CA1130706A - Process for determining bacterial endotoxin and reagents used therefor - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

This invention relates to a process for determining a bacterial endotoxin using an amoebocyte lysate of horseshoe crab and/or a pro-clotting enzyme separated from the lysate and (B) a peptide-type substrate of the formula R1 - Gly - Arg - R2 wherein R1 represents a member selected from the group consisting of an L-amino acid moiety whose N-terminal is protected by a protective group, a peptide moiety consisting of an L-amino acid and protected by a protec-tive group at its N-terminal, a D-amino acid substituted L-amino acid moiety, and a D-amino acid substituted peptide moiety consisting of an L-amino acid, and is bonded to the amino group of the glycine moiety ex-pressed by Gly through a peptide bond; and R2 represents a moiety which is bonded to the C-terminal of an L-arginine moiety expressed by Arg through an acid amide bond and/or ester bond and can be enzymatically hy-drolyzed in the presence of the material (A) and the endotoxin to liberate R2H, and/or its mineral acid salt, and detecting the resulting R2H in which R2 is as defined above. According to the invention, various advan-tages such as improved reliability, improved measuring sensitivity, ease of measurement and rapid measurability can be achieved over conventional processes for endotoxin determination utilizing the above lysate or enzyme, and the measuring sensitivity of the conventional processes which is about 10-3 to 10-4 µg/ml can be increased to about 10-6 to 10-9 µg/ml.

Description

~3a~7~6 ~ hi~ inventlon relate~ ~o a proce~s for determining a ba~t~rial endotoxi~ (freguently abbreviated as "endotoxin"
~n th~ present application) utiliz~ng an amoobocy~e ly~ate of horseshoe crab and/or a pro-clotting enzyme separated ~ro~ the l~sate, a~d to a ¢ombination of rea~ents used therefor. Accord$n~ to the pre~ent in~entlo~, ~arious ad-~antage~ au¢h as improved reliability, impro~od moasuring sen~it~ity, ea~e o~ mo~surement and rapid ~oasurability can be achie~d o~er conventional ~roce~sos for endotoxin dot~rmin~tion utill~ing the abo~e lysate or c~zy~e, and the measurin6 sensiti~lt~ of the conventlonal ~rocosses whi¢h i~ about 10 3 to 10-4 ~g/ml aan be increa~ed to about 10 6 tQ 10-9 ~g/ml.
Horse3hoo ar~b~ ar¢ agu~tlc animals bolon~in6 to . the ph~lum Arthro~oda, tho alass Mero~tomata, the Subcla~s Xipho~urida tho ordor Iimuli~acea, and aro sclenti~loally intere~ting a~ lng rossil~. ~h~ living ~peaio~ i~alude o~e g~uc-o~e ~eoies (IimNlu~ polyph0mus) alon~ the Atlantio coa~t or~h~ U. ~. A.~ and t~o genu~-threo ~ccle~
~ear the ~outh ~t ~oe~t o~ the A~i~n Contlnent includln~
Jap~ ChinA~ Mal~d a aod Phll~ppinos. I~ Japan, Jap~neso hors~hoe ~rab (~a~hypleus tridentatu~) lnhabits part o~
the Seto Inland S~a ~d part o~ ~orthern K~u~hu~
Ihe oix~ulatory oy~t~m o~ hor~eshoe crab i~ an 25~ o~n ~a~a~lar ~y~e~ and lts blood contalns amoebocyt0s.
N i~ known that whcn a Gram-ne~ati~a b~a¢terium or an endotoxin ~8 added:to a ~ w ~e~s~on Or a~oebocyte3 in a bu~fer, granul~a ln tha ~mo~boc~tes ~oon ~ani3h, and the a ~bo~ytcs get o~t o~ th~ir ~ha~e and fuBe~ and in the : - 2 ~

