Immunoassays 117

Cartridges were placed in PREP I and automatically extrac- ted, washed with l ml of water, and eluted with 1 ml of acetone. The dried extracts were reconstituted in 100 pl of the LC mobile phase, and 50 I,tl injected into the chromatograph.

The peak height ratios of drug to internal standard were calculated and calibration curves formed. Concentrations of drugs in patient samples were calculated by comparison with the calibration curves.

Results. The absolute extraction of the benzodiazepines and their principal metabolites with the PREP I was studied at two concentration levels. At a concentration of 2 pmole - 1- ~ the extraction of the drugs averaged 98 % recovery with a coefficient of variation of 4 % (n : 10). At the 80 pmole �9 1-1 level, the extractions averaged 90 % with a variation of 8 % (n = I0). The LC calibration curves obtained with extracted standards were linear to concentrations of at least 70pmole- l -1 . The GC calibration was linear to concen- trations of 15 pmole �9 1- l.

Patient samples were analyzed and compared to results obtained with the GC method. A linear regression analysis of the data for diazepam and chlordiazepoxide gave correlation data shown in Table 1.

For chlordiazepoxide samples above 30gmole-1 1, the GC calibration was extrapolated but gave values significantly lower than the LC-PREP I method, indicating a possible non- linearity in the GC method at high concentrations.

Conclusions. An automated sample processor (PREP TM I) can be applied to the extraction of benzodiazepine drugs and

Table 1

n r Slope Inter- Range cept (gmole

1-1 )

Diazepam 15 0.987 0.966 -0 .21 0 - 5 7 Chlordiazep-

oxide 10 0.981 0.928 0.04 0 - 2 0

metabolites from blood serum. The absolute extraction recoveries are greater than 90 %. The dried extracts can be used for analysis by techniques such as liquid chromatog- raphy. Comparison of the LC results to a GC method with manual liquid-liquid extraction was excellent for concen- trations up to 30 ~tmole. 1 1

References

1. De Silva, J. A.: Anal. Chem. 48, 10 (1976) 2. Rosano, T. G., Brown, H. H. : Clin. Chem. 25, 550-554

(1979) 3. St. Onge, L. M., Dolor, E., Anglim, M. A., Least, C. J.:

Clin. Chem. 25, 1373-1379 (1979) 4. Williams, R. C., Viola, J. L. : J. Chromotogr. 185, 505 -

513 (1979)

Immunoassays Immunoassays

A16

Fresenius Z. Anal. Chem. 301, 117-118 (1980) - ~) by Springer-Verlag 1980

Determination of Free Lipid A by Enzyme-Immunoassay (ELISA)

P. C. Fink and J. Kozak

Med. Hochschule Hannover, Inst. f. Klin. Chem., Karl-Wiechert-Allee 9, D-3000 Hannover 61

Bestimmung von freiem Lipid A mit einem Enzym-Immunoassay (ELISA)

Key words: Best. yon Lipid A, Endotoxin; Enzym-Immuno- assay; ELISA

Introduction. The endotoxic activities displayed by serologi- cally distinct lipopolysaccharides (LPS, endotoxin) are gene- rated by the Lipid A component of the LPS molecule [4]. The Lipid A moieties of exogenous and enterogeneous LPS share

common immunodeterminants, which elicit the production of cross reacting, highly specific anti-Lipid A antibodies [5]. The level of those antibodies may provide a more reliable diagnostic tool for measuring simultaneously different LPS chemotypes, than the Limulus amebocyte lysate test [2, 3]. The present procedure of detecting the hidden antigenic sites of Lipid A in intact LPS by cleaving the linkage between polysaccharide and Lip idA appears to be unsuitable for clinical use [4]. Therefore, it may be more practical to quantitate anti-LipidA antibody or polysaccharide free LipidA in biological fluids. For testing this, anti-LipidA antisera were prepared and free Lipid A was measured by use of a heterogeneous enzyme-immunoassay technique (ELISA) [6].

