CA1059901A - Direct radioimmunoassay for antigens and their antibodies - Google Patents
Direct radioimmunoassay for antigens and their antibodiesInfo
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- CA1059901A CA1059901A CA225,016A CA225016A CA1059901A CA 1059901 A CA1059901 A CA 1059901A CA 225016 A CA225016 A CA 225016A CA 1059901 A CA1059901 A CA 1059901A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
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- Urology & Nephrology (AREA)
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- Biotechnology (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
A B S T R A C T
A method for direct radioimmunoassay of antigens or their associated antibodies utilizing a coated test apparatus comprising adding a serum to be tested for an antigen to test apparatus coated with its antibody; incu-bating the tubes for from 0.5 to 42 hours; aspirating the contents and washing the same with a Tris-HCl and sodium azide mixture; adding purified I125 labeled antibody into the tube and incubating for from 1 to 24 hours; aspirating and washing the contents; and counting the time for I125 radiation, A similar assay for the antibody may be con-ducted utilizing an antigen coated apparatus and I125 labeled antigen. The method for coating these apparatus such as tubes comprises adding the antigen or antibody solution in a Tris-HCl, sodium azide solution into a suitable test tube or well, incubating the tubes at room temperature, aspirating and washing the contents and storing at from between 2 and 25° C. until use.
A method for direct radioimmunoassay of antigens or their associated antibodies utilizing a coated test apparatus comprising adding a serum to be tested for an antigen to test apparatus coated with its antibody; incu-bating the tubes for from 0.5 to 42 hours; aspirating the contents and washing the same with a Tris-HCl and sodium azide mixture; adding purified I125 labeled antibody into the tube and incubating for from 1 to 24 hours; aspirating and washing the contents; and counting the time for I125 radiation, A similar assay for the antibody may be con-ducted utilizing an antigen coated apparatus and I125 labeled antigen. The method for coating these apparatus such as tubes comprises adding the antigen or antibody solution in a Tris-HCl, sodium azide solution into a suitable test tube or well, incubating the tubes at room temperature, aspirating and washing the contents and storing at from between 2 and 25° C. until use.
Description
1C~5990~
This application is related to Canadian Patent Application No. 158,644, filed December 12, 1972.
Backg~ound of the Invention This invention relates to a diagnostic method for the radioimmunoassay of antigens and their antibodies and a method for coating apparatus useful in the above determination. More particularly, this invention relates - to a direct method for determining hepatitis associated antigens or their antibodies and is also directed toward a method for preparing diagnostic apparatus suitable for use in the same.
Although there have been methods for determining the presence of antigenically active macromolecules such as intact viruses, virus capsids, virus subunits, bacteria, membranes, cell walls, hormones, etc., there has been a lack - of a simple, yet sensitive, test method and apparatus for - 15 determining the presence of these materials. Viral hepatitis, including so-called serum hepatitis, which is a relatively common disease, has not been heretofore easily detected by a sensitive test which is both specific and reproducible for quickly determining whether or not the sera from a patient or a donor contains hepatitis associated antigens or antibodies.
Furthermore, radioimmunoassay techniques have been developed in the past for various antigen-antibody materials;
however, these radioimmunoassay techniques such as disclosed in articles by Kevin Catt et al in the Journal of BiochemistrY, 1966, Volume 100, pages 31c and 33c and in Science, ~ 59 9 ~
Volume 158, page 1570, 1967, are an indirect radioim~unoassay technique wherein the amount of antigen present in roughly inversely proportional to the amount of radiation emitted by the tracer material. These procedures required the use of correlation tables and other materials which generally rendered the resu~ts less than reproducible and exact.
Wide, Karolinska Simposia, 1st Simposia, September 23 to 25, 1969, University Hospital, Upsila, Sweden, pages 207 to 214 discloses a radioimmunoassay wherein an allergen is absorbed on a plastic polymer and reagin is bound immuno-chemically to the solid phase allergen and then labeled anti-reagen is bound to the reagln.
Briefly, it has been discovered that the above-noted difficulties, i.e., lack of reproducibility and exactness, have been overcome by utilizing the method of the precent invention. Briefly, in one application, the method of the present invention comprises contacting an unknown serum sample with an antibody coated implement, incubating the test implement and serum for a period of from 0.5 to 42 hours, aspirating and washing, contacting an Il 5 labeled antibody with the serum and coated the test apparatus and incubating for from 1 to 6 hours, aspirating and washing, and counting the tube for I gamma radiation.
It is therefore the principal object of the present invention to provide a novel method for the direct determination of antigens and their antibodies.