1~3~7Q6 meantime, the cells or endotoxin is surrouDded by a ribrous ~el. qhi~ phenom~non is a bodg defense reaction of horseshoe crab, and is known as a common nature of horseshoe crabs irre-spective of their ~pecies. An extract (termed "amoeboc~te lysate") obtained by treating the amoeboc~tes w~th a hypotonic 801ution forms a gel by reaction with a tiny amount (about 10 3 to 10 4 ~g/~l) o~ eDdotoxin. Methods ~or microdetermination of endotox$n utilizing the gellin~ phenomenon which occur~ by the reacbion of the amoebocyte lysate of horseshoe crab with a tiny amount of endotoxin have been develo~ed, and already come into use ln the flelds of medlcine, pharmaceutical ~cience and environmental hy~ienics. It ha~ been widely recogn$zed that there is a fairly high corr~lation between the ~easured value of endotox$n utiliziDg a golliD6 reaction b~twe~n tho amoebo¢gte l~ate and aD endotoxiD (especially ~n enflotox$n of a Gram-ne6ative bacterium) and the mea~ured value of a pyro6en to~t uslng rabbits for oxample, if the p~ro~en used 18 an Qndotoxin. ~his fact has been utilized, for exam21e~ iD a pr~l$miDary port$on of the pyrogen te~t or in 9ual$ty control in the manuracture o~ in~ectablo pharma-aeutiaals for prevention of a p~ro6enic aacideDt caused b~
the ¢ontamination o~ ph~armaceuticals b~ pyrogen.
K~own conventional method~ for determinat~on of endotoxin~ usin6 the amoebo¢~te l~sate of hors~shoe crab include a method in whi¢h the hardness of the gel formed as a re~ult or the gelling rea¢tion i~ evaluat~d visuallg by the nakod eye, a method wherein the gelling time is measured, a method i~ whiah ~hang~ in turbidity are traced by using a turb$dlmeter ~des¢ribed ~or example, ln N. S. Young et al.

