Note: Descriptions are shown in the official language in which they were submitted.
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FIELD OF INYENTION
_
~ The present invention provides an improved method for assaying
complement fragments C3a, C4a and C5a or the des-Arg derivatives
thereof and a mercantile kit useful in performing said immunoassay.
BACK6ROUND OF THE INVENTION
The complement system of humans and other mammals involves more
than 20 components which participate in an orderly sequence of reac
tions resulting in complement acti~ation, Numerous studies indicate
that the complement system is a fundamental element of normal host
defense mechanisms. As a consequence, complement activation is com-
monly associated with a variety of pathological states such as certain
malignancies, myocardial infarction, systemic lupus erythematosis, and
adult respiratory distress syndrome. Because of these correlations
clinical laboratory methods that detect complement activation are use-
ful in diagnosing certain disease conditions.
Complement activation can occur by either of two primary modes
known as the "classical" pathway and the "alternative" pa~hway,
respectively. These different pathways are generally distinguished
according to the process which inîtiates complement activation. Acti-
vation v~a the classical pathway is usually associated with an immuno-
logic stimulus whereas activation via the alternative pathway is mo~t
commonly associated with non-immunoloyic stimuli. Regardless of the
initiating stimulus both pathways converge, followed by the conversion
of the C3 component of complement into its C3a and C3b fragments.
This cleavage of C3 into its subcomponents is considered to be one of
the significant events signalling activation of the alternate comple-
ment cascade. Following the conversion of C3, a Cs convertase enzyme
complex is formed.- This enzyme cleaves the Cs component to yield the
fragments Csa and Csb. Complement activation by the classical pathway
mechanism îs uniquely characterized by the fact that this route leads
to the conversion of the C4 component to its fragments C4a and C4b.
The physicochemical and physiological properties of the cleavage
products C3a, C4a and C5a, termed anaphylatoxins are well known. Each
is a potent bioactive polypeptlde and plays a key role as a mediaeor
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of acute inflammatory processes~ hmong these three anaphylatoxins Csa
alone is uniquely characterized by its abil~ty to interact with white
blood cells. Both C3a and C4a are rendered inactive in vivo by con-
version to their respective des arginine derivatives (C3ades Arg or
C3aj, C4adeS Arg or C4aj) by a serum carboxypeptidase. Human Csa, on
the other hand, is converted to C5ades Arg by this serum carboxypepti-
dase only after all available white blood cell binding sites for C5a
have been saturated.
Conversion of the human complement components C3 and C5 to yield
their respective anaphy1atoxin products has been implicated in certain
naturally occurring pathologic statles including: autoimmune disorders
such as systemic lupus erythematosis, rheumatoid arthritis, malig-
nancy, myocardial in~arction, Purtscher's rekinopathy, and adult
respiratory distress syndrome. In addition, increased circulating
7evels of C3a and C5a have been detected in certain conditions associ-
ated with iatrogenic complement activation such as: cardiopulmonary
bypass surgery, renal dialysis, and nylon Fiber leukaphoresis. Ele-
vated levels of C4a anaphylatoxin are commonly associated with the
autoimmune disorders mentioned above. Therefore9 the ability to quan-
titatively measure the circulating levels of these anaphylatoxins ortheir des-Arg derivatives is of great utility in diagnosing a variety
of important pathological conditions. Additionally, the ability to
measure levels of C4a or C~ades Ar9 enables one to determine the path-
way by which complement activation occursO This facility enables one
not only to determine the precise mechanism of complement activation
but also whether a patient's natural immunological defense mechanisms
are functional~
Until the development of the radioimmunoassay (RIA) method of
Tony E. Hugli and Dennis E. Chenoweth reported in ''Immunoassays: Clin-
ical Laboratory Techniques for the 1980s," 443-460, Alan R. Liss,
lnc., New York, NY (198D), measurement of the anaphylatoxins C3a, C4a
and C5a or their des-Arg derivatives had only been achieved when the
levels of these factors were relatively elevated, for example, when
the disease process had reached an advanced stageO The RI~ techniques
of Hugli and Chenoweth permit quantitative measurement of trace
amounts of the anaphylatoxins or their des-Arg derivatives and hence
provide a 5ensitive diagnostic tool. However, the means known hereto-
fore for measuring these factors have been frought with a significant
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problem associated with the requirement that the C3, C4 and C5 plasma
precursors of the anaphylatoxins must be removed from the biological
fluid to be tested. This stringent requirement is predicated on the
observation that the anti~odies raised to the anaphylatoxins possess a
significant cross-reactivity with their respective plasnla precursor as
C33, C4a or C5a is a part of the parent molecule C3, C4 and C5,
respectively. Because of this unavoidable cross-reactivity it is
imperative that the precursor which exists in relatively high concen-
trations in serum and plasma be completely removed to avoid detecting
artifactually elevated levels of the anaphylatoxins which are normally
present in only trace amounts. Prior known methods of separating the
anaphylatoxins from their plasma precursors involves diluting the
plasma with sodium chloride and acidifying with hydrochloric acid and
subsequently neutralizing the recovered serurn sample. See Hugli and
Chenoweth cited above. The present invention prov~des a novel and
simplified means of quantitatively removing the plasma precursor of
the anaphylatoxin from the biological samples yet simultaneously
permitting a quantitative recovery of the low molecular weight anà~
phylatoxins, C3a, C4a and C5a or the des-Arg derivat~ves thereof.
