Purification ofspecific precipitinogen and extraction

Journal of Clinical Pathology, 1978, 31, 370-377

Purification of specific precipitinogen and extractionof endotoxin from Haemophilus influenzae1

J. C. VAN DER ZWAN, J. DANKERT, K. DE VRIES, N. G. M. ORIE, ANDH. F. KAUFFMAN

From the University Hospital, Groningen, The Netherlands

SUMMARY After purifying a Haemophilus influenzae precipitinogen from endotoxic activity by meansof ultracentrifugation, column chromatography (Sepharose 6B), and ion exchange chromatography(DEAE Sephadex A25) a fraction was obtained which still contained a specific precipitinogen thatwas virtually free of endotoxin. Furthermore, during the chromatographic procedures fractionswith a high and a low molecular weight endotoxic activity were found. The limulus lysate test wasmore sensitive in the high molecular weight fractions and the LD50 in mice in the low molecularweight fractions with endotoxic activity.

The non-encapsulated Haemophilus influenza (HI)is the most common pathogen in the bronchial treeof patients with chronic non-specific lung disease(CNSLD) (Mulder, 1938; Brumfitt et al., 1957).When the effect of H. influenza precipitinogen inskin test and bronchial provocation was studied itwas difficult to differentiate between an immuno-logical and a toxic mechanism in the reaction to thetest substance (van der Zwan et al., 1975). It hasbeen suggested that endotoxin is present in H.influenzae (Branefors-Helander, 1973; Denny, 1974;van der Zwan et al., 1975). A lipopolysaccharide hasrecently been isolated from H. influenzae and definedchemically (Zoon and Scocca, 1975). Therefore aspecific precipitinogen, as defined by Omland (1964)and May (1965), without endotoxic activity andalso a purified endotoxin is necessary in order to beable to identify the immunological and toxic reactionseparately.A purified precipitinogen is obtained by the

methods described below. For extracting lipopoly-saccharides from rough growing bacteria the methoddescribed by Galanos et al. (1969) was used. Thelimulus lysate test (LLT) was used to measure theendotoxic activity of the extracts, because it isrelatively easy to perform and highly sensitive. Anin-vivo system was required since the bacterialcompounds were to be used in man. For that purposethe mortality in mice after potentiation with actino-mycin-D was used (Pieroni et al., 1970).

'Dedicated to the late Professor J. R. MayReceived for publication 28 September 1977

370

Methods

CULTUREA rough growing, non-encapsulated strain of H.influenzae from a patient with chronic purulentbronchitis (confirmed by The Netherlands NationalHealth Laboratory Service, strain number BBD5048) was cultured at 370C on Levinthal plates(Oxoid Ltd). After 24 hours the micro-organismswere transferred to a Microform Fermentor (NewBrunswick Scientific Co) containing 12 1 Levinthalbroth. The inoculum was cultured for 20 hours at370C with an aeration of 1 I/min and stirred at 100rpm. By taking samples from the culture at intervalsof four hours it was shown that the pH remainedbetween 7-1 and 74. Control of the culture waschecked by a Gram stain and by culture on Levinthaland blood-agar plates (Oxoid Ltd). After centri-fugation of the culture at 1900 g for 15 minutes theresidue was washed three times in 12 5 mmol(6 25 mEq/l) MgCl2 to remove the remaining broth.The weight of the pellet was considered to be thewet weight of the bacteria.

IMMUNOLOGICAL METHODSPrecipitinogens in the different fractions wereidentified by immunodiffusion according to Ouch-terlony (1962). When the immunodiffusion waspositive immunoelectrophoresis was performed bythe method of May (1968), which detects antibodyto the specific and non-specific antigens of H.influenzae. The sera used were from patients withCNSLD who had H. influenzae in their sputum. The

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Purification ofspecific precipitinogen and extraction ofendotoxin from Haemophilus influenza

sera contained either specific or non-specific, or both,precipitins against H. influenzae (May, 1968).

ENDOTOXIN DETECTIONLocalisation of endotoxin was attempted by stainingon the immunoelectrophoresis slide, using Sudanblack for the lipid and Shiff's reagent for thesaccharide moiety of the endotoxin (Uriel, 1964).The LLT was used for in-vitro activity (Rojas-

Corona et al., 1969). The result was read after twohours and considered to be positive when a firm gelhad been formed.