113~7~6 J. Clin. InYe~t: 51 1790~1972) and E. T. Yin et al. B.B.A.
261, 284(19~2)J and a clotted protein determinatioD method rde~cr~bed for ex~mple, in M. Niwa et al. JPD. J. Med. ~ci.
Biol. 27, 108-111(1974)), the first method being most freguently u~ed~ ce these prior ~ethods all utilize the gellation phenomenQn, they have the defect that the end point of gellation or flocculation i8 difficult to evaluate.
The dirricult~ is in¢rea~ed when the assag 8ample ha9 a high vi~¢osity. ~he measurin~ sensitivity of endotoxin by these conveDtional methods i8 at best about 10 ~ to 10 4 ~g/ml although it can vary according, for example, to the activity of the a~oebocyte lysate, the type of endotoxin, and the means and standards of evaluation, and it has been desired to provide a determination method having higher s~nsitivit~.
~hese prior techniques are advantageous i~ sensi-tivity~ rapidity and simplicity o~er the method of determining endotoxiDs by a pyrogen test or fatal test u~in~ an experimental animal. Howe~er, since they rely on the dlreot or indirect determinatlon of the de~ree of ~ella~lon, thoy cannot ~ubstantially avoid retardation or inhibitlon oX gellatio~ in the presence of a ~ellation iDhlbitor in the a~ay sample or aCceleration of gellation in the ~rese~ce of a certain protea~e in the assay sample.
Ihe prose~t inventor~ have worked on the gellation meohanism of the amoebocyte lysate of horseshoe crab, and found that an endotoxin can be detected and determin~d with superior reliabllity and ~ery hi~h sensitivity on the basi~
of a Dew mechanism which quite differs from the gellation 113~7~116 phenomenon utilized in the prior methods.
qhe in~Qstigations of the pre~ent in~entors led to the di~covery that when tA) a material selecte~ from an am~eboc~te lysa~e o~ horse~hoe crab a~d a pro-clotting eDz~me separated from the lgsate and (B) a peptide-type substrate of a 3pecified ~tructure are contacted with an assa~ sample, a specified terminal ~tructure portion o~ the substrate (B) i~ freed by enzymatic h~drolysis ir the assay ~ample contains an enaotoxin, and that the amount Or the freed termiD~l portlon increases with increasing amount Or the endotoxin present in the assa~ ~amplo. They al~o found that the 3ub~tan¢e freea b~ this hydrolysis caD be detect~d and deter~ined, and the measureme~t sensiti~ity is ~o good a~ to eDable a tiny a unt (about 10 6 to about 1~9 ~g/~l) o~ an endotoxin to be detected and determined.
It was al~o ~ou~d that the p~ptlde-type sub~trate (B) must have a ~tructure in which L-amiDo acids are connected in th~ order of ~rginln~ (~rg) and 61ycine (Glg) starting at the C-end of the terminal portion to be cut, aDd that the peptlde-tgp~ substrate (B) of thi~ specified structure, iD
the pr~sence o~ an endotoxin, acts on the pro-clotting enzyme present in the amoeboGyte ly~ate of horseshoe crab or separ~ted from it, and specifia~lly uDdergoes enzymatic hydrolysis by the generated acti~e enzyme and thu~ free~ the terminal portion. It wa~ al~o found that the amount of the terminal portion to be freed show~ a good correlation with the amount of the endotoxin within a certain range. and the amount of the terminal portion freed increases proportionally to th~
increa~e Or the e~dotoxin content. qhey al80 found that ~13~76~6 ~ince only the pre-clottin~ e~zyme i~ activated in the presence of endotoxin quantitati~ely and i8 involved iD the cu~ting of the t¢rminal portion of the peptide-type ~ub~trate (B), the endotoxin can be co~veniently detected and determined wit~out ang adver~e effect of other protease~ and/or eQterases which may be pre~ent in the amoebocyte lysate or t~e enxgme sepa-rated ~rom the l~sate or other proteases and/or esterase~
which may be present in the a~ay ~ample.
~he detalled meohanism of the activation of the pro-clotting enzyme ha~ not been entirel~ known, but it i~
pre~umed that the pro-¢lotting enzyme (an amida~e precur~or and/o~ an e~tera~e precursor) in the a~o~bocyte lysate i8 a¢~i~ated by tho a¢tion of endotoxin and thus converted to a clotting enzyme (amidAse-like substance ~nd/or estcra~e-like ~ub~t~nce)~ and that the actl~ated en~yme slecti~olya¢t~ on the terminal portion Or the peptide-type sub~trate (B) of the 8pec~ fi~d Jtructure to cut ~nd free it.
It is an ob~eot of th~ 8 iDveDtion therefor~ to provide a ~uperlor prw e~e for detecting and/or aetermiDin~
endotoxin~ u~ilizing the amoebocyte lysate of horseshoe crab and/or a pro-clottiDg enz~me separated from the l~ate.
Another obJe¢t of this iDvention i8 to provlde a ~ombination of reA6eDta usod in thi~ procos~.
: ~he abo~e and other ob~ect~ and advantage~ Or the lnveDtio~ ~111 becomo more apparent from the following de~crlptioD.
According to the preseDt invention, there ar0 provided a proce~s ~or determini~g a bacterial endotoxin, which compri~e~ contaCt~ng an a88ay sample with (A) a material ~13~76~16 selected from t.he OEoup consist m g of an amoebocyte lysate of horse~hoe crab and a pro-clotting enz~me separ~ted from the l~ate, ~nd (B) a peptide-type ~ubstrate of the formula Rl - Gl~ - Arg - R2 (1) wherein Rl represent~ a member selected from the group consi~ting of an I-amino ac~d iet~ who~
~-terminal $8 protected by a protective OEoup, a peptide moiety consisting of an I_amino ac~d and protected b~ a protective group at $ts N-terminal, a D~amino acid substituted l_amino acid moiety, and a D-amino acid substituted peptide moiety consisting of an l_amiDo acid, and is bonded to the amiDo group of the glycine ioty expressed by Gly throuEh a peptide boDd; and R2 represent~
a moiety which i9 boDded to the C-term$nal of sn L-argi~ine moiety expre~ed by Arg through an acid amid~ bond and/or an ester bond and can be enz~matically hydrolyzed ln tho presence Or the material (A) A~d the endotoxin to liberato R ~ , ~d~or it~ mlneral aoid salt, detecting the re~ulting R ~
in whl~h R2 i~ as de~ined above, and if desired, deter~ining it~ and a rea6ent ~or detecting or determining an endotoxin which compri~es a co~bination of the componQnts (A) and (B).
~he amoobocyte lysate of horsesho~ crab used in this invention ca~ be obtained by a known procedure which involve~ treating amoeboc~tes contained in the blood of horse~hoe crab with a h~potonic solution ~or exampla, E. ~.
Yin et al. B.B~A~ 261, 284 (19?2 ) . and S. Nakamura et al. J.
Biochem., 80, 1011-1021 (1976-)). It ~ also available ~ ~!

