determination of anti-lipid a and lipid a by enzyme immunoassay
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Immunobiol., vol. 158, pp. 380-390 (1981)
Institute for Clinical Chemistry, Medical School, Hannover!, and Max-Planck-Institut fiir Immunbiologie2, Freiburg/Breisgau, Federal Republic of Germany
Determination of Anti-Lipid A and Lipid A by Enzyme Immunoassay
PETER C. FINK 1 and CHRIS GALANOS2
Received December 17, 1980 Accepted January 24,1981
Immunizing rabbits and goats with acid-treated Salmonella minnesota R595 bacterial cells, that expose lipid A on their surface, an IgG anti-lipid A antibody response could be obtained. In passive hemolysis test, using lipid-A-coated sheep red blood cells and complement, the highest IgG anti-lipid A antibody activity was found in immunized rabbits, while goats produced a relatively weak antibody response against lipid A. The passive hemolysis test showed that IgG anti-lipid A antibody activity was completely abolished by preceding preincubation with lipid A. No reactivity of IgG anti-lipid A antibodies was found with the tested intact lipopolysaccharides from one Salmonella abortus equi and one Escherichia coli strain. Quantitative analysis of free lipid A was carried out by the enzyme-linked immunosorbent assay (ELISA). In sandwich antigen type ELISA, using goat IgG anti-lipid A antibodies bound to the wells of polystyrene microassay plates, rabbit IgG anti-lipid A antibodies and peroxidase-labeled goat anti-rabbit antibodies (PGRA), the measuring range for free lipid A (0.001 to 1 ~g/ml) correlated significantly (2p < 0.01) with absorbance. In sandwich antibody type (SAB) ELISA binding lipid A to the solid phase, and using rabbit IgG anti-lipid A antibodies and PGRA insignificant correlation could be found for the same measuring range (2p > 0.1). Comparison of passive hemolysis test and SAB type ELISA to determine IgG antilipid A antibody activity showed that ELISA was more sensitive for measuring anti-lipid A antibody activity (from antibody dilution of -log: 2.43 to 3.7) than the passive hemolysis test.
Gram-negative septicemia is known to be a clinical entity with a high
mortality rate (1-3). Many pathological alterations appearing on various organs (e.g. lung, kidney, liver, brain) (4-9) in process of gram-negative infections have been assigned to the deleterious effects of lipopolysac
charides (LPS, endotoxin) which are released from the cell wall during
bacteriolysis. The detection of LPS in peripheral blood may be carried out
by bioassays such as the Limulus amebocyte lysate test (10, 11), USP rabbit
Abbreviations: LPS = lipopolysaccharide; USP = United States Pharmacopeia; PBS = phosphate-buffered saline; SRBC = sheep red blood cell, VBS = veronal-buffered saline; PHT = passive hemolysis test; PHIT = passive hemolysis inhibition test; ELISA = enzymelinked immunosorbent assay; SAB = sandwich antibody; SA = sandwich antigen; PGRA = peroxidase-labeled goat anti-rabbit antibody.
Anti-Lipid A and Lipid A . 381
pyrogen test (12), histamine hypersensitization test (13), and the chick embryo cytotoxicity test (14). In these assay systems which measure the biological activity of endotoxin rather than endotoxin per se, the question of specificity often arises. The clinical value of other endotoxin-sensitive immunoassays (15, 16) may be curtailed by their limited applicability because of their restricted specificity for antigenic ally (O-antigen) defined endotoxins.
Recent studies by several authors (17) have demonstrated that the lipid component of LPS, namely lipid A, encompasses both the toxic site and the least variable antigenic part of the LPS of numerous gram-negative bacteria. In addition, common immunodeterminants in lipid A were found to elicit the production of cross-reacting highly specific anti-lipid A antibodies (18-20). While the procedure of detecting the cryptic antigenic sites of lipid A in intact LPS by cleaving the 2-keto-3-deoxy-octonate trisaccharide linkage between polysaccharide and lipid A appears at present to be unsuitable for clinical use, the determination of anti-lipid A antibodies in serum may provide an appropriate diagnostic tool since the presence of such antibodies in certain levels would reveal contact of the organism with LPS regardless of serospecificity.
To establish a suitable assay system for anti-lipid A antibodies, antisera were raised in rabbits and goats, and purified anti-lipid A IgG was compared in the passive hemolysis test and in an enzyme immunoassay (ELISA).
Materials and Methods
Preparation of lipid A antisera
Antisera with specificity against lipid A were raised in rabbits and goats by repeated immunizations with acid-treated Salmonella minnesota R595 bacteria coated additionally with free lipid A as described elsewhere (20). Rabbits received 100 [.Ig, 200 [.Ig, 300 [.Ig and 500 [.Ig lipid A vaccine on days 1, 4, 7 and 11, respectively, administered intravenously in 1 ml physiological phosphate-buffered saline (PBS, 0.01 molll, pH 7.2). On day 39 the animals were boostered with 500 [.Ig vaccine, administered as above, and 6 days later blood was collected from the marginal ear vein.