In-vivo endotoxic activity was studied in femaleSwiss mice, weighing between 15 and 25 g, by usingthe potentiating effect of actinomycin-D (Pieroniet al., 1970). The LD5o was estimated by the methodof Reed and Muench (1938). To compare thecontent of endotoxin in the different preparationsend-point titrations were carried out according toJorgensen and Smith (1974). A reference endotoxinfrom Escherichia coli had an end-point titration at10-7 mg/ml solvent.

ENDOTOXIN EXTRACTION

The method of extracting endotoxin, which isspecially suited for rough-growing bacteria, was byusing a monophasic extraction mixture consistingof aqueous phenol, chloroform, and petroleum ether(Galanos et al., 1969).The fractionating procedure was as depicted in

Fig. 1.

EXTRACT PREPARATION

The bacteria were disrupted by ultrasonic disin-tegration (MSE) as described by May (1968) usinga buffer of 12-5 mmol (6-2 mEq/l) MgCI2 to stabilisethe endotoxin in its high molecular weight form(Greer et al., 1973). Ultracentrifugation was carriedout at 200 000 g for 30 minutes to remove allparticle-bound endotoxin. Since the supernatantstill had considerable endotoxic activity a furtherseparation was tried by means of chromatographicprocedures-

6g wet weight bacteria

resuspension in 60 ml 12.5mM MgC12

-Itrasonic desintegration 3.5 min/10 ml suspension at 4'C

ultracentrifugation at 200.000 g/30 min.

+ 5 ml supernatant (= CF) decanted

concentration in a diaflowmeter till 3-5 ml

centrifugation at 2000 g/20 min.

residue removed clear supernatant

Sepharose 6B. PH 7.2

Fract. 11 Fract. III Fract. IV

DEAE-Sephadex A25: pi, 8

Filtration I

Fr. III/S Residue + 0,5 M NaCI

Fr. III/ASl ~ ~ ~ ~ ~~~~~I ,

|~~ ~ ~~~~~~~ I

DEAE-Sephadex A25; pH 8

Filtration

Residue + 0,5 M Na Cl

CF/S CF/AS

dialysis against running tap water/48 hours

Iyophilisation. I

105 mg 136mg 18 mg

Diagram offractionatingprocedures.

residue removed

IFract. I

Il

21 mg

Fig. I

18 mg 85 mg 57 mg

a

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J. C. van der Zwan, J. Dankert, K. de Vries, N. G. M. Orie, and H. F. Kauffman

CHROMATOGRAPHYColumn chromatography was carried out usingSepharose 6B as a carrier (column diameter 2.5 cm,volume 450 ml). The supernatant obtained afterultracentrifugation was concentrated in a diaflow-meter with a filterpore of 10 m,u to a volume of3-5 ml. This concentrate was centrifuged at 2000 gfor 20 minutes to remove denaturated material andwas brought on the column. The eluate was sampledby means of a LKB fraction collector and measuredat 276 nm by an Uvicord (LKB). After chromato-graphy the eluate was pooled as follows: fraction I

(± 60 ml), fraction II (± 36 ml), fraction III (± 132ml), and fraction IV (± 78 ml), (Fig. 2). The placeof the absorption maxima of the fractions werereproducible for different batches, although thesharpness of the peaks differed slightly. In additionion exchange chromatography was performed usingDEAE-Sephadex A25 (Davies et al., 1974). Thepooled fractions were dialysed during 48 hoursagainst running tap-water and lyophilised.

0 50 100 150 200 250 300mi

Fig. 2 Absorption maxima afterpassage ofparticle-freecytoplasmic fraction ofH. influenzae via a column withSepharose 6B.