SUMMARY OF INVENTION
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The present invention provides a novel method for removing sub-
stantially all traces of complement precursors or components 03, C4
and C~ from samples o~ biological fluids and recovering from said
fluids complement fragments C3a, C4a and C5a or the des-Arg derivci-
tives thereof without interfering with the immunogenicity of saidfragments which comprises combining equal volumes of the biological
fluid sample and a ouffered solution of 0.8 to 1.6~ of an acridine
derivative selected from the group consisting of acrinol 9 acriflavine,
acriflavine hydrochloride and aminacrine, incubating the mixture for
from one minute to 2 hvurs at about 25C and recovering the super-
natant from the resultant- precipitate.
~ he present invention also provides a novel and improved method
for quantitatively measuring complement fragments C3a, C4a or Csa or
the des-Arg derivatives thereof in a biological sample which comprises
combining equal volumes of the biological sample and 0.8 to 106% of an
acridine derivative selected from the group consisting of acrinol,
acriflavine, acriflavine hydrochloride and aminacrine, incubating ~he
mixture for from one minute to 2 hours at about 25~C, recovering the
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supernatant from the resultant precipitate~ and incubating the
supernatant with a known amount of a labeled complement fragment
selected from C3a, C4a or Csa or the des-Arg derivative thereof and a
known amount of an antibody which recognizes sald labeled complement
fragment or des-Arg derivative thereof, separating the free labeled
complement fragment from the bound labeled complement fragment~
measuring the amount of labeled complement fragment in either the free
or antibody bound complement component, and determining the
concentration of complement fragment in the biological sample by
reference to a standard curve.
The present invention also provides a novel kit wherein the com-
ponent parts are assembled for use in assaying samples of biological
fluids for complement fragments C3a, C4a and Csa or the des-Arg deriv-
atives thereof which comprises a ~irst container having therein a
buffered solution of 0.8 to 1.6% solution of an acridine derivative
selected from the group consisting of acrinol~ acriflavine, acriflav-
ine hydrochloride and aminacrine, a second container having therein.a
labeled reagent selected from labeled complement fragments C3a, ~4a or
Csa or the des-Arg derivatives thereof; and a third container having
therein an antibody reagent selected from antibody which recognizes
complement fragments C3a, C4a or C5a or the des-Arg derivatives
thereof.
DETAILED DESCRIPTION OF INVENTION
.
As used herein acrinol is taken to mean 2-ethoxy-699-diamino-
2~ acridine or the lactate monohydrade salt thereof; aeriflavine means amixture of 3,6-diamino-10-methylacridinium chloride and 3,6-diamino-
acridine contairing when dried at 105C for ~wo hnurs not less than
13.3% and not more than 15.8~ of Cl; acriflavine hydrochloride means a
mixture of the hydrochlorides of 3,6-diamino-lO-methylacridinium
chloride and 3,6-diaminoacridine containing when dried for one hour at
105C not less than 23X and not more than 23.5% of Cl; and aminacrine
means 5-aminoacridine .