~3~7~t6 commercially under the registered trademark~ Pregel (Teikoku ~ormone Co., Ltd., Japan) 9 ` Pyrotest (Difco Ra~o., U. 6~ A.), Pyro~ent (Mallinckradt Chem. Works~ U. S. A.), P~rostat (Worthington, U. S. A.), LAL (Haemachem~ U. S. A.), and Iimulus Amebocyte Iysate (~L) (Microbiological Associates, U. S. A.).
~ he resulting Qmoebocyte lysate may be sub~ected to such a procedure as column chromatography, electrophoresis, isoelectric fractionation (Electro-rocuslng~ a tradename LKB), a~init~ ¢hromato~raphy, or gel filtrat$on t~o separate pro-¢lottinE e~zyme in the lysate. $he separatlng procedure ~8 described~ for oxample, in N. S. Young et al., J. Cli~.
In~e~t. ~, 1790 (1972), J. S. Salivan et al., B.B.R.C. 66, 848 (1975), J. Y. Tai ~t al., J. Biol. Chem. 252, 2178 (1977), an~ S. ~akamura et al~. J. Biochem. 80, 1011 (1976), and can be used to produce the pro-clotting e'nzyme used iD the present in~ention.
I~ tho clottable protein containe~,in the amoebocyt~
lysato iB removed by U~iDg a gel filtration proceduro, the Aenslti~ity of as~ay aan be increased to at lea~t 10 times.
Example9 o~ the protective group at Rl in the ~eptldo-t~pe ~ubstrate (B) expressed bg formula (1) are x-N_bensoyl group~ a-N-carbobenzoxy~ N_tert.-butoxycarbonyl and p-tolu~ne~ulfon~l ~roupH. Specific examples of Rl are B~-Ile-Glu(-r-OMe)-~ Z-Ile-Glu(-r-OMe)-, ~o~-Ile-Glu-, Z-Ile-Glu-, Bz-Val-, Boc-Val-Ieu-, Bz-Val-Leu-, (D-amino acid moiety)-Ile-Glu-~r-OMe)-, (D-amino acid iety)-Val-Leu-, Bz-Val-Ser-, (DLamino a¢id iety)-S~r-Gly-Val-Ser-&ly-Arg-, Bbc-Val-Ser-, Boc-Iou-~ Boc-Ser-, Z-~eu-, Z~Ser-, Z-Val-, 1:~L3~7~6 (D-amino acid moietyJ-V~l-Leu, (D-amino acid moiety)-Val-~er-, (D-amino acid moiet;sr ~ u~, and (D-aminG acid moiet;y )-~er-.
In the abo~e and ot~er exempllficatio2ls ira thi~ application, Bz repre~eD~ a be~zoyl group; ~, a carbobenzoxy g~oup, Boc~
a tert-butoxycarbo~yl group; 108~ a p-toluenesulfonyl group;
Me, a methyl group~ Ile, I_isoleucine; Glu, I_glutamic acid;
Val~ L-valine; ~er, I_serine; and Leu, I_leucine.
R2 iD the pe~tide-type sub~trate (B) of formula (1) is a protective group for the C-terminal of the L_arginine moiety expres~ed by Arg, and i8 bonded to the C-terminal by aD acid amide bond and~or an ester bond. In the presence of the component (A) and the endotox~n, the acid amide bond and/or ester bond undergoe~ the action of enzymatia hydrolysi~
to liberate R2H. According to the method of thi~ invention, the pre~ence or absence (when R2H i~ not liberated) of an endotoxin can b~ know~ by detectlng the liberated R2H. The amount of the endotoxin can be known by determini~g the amount of the R ~. Hence~ R2 m~y be a moiety capable of permitting th~ deteotion and/or determinatlon of the liberated R2H. Any desired ~pecies of R2 whloh can be detected by, for example, a phy~iaal or chemioal meaDs can be sel~cted for u~e in this iDv~tion. A suitable d~tecting meaDs is one i~ whiah R2 ¢~pable o~ generatin6 chromogeDic R2~ is u~ed, ~nd the presence and amouDt of R2H is determined by 8~ optical meaD~ 3uch as the measurement of it~ ~bsorbance.
Example~ o~ R2 suitabl~ for use in this procedure are a para-nitroanilid~ group (PNA) which generates a compound R2H
formiDg a ~ellowi~h orange color, a 5-nitro~a-Daphthylamide (5-~NA) which senerates a compound R2H forming an orange _ 9 _ :1 1~3~7:~6 yellow color, and ~-naphth~lamide (~-NA), ~-naphthgl ester ~-NE), ~-naphthyl e~ter (~- Æ ), indoxyl e3ter (INDE), ~-methyl indoxyl ester (MINDE), (4-methyl~umbellife~yl est~r and resor~in ester which generate fluoresce~t compounds R2H^