' `1059901 It is a further object of the present invention to provide a method for coating diagnostic apparatus for use in radioimmunoassay determinations.
It is a still further object of the present invention to provide a method for quickly and accurately determining the presence of hepatitis associated antigens or antibodies in sera.
Still further objects and advantages of the diagnostic method for direct radioimmunoassay and method for coa~ing test implements useful in the same will become more apparent from the following more detailed description thereof and the following attached drawing wherein:
The drawing is a cut-away view of a coated test implement useful in the method of the present invention.
The drawing shows a test tube shaped test implement 1 with a coated portion 2. Coated portion 2 has a coating of an antigen or its antibody preferably located as shown, i.e., in the bottom of the tube. Although the drawing shows one embodiment of apparatus suitable for use in performing the method of the present invention, the method of this invention should not be limited thereto. Other solid state devices may be employed such as inserts as disclosed in copending Canadian Patent Application No. 158,644, filed December 12, 1972g discs, spherical beads or particles, and the like, all of which may be coated with an antigen or antibody.
~ ` 1059901 Coated portion 2 is coated with either an antigen or an antibody depending on the material to be tested.
The antibody or antigen can be affixed to the test apparatus by contact coating from solution or any other means, the ~ manner of affixation not being critical. Since the method i5 similar with regard to almost all antigens and antibodies, the process for coating these tubes will be described with reference to a particular hepatitis associated antibody, i.e., anti-Australia antigen. A solution of anti-Australia antigen having a concentration of from about 1 to about 100 micrograms of protein per ml. is prepared from an antl-Australia antigen serum in from about 0.005 to about 0.02 molar Tris-HCl, i.e., 2-amino-2-hydroxymethyl-1,3-propanedîol-HCl. The Tris-HCl buffers the solution to a pH of from about 7.1 to about 9.5 together with from about 0.01% to about 0.05% sodium azide. This anti-Australia antigen solution is then coated on the tube surfaces and incubated at room temperature for from 6 to 72 hours and preferably for from 12 to 48 hours. These coated tubes are then washed with from about 0.005 to about 0.02 molar Tris-HCl at a pH of 6.9 to 8.4 plus from about 0.01% to about 0.05% sodium azide. Following this washing and rinsing step, the test implements may be stored at 4C. until necessary for use for radioimmunoassay. For additional details for methods of preparing antigen or antibody or the affixing of such to tubes or other apparatus, reference can be made to the ~~
publication by ~. M~ Ling and L. R~ ~verby, ~Preval~nce of Hepatitis B Antigen as Revealed by Direct Radioimmunoassay with I125 Antibody", the Journal of Immunolo~y, Volume 109, ~o. 4, October 1972.
It is preferred to utilize an 0~01 molar solution of Tris-HCl and 0.02% sodium azide buffered at a pH bf 7~1 for both the incubation medium and the washing medium.
The amount of antibody or antigen coated in the tubes i9 not critical since the test is run each time in comparison with at least one blank test~ No standard curves or charts are necessary for the test of the present invention; therefore, no specific amount of antibody or antigen in the coating is required as long as two similar tubes are used.
~ Although the coating method has been described with reference to a coated tube, the coating method may be utilized to prepare coated lnserts, beads, or any apparatus for use with wells, etc. by dipping the inserts in the antigen or antibody solution and following the remaining procedure. Likewise, while generally described in terms of coating of a tube, the antibody or antigen can be affixed to the test apparatus to provide a layer of the antibody or antigen, using any suitable methQd for achieving bonding or attachment thereto. The time required for incubating the affixed antigen or antibody to the test apparatus is not critical and may be as low as about ten minutes. However, where a coated tube is used, production processes dictate that a period of 6 to 72 hours is preferred.
Antigens and antibodies which may be determined by mean~ of the method of the present invention include:
various intact viruses, virus capsids, virus subunits, bacteria, membranes, cell walls, various hormones, gamma globulins, etc. The only requirement with regard to the above materials i8 that the materials havè a ~inimum of two antigenically active sites. Furthermore, antigens and antibodies having multiple combining sites are detectable even in the presence of their re3pective antibodies and antigens, provided a minimum of two free combining sites remain available. Although the radioimmunoassay method of the present invention is useful for ~etecting the above class of materials, it is especially well adapted, and this is a preferred embodiment of the present invention, for the determination of the presence of hepatitis associated antigens and antibodies, such as Australia antigen and anti-Australia antigen.
While the radioimmunoassay method of the present invention has been briefly described above, the method will now be described with reference to the specific materials and steps necessary for conducting the direct radioimmunoassay technique for determining the presence of the hepatitis associated antigen, Australia antigen.