CHEMICAL ANALYSISThe fractions were characterised according to theirprotein, lipid, and carbohydrate content. Theamount of the protein in the various fractions wasestimated by the method of Lowry (Lowry et al.,1951). The lipid content of the unsaturated moietywas assessed as described by Hoefimayr (Hoeflmayrand Fried, 1967). For the estimation of saturatedfatty acids the fractions were extracted in chloroformand dried in a nitrogen stream. For the thin-layerchromatography Kieselgel (Merck Ltd) was used(thickness 0-25 mm). A mixture of petroleum ether40/60 and acetone in a ratio of 85:15 v/v (MerckLtd) was used for development. The results were readwith ultraviolet light (350 nm) and graded from - to+ + +. Anthrone reagent was used for detectingcarbohydrate, which was quantified in amountsequivalent to galactose (Toennies and Kolb, 1964).When repeated the results varied by 5% to 12 % forthe protein and lipid content and by 5% to 18%for the carbohydrate.

Evaluation of procedures and results

PURIFICATION OF PRECIPITINOGENThe whole H. influenzae bacterium showed a highendotoxic activity (Table 1) and was obviouslynegative in the precipitation tests. Since endotoxinis a component of the inner cell wall (de Petris, 1967)ultrasonic disintegration and ultracentrifugationwere performed and a particle-free cytoplasmicfraction (CF) obtained. This was still considerablycontaminated with endotoxin but it contained thespecific and non-specific antigen, as was shown inthe IEF (Table 1).

Since it could be expected that the endotoxin hada high molecular weight (Luderitz et al., 1966)column chromatography was carried out. Fig. 2shows the elution pattern of 3.5 ml of the concen-trated CF when brought on a Sepharose B6 column.

Table 1 Endotoxic activity (measured in vivo and in vitro) andprecipitinogen content in the lyophilised H. influenzaefractions expressed in tsg ofE coli endotoxin equivalents

Fractionating procedures

None Chlorof. ether Ultracentr. Sepharose 6B DEAE-Sephadex Sepharose 6Bphenol extract A25 DEAE Sephadex

A25

Bacteria Endotoxin CF Fr. I Fr. II Fr. III Fr. I V Specific Non-specific III/S III/AS

LD,0 in mice +actinomycin 2-5 25 0-2 2 0.5 0.03 0-02 0.07 2 0.003 0-014

End titration limulusassay 100 1000 1 1000 0-1 0-1 0-1 0-1 0.1 0-001 0-1

PrecipitinsSpecific 4- - + + - ++.% +Non-specific - +++ ++ +++ + , , V_.

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Purification ofspecific precipitinogen and extraction ofendotoxinfrom Haemophilus influenzae

Each of the four main absorption maxima (I/IV)was pooled and tested for endotoxic activity and thepresence of specific and non-specific precipitinogen.The main endotoxic activity was concentrated infraction I, whereas fractions II-IV had a much loweractivity. The main content of specific precipitinogenwas found in fraction III. This indicated that toxicitydoes not depend on the presence of specific preci-pitinogen.

Because the separation was only partial anothermethod was used. Immunoelectrophoresis followedby lipid and saccharide staining suggested that thecharge of the lipopolysaccharide moiety wasnegative, as opposed to that of the positively chargedspecific precipitinogens (Fig. 3). Therefore ionexchange chromatography was carried out (Davieset al., 1974), using a cytoplasmic fraction.The first eluate (CP/S) contained only the posi-

tively charged fraction. The second eluate (CP/AS)with the negatively charged fraction contained alsocomponents of the positively charged fraction. Thiscontamination was slightly variable. When fractionIII obtained by column chromatography was usedin the ion exchange chromatography instead of theCF fraction a fraction (III/S) was obtained. Thisshowed a very low endotoxic activity (Table 1)while still containing a high concentration of thespecific precipitinogen as demonstrated in theimmunodiffusion. It showed a reaction of identitywith H. influenzae extract prepared as described byMay (1968) (Fig. 4). It could be that endotoxinitself is a precipitinogen. Therefore endotoxin,prepared as described by Galanos et al. (1969), wastested in the immunodiffusion against sera withspecific or non-specific precipitins against H.influenzae. No precipitate was formed. Therefore

Fig. 4 Lines ofidentity between May antigen (twoopposite wells) andpurified H. influenzae antigen (two wellsin between, bottom left). Centre well contains serum of apatient with mainly specific H. influenzae precipitins.

we concluded that endotoxin did not contribute tothe precipitate as shown in Fig. 4.

