CA1049405A

CA1049405A - Test method to determine pulmonary embolism

Info

Publication number: CA1049405A
Application number: CA253,011A
Authority: CA
Inventors: Emanuel Silverstein
Original assignee: Individual
Current assignee: Individual
Priority date: 1975-05-20
Filing date: 1976-05-20
Publication date: 1979-02-27
Also published as: BE842041A; IT1123653B; GB1514639A; JPS6026979B2; NL7605393A; JPS521016A; FR2311844A1; DE2622588A1; FR2311844B1

Abstract

A B S T R A C T
Detection of an antigen in circulating blood which is normally present in the lung signals the presence of pulmonary thromboembolism.

Description

49~5 BACKGROUND O~ INVENTION

Pulmonary thromboembolism is a highly prevalent disease associated with repetitive episodes of increasing peril, and is one of the leading causes of death. While prompt recognition is crucial since rational and frequently effective therapy is available, clinical diagnosis is difficult ; and frequently incorrect. There is a need for a diagnostic indicator which is available on a larger scale than present radiologic methods which not infrequently are indeterminate.
~uch a diagnostic indicator could serve both as a screening method and also to complement information from radiologic methods. Since recognition of pulmonary thromboembolism is a major medical problem world-wide, a simple screening test j for it would have widespread use and impact in medical manage-ment of the probleM.
It has now been discovered that the presence of an antigen in human serum provides a safe, reliable and effective basis for confirming the presence of pulmonary embolisms.

THE INVENTION
An antigen which is normally found in human lungs spills over into blood serum in the presence of a pulmonary embolism. The antigen can be used to stimulate the production of an antibody in animal blood. The antibody, in turn, can be used to detect the presence of the antigen in serum, thereby confirming the existence of the pulmonary embolism.
The invention therefore, comprises a test method for determininy the presence of a pulmonary embolism in a human by irst utilizing normal lung tissue to stimulate the production of an antibody in animal serum, and thereafter incubating the serum containing the antibody with the human serum to be tested. The positive presence of a pulmonary

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D4~ 5 embolism is signaled by the occurrence of an immunological reaction.
In accordance with one aspect of this invention, there is provided a method for determining the presence of a pulmonary embolism in a human which comprises utili~ing human lung tissue to stimulate the production of an antibody animal serum, absorbing said serum with normal human serum to remove extraneous antibodies, separating resulting antiserum, incubating said antiserum with human serum to be tested and determining the positive presence of a pulmonary embolism by the occurrence of an immunological reaction.
In accordance with a further aspect of this invention, :
there is provided a test method for determining the presence of ;a pulmonary embolism in a human which comprises incubating human serum to be tested with an antiserum containing an antibody produced in animal serum by stimulation with an antigen contained in human lung tissue and determining the positive presence of a pulmonary embollsm by the occurrence of an immunological reaction.
In accordance with yet a further embodiment of this invention, there is provided an antibody preparation containing an antibody reactive with the antigen normally present in human lung tissue which appears in the serum of humans afflicted with pulmonary embolism.
In a further embodiment of this invention, there is . provided an antigen which is a protein having an apparent molecular weight of about 210,000, being negatively charged in aqueous media, having an isoelectric point above 5, giving a single coomasie blue staining band in polyacrylamide electrophoresis, being free of lipid or carbohydrate segments;
the said antigen being further characteri~ed by an ability to ~ ~ - 2a -, :,' ~

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~4~4~5 stimulate antibody production in animal serum so as to produce an antiserum which when incubated with serum from a human afflicted with pulmonary embolism will indicate the presence of the said embolism by the occurrence of an immunological reaction.

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~ 94~5 Isolation of ~nti~en Compositions and Preparation of Antibod Y.
While substantially any animal can b~ em~loyed for the production o~ antibody, as is the case witn such pro-cedures, for convenience the procedure will be described as applied to the utilization of a rabbit. Those s~illed in the art will recognize that larger animals such as goats, horses and cattle can be similarly employed; and, in fact, would be employed for production of large amounts of antibod~.

