Note: Descriptions are shown in the official language in which they were submitted.
1 This is a divisional applicat-on of ~anadian patent
application serial number 366,572 filed on December 12, 1980.
BACKGROUND OF THE INVENTION
Field oE the Invention
_
The presen-t inven-tion generally relates to a biolog~
ical test method and more particularly to an improved method of
measuring bacterial endotoxins in bloo~ -fractions, particularly
hurnan blood fractions, and an improved method of stahilizing
king crab arnebocyte lysate.
Prior Art
Various method~ have ~een devised for the detection
of bacterial endotoxins in hllman and animal blood. One of the
newer methodsinvolYes the use ~f Limulus arnebocyte lysate. In
that method 7 the lysate is contacted with an endotoxin-contain-
ing source such as a hurnan blood frac-tion which has been prev~
iously ex-tracted ~ith chloroEorm or the like or diluted substant-
ially wi-th water to reduce the concent;ration of an inhibitor in
the blood to below tlle level which would subs-tantially impair
the desired yelation of the lysate ~y blood endotoxins. The
previously diluted o~ purified bloocl frac-tion is mixed with the
lysate and the endotoxins in the blood cause the lysate to ~orm
a clot~
Unfortunatel~ test results have varied widely, due to
the varia~le nature o~ the lysate. However, advances have bPen
recently made in the purifi~ati.on o~ the lysate to improve -the
test results. See for example, U.S. patent No. 4,107,077 wherein
a mern~er oE a selected group of organic solvents is utilized to
extract inhibitors from the lysate in order -to improve the sen-
si-tivity oE the lysate to ~lood endotoxins. The ~irmness and
extent of the clot fo~ned by -the gelation reaction is measured
su~jeGtively in this test by viewing the same, in some cases
while in~er-tiny the tube containing the clot. Therefore a true
1 ~uanti-tative determination of the concen-tration of endotoxin
cannot be made utilizing this method. Moreover, the method
requires skilled personnel, is not always accurate~ and takes a
conslderable length of time to perEorm, of the order of 45-~0
minutes.
Bacterial endotoxins are produced by Gram negative
hacteria, many of which are very dangerous or deadly in human
~eings and animals Symptoms range from mild to high fever and
in many cases dea-th results~ It is extremely important in order
to promptly initiate the proper medical treatment to identify
as soon as possible the fact that endotoxins are present in the
~lood fraction sample and, if possible, the concentration of the
endotoxins; The previously described gelation reaction test
method is deficient hecause of the considerable length of time
necessary to carry it out, because it does not accurately measure
endotoxin concentration, ~ecause it is difficult to standardize
and ~ecause it requires highly skilled experienced personnel to
perEorm it
A new chromogenic substrate method for assaying bacter-
ial endotoxins using Limulus ame~ocyte lysate is described inpages 20~-22Q of "Biomedical Application of the Horseshoe Crab
~l~79] Allen R. Lis, Inc. That method is specified as not being
applica~le to ~lood and ~lood fractions because oE the inhibitors
in the blood, Instead, the disclosure is directed to the testing
oE bacterial s~lutions containing endotoxins such as might be
the case, for example, in testing food for contamination. German
Auslegeschrift 27 40 323 discloses a simllar process to the one
described in the ahove-indicated literature reference. ~he
test specimens utili~ed in the disclosure in the Auslegeschri-Et
are solutions derived from ~acterial sources other than blood.
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1 Such procedures have no-t been utili~ed in testiny for blood
endotoxins.