COMPARISON OF METHODS USED FOR ENDO-TOXIN DETECTIONIn vitro the LLT shows a high endotoxic activity forthe whole bacterium, the high molecular weightfraction I, and the extracted lipopolysaccharide(Table 1). The lower molecular weight fractionscontain less endotoxic activity.The in-vivo test confirmed the in-vitro findings.

Fig. 3 From top to bottom: lipid moiety in Sudan black staining, lipopolysaccharide moiety in PASstaining, and precipitinogen content of H. influenzae antigen after immunoelectrophoresis.

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J. C. van der Zwan, J. Dankert, K. de Vries, N. G. M. Orie, and H. F. Kauffman

After a separation via DEAE-Sephadex A25 thein-vivo test confirmed that the main lipopoly-saccharide activity is found in the negatively chargedfraction, as was suggested by staining techniques.Endotoxin of E. coli (Jorgensen and Smith, 1974) isused as a standard; the endpoint in the titration inthe LLT is found at 10-7 mg.The LD5o for E. coli endotoxin in mice after

potentiation with actinomycin-D is 10-6 mg(Pieroni et al., 1970). If the amount of lyophilisedmaterial used in both experiments is expressed inmg necessary to obtain an endotoxic activityequivalent to 1 jug E. coli endotoxin the sensitivitiescan be compared (Table 2). In the high molecularweight fractions the LLT is more sensitive than thein-vivo method, whereas for the low molecular weightfractions the reverse is found.

Table 2 Amount of the various fiactionis expressed in mzglyophilised material/ml solvent containing an endotoxicactivity equivalent to I tig coli endotoxinas estimated in the limulus lysate test (LLT) comparedwith the L D50 in mice after potentiation with actinomycin-D

Compounds LLT Mouse L D,0 LDO/LLT

Bacterium 0 01 0 4 40Endotoxin 0 001 0 04 40Cytopl. fraction 1 5 5Fraction I 0 001 0 5 500Fraction I I 10 20 2Fraction III 1( 30 3Fraction IV 10 5 0-5Specific fraction 10 15 1-5Non-specific fraction 10 0 5 0(05

The results of the chemical analysis and themolecular weight of the fractions are summarised inTable 3. It was shown that fraction I, II, and III hadan increasing protein/carbohydrate ratio. The lowmolecular weight fraction IV, however, had a lowprotein/carbohydrate ratio. Unsaturated lipids weredetected in the bacteria and in fraction IV, whereasthe saturated fatty acids were mainly visualised inthe bacteria, fraction 1, the fraction IV.

Discussion

Gram-negative bacteria are commonly used in skinand bronchial provocation tests (Barr et al., 1965;Brown, 1934; Haj6s, 1963; Haj6s and Hajds, 1964;Herxheimer, 1951; Hirdes, 1969; Ricci et al., 1967).Seemingly, however, no thought was given to thepossibility that the reaction could be due to endo-toxin rather than an antibody-antigen mediatedreaction. For detecting endotoxin the LD50 in miceafter actinomycin-D was used as an in-vivo method.

Despite the limitations of the LLT's non-specificity(Elin and Wolff, 1973; Stumacher et al., 1973;Sullivan and Watson, 1974; Wildfeuer et al., 1974;Weckesser et al., 1974) its usefulness is well estab-lished (Cooper et al., 1971; Jorgensen and Smith,1973; Levin et al., 1970). To what extent particle sizeand electrophoretic mobility of compounds were

related to endotoxic activity was unknown. Thepurification of the H. influenzae precipitins gave us

the opportunity to find out.To separate the specific precipitinogen from endo-

toxin we used the following characteristics of thelatter: (l) its localisation in the cell wall, (2) its highmolecular weight, and (3) its electrophoretic mobility(Fig. 1). After centrifugation the supernatant provedto be contaminated with water-soluble free endo-toxin (Jorgensen and Smith, 1974), probably due tothe ultrasonic disruption. The cell wall itself was

completely removed from the cytoplasmic fraction.The highest concentration of endotoxin was foundin fraction I after gel filtration by Sepharose 6B.The fractions H1-IV, however, still had a considerableendotoxic activity. Since the molecular weight ofthese fractions is below that of the endotoxin(Table 3) when prepared by the phenol-waterextraction (Ltderitz et al., 1966) this activity isprobably the result of fragments of the endotoxin,as described by Marx et al. (1968).