Lung washings were prepared by first extracting 50 to 100 gram samples of normal human lung with isotonlc saline (0.85% aqueous NaCl~. The lung had been ob.ained following surgical remo~al for carcinoma. Four e~tracts o about 50 ml each were lyophilized and redissolved in about 10 ml of isotonic saline and applied to a Sephadex G-200 col~n having a 2.5 cm diameter and a bed volume of 260 ml. Ten ml fractions were collected at a flow rate of 10 ml/hr at a te~perature of 16C.

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The column was developed with 0.1M Tris-Cl, pH 7.4-lM NaCl.
Fractions were assayed for their absorbance at 280 nm, a characteristic absorbence feature of protein molecules. The 2~ initial excluded peak, the surfactant fraction, ~fractians 10-25) w~re pooled and split into 5 ml portions. One such portion mi~ed with an equal portlon of Freund's adju~ant is used for the development of antibody in~raboits.

, ; In an alternate method for preparing the antigen .. . .
contalning mixture, 0.4 to 1 g portions oî numan lung similarly obtained and held, upon removal, at 0C and thereafter at -75~ to 85C were cut into small pieces and homogenized at 0C in 3 ml of 0.1 M potassium phosphate at p~ 7Ø An alter-nate homogenizin~ ~ehicle is 3 ml of 0.25~ sucxose-1~1 potassium phosphate at p~7.4.

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. ~ ~ . . . -~4~405 Homogenization is effected by grinding in a glass tissue grinder with a motor driven Teflon pestle or in a motor driven Lourdes stainless steel homogenizer. Additional buffer is gradually added to increase the volume to lS ml.
The mixture is then added to an equal volume of Freund's adjuvant and blending is continued at 0C to form a thick creamy mixture. This suspension may be used to stimulate antibody production.
- About 5 ml of the antigen containing medium was injected subcutaneously into adult white male rab~its, gen-erally at one, but sometimes up to fou~,separate sites.
Injections were repeated at about two week intervals for about sixteen weeks. At the end of the series, the rabbits were bled by cardiac puncture or from the ear vei~. The blood was allowed to clot and the serum separated by centrifugation for 10 minutes at 2000 rpm.
The extraneous antibod~es in the rabbit serum are remo~ed by absorption with the normal human serum. This was - effected by mixing the normal human serum with the rabbit serum ih volume ratios of from about 0.03 to 1 to 0.1 to 1 human to rabbit. The mixture is stirred at room temperature for 30 minutes and then stored for about 16 hours at 4~. The mixture was centrifuged at 10,000 g for 30 minutes at 4C
to precipitate the reaction products of the extraneous antigen-antibody reactions. Since normal human serum was employed, the desired antibody did not react, and remained in the supexnatant. The procedure was repeated several (about 3) times tD insure as complete remo~al as possible of poten-tially interfering antlbodies. The flnal supernatant is used as the testin~ composition.
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9~s ~-~ This testin~ composition will give an immunological reaction when incubated with human lung supernatant (prepared as described hereinafter), the lung surfactant fraction, or serum ~f patients positively diagnosed by pulmonary scan for the presence of pulmonary embolism~ It does not however react with normal human sera. It appears then, that the antigen is normally present in the human luny, is not normally present in normal human sera, but does appear in the sera of patients afflicted wit~ pulmonary embolism.
Human lung supernatant was prepared by a 90 minute centrifugation at 100,000 g at 4C in an ultracentrifuge of human lung ~ieces homogenlzed in a 1:1 suspension with 0.25 M
sucrose - 1 m.'~ phosphate at pH 7.4.
The antigenic preparations, that is the surfactant fraction, isolated from the Sephadex G-200 columns can be further purified. To do so, the surfactant fractions are dialyzed against deionized water for 24 hours, lyophilized and the residue dissolved in 0.1 M Tris-Cl, p~ 8.6~ One ml of the dissolved lyophilizate is subjected to ion exchange on a DEAE cellulose column (2.5 x 66 cm) and elu~ed at a flow rate of 0.33 ml/min with a gradient between equal volumes of 0.1 M Tris-Cl, pH 8.6 (mixing chamber) and 0.1 M Tris-Cl, p~ 8.6 plus l~S NaC1 (reservoir). Ten ml fractions are collected, lyophilized and dissolved in 0.5 ml of deionized water. A sample as small as 3 microliters will give a positive test with rabbit antibody serum by microdifusion.
.
When human lung surfactant fraction, human lung 100,000 g supernatant and human serum from a patient positive for pulmonary embolism ~y perfusion scan were tested against rabbit serum antibody by standard microimmunodiffusion, macroim~unodif~usion and electrophoresis techniques, all three .. ...
materials indicated the presence o~ a common antigen.