Examples Or ~uitable peptide-type substrates (B) of formula (1) ares Bz-Ile-Glu(~-O~e)-Gly-Arg-PNA, ~o~-Ile-Glu-Gly-Arg-PNA, Boc_Val_Leu_Gly_Arg_PNA, Bz-Val-Gly-Arg-PNA, Bz-Val-8er-Gl~r-Ar6-5-NNA, ,.
Bz-Val-Leu-Gly-Arg-~-NA, ~o~-Ile-Glu-Gly-Arg-fl-N~, Boc-V~l-Gly-Arg-INDE, Z-Val-Ieu-Gl~-Ar6-4-methyl umbelliSeryl ester, Bz-Val-~er-Gl~-Ar6-re~orurin ester, D-Val-~er-Gl~-Val-Ser-Gly-Arg-MINDE, Boc-V~ er-al~-Arg-PNA, Boc-Val-Gly-Ar~-PNA, Bo¢-L~Gl;y-Arg-PNA, Boc-~er~-o-B~)-Gly-Ar~-PNA, D_Val-l_L~u-Gï;5r-Arg-5NlNA
~LV~l-L_I~u-Gl~-ArK-~-NA~ and DLVal-I_Ieu-Gl~-Ar6-Re~orufin~
~ome oS these ~ub3tr~tes (B) are commerciall~ ~
available, and ~om~ c~n b~ prepared by a combinatio~ of u8~al peptlde ~ynthe~lz~ng method~. Basicall~, the~ can be prepared, for example, b~ the follow~ng two ~ethod~. One _ 10 --' 113~7i~6 m~thod i8 a ~tepwi~e method which compri~e~ coupling a com-pound of R2 in formula ~1)A with ~rg, and then ~ucces~ively coupliDg Gly and the other amiDo acid residues. The other method compri~e~ ~ormlng an amlno acid arrangement of the de~ired peptide ~tructure stepwise, and finally coupling a compound R2 with th~ C-terminal of Arg of the peptide structure.
Examples o~ available coupliD~ method~ are the DCC mcthod d~eloped by J. C. SheehaD et al. ~Jacs 77, 1067 ~1955) and Jacs~ ~ 1367 (1956)~ and it3 improvement, a mixed acid ~nhydridc ~ethod which comprises fprming an acid anhydride o ethglchlorocarboxylic acid or i-butylchlorocarbo-xylic acid with an amino acid, and condensing the amine component, and the HO~u-DCC method which comprise~ reac~ing an,aold ao~poneDt with N-ox~uccinimide to form N-oxy-succinimide ester, and condensing it by addiDK an amine component and DCC.
~ he guanidine group of Arg can be protected by a nitro eTOUp; tho -OH group of Ser, by a benzyl group; and th~ r-carboxyl group Or glut~mic acid, as a methyl e~ter or a bon,~yl e~ter.
Ih~ mea~urement by an optical means ~8 described above i~ ba~ed o~ the fact that R2H formed by enzymatic h~drol~is ha~ quito a diff~rent absorption ~pectrum from that of the sub~tra~e (B). Thi~ measuring method has greatly advanced i~ recent years as an assay method for enzymes usin~
chromogenic substrate~ (~ew Method for the Analysi~ of Coagulation U8ing Chro ge~ic Substrates: Proceedings of the S~mposium o~ the Deut~che Gesellschaft f~r Kl~nische ( 1~3'~37~6 Chemie Titisee, Breis~an, We~t Germany, July 1976, Editor I~ Witt Walter de Gruyter-Berlin, New York 1977).
qhe mea~urement method is de~cribed in ~ore detail by referring to the u~e of Bz-Ile-Glu(-r-OMe)-Gl~-Ar~-PNA-HCl a~ a sub~trate~
~h~3 sub~trate ha~q an abqorption maximum ha~ing a molecular extinction coeficient of 12000 at 316 nm, and its absorption near 405 Dm i~ not clear and i8 ver~ little.
On the other hand~ para-nitroanillne formed and liberated from the ~ubstrat~ by enzymatic hydroly~i~ ha~ an ab-~orption maximum of a molecular extinction coeicient of 13200 at 380 nm, and a clear and great absorption having a molecular extinction coeficient o 9620 at ~05 nm.
Hence, the amount o~ the para-nitroaniline ~9 measured and determined by the ~pectrophotometric method at 405 nm, and the amount of tho endotox~D proportional to the amount o the para-nitroa~ e CaD be ea~ determined by using a standard curve (calibratioD curve) prepared in advance U8iDg a ~tandard samplo oont~lniD6 a knowD amount of endotoxin.
~he follow~ng compounds having 1uorescence which are 6oneratod by en~matic h~droly~is can bo determined by an ordinary 1uore~aent spQctrophotometer by ~electin~ the excitation wavelength (ex.) and the measurement wavelength eI. ) a~ tabulated below.