Interestingly, the sensitivity for the endotoxin inthe high molecular weight fractions, as found in theH. influenzae bacteria and fraction 1, was about 10:3

Table 3 Molecular weight ofthefractions obtained in column chr-omatography Sepharose 6B. Content of protein aildcarbohydrate expressed as a ratio. Unsaturated lipid in percentage oforiginal amount in dry weight. Saturated fatty acidgradedfrom 0 to + + + in thin-layer chromatographyProcedures Molecular Protein Protein/ Unsaturated Saturated fatty aidl

weight ( %) carbohydrate lipids

Bacterium 54 2-1 4Sepharose 6B

Fraction >340000 27 2-4 NDFraction II 210000 30 3-4 NDFraction III 44000 52 11 6 NDFraction IV 23000 7 1-8 2

DEAE-Sephadex A25Specific fraction Ill S 24 5 2-9 NDNon-snecific fraction III AS 33 2-3 ND

N D) = not detectable.

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Purification ofspecific precipitinogen and extraction ofendotoxinfrom Haemophilus influenzae 375

times higher in the LLT when compared with thetoxicity in mice. This discrepancy was not found inthe low molecular weight fractions II and III andonly partly found in fraction IV (Table 1).

This suggests that fragmentation of the endotoxinmolecule results in a diminished endotoxic activityin mice. Nowotny et al. (1975) studied a poly-saccharide-rich fraction from Salmonella endotoxinwith an average molecular weight of about 10 000which gave neither a mortality in mice nor a positiveLLT. Ng et al. (1974), however, found a glycolipidfrom rough growing bacteria with a molecular weightbetween 15 000 and 18 000 with a high endotoxicactivity when measured in the LLT and in mice.The clotting reaction in the LLT is probably an

enzymatic process (Young et al., 1972). The com-ponent in the lipopolysaccharide responsible for thereaction is unknown. The toxic effect in animals iscaused mainly by 'lipid A' (Milner et al., 1971), butpolysaccharides play an important role as well(Westphal and Luderitz, 1954).

Tables 1 and 3 show that a compound present infraction IV with an average molecular weight ofabout 23 000 was still toxic for mice. The positiveLLT and the potentiating effect of actinomycin-Dsuggest an endotoxic activity. The fraction had avery low protein/carbohydrate ratio, but some lipidcompounds and unsaturated fatty acids weredetectable (Table 3). The glycolipid demonstratedby Ng et al. (1974) might be present in this fractionand be responsible for the clotting reaction. Sincewe were dealing with unpurified endotoxic prepara-tions it was impossible to correlate the chemistrywith either mortality in mice or clotting propertiesin the LLT.The separation obtained with the DEAE-Sepha-

dex showed a difference in sensitivity of both testmethods in the positively charged and the negativelycharged fraction, which was partly caused by theinsolubility of the former. This difference suggeststhat compounds difficult to resolve should be testedin vivo and in the LLT.

Conclusion

To discriminate between an immunological and atoxic mechanism a specific precipitinogen andendotoxin had to be prepared from H. influenzae. Acombination of ultracentrifugation, column chroma-tography (Sepharose 6B), and ion exchangechromatography (DEAE-Sephadex A25) resulted ina bacterial extract that contained the specific pre-cipitinogens and a very low endotoxic activity.Aqueous phenol, chloroform, and petroleum ether.extraction were performed to obtain endotoxin.

Both the LLT and the mortality in mice after

actinomycin-D potentiation proved to be sensitiveand reproducible methods for detecting endotoxicactivity in bacterial compounds. It was shown thatthe low molecular weight fractions from H. influenzaestill contained endotoxic activity. The LLT provedto be more sensitive in the high molecular weightfractions, whereas the in-vivo method was moresensitive in the low molecular weight fractions. Wecan offer no explanation for this phenomenon. Theuse of both methods together seems justified.