In practicing the present invention the sample of biological
fluid can be any bodily fluid such as, ~or example, serum, plasma,
urine or cerebrospinal fluid. When the biological fluid employed is
plasma or cerebrospinal fluid EDTA is added thereto.
Equal volumes of the biological fluid and 0.8 to 1.6% of the acri-
d;ne derivative are conbined for a final concentration of 0.4 to 0.8~
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of said acrid;ne derivative. The preferred flnal concentration of
acridine derivative ;s 0.6%. The acrid;ne derivative-protein complex
is buffered to a pH of about 7.4. Any buffer which does not contain
chloride ;ons is suitab1e, for example~ carbonate buffers, phosphate
buffers, or HEPES (N-2-hydroxyethylpiperazineethane sulfonic acid) may
be e~ployed. Preferably the molarity of the buffer is between 0.05 to
O.lM with a pH of from 6.8 to 7.8. The preferred buffer for use in
practicing the present invention is 0.05M phosphate buffer, pH 7.4.
Once the biological fluid and acridine derivative are combined a
precipitate forms almost immediately. However, the complex preferably
should be permitted to incubate for at least about 20 minutes to
effect maximum separatlon of the complement precursors C3, C4 and C5
from their activated fragments. We have found that precipitation of
the complement component is complete within about one minute to 2
hours. The complex may be permitted to incubate longer than one hour
if necessary, however, no beneficial additional separation of protein
is expected to occur beyond one hour. Incubation is carried out àt
room temperature, that is, about 25C.
Removal of the precipitate is conveniently accomplished by cen-
trifuging the acridine derivative-protein complex dt about 3~000 to
7,000 x 9 for about 1~ tù 20 minutes then decanting the supernatant
which contains the complement fragments C3a, C4a and C5a or the
des-Arg derivatives thereof. As indicated hereinabvve9 complement
fragments C3a, C4a and C5a are rendered inactive in vivo in serum and
plasma by carboxypeptidases by conversion to their respective inactive
des-Arg derivatives. Swch conversion also occurs in vivo in urine.
Hence, the supernatant recovered from the novel acridine derivative-
precipitation method of the present invention will contain primarily
the des-Arg derivative of the complement fragments`C3a, C~a and C5a
rather than the activated form of said ~ragments when the biological
fluid is plasma, serum or urine. For diagnostic purposes measurement
of the inactive des-Arg derivative is just as meaningful as the direct
measurement of the active forrn of the complement fragments.
The recovered supernatant is used without further treatment or
processing in the immunoassay procedure. We have found that the
presence of the acridine derivative in the test samples does not
interfere with the antigenicity of the complement fragments or the
des-Arg derivatives thereof, thus providing a unique and greatly
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simplified means of assaying for sa1d fragments.
The immunoassay procedure is not substantially different from
known immunoassays for C3a, C.,a and Csa or the des-Arg derivatives
thereof. Equal volumes -of the test sample to be assayed, labeled
complement fragment G3a, C4a or Csa or the des-Arg deri~ative thereof,
and antibody which recognizes said complement fragment are combined
and incubated after which the antibody bound and unbound, or free,
labeled complement fragment are separa~ed and measured to determine
the amount of complement fragment in the test sample by comparison to
standard curves. An assay buffer is generally added to the incubate.
We have found a buffer comprising HEPES, protamine sul~ate, thimerosol
and gelatin to be particularly useful.
Purification of the complement fragment C3al C4a or Csa or the
des-Arg derivative thereof for use in raising antibody thereto, and
for use in generating standard curves as well as for preparing labeled
complement fragment can be achieved by the general method described in
J. Biol. Chem. 256, 8685-8~9~ (1981). We have found that this proce-
dure can be modi~ied an~ improved by substituting the acridine deriva-
tive-precipitation technique described hereinabove for the hydrochlor
2C ic acid precipitation technique.
Antibody to complement fragments ~3a, C4a, C5a or the des-Arg
derivatives thereof is r~ised as generally described by Hugli, et al.,
J. Biol. Chem. 250, 1472-1478 (1975;, and in J. Biol. Chem~ 25Ç, 8685-
86~2 (1981~.