~3~7~6 Excitation Measurement Compound R H Structural formula wavelength wavelength - (ex.; nm) (em.; nm) ~-Naphtylamine ~ NH2 335 410 OH

~-Naphthol ~ 330 460 - 470 ~-Naphthol ~ OH 330 410 Indoxyl ~ 395 470 1, .

' N-methylindoxyl ~ 430 500 c~l3 4-Methyl ~ cl-l3 umbelliferone O ~ O ~ OH 330 450 ResorUfin 0 ~ 0 ~ 580 This optical means can bo utilizcd in thc visible rcgion, the ultra-violet region~ thc fluoresccnt rcgion, etc. dcpclldillg ul)on the selection of the compound expressed by R~ll.
The invention is furthcr illustrated in the following examples and in the accompanying drawings. Of the drawings, Figure 1 relates to Example

2 below and is a graph showing correlation between the concentration of the endotoxin and the absorbance of the resulting PNA. Figure 2 relates to Example 4 and is a graph showing correlation between the relati,ve fluores-cence (%) and the amounts of the endotoxin.

Preparation Example 1 113~7~6 Preparation of amoebocyte lysate:
About 100 ml of a hemolymphatic fluid was extracted from Tachypleus tridentatus (body weight about 2 kg), a Japanese horseshoe crab) in accord-ance with the method - 13a -~3'~7'~6 di3closed in Jap~ne~e Patent Publication No. 40131/76 while ~tri¢tly avoiding contamlnation. Amoebocytes were separated by ce~tr~fugal separation, and wa~hed wlth a 3% agueou~
~olution of ~odl~m chloride to ob~ain amoebocyte pellet~.
To the amoeboc~te pellets was added distilled water or buffer ttris-XCl, 0.05 M; CaC12, O.OOlM; NaCl 0.15 M; pH 7.2~ in an amou~t oDe-tenth the volume of the starting hemolymphatic fluid . ~he mixture was well stirred by a sterilized homogeni~er, frozen and melted, and then centrifuged for 15 minute~ at a speed of 5000 rpm to form amoebocyte lysate ~achgpleus (AL~
for short).
Preparation Example 2 ~ he hemolymphatic fluid of Limulus polyphemus, a horse~hoe crab occurri~g in U. ~. A., was treated in the same wa~ ~ in Preparation Example 1 to afford amoebocyte ly~ate Iimulu~ (ALL for short). ~he ALL wns gel-filtered using ., . ~ Sophadex G 50 by the method of N. ~. Young, J. Clin. In~est., ~ 1790 (1972) to ~orm a fraction I co~tainin~ an amidase preour~or (AII-FI for ~hort).
Exam~le 1 An cndotoxi~ of Salmonella minesota R 595 prepared by the method of H~ Niwa et al., Japan J. Med. Sci. Biol.
26~ 20 (1973) was cau~ed to act on AL~ and ALL of horse~hore cr~bs obtained in Preparation Examples 1 and 2, and the 25 activity of re9ultin6 amidase was measured by using variou~
~ynthetic substrate~. ~he results are shown in ~able I.
Ihe method of mea~urin~ the acti~ity of amidase on ALT ~nd ALL wa~ a~ follows:
A m~xture aoDsi3ting of 0.8 ml of 0.1 mM synthetic i -- 14 _ f ff p~ r) ~R~;

113~7~6 ~ub~trate di~ol~ed in O.lM Tris-HCl buffer (p~ 8~0), 50 ~1 Of 0~5M MgC12~ and 20 ~1 Of a 0~1% eDdotoxin solution was pre-~ncubated at 37C ~or 3 minute~. Then, 5 to 20 ~1 of the ly ate was added ~nd well mixed. ~he mix~ure wa~
incub~ted at 37C for 15 minut~. A~ter lncubation, 100 l~l of glacial acetic acid was added to terminate the reacti.on.
~he absorba~ce of thQ re`sulting p-n~troanili~e at 405 nm was mea~ured.
~ble 1 shows the amount iD n moles of the p-nitroaniline calculated from the ab~orbance, aDd it~ relativ~
~mount iD ~ercent with the amount of p-nitroaniline det~rmined w~th re6ard to the 3z-Ile-Glu(-~-OMe)-Gl~-Arg-PNA substrat~
being taken as 100.
~able 1 .. . __ ___ . . . _ No. ~ubstr~te A30~nt Xelati~e Alount Relative (n activity (n activity _ ,.. _ ~ . moles) t%~ mole~) (%) 1 Bs-Ile-Glu~Y-OMe)-Gly-~rg- 17.7 100 4.~ 100 2 To~-Ile-Glu-Gl~-Arg-PNA 7.3 ~1 14.1 318

3 Ebc-Val-Ieu-Gly-Arg-PNA 11~0 62 19.1 431

4 ~z-Val-Gly-Arg-PNA 12.4 7o 10.1 227 Bz-Phe-V~l-Ar~-PNA 0.5 3 0.2 5 6 Z_Gly-Pro-Ar~-PNA 0.5 3 0.4 9 7 H.D.Phe-P~p-Arg-PNA _ _ o.3 6 8 H.Glu-Gly-Arg-PNA <0.1 ~ 1< 0.1 ~ 3 9 H.D Val-Ieu-Arg-PNA <0.1 < 1~0.1 ~ 3 H.D Val-Ieu-~ys-PNA ~0.1 < 1~0.1 ~ 3 11 D Pro-Ph~-Arg-P~A <0.1 ~ 1<0~1 < 3 .