It would therefore be highly desirable to be able to
provide an improved method o:E determining bacterial endotoxins
in human and ani'~al ~lood. Such method should ~e rapid r repro~
ducihle, simple -to conduc-t and inexpensive and should preEerably
result in a quantitative determination o~ exdotoxin concent-
ration. It would also be highly desirable if the method could
employ standardized measuring, eguipment utiliza~le by relativel~
unskilled personnel~
It has also heen found that, althouyh lyophilized
lysate xeta.ins its potency under suitable storage conditions
henever liquid }~in~ cra~ amehocyte lysates are used~ whether
original or reconstituted from the powder form, they tend to
deteriorate in potency rapidly~ Thus, -their reactivity to ,
bacteric~l endotoxins sharply and progress,ively decreases with
~ime ~efore and during tests~ Accordingly, varia~le test results
are a common occurrence, especially when utilizing liquid lysates
oE different ages, 'It would therefore be highly desira~le if
a method could be devised to improve the s-tability of the liquid
lysates in order to increase the accuracy of tests invo~ving
their use t
SUM~A Y OF THE INVENTION
The present inventi.on satisfies all of the foregoing
needs. Thus~lan impro~ed method of detecting the presence and
ooncentration of bac-terial endotoxins in human and animal ~lood
is provided t ~.t involves rea,cting a human or animal blood
ser~lm and/o:r plasma ~ract.ion which may contain bacterial endo-
to,xin wi.th kiny carb amebocyte lysate, speci.Eically Limulus
amebocy-te lysate, and a selec-ted substrate which contains a
bound colorimetric indicator capable o-f ~eing split -Erom the
substrate by an enzyme which can be yenera-ted in -the lysa-te by
the endotoxin, Thus, the endotoxin is capable of'conver-ting
proenzyme in the lysate to -the desired enzyme~ The concentration
of colorimetric indicator split from the substrate by the enzyme
can be colorimetrically measured in standard equipment and is
directly proportional to the concentration of endotoxin in the
blood fraction being tested.
It has been determined that chromogenic substrates of
the type, for example, which are disclosed in U~S~ patent No.
4,028~318 are suitable for use as the chromogenic su~strate in
the presen-t method~ Such substrates are represented by the gen-
eral formula: Rl~Al-A2 Gly-Arg-NH-R2 or its salts, where Rl is
hydrogen or alkanoyl having from 1 to 12 carbon atoms or cyclo-
hexylcar~onyl or benzoyl or henzoyl substi.tuted with one or two
halogen atoms, methylamine or phenyl groups or benzenesulphonyl
or toluenesulphonyl, R~ is nitrophenyl or naphthyl or nitro-
naphthyl or methoxynaphthyl or quinolyl or nitroquinolyl r Al is
~0 a single bond or one of the amino acicls Gly, Ala, Val~ Leu~ Ileu,
Pro~ Met, Phe or Tyr, and A2 is one of the amino acids Glu, Gln,
Asp, or Asn~ Other suhstrates such as are disclosed in Volume
Z~, pages 209-220, ~1979,) of Progress In Clinical And Biological
Research may be employed.
It hlas been found that it is unnecessary to purify the
hwnan or animal ~lood fraction to remove inhibitors of a gelation
reaction, ~etween the lysate and ~lood endotoxin in order to
carry out the presen-t met~od~ Nor is it necessary to greatly
dilute the blood fraction to reduce the inhibitor concentration
to below an effective level. At least one of these procedures
ia~5
1 has been necessary heretofore whenever Limulus amebocyte lysate
has heen used to detect bacterial endo-toxins.
A method has also been found to prevent a decrease in
potency, that is a decrease in effectiveness, of the lysate,
The method involves the use of a selected concentration of
heparin. That concentration is enough to accomplish the stab-
ilization of the lysate hut insuff:icient to inhibit the endo-
toxin-lysate reaction which genera-tes the colorimetric indicator-
splittincJ enzyme. Such heparin concentration normally is in the
range of about ~ 0.4 standard units of heparin per milliliter
of the lysate, where the lysa-te contains 15-25 mg of lysate ~on
a dry basis) therein.
With the present method, it is possible to conduct the
desired endotoxin test within a 15 minute period, including mix-
ing oE the ingredients, allowing them to react and making the
colorimetric determination. This 15-minute period is much shorter
than required for previous test methods for bacterial endotoxins,
Moreo~er, the present method simply and accurately measures the
concentration of the endotoxin to indicate the exten-t of the
Gr~n negative bac-terial inEec-tion. Further fea-tures are set
forth in the following detailed description,
DETA~CLED l~ESCRIPTION
A human or animal blood sample is tested in accordance
with the present me-thod. Althouyh a whole blood sample could
be used, the color of the blood would tend to interfere with the
colorimetric reading so that it is preferred -tha-t the blood
utilized be a fraction such as blood serum and/or hlood plasma.