The label utilized in the labeled complement fragment or the
des-Arg derivative thereof can be any substance capable of detection
by physical or chemieal means. Radioisotopes such as tritium iodine-
131 and iodine-125 are useful with l25I being preferred. The 125I
labeled material can be prepared by various means generally known in
the art, such as, the solid-phase lactoperoxidase method. A preferred
method employs the use of TCDG (1,3,4,6-tetrachloro-3a,6a-diphenyl-
glycouril) in phosphate buffered saline.
The antibody bound complement fragment can be separated from the
free or unbound complement fragment by various means commonly employed
in immunoassay procedures. For example, this separation can be
achieved by treatment with polyethylene glycol ~Desbuquois, 8. and
Aurback, G.D., J. Clin. Endocrinol. Metab. 33, 732 (1971)3 or IgG Sorb
or by contacting the incubate with a second antibody. The second
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antibody, which is prepared by standard procedures~ for example, as
described in Daughaday, et al~, "Principle of Competitive Protein
Binding Assay", J.B~ Llppincott~ Philadelphia (1971~ recognizes the
complement fragment-antibody complex contained in the incubate. Use
of the second antibody technique is particularly preferred~
Standard curves are derived essentially by performing the above-
described assay procedure using known quantities of complement frag-
ment C3a, C4a or C5a or the des-Arg ~derivative thereof in plaçe of the
test sample. The assay procedure described herein is highly sensi-
tive, and we have found it particularly useful to develop standard
curves for concentrations of the complement fragments ranging from 1.0
ng to 25 ng.
The following examples further illustrate the invention~ In
Examples 3-5 acrinol is used as the precipitating agent and is the
preferred acridine derivative of the present invention.
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Example 1 Purification o~ complement Fragments or khe des-Arg
derivative
From 2 to 4 liters of serum was activated at 37DC by addition of
boiled yeast (20 mg/ml serum) and allowing the mixture to stand ~or 45
minutes to one hourn Equal volumes of ac-ti~ated serum and 0.8 to 1.6%
acrinol, bu~fered to pH 7.4 with 0.05M phosphate buf~er, were combined
and let stand for 30 minutes at 25C a~ter which the mixture was
centrifuged and the supernatant was decanted. The supernatant was
dialyzed against running water overnight at 5~-8~C then gel filtered
1~ on a p-60 column ~quilibrated with O.lM ammonium formate, pH 500~ lhe
fractions containing complernent fragments C3a, C4a or C5a or the des-
Arg derivatives thereof were pooled and applied to an SP-Sephadex*col-
umn equilibrated with O~lM ammonium formate~ pH 5.0, rhe column was
eluted by a linear gradient of 0~1M to 0.8M ammonium ~ormate, pH 7Ø
The fractions of C3a, C4a and 05? or the des Ar~ derivatives thereof
were collected and appl-ied individually to separate CM-cellulose col-
umns and eluted by a gradient of 0.15M to 0.43M ammonium formate, pH
7.0, at a flow rate of 75 ml/hour. Each of the complement components
C3a, C4~ or C5a was pooled with its respective des-Arg deriva~ive
recovered ~rom the column and lyophilized then redissolved in water
and dialyzed against 1% acetic acid. A final lyophilization and
resuspension ir water of about 10 mg/ml provided each fragment ready
for use~
Example_2 1251 Complement C3a or the des-Arg deriYative thereof
To an incubation tube was added 50 ~g of complement fragment C3a
or the des-Arg deri~ative thereof, 150 ~1 of phosphate bu~fered
saline, 100 ~9 of TCDG and lmCi (10 ~1~ 125~ sodium. The phosphate
buffered saline was prepared as a stock solution comprising 81 my
sodium chloride, 39 mg anhydrous sodium biphosphate, 199 mg sodium
phosphate dibasic heptahydrate reagent and 10~ ml glass distilled
- deionized water. 7he mixture was incubated for 20 minutes at about
25C then transferred to a chromatography column equilibrated with
phosphate buffered saline with gelatin which was prepared as a stock
solution comprising 143 9~ sodium chloride, 200 mg thimerosol NF? 6 g
anhydrous sodium biphosphate~ 34 g sodium phosphate dibasic hepta-
hydrate reagent, 17 ~ gelatin and glass distilled deionized water to a
volume o~ 17~5 liters. Radioactive fractions of 0~ mt each were
collected and diluted to give 20,000 to 60,000 cpmlO.05ml in phosphate
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buffered saline with gelatin.