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9~5 Microimmunodi~fusion was carri~d out by pouring onto a plastic microscope slide 2-5 ml o 1.5~ Difco special noble agar in 38 ~M barbital buffer, pH 8.2. Wells and troughs were cut into the agar, The antibody preparation was addcd to the troughs, and antigen preparations were added to the wells.
Macroimmunodiffusion was accomplished by preparing a 100 by 1.5 cm Petri dish with an agar layer from 8 ml of the same agar as in the micro techni~ue, and then cutting the wells and troùghs.
Both types of diffusion were carried out by incuba-tion at 37C for 24 to 72 hours~ The gels were washed for about 24 hours wi~h several changes of normal saline. The lines of precipitation were visualized by staining with 1%
amido black dye solution in water, methanol, acetic acid, 5:5:1 by volume.
Optimum concentrations of antibody and antigen preparations were determined by varying the concentration of antigen in welis with constant antibody in troughs. A few simple observations of the precipitin lines is sufficient.
Immunoelectrophoresis was carried out on 7.5 by 2.5 ~m glass or plastic slides. Agar buffered as in ~he dif~usion techniques was poured and wells formed on opposite sides o the middle. ~Antigenic preparations were placed in the wells and electrophoresed for about 2 hours at room temperature at a constant voltage power supply such that the potential dif~er-ence across the slides was 4a volts. The gel slides and buffer .
, reservoirs were connected by microporous membrane wicks. After :i ~lectrophoresis, troughs were out out and the antibody composi-` ~ tion or antiserum addedO Incubation and staining were as ;~ described or the`immunodiffusion procedures. The preci;piti~
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reaction, as evidenced by the precipitation took place on the anode side of the startlng well. ~-s As is clear from the above, the antigen may be ~eparated from lung tissue by extraction. Saline or other extractants may be employed~ It may thereafter be puri~ied by chromatographic techniques, although it is not necessary to do so since relatively crude suspensions of lung homogenates can be usefully employed. Any of these lung tissue prepara-tions can be used to stimulate antibody produc:tion~
While the principal Lmmunological reaction used to describe the test method of this invention has been the precipitin reaction in agar using immunodiffusion or electro-phoresis, it is evident that other procedures can also be employed. These include, for example, complement fixation and hemagglutination. The antigen may also be tagged or labeled with a detectable radioactive element such as 1 lI; I, C or S. Labeling may similarly be effected with a fluorescent material such as fluorescein, rhodamine or auramine, or with an enzyme such as peroxidase, ~-glucouroni-..
dase, urease or the like. The antibody-antigen reaction can then be detected colorimetrically or by radio counting procedures.
: For convenience, the antigen of this invention has been tentatively named the I~ antigen. In an electrophoretic study of 106 patients studîed by pulmonary scan for suspended pulmonary thromboembolism, 67~ of those diagnosed as positive by the scan had detectable LA antigen compared to only 12 to 14% of patients with negative scan. The results may actually be better than indicated because it is generally recognized . ~ .
that the scan technique gives a number of false positives and negati~es.

The following non-limiting example illustrates the preparation of purified antigen, and the testing thereof to , ~-~ establish certain of its chemical and physical properties, .: ' , ' ~. . . . .
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An analysis of the example indicates that the antigen is a protein with an apparent molecular weight of about 210,000, is negatively charged in aqueous media at pH 8.2, and has an isoelectric point above 5. In polyacrylamide gel electrophoresis, it gives a single coomasie blue staining band. It is free of detectable lipoprotein or glycoprotein; that is, it contains no apparent lipid or carbohydrate segments.
The purified antigen, or antigenic preparations are of value because when utilized to stimulate antibody production fewer contaminating antibodies are produced so that the initial absorption with normal human serum which is referred to above can be omitted. Moreover, wi-th purified antigenic compositions, a higher concentration of antibody is produced in the animal serum. Furthermore, the sensitivity of the radioimmunoassay test can be markedly improved.