We thank the Department of Medical Microbiology(Professor J. B. Wilterdink) and Messrs H. Jans andJ. Staal for their assistance and for supplying themedia. We also thank Mr B. H. P. Hazenberg forcaring for the cultures, with the help of Miss E. Sluis,and doing the immunoelectrophoresis together withMiss G. Bergsma. The Limulus lysate test wascarried out by Mr A. Feringa (Department ofHospital Epidemiology). Mr J. Berends (CentralAnimal Laboratory) and his staff assisted in theanimal tests. Chemical analyses of the bacterialcompounds were carried out with the help of MessrsW. Sluiter, L. Ruinen, J. G. Bouman, and theirstaff. Mr S. Pasma prepared the figures. Mrs H.Jipping-Anholts typed the manuscript. ProfessorA. Kroon read it carefully and Dr Chris W. Clarke(Brisbane, Australia) revised it.

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The March 1978 IssueTHE MARCH 1978 ISSUE CONTAINS THE FOLLOWING PAPERSUltrastructure of Ebola virus particles in humanliver D. S. ELLIS, D. I. H. SIMPSON, D. P. FRANCIS,J. KNOBLOCH, E. T. W. BOWEN, PACIFICO LOLIK, ANDISAIAH MAYOM DENG

Evaluation of the Microcult system for isolating andidentifying Neisseria gonorrhoeae R. J. WILLIAMS,C. S. RATNATUNGA, J. M. T. HAMILTON-MILLER, ANDW. BRUMFITT

Chemotactic activity of cerebrospinal fluid inpyogenic meningitiS B. M. GREENWOOD

Outbreak of infantile enteritis caused by entero-toxigenic Escherichia coli 06.H16 B. ROWE, R. J.GROSS, S. M. SCOTLAND, A. E. WRIGHT, G. N. SHILLOM,AND N. J. HUNTER

Flavobacterium meningosepticum as an opportunistRAMA M. MANI, K. C. KURUVILA, P. M. BATLIWALA,P. N. DAMLE, G. V. SHIRGAONKAR, R. P. SONI, ANDP. R. VYAS

Some interesting isolates from a diagnostic labora-tory P. L. LIM

Effect of Tween 80 on incubation period of Lacto-bacillus casei assay of serum folate G. B. TENNANT

Polyene antibiotics in assessing significance ofantistreptolysin 0 activity K. C. WATSON ANDE. J. C. KERR

Paraganglioneuroma of the duodenum: an evolu-tionary hybrid? T. COONEY AND E. C. SWEENEY

Significance of a-subunit HCG demonstrated inbreast carcinomas by the immunoperoxidasetechnique ROSEMARY A. WALKER

IgM lambda cytoplasmic crystals in three casesof immunocytoma: a clinical, cytochemical, andultrastructural study W. W. FEREMANS, P. NEVE,AND M. CAUDRON

Cerebral involvement in multiple myeloma: casereport J. MCCARTHY AND S. J. PROCTOR

Congenital syphilis in aborted second trimesterfetus: diagnosis by histological study HERBERTBRAUNSTEIN

Internal sphincter and haemorrhoids : a pathologicalstudy M. T. HAQQANI AND B. D. HANCOCK

Abridged differential leucocyte counts provided bya Coulter Channelyzer in a routine haematologylaboratory PATRICIA A. WYCHERLEY AND M. J.O SHEA

International Committee for Standardization inHaematology: Protocol for type testing equipmentand apparatus used for haematological analysis

Staining properties and stability of a standardisedRomanowsky stain P. N. MARSHALL, S. A. BENTLEY,AND S. M. LEWIS

Measuring IgG anti-A/B titres using dithiothreitol(DTT) R. C. KNIGHT

Technical methodsA multiple sampling device for the mass screeningof serum samples for hepatitis B surface antigenC. H. CAMERON AND J. A. J. BARBARA

An immunofluorescent method for detecting anti-bodies against viridans streptococci in Streptococcusviridans endocarditis D. C. SHANSON AND CAROLHINCE

Letters to the Editor

Book reviews

Copies are still available and may be obtained from the PUBLISHING MANAGER,BRITISH MEDICAL ASSOCIATION, TAVISTOCK SQUARE, LONDON WC1H 9JR, price £3.00, including postage



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