This raction need not he trea-ted in any way, such as hy heating,
or by dilutiny it with water or by extracting it with an oryanic
solven'- such as chloroform to remove reduce the concentration oE
inhibi-tors in the blood which would interfere with a yelation
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1 reaction ~ith the king crab amebocyte lysate. Previous chromo-
genic methods for detecting endotoxin have been :indicated as in-
applicable to blood because of the presence of such inhibitors
and/or have indicated that purification of the blood to remove
or reduce the inhibitor concentra-tion would be required.
Nevertheless, it has been discovered that such puriEication or
dilution is not necessary in the present method.
In carrying out the present method, the blood sample
which preferably is a human blood sample, although the method
is applicable to animal blood samples, is mixed with a king crab
amebocyte lysate, preferably Limulus amebocyte lysate. However,
lysate from other forrns of horseshoe crabs such as Tachypleus
tridenta-tus, the Japanese horseshoe crab, can be used. The
lysate can be prepared in accordance with conventional procedures.
Procedures such as are disclosed in Journal of Clinical Invest~-
gation, Yolume 51, July 1972, Bul].etin of Johns ~opkins Hospital,
Volume 115, pages 265-274 (1964) and Proceedings of the Society
for Experimental Biochemical Medicine 7 Volume 137, pages 334-342,
can be used to recover the lysate. Briefly, the conventional
~o procedure usually involves withdrawinq crab blood by sterile
needle from the crab heart, placing the blood into a mixture
which pre~ents aggrecJation o~ the blood cells and premature lysis
of the cells, separating the ame~ocytes from the remainder of
the blood by centrifuga-tion, lysing the amehocytes ~y mechanical
breaking, freeze-thawiny or by osmotic lysis in pyrogen-free
distilled water in a volume ratio of water to cells of about
3--6:1 or the like. The lysate thus obtained is centrifuged free
of the broken cells and usually freeze dried, that is, lypho-
philized to preserve it. When it is ready for use, the lysate
powder is diluted approximately 50 volumes of sterile pyrogen-
free distilled water to bring it to a solid concentration per
ml, of about .02 gm In the present method, lysate is used which
1 may either be one freshly prepared or one which has been pre-
served, usually in lyophilized po~der orm, and which has been
~econstituted or which during the preparation of the reaction
mixture is directly added as a powder to the liquid mix-ture. In
any event, the lysate is present in a solid concentration in
the reaction mixture of a~out 1~50 to a~out 2.50 gm/100 ml
The substrate utilized in the present method contains
a selected colorimetric indicator capable of being split off
from the substrate by lysate enzyme produced by conversion of
proenzyme by endotoxin in the ~lood s~mple. For such purposes~
any suitable substrate genarally of the type descri~ed in, for
example, U.S. Patent No. 4,028,318 can be utilized~ Howe~er,
certain of such substrates have been found to be more ad~an-
tageous than others. In this regard, a su~strate characterized
by the general formula Bz Ile~Glu-Gly-Arg-pNA is preferred~ This
substrate comprises benzoyl isoleucine-glutami~ acid-glycine-
arginine-p~nitroanilide. Other suitable su~strate is acetate
isoleucine~glutamic acid-glycine-argin;ne-p-nitroanilide. A
third useful substrate is benzoyl-valine-glycine-arginine-p-nitro-
analide. Further suitable su~strates can easily be d~terminedby minLmal experimental testing~ Such substrates may contain,
for example~ 2~naphthyl--amide as the color indicator instead of
the p nitroanilide~
In accordance with the present m~thod, the desired test
reaction is effected by mixing together the blood fraction
sample, the amebocyte lysate and the selected substrate in suit-
able relative concentrations. In carrying out the method human
blood serumcan, for example~ ~e added to a reaction zone such as
a sterile test tube, can then be diluted with about five to ten
volumes of pyrogen~free sterile water and powdered Limulus
~r`~ -7-
1 amebocyte lysate or o-ther king cra~ amebocyte lysate in a con-
centration of abou-t 1O50 -to about 2.50 gram/100 ml. can then be
added to each such -test tu~e. Each test tube is supplied with
0.1 ml of the lysate solutionO The temperature o:E each test
tube is preferably kept within a suita~le range, for example,
35--40C~, most preferably abou-t 37C. This mixture is thoroughly
mixed together and incubated for about 7-9 minu-tes~ preferably
about 8 minutes.