~13i~?76! 6 The ~ynthotic ~ubstrates 1 and 2 ~hown in Table 1 are specific ~or blood coagulation factor X~, and are very interesting in v~ew of the fact that they are specific also for amidase resulting ~rom the acti~ation of an amoebocyte lysate of horse~hoe crab by an endotoxin. ~he sgnthetic sub~trate 3 i~ a substrate synthe~ized by reference to the amino acid arr~ngem~nt .~... Asp-Glu-Pro-Gly-Val-Ieu-Gly-Arg- ~.... (A-Chaln) at the cleavage 9ite or coagulogen cau9~d b~ a clotting enzyme. ~he ~ynthetiG substrate 4 i9 a substrate for urokins~e. ~he substrate~ 5, 6 and 7 are substratos for a_thromb~D and do not have the e~sontial amino acid structure in accordance with thQ preseDt iD~ention.
~he s~nthetic substrate 8 does not meot the requirement of the Rl moiety of th~ component (A) used in this iD~oDtion.
~he ~ynthetic sub~trates 9 a~d 11 are for kalli~rcin, and the synthetic sub~trate 10 is for plasmln. ~hese ~ubstrate~
9 to 11 ~o Dot undergo the actioDs of enzymès in a blood coagulation-fibrinoly~i~ systom, an~ ther~rore, can permit the dotectioD aDd ~atermination Or an oDdotoxiD b~ a gellation phenome~on utili~in6 an a~oeboc~te l~sate of horsoshoe crab without tho def~ct of the prior methods (the preseDco of ~ariou~ en~mQ system3 pres~nt in the blood cause~ the non-speci~ic gellation of the a ebocyte l~ate, which in turn lea~ to the failuYe of detecting and de-termining the endotoxin?, and t~e determiDatioD of anendotoxin in protea~e preparatioD~.
Example 2 U~ing AL~, the endotoxin Or ~almonella minesota R595, and Bz-Ile-Glu-Gly-Arg-PNA, the correlation between 7~!6 I

the concentration of the endotoxin and the absorbance o~ ~he resultln~ P~A was examined, ~able 2 and Figure 1 show the absorban~ of P~A at a ~ub~trate concentration of 0.05 ~ !
after the reaction mixture wa~ incubated for 15, 30, and 60 minute~
~able 2 . ~
, . . _ Concentration ~bsorbance of PNA after an iI cubation time of of endotoxin 15 minute~ 30 minutes 60 minute~
I ( ~ ~ _ 1 0.38 0.38 0.38 lo~l 0,34 0~7 0.~8 10-~ 0.23 0. 34 0. 37 lQ-3 0.07 0~25 0.35 10-4 0.01 0.13 0.30 10-~ 0.00 0.05 0.18 10-6 l 0.01 0.08 10-7 0.00 0.0 10-8 0.01 ~....... . .
A3 i9 seen from Table 2 and ~'igure 1, the absorbance of P~A increase~ with increa3ing concentration of the endo-toxin, and the aorrelation between the concentration of the endotoxin and the absorbance Or PNA is ~traightly linear within an endotoxin concentration ran~e o~ 2 x 10 7 to 2 x 10 4 ~/ml. ~hi~ shows that the method of thi~ invention has high ~en~itivity and stability as compared with the dete¢tion limit of th~ con~entional endotoxin determlning method which i~ 10 3 to 10 4 ~g/ml.

~3~7~6 Exa~ple ;7s Using ALI~FI, ~nd ~Ile-Glu-Gly-Arg-PNA, endotoxins derived from ~ariou~ species of bacteria were determined. The raD6es of the concentrations of the endo-toxi~ that could be detected were determined from thecorrelation ch~rt of the concentrations of variou~ endotoxin~
versu~ the ab~orba~ce o~ PNA which was prepared in accordance with hxample 2. The re~ult9 are shown in Table ~.
~able 3 , .
~ndotoxin~ E~dotoxin concentr~tions (~g/ml) , . . _ Endotoxin derived from 2 10 5 to 2 10 7 (5 x 10-4) Salmonolla mine~ota R595 x x . . _ E~ col~ UK~B 6 x 10 5 to 6 x 10 7 (2 x 10 4) ~higella K3 2 x 10 4 to 2 x 10-6 (~ x 10-3) ., E. coli Olll:B4 5 x 10 4 to 5 x 10 6 (3 x 10 3) , ....... .... _ ..
P~- aeru6inosa 2 x 10-3 to 2 x 10-5 (5 x 10-1) .
Note~ ~he rigures in the parenthe~e~ aro tho co~centration~ of the endotoxins which were determiD~d by the conventional gellation ~e~hod.

