Blood samples were also obtained from :
1. Thirty-five healthy children being followed up after routine surgical procedures such as hernia repair or being investigated for short stature.
2. T w e n t y - o n e i n p a t i e n t s i d e n t i f i e d by t h e haematology laboratory as having a PMNL count greater than 10 xlO^/L and defined as high PMNL count controls; they either had a septicaemic illness or other infection and all had a normal plasma creatinine concentration, blood samples were obtained within 48 hours of admission and prior to or within 24 hours of instituting therapy.
3. Thirty-five children with chronic renal failure w h o h a d a g l o m e r u l a r f i l t r a t i o n r a t e less t h a n 15 ml/min/1.73 m^ SA.
Chapter 7: Elastase and Neutrophil Activation
7.2.3 Methods
Blood samples were collected into potassium EDTA (ethylenediaminetetraacetic acid) tubes, and the plasma separated by centrifugation at 2000g within 30 minutes and stored at -70°C until assayed.
(a) a l - A T C o m p l e x e d E l a s t a s e E n z y m e —L i n k e d Immunosorbent Assay (ELISA) . A modified assay for the
measurement of al-AT complexed elastase was used (257). The first antibody was sheep anti-human elastase antibody (ICN Immuno biologicals, High W y c o m b e , England) which specifically recognises leucocyte elastase found in the azurophilic granules of human neutrophils. The antiserum was produced by immunising sheep with purified protein obtained from the peripheral leucocytes of a patient with c h r o n i c m y e l o i d leukaemia. M i c r o t i t r e p l a t e s (Nunc- Immunoplate Gibco ERL, Paisley, Scotland) were coated with 100 jitL antiserum (10 /xg/ml) in bicarbonate buffer (50 mM, pH 9.6) by incubation overnight at 4°C. All subsequent incubations were carried out at room temperature with volumes of 100 /xl and the plates were always washed four times with 150 mM Dulbecco's phosphate-buffered saline pH 7.2 (PBS) containing 0.1% 'Tween 2 0 ' (BDH Limited, Porte, England) between stages. Standards or samples diluted 1:80 in PBS were then added and incubated for two hours. The second antibody, horseradish-peroxidase-conjugated sheep
Chapter 7: Elastase and Neutrophil Activation
anti-human al-AT (The Binding Site Limited, Birmingham, UK) diluted 1:1000 in PBS, was added to each well and incubated for two hours. The substrate (1.0 mg/ml O- p h e n y l e n e d i a m i n e and 0.03% ^2^2 10 0 m M c i t r a t e - phosphate buffer, pH 4.0) was added and incubated for 20 minutes. The reaction was stopped by the addition of 50 jLtl of 2 M H g S O ^ . The absorbance was measured at 492 nm in T i t e r t e k m u l t i s c a n plus (Flow L a b o r a t o r i e s Limited, R i c k m a n s w o t h , England) ELISA reader. S t a n d a r d s were obtained by adding increasing amounts of reconstituted elastase 2.5 /xg/ml in PBS to a constant volume of control plasma, giving final elastase concentrations of 50-16,000 ng/ml. A typical standard curve obtained is shown in Fig. 7.1. The c o n t r o l p l a s m a was h a r v e s t e d on a s i n g l e occasion, aliquoted into small portions, and frozen at -70°C. The concentration was expressed as a function of the added elastase. The units obtained by these standards are arbitrary because of the unknown concentration of al- AT initially present in the control plasma.
(b) F r e e E l a s t a s e ^ Free e l a s t a s e a c t i v i t y was measured enzymatically using a method adapted from Johnson
et al ( 2 5 8 , 2 5 9 ) . T h e s y n t h e t i c p e p t i d e methoxysuccinyl-ala-ala-pro-val-p-nitroanilide (MSAAPV- pNA) (Sigma Ltd, Porte, England) was used as substrate.
Chapter 7: Elastase and Neutrophil Activation
This substrate is specific for human leucocyte elastase a nd d o e s not r e c o g n i s e p a n c r e a t i c e l a s t a s e . S a m p l e activities were determined by reference to a standard curve obtained with human leucocyte elastase (Sigma) made up in a 100 jitl of 100 mM HEPES (Ng-hydroxyethylpiperazine- ng-ethanesulphonic acid) buffer p H 7 .5 containing 0.5 M NaCl and 10% dimethylsulphoxide added to 1 ml of 1 mM substrate made up in the same buffer. One unit was defined as the change in absorbance of the optical density at 410 nm of 0.001/min at 25°C over 10 minutes. The elastase standard concentrations ranged from 1 to 400 ng/ml. A concentration of elastase of 1 ng/ml produced a change in o p t i c a l d e n s i t y of 0 . 1 6 / 1 0 m i n u t e s , w h i c h w a s significantly greater than 0.04/10 minutes of the blank buffer.
7.2.4 Statistical Analysis
D a t a w e r e t e s t e d for n o r m a l or log n o r m a l distribution using normal probability plots. Means were compared by Student's t-test and regression analysis was performed using an ordinary least square regression using one independent variable for a linear model.
Chapter 7: Elastase and Neutrophil Activation
7.3 Results
7.3.1 al-AT Complexed Elastase Levels in Different Groups