Material and Methods. Goat and rabbit anti-Lipid A antisera were obtained after immunization with Lipid A vaccine. The igG fraction of heat-inactivated antisera was prepared follow- ing ammonium sulphate precipitation by elution from a DEAE-A 50 cellulose column. After immunoelectrophoretic

118 Fresenius Z. Anal. Chem., Band 301 (1980)

Fig. 1. Immunoelectrophoresis indicating rabbit IgG and goat IgG purified by ammonium-sulphate precipitation and DEAE-Sephadex A-50 chromatography, a First concen- trated fractions of rabbit anti-LipidA antiserum eluted by PBS, pH 7.2. b Polyspecific goat antiserum to rabbit serum. c Monospecific goat antiserum to rabbit IgG. dFirst con- centrated fractions of goat anti-Lipid A antiserum eluted by PBS, pH 7.2. e Polyspecific rabbit antiserum to goat serum. fMonospecific rabbit antiserum to goat IgG

testing and adsorption with sheep cell erythrocytes (SCE),'the purified anti-LipidA antibodies were assayed for biological activity using the passive hemolysis test (PHT). Thereby, SCE sensitized with Lipid A were incubated with serially diluted anti-Lipid A antibodies; haemolysis was recorded after add- ing complement. To determine the specificity of anti-Lipid A antibodies, passive haemolysis inhibition tests (PHIT) were employed. The anti-LipidA antibodies were preincubated with several endotoxin standards or Lipid A and the loss of

~haemolytic activity was measured. Antibody titers were expressed as the negative logarithm of the highest dilution giving 50% haemolysis (e.g. 1:32 = - l o g 1.51). To quan- titate Lipid A different "sandwich" type ELISA were applied. Performing "sandwich antibody" (SAB) type ELISA, micro- titration plates were coated with serial dilutions of free LipidA. Washing steps (PBS-Tween) and incubation with human serum albumin followed. To the coated solid phase, rabbit anti-Lipid A antibodies and swine peroxidase labelled anti-rabbit antibodies (SPARA) were added stepwise. Unbound SPARA was washed off and bound SPARA was quantitated photometrically at 450 nm after adding enzyme substrate (5-amino-2-hydroxy-benzoic acid). For "sandwich

antigen" (SA) type ELISA, serial dilutions of Lipid A were pipetted to solid phase goat anti-LipidA antibody. The binding of the second rabbit anti-LipidA antibodies to free determinants of Lipid A was assayed by determining bound SPARA.

Results and Discussion. As shown in Fig. 1, the immunoelec- trophoretic characterization of the eluted fractions from goat and rabbit anti-Lipid A antisera showed no other plasma- proteins than IgG immunoglobulin. Performing PHT to determine anti-LipidA activity, the highest titer found at 50 ~ hemolysis was produced from immunized rabbits ( - log 2.43), while goat anti-LipidA antibodies exhibited titers ranging from - l o g 1.23 to --log 2.73. The specificity testing of anti-Lipid A antibodies by use of PHIT revealed that intact LPS could not neutralize anti-Lipid A activity of the immune sera. In contrary, LipidA preincubated with goat or rabbit anti-LipidA antibodies abolished their haemolytic activity completely, indicating their high specificity to Lipid A. Carrying out SAB type ELISA, the sensitivity in detecting free Lipid A was in the 0.250-25 gg/ml range. Beyond the linear portion of the measuring range, concomitantly with increasing concentrations of Lipid A, a significant decrease of detectable colouration occurred. This phenomenon may be due to decreased complexation between LipidA antigen excess and available anti-Lipid A antibody [1]. The SA type ELISA was found to be significantly more sensitive than SAB type ELISA detecting Lipid A in the 0.001 - 1 0 gg/ml range. However, this method has a restricted measuring range, apparently dependent on the reduced sensitization rate of solid phase with anti-Lipid A antibody.

Studies are in progress on the detection of naturally occurring anti-Lipid A antibodies in human sera using these anti-Lipid A antisera and a competitive ELISA technique.

Acknowledgements. This paper is dedicated to Prof. F. Hart- mann. This work was supported by grant Fi 281/1 from the Deutsche Forschungsgemeinschaft.

References

1. Beckman Instruments: Immunochemistry System 2,6 (1978)

2. Elin, R. J., et al. : Engl. J. Med. 293, 521 (1975) 3. Elin, R. J., Wolff, S. M.: J. Infect. Dis. 128, 349 (1973) 4. Galanos, C., et al.: Int. Rev. Biochem. 14, 239 (1977) 5. Galanos, C., et al. : In: Biomed. Applic. of Horseshoe Crab

(Limulidae), published by Alan R. Liss, p. 321. New York 1979

6. Schuurs, A. H. W. M., Van Weemen, B. R. : Clin. Chim. Acta 81, 1 (1977)