It c~n be ~een from Table 3 that the method of thi~ inventioD ¢an detect and determine bacterial endotoxins with a sen~iti~ity 100 to 1000 times as high as that of the conventio~al gellation method using ALL, and permits the detection of endotoxin3 u3ing an amoebocyte lysate of horseshoe crab.

~13~7(~6 Example 4 A ~pecifl¢ ~epti~e fluorogenic substrate D_Val-I-Leu-Gl~ Arg-Re~orufin e~ter (ra~28_13.8 C~0.46 in 8~' DMS0 ) wa~ first dis~olved in dimethylsul~oxide (DMS0, ana-lytical reagent), ana the solution wa3 diluted to gi~e a final concentration of 0.1 mM u~ing 50 mM Tris-HCl Buffer (pH 8~0) containing lOO ~M NaCl and 10 mM CaC12.
U~ing a fluorescence spectrophotometer (for example, Hitachi, del MP~-2A)~ 2.5 ml Or the 0.1 mM ~ubstrate buffer ~olution wa~ added to a cuvette and preincubated at 37C for 2.5 minutes.
10 ~1 of ALI-Pl (OD280Ø3/ml) activated with endotoxin (E. Coli OlllsB4) wa~ added, and mixed immediately.
~he r~corder for mea~urement of excitation at 540 nm and emi~ion at 580 nm wa~ ~tarted.
~he inaroa~e of the relative fluore~¢ence (%) wa~
read at re6ular time interval~ after a lapse of 100 ~econd~
The a~y wa8 perform~d according to the varying ~mountJ of the endotoxin., ~he instrumont wa~ standardized ~o that A 10 I~M ~olutlon o~ Re~orufi~ in 0.1% DMS0 wo~ld Eive 1.0 rel~tive fluore0cence unit. ~I!he re~ults are ~hown ln Figure 2.

Claims (4)

WHAT WE CLAIM IS:

1. A process for determining a bacterial endotoxin, which comprises contacting an assay sample with (A) a material selected from the group consisting of an amoebocyte lysate of horseshoe crab and a pro-clotting enzyme separated from the lysate, and (B) a peptide-type substrate of the formula R1 - Gly - Arg - R2 wherein R1 represents a member selected from the group consisting of an L-amino acid moiety whose N-terminal is protected by a protective group, a peptide moiety consisting of an L-amino acid and protected by a protective group at its N-terminal, a D-amino acid substituted L-amino acid moiety, and a D-amino acid substituted peptide moiety consisting of an L-amino acid, and is bonded to the amino group of the glycine moiety expressed by Gly through a peptide bond; and R2 represents a moiety which is bonded to the C-terminal of an L-arginine moiety expressed by Arg through an acid amide bond and/or ester bond and can be enzymatically hydrolyzed in the presence of the material (A) and the endotoxin to liberate R2H, and/or its mineral acid salt, and detecting the resulting R2H in which R2 is as defined above.

2. The process of claim 1 wherein the protective group is a group selected from the class consisting of an .alpha.-N-benzoyl group, an .alpha.-N_carbobenzoxy group, an N-tert.butoxy-carbonyl group and a p-toluenesulfonyl group.

3. The process of claim 1 wherein R2 is a member selected from the group consisting of para-nitroanilide, 5-nitro-.alpha.-naphthylamide, .alpha.-naphthylamide, .alpha.-naphthyl ester, .beta.-naphthyl ester, indoxyl ester, N-methyl indoxyl ester, (4-methyl)umbelliferyl ester and resorufin ester.

4. A reagent for the detection or determination of an endotoxin, comprising (A) a material selected from the group consisting of an amoebocyte lysate of horseshoe crab and a pro-clotting enzyme separated from the lysate, and (B) a peptide-type substrate of the formula R1 - Gly - Arg - R2 wherein R1 represents a member selected from the group consisting of an L-amino acid moiety whose N-terminal is protected by a protective group, a peptide moiety consisting of an L-amino acid and protected by a protective group at its N-terminal, a D-amino acid substituted L-amino acid moiety, and a D-amino acid substituted peptide moiety consisting of an L-amino acid, and is bonded to the amino group of the glycine moiety expressed by Gly through a peptide bond; and R2 represents a moiety which is bonded to the C-terminal of an L-arginine moiety expressed by Arg through an acid amide bond and/or ester bond and can be enzymatically hydrolyzed in the presence of the material (A) and the endotoxin to liberate R2H.