The substrate is then added to each test tube~
preferably in a relative concentration of about four to six,
most preferably about five, volumes per volume of undiluted serum.
The resultiny mixtllre is then mixed together and incu~ated for
a~out 2-4 minutes, perferably ahout 3 minutes, at the desired
previously indicated temperature of a~out 37C. for a -total re-
action time (initial incubation plus final incubation) o~ approx-
i.mately 10-12 minutes, preferably about 11 minutes, whereupon
acetic acid, for example, 1 N, is added -to each test tube in a
vol.ume sufficient to stop the reaction from continuing, usually
10~ ~1. The mixture is then left in each test tube and read on
a colorimeter at 405 nM. The concentration of the endotoxin is
calculated from a standard curve prevlously prepared from known
dilutions of a standard enaotoxin.
I-t has been found that the reaction rate~ that is the
rate at which the splitting of the color indicator from the
suhstrate occurs, increases linearly with increasing concentration
of endotoxi.n i.n the 0.01-0.1 ng per ml. range. Further details
are indicated in the Example below:
EXAMPLE _I
A human serum sample suspected of containing endo-toxin
from E. Co:Li gram negative hacteria is diluted with 10 volumes of
1 sterile pyrogen-Eree water and 100 ~1 thereof is added to a
sterile pyrogen-free test tube. Limulus amebocy-te lysate recon=
stituted from the lyophilized powder in pyrogen-free sterile
water to a solids weight concentration of about 2 gram/100 ml.
is added in a 100 ~1 volume to the same test tu~e, and the mix-
ture is incubated at 37C. for 8 minutes. 500 ~1 of benzoyl-
isoleucine-glutamic acid-glycine-argin:ine-p~nitroanilide color
indicator is then added to the test tu~e and stirred into the
mixture for 3 minutes at 37C " whereupon 100 ~1 of 50% ace-tic
acid is added to the tes-t tu~e and the mixture is immediately
read in a colorimeter at 405 nM. The reading is 0,5 O.D. This
reading is compared with a s-tandard curve previously prepared
from known dilutions of a known concentration of endotoxin,
sufficient lysate to provide the enzyme color splitting as prev-
iously described, and the same color indicator. The reading
indicates the presence of endotoxin in a concentration of 0,05
ng~ml,, establishing the presence of E, Coli bacterial infection
and an indication of its severity.
Xn a parallel test r the same human blood serum is used
but in a 10 ~1 undilllted amount added to 200 ~1 of lysate at
one-half o-F the usual concentration, i.e,, about 1 ym/100 ml.
The other test parameters are held the same. The test results
are comparable. So are test results using -two-fold and four-
fold dilutions of -the serum, The only effect dilution of -the
serum has on the test is to change the sensi-tivity of detection
of the -test, greater dilutions increasing the minimal amount of
endotoxin concentration which is detectable~ At a 1:10 dilution
of sample in water, the normal detection limit of 0,01 ng of
endotoxin is changed to 0.1 ng/ml.
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1 EXAMPLE II
The method of Example I, first test, is followed,
except that the subs-trate is ace-ta-te-isoleucine glutamic acid-
glycine aryinine p-nitro-anilide used in a 500 ~1 concentration.
The same results as were obtained in Example I are found using
the same human serum as used in Example I. A readiny of O.5
at ~05 n~l is obtained in a colorimeter, indicating the presence
of the endotoxin in a concen-tration of about 0.05 ng/ml.
Compara~le results are obtained in a parallel test
substitutin~ ~enzoyl-valine-glutamic acid-glycine-arginine-p-
nitroanilide as the substrate listed above in this Example.
IMPROVED LYSATE SENSITI~ITY
.. . ._
Inasmuch as the king crab amebocyte lysate tends to
lose sensitivity over a period of time commencing with its re-
constitution from the powder or from its ori~inal preparation,
it is desirable to find a way of restorir.g that sensitivity
and/or preventing its decrease. The sensitivity loss is charac
terized by a gradual reaggrega-tion of an active component in the
lysate and a decreased response to endotoxin. When a series of
tests are run utilizing lysate or when a single test is run ~y
a method which requires the use of lysate over an extended period
of time such as one or more hours,noticeable decreases in lysa-te
sensitivity occur and contribute to a decrease in the accuracy
~nd effec~iYelless of any test employing the lysate.