EXAMPLE
A total of 340 g of human lung tissue at 0C was cut into small pieces and homogenized in 4 volumes of 0.01 M Tris-0.25 M sodium chloride at pH 7.6 in a blender for 5 minutes at 30 second intervals at 3 to 5C. The homogenate was centrifuged at 6,000 g for 60 minutes at 5C, and the supernatan-t liquid centrifuged for 2 hours at 45,000 g at 5C.
The resulting supernatant liquid was then subjected to salt fractionation.
Ammonium sulfate fractionation was achieved by slow addition of saturated ammonium sulfate with stirring at 0 to 5C to 30% saturation. The liquid was adjusted to pH 7.8 with 5Nsodium hydroxide, stirred for 2 hours, and the resulting precipitate removed at 6,000 g. The supernatant was then brought to 50% saturation with ammonium sulfate in the same manner, and the precipitate recovered by centrifugation at 6,000 g for 30 minutes.

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The precipitate was dissol~ed in one quarter of the original homogenate volume of normal saline and dialyzed e~haustively against the same solution at 4C for about 24 hours.
(The normal saline may be replaced with 0.01 M Tris-0.25 M
sodium chloride.) The dialyzed solution was clarified by centrifugation at 6/000 g for 60 minutes and the precipita-te discarded.
The supernatant was subjected to the exact same procedure with 30% and 50~ ammonium sulfate followed by dialysis and the resulting dialyzed solution adjusted to pH 3.0 by slow addition of l N hydrochloric acid with gentle stirring during the period of about l hour. The solution was centrifuged at 6,000 g for 60 minutes to separate insoluble precipitate which was then discarded.
The acidified solution was brought to 50~ ammonium sulfate saturation by slow addition of an equal volume of saturated ammonium sulfate with constant stirring. Stirring was continued for 2 hours, and the mixture centrifuged for one-half hour at 6,000 g. The precipitate was dissolved in an equal volume of 0.01 M Tris-0.25 M sodium chloride at pH 7.4. The acidification and ammonium sulfate precipitation steps were -repeated and the precipitate again dissolved in one-quarter the initial volume of the Tris buffer. The solution was dialyzed against the same solution, and centrifuged at 6,000 g for l hour to remove the precipitate.
The solution was subjected to gel filtration on a sepharose 6B column (2.6 by 85 cm previously equilibrated with Tris buffer at pH 7.4), at a flow rate of 30 to 35 ml/hr. at 5C. The 2.5 ml eluted fractions were tested by immunoelectro-phoresis -to locate LA antigen which appeared in fractions 95 to 113, and represented about 9.9% of the protein applied as measured by absorbance at 280 nm. Protein recovery was 98.6%.

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~4~ D5 The LA antigen containing fractions were pooled and e~haustiYely dialyzed against deionized ~a~er at about ~C.
Precipitate was removed by centrifugation at 6,000 g for 1 hour.
The supernatant was dialyzed against Tris buffer, pH 7.4, and centrifuged at 6,000 g for 1 hour to remove any precipitate.
The volume was reduced by blowing a stream of air on the solution in a dialysis bag, and the solution subjected to sepharose 6B column gel filtration twice. The LA antigen con-taining fractions were identically placed in all three gel filtrations, and were identified by immunoelectrophoresis.
The LA antigen containing fractions were pooled, concentrated and dialyzed against 0.01 M potassium phosphate at 4C for a period of about 10 hours at pH 6. The dialyzate was centrifuged at 6,000 g for 30 minutes. The supernatant solution was applied to a carboxymethyl cellulose column (2 by 18 cm, 56 ml bed volume, previously cycled with 0.5 N sodium hydroxide, water, 0.5 N hydrochloric acid, 0.5 N sodium acetate at pH 4.8 and 0.06 M sodium acetate) and equilibrated with 0.01 M potassium phosphate buffer at pH 6. The solution was eluted stepwise with separate fractions of 0.01 M potassium phosphate buffers at pH
6.0, the initial buffer solution being free of sodium chloride, and subsequent solutions containing respectively 0.05 M, 0.1 M, 0.15 M and 0.2 M sodium chloride. The eluted fractions (each 2.5 ml) were analyzed by immunoelectrophoresis, and the LA
antigen was found in the fractions containing 0.15 M sodium chloride. (Immunoelectrophoresis here and in the previously described analyses was carried out using rabbi~ antihuman lung antiserum.) The fractions containing the antigen were pooled, reduced in volume, and dialyzed against 0.01 M potassium phos-ce~tri f~.