Goats were immunized on day 1 with 100 [.Ig lipid A vaccine. The antigen suspension (100 [.Ig in 2 ml PBS) was emulsified with 2 ml incomplete Freund's adjuvant (Behringwerke, D-3550 Marburg), and 1 ml was injected subcutaneously at four different sites. At monthly intervals 3 further booster injections were carried out with 500 [.Ig vaccine in incomplete Freund's adjuvant as described above. Twelve, twenty and twenty-five days after the last booster injection, blood was drawn from the jugular vein.
All blood samples after allowing to clot were centrifuged at 1900 g for 20 min at 4 e, heatinactivated for 30 min at 56 e and absorbed with packed sheep red blood cells (SRBC) (Biolog. Arbeitsgem. D-64202 Lich) (0.2 ml packed cells/5 mllipid A antiserum) for 30 min at 37 e. The cells were pelleted at 1000 g for 10 min at 20 e, and the serum supernatant was stored at - 60 e until use.
382 . P. C. FINK, and CH. GALANOS
Chromatographic isolation of IgG immunoglobulins
A crude gamma-globulin fraction was obtained from rabbit and goat lipid A antisera by two precipitations with 50 % saturated ammonium sulfate. The final precipitate was dissolved in PBS (II, of the original serum volume) and dialyzed overnight against 51 PBS. The IgG fraction was prepared by elution from a DEAE-Sephadex A 50 column (2.6 X 40 cm) (Deutsche Pharmacia, D-7800 Freiburg) by PBS and concentrated by Minicon B 15 (Amicon, D-5810 Witten) (21). The protein content was determined spectrophotometrically according to LAYNE E. (22).
Immunochemical characterization of the eluted proteins
The purity of the rabbit and goat IgG preparations was tested by immunoelectrophoresis using polyspecific goat anti-rabbit plasma protein, rabbit anti-goat plasma protein (Medac, D-2000 Hamburg) and mono specific goat anti-rabbit IgG (Paesel, D-6000 Frankfurt) and rabbit anti-goat IgG (Byk-Mallinkrodt, D-6057 Dietzenbach-Steinberg).
Determination of anti-lipid A activity and specificity of the IgG preparations
Anti-lipid A antibody activity was measured by the passive hemolysis test (PHT) using lipid A sensitized SRBC as described elsewhere (20, 23). Briefly, washed SRBC (0.2 ml packed cells in 5 ml PBS) were incubated with alkali-treated lipid A (40 fll, 2 mg/ml) for 30 min at 37C. They were washed three times with PBS and the pellet resuspended in 11 ml of veronalbuffered saline (VBS, 0.02 molll, pH 7.3 containing 15 mmolll CaCI2 and 0.5 mmolll MgCI2). To serial dilutions (in 0.1 ml VBS) of the rabbit and goat IgG preparations, VBS (0.8 ml), lipid A sensitized SRBC (0.5 ml) and guinea pig complement (0.1 ml diluted 1 : 12 with VBS) (Behringwerke, D-3550 Marburg) were added and the samples incubated for 60 min at 37C. Identical samples containing instead of sensitized, normal SRBC served as controls. In addition, samples from which IgG, complement or both were omitted, as well as blanks containing IgG or complement alone were included as controls. After incubation the cells were centrifuged at 1900 g for 20 min at room temperature and hemolysis in the supernatants measured at 546 nm. Anti-lipid A hemolytic activity was expressed as the negative logarithm (-lOgIO) of the dilution at which 50 % of sensitized SRBC occurred (e.g. -log ['132] = 1.51).
In the passive hemolysis inhibition test (PHIT) the rabbit IgG anti-lipid A preparations were preincubated with serial dilutions of lipid A (100 ng - 10 pg/ml) or LPS from Salmonella abortus equi (Novo-Pyrexal forte) (Hermal-Chemie, Kurt Hermann, D-2057 Reinbek b. Hamburg) or E. coli 055: B5 LPS (Difco laboratories, Detroit, Michigan, USA) (100 ng-10 pg/ml) respectively for 60 min at 37C. After centrifugation, the supernatants were processed under identical conditions as in PHT, and hemolysis was determined.
Quantitation of anti-lipid A and lipid A by ELISA
Using rabbit and goat anti-lipid A antibodies of known hemolytic activity, two types of ELISA were carried out in microtitration plates (No.: M129 A, MicroElisa Reader AM 115, Compuprint 700, Dynatech, D-7310 Plochingen) as described elsewhere (24, 25) with some modifications. In sandwich antibody (SAB) type ELISA, the solid phase was sensitized with alkali-treated lipid A (1 flg/0.2 ml) dissolved in carbonate-bicarbonate buffer 0.05 molll, pH 9.7 for 60 min at 37C. After washing three times with PBS/Tween 20R, the wells were incubated with bovine serum albumin (BSA, 5 gil) (Behringwerke, D-3550 Marburg) for 30 min at 37C to reduce non-specific binding. Subsequent to three washings with PBSI Tween 20R the wells were incubated with serially diluted rabbit anti-lipid A antibodies (1 : 10 to 1 : 5000) (3.68 mg/0.2 ml) for 60 min at 3