative control sera and positive patient sera. The antigen was coated onto microtitre plates (Nunc). Test or control serum samples were added in duplicate to neutrophil antigen- coated plates. For detection, peroxidase conjugated anti h u m a n IgG and IgM (Sigma) w e r e a p p l i e d , and a f t e r
i n c u b a t i o n and w a s h i n g a s u b s t r a t e was added. A f t e r fu r t h e r i n c u b a t i o n the r e a c t i o n was s t o p p e d and ODs measured using an ELISA reader. The OD values of the standard sera at various dilutions were used to construct a standard curve of arbitrary units, from which the ANCA c o n t e n t of each t e s t s e r u m c o u l d be e s t i m a t e d . The arbitrary units were set as 64 and 32 for the IgG class ANCA-positive standard serum and IgM class ANCA-positive standard serum, respectively. The normal range for IgM for this assay is 0.5 to 9.1 units and for IgG 0.5 to 9.0 units.
Chapter 8: IL-8 and Neutrophil Activation
8.2.5 Statistical Analysis
Fishe r ' s Exact test was use d to d e t e r m i n e the significance of the IL- 8 results in the different groups
compared to normals. The Mann-Whitney test was used as a test of significance of difference between the medians of the different groups. The correlations between lL- 8 and
al-AT-E, and l L- 8 and PMNL count w e r e e x a m i n e d n o n
p a r a m e t r i ca lly u s i n g S p e a r m a n ' s r a n k c o r r e l a t i o n coefficient, al-AT-E and PMNL counts were log-normally distributed, and geometric means were compared using Student's t-test on log-transformed data.
8.3 Results