~e~
(a) A mixture of 0.45 ml act1vated plasma, 0.45 ml of 0.8% acri-
nol in 0.05M phosphate buffer, pH 7,4, and Ool ml distilled water was
reacted at about 25C for 20 minutes then centrifuged at 1700 x 9 for
lO minutes. The supernatant was collected. A mixture of 50 ~l of
assay buffer prepared as described hereinabove9 50 ~l of supernatant
obtained above, ~0 ~l of l25I complement fragment C3a or the des-Arg
deriYatiYe ther~of and 50 ~l of complement fragment C3a rabbit anti~
sera was incubated for 30 minutes at about 25C after which 50 ~l of
goat anti-rabbit antisera was added and mixed well. The mixture was
incubated for an additional 30 minutes at about 25C after which 2 ml
of isotonic saline was added. Ihe mixture was cen~rifuged at 2noo g
for 10 minutes and the supernatant decanted. The radioactivity of the
l~ pellet was 4555 cpm.
(b) The foregoing procedure was performed only the acid-precipi-
tation technique of Hugli and Chenoweth described in Immunoassays:
Clinical Laboratory Techniques for the 1980s: 443-460 was substituted
fo~ the acrinol precipitation step. The radioactivity of the result-
ant pellet was 4521-4757 cpm demonstrating that the acrinol precipita-
tion technique removes intact complement protein precursor as effect-
ively as treatment with hydrochloric acid followed by base neutraliza-
tion providing a greatly improved simplified assay procedure~ Also
the results of the two experiments indicate that the presence of
acrinol does not affect either the first or the second immunological
reactions.
In generating standard curves for complement fragment C3a or the
des-Arg derivative thereof using 1.0 ng9 2.5 ng, 5.0 ng~ 10 ng, 25 ng
and 50 ng quantities of C3a we found that use of thè acrinol precipi-
tation step in place of the acid precipitation-base neutralization
step of Hugli and Chenoweth increased the sensitivity of the immuno-
assay by a factor of 12 as evidenced by the fact that the range of
detection became shorter and the slope %B/Bo (Standard Counts/Bound
Standard) was increased from -2.3 to -4Ø
Example 4
The assay as described in 3(a) above was performed using acivated
plasma samples to which known quantities of complement factor C3a or
the des-Arg derivative thereof ranging from 2 to 20 ng was added. The
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results indicate that there was 100~ of the known C3a concentrations
in each of the "spiked" normal sampies assayed.
Example 5
The essential reagents for the performance of the immunoassay of
the present invention are assembled into a mercantile unit as a kit.
The kit comprises multiple containers7 such as, bottles or other suit-
able containers as follows:
(a) a first container having therein a buffered solution of 0.8
to 1.6% acrinol;
(b3 a second container having therein a labeled reagent selected
from I25I labeled complement ~ragments C3a, Clla or Csa or the
~es-Arg deri~atives thereof;
(c) a third container having therein rabbit antisera to the par
ticular complement fragment,
and which optionally contains the following additional reagents:
(d) a container having therein assay buffer, prepared as
described hereinabove;
(e) a container having therein second antibody which binds thP
product of the initial complement fragment-antibody reaction,
preferably goat anti-rabbit antisera;
~f) a oontainer having therein complement fragment standard, 25
ng;
(g) a container having therein complement fragment standard, 10
ng;
(h) a container having therein complement fragment standard, 5
ng;
(i) a container having therein complement fragment standard, ?.5
ng;
(j) a container having therein complement fragment standard, 1.0
ng.
In each of (f) thrQugh (j) the complement fragment is either C3a9
C4a or C5a or the des-Arg derivative thereof depending on whioh oom-
plement fragment is being assayed.
Although the foregoing examples are limited to the separation and
assay of complement fragment C3a or the des-Arg derivative thereof the
novel separation and assay procedure of the present invention is
equally appliciable to complement fragments C4a and Csa or the des-Arg
derivatives thereof.
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