Howe~er, it has now ~een found that t~e addition of
small amounts of heparin to the lysate reverses or prevents the
loss of sensitivity. At high concentrations of heparin, the
heparin inhibits the lysate reaction with endotoxin and therefore
such high concentrations of heparin must be avoided. However r
just ~elow the level where all such inhibition ceases, the heparin
-lQ~-
1 stabilizes the lysate against loss of sensitivity, The range
of efEectiveness of the heparin has been de-termined -to be about
0,1 0,8 standard units of heparin per ml. of the lysa-te, where
the lysate has a concen-tration on a clry solids basis of about
1,5 to 2.5 gm/100 ml~ A standard unit of heparin is defined
in the Pharmacopoeia of the United States of America as the
minimum guantity of USE~ sodium heparin which, when added to 0.8
ml~ of saline T.S. r maintains fluidity in one ml. of prepared
plasma for one ho~lr after the addition of 0.2 of calcium chloride
solu-tion (1~ volume concentration).
No other means have ~een found to be effec-tive to
sta~ilize the lysate against loss of sensiti~ity. It will be
understood that this method of sta~i]izing the lysate is useful
in ~arious t~pes of applications including ~ut not limited to
the improved chromogenic method of the present invention. The
following example illustrates the improved effects o~tained
through the use of heparin-sta~ilized lysate in comparison with
unsta~illzed lysate in the chromogenic method of the present
inYentiOn.
F,XAMPLE I I I
~ first sample ~as forrned by reconstitu-ting one ml of
lyoph~lized Limulus ame~ocyte lysate (2 gm/100 ml.) with 1 ml
of pyrogen~free sterile water containing 0~1 units of heparin.
A second sample was formed ~y adding together 1 ml oE the same
lyophili2ed Limulus ame~ocyte l~sate as was used in the first
sample but reconstituted with 1 ml of pyrogen-free steriIe water
containing no heparin~
In parallel tests, 0.1 ml of each sample was then
mixed in a ster;le pyrogen-free test tube with 0.1 ml of 0.05
mg per ml standard endoto~in and was incu~ated -therein at 37 C.
1 for 10 minutes. In each case, 0.5 ml of the same colorimetric
indicator substrate utilized in Example I was added with stirr-
ing to the test tube and the resultant mix-ture was incuba-ted
for an additional 3 minutes, whereupon 0.1 ml to 50Q~ ace-tic
acid was then added to each solution to stop the reaction. The
color of the resulting solution was in each case read at 405
nM in a colorimeter over a wa-ter blank. The same procedure was
repea-ted each hour for eight hours to determlne the extent of
loss of sensitivity of the heparin stabilized samples in com-
parison with the unsta~ilized samples.
I-t was determined that in the case of the heparin
stabilized samples, an initial optical. density of about 0.74
remained substantially constant for approximately four hours
and decreased only slightly to a~out 0.70 over the remaining 4
hours, In contrast~ the unstabilized samples had an initial
optical density of n, 62 which decreased to 0.60 in one hour,
further decreased to ahout 0~-50 in three hours and by the end of
the 8th hour had declined to 0.30..
The test results clearly indicate that the heparin
sta~ilized lysate samples had ~etter sensitivity initially and
that the sensitivity remained substantially cons-tant throughou-t
an 8 hour period, whereas the unsta~ilized lysate samples ex-
hi~ited a decline in sensitivity almost immediately and tha-t the
sensitivity ~y the end of the 8t~ hour was less than 50~ of the
initial sensitivity of the samples. Accordingly~ it is clear
that introduction of the heparin into the l.ysate ena~les the
l~sate to ~e used o~er a long period of time without impairmen-t
of lysate sensitivity and without producing fluctuating tes-t
results, which interfere with an accurate quantitati~e determin-
ation of endotoxin levels~
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1 Various o-ther changes, modiEications, alterations and
additions can be made in the methods of the present invention,
their steps and parameters. All such changes, moclifications,
alterati.ons and changes as are within the scope of the appended
claims form part of the present invention.
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