phate at pH 6.8. The diaLyzate was ~ to remove precipitate~

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- ` 1(1 9~9LC~5 The supernatant solution ~as applied to an 0.8 by 15 c~ ~rushite-hydroxyapatite colume (Analy-tical Biochemistry 59 16-23, 1974~ previously equilibrated with 0~01 M potassium phosphate at pH 6.8, and eluted stepwise with 0.01, 0.1, 0.2 and O.4 M po-tassium phosphate at pH 6.8. Fractions (2.5 ml) were tested by immunoelectrophoresis as previously described. The LA antigen was located in the fractions eluted with 0.2 M
potassium phosphate.
The fractions were pooled, reduced in volume, and dialyzed against Tris buffer at pH 7.4 at 4C for about 10 hours.
The LA antigen in the dialyzate was analyzed. It is a protein which reacts immunologically with rabbit antihuman lung antiserum. Its molecular weight by sepharose 6B fil-tration is approximately 210,000. Upon polyacrylamide gel electrophoresis ~r s~ 9 nn~1s / by the method of Ornstein and Davis (An-a~ of the New York Aca,demy of Science 121, 321 and 404; 2.5 milliamp per tube, 1.5 ; hours running time) it gives a single coomasie blue staining 7~ b~
b~ about 5 mm from the top of the gel which was about 5 cm in length.
The material does not contain detectable lipoprotein by Fat Red 7B staining (Corning Fat Red 7B Stain Set, Catalog No. 470126, July, 1974 instruction sheet, Corning ACI 490 San Antonio Road, Palo Alto, Calif. 94300).
Additionally, it is apparently free of glycoprotein when tested with Alcian Blue stain following the procedure des-cribed in Analytical Biochemistry 49, 607, 1972).

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Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for determining the presence of a pulmonary embolism in a human which comprises utilizing human lung tissue to stimulate the production of an antibody in animal serum, absorbing said serum with normal human serum to remove extraneous antibodies, separating resulting antiserum, incubating said antiserum with human serum to be tested and determining the positive presence of a pul-monary embolism by the occurrence of an immunological reaction.

2. A test method as in Claim 1 wherein the immuno-logical reaction is a precipitin reaction.

3. A test method as in Claim 2 wherein the reac-tion is identified by microimmunodiffusion.

4. A test method as in Claim 2 wherein the reac-tion is identified by macroimmunodiffusion.

5. A test method as in Claim 2 wherein the reac-tion is identified by electrophoresis.

6. A test method as in Claim 1 wherein the animal is a rabbit.

7. A test method for determining the presence of a pulmonary embolism in a human which comprises incubating human serum to be tested with an antiserum containing an antibody produced in animal serum by stimulation with an antigen contained in human lung tissue and determining the positive presence of a pulmonary embolism by the occurrence of an immunological reaction.

8. A test method as in Claim 7 wherein the immuno-logical reaction is a precipitin reaction.

9. A test method as in Claim 8 wherein the reac-tion is identified by microimmunodiffusion.

10. A test method as in Claim 8 wherein the reac-tion is identified by macroimmunodiffusion.

11. A test method as in Claim 8 wherein the reac-tion is identified by electrophoresis.

12. A test method as in Claim 7 wherein the animal is a rabbit.

13. A test method as in Claim 7 wherein the anti-serum is preabsorbed with normal human serum prior to incu-bation with the serum to be tested.

14. An antibody preparation containing an antibody reactive with the antigen normally present in human lung tissue which appears in the serum of humans afflicted with pulmonary embolism.

15. An antigen which is a protein having an apparent molecular weight of about 210,000, being negatively charged in aqueous media, having an isoelectric point above 5, giving a single coomasie blue staining band in polyacryla-mide electrophoresis, being free of lipid or carbohydrate segments; the said antigen being further characterized by an ability to stimulate antibody production in animal serum so as to produce an antiserum which when incubated with serum from a human afflicted with pulmonary embolism will indicate the presence of the said embolism by the occurrence of an immunological reaction.

16. A test method as in Claim 1 wherein the reaction is identified by radioimmunoassay.

17. A test method as in Claim 7 wherein the reaction is identified by radioimmunoassay.