-lOS9901 FirstJ a measure sample of plasma or blood to be tested for hepatitis associated antigen is placed in an anti-Australia antigen or antibody coated tube. The material is incubated for a period of from 0.5 to 42 hours at room temperature and preferably for from 12 to 24 hours. The coated material is washed with the buffer mixture, i.e., Tris-HCl and sodium azide or with distilled or deionized water. A measured amount of purifLed I125 labeled anti-Australia antigen is then added to the tube or test receptacle well and the tube or insert in contact therewith is incubated for an additional 1 to 24 hours at room temperature and preferably for from 1.5 to 6 hours.
Following thls incubation, the contents are washed utilizing the buffer mixture or distilled or deionized water and the tube is placed in the well of a counter which is capable of counting gamma radiation. Background controls in duplicate are run simultaneously utilizing a normal plasma in place of the tested plasma and tested iD a similar manner. If the unknown plasma has a higher count rate than the backgroundJ
it is considered hepatitis associated antigen positive.
Generally, it is preferred to utilize a counting time of one minuteJ howeverJ if a sample is quite close to the upper limit of the controlJ a longer counting time up to ten minutes may be utilized in order to obtain exact .
~ 25 counting results.
, ~ .
~ - 8 -~05990~
As noted above, the incubations are generally conducted at room temperature although slight warming up to about 35 C. may be utilized to shorten the incubation time. Where a short incubation time is desirable as a ; 5 means of reducing the total time required to conduct the test procedure, the incubation time can be greatly reduced ; by increasing the reaction temperature to from about 35 to 55C., especially from 38 to 50C. J with about 45C.
~; being most preferred. Use of the foregoing higher temper-atures can effectively reduce the incubation time to about l/6 to 3 hour~, especlally about 1 to about 2 hdurs. For example, in the procedure described above, after the plasma or blood to be te8ted is placed in the coated tube, or other apparatus, the incubation time can be greatly reduced to about two hours by incubating at about 45C. rather than at room tenperature. After incubation, the plasma or blood sample is removed from the test apparatus and rinsed with distilled , or deionized water, the washing procedure being preferably repeated. Labeled antibody is then added to the test apparatus and the apparatus incubated at about 45C. for about one hour. At the end of the hourJ the antibody~solution is removed from the test apparatus which is then rinsed with distilled or deionized water as previously described. The test apparatus is subsequently placed in a suitable well-type gamma scintillation counter and the count rate deter-mined. The presence or absence of antigen in the plasma or blood sample is then determined by relating net counts ~' _ g _ ~ ~059901 per minute of the unknown sample to net counts per minute of a negative control sample.
To determine the presence of an antibody in a sample, the procedure is reversed by instead affixing to the test apparatus a suitable antLgen. As described, the sample is then placed in contact with the apparatus having an antigen layer whereby any antibody in the sample will bond to the antigen. After washing, a suitably labeled antigen is then placed in contact with the apparatus to bond the labeled antigen to the antibody being measured.
~: .
The radioactivity is then measured as previously described ; to determine the presence or absence of antibody.
While an incubation temperature of from about 35 to 55C. can be employed as a means of reducing the incubation time, incubation temperatures greater than 55& .
and especially 60C. are undesirable 9ince activity drops off rapitly because of the destrùction or denaturing of the reagents which is believed to occur at these temperatures, i.e., the layer of antigen or antibody becoming disassociated from the test apparatus, as well as denaturing of the labeled - antiboty or antigen.
Short incubation times are desirable since rapid completion of the test procedure is permitted. In the collection and use of human blood for example, two to three hour incubation periods permit the collection, testing and use of blood, all within the same day. As used herein, the term "deionized" water is taken to mean water where the ions of any impurities have been removed by any conventional means.
~059901 Generally, the buffer medium ~ontains from about 0.005 to about 0.02 molar Tris-HCl and from about 0.01 to 0.05% by weight sodium azide at a pH of 6.9 to 8.4. The preferred buffer comprises 0.01 mo~ar Tris-HCl and 0.02X by weight sodium azide.
Generally, tne tests are run using undiluted blood serum or plasma, however, if samples are limited, a suitable dilution of the sample in normal serum or plasma, such a~ bovine serum albumen or in a buffer mixeure, such as a mixture of Tris-NCl and sodium azide, a mixture of Tris-HCl, sodium azide and 1% bovine serum albumen, etc.
may be used.
Also, although the method of the present invention has been-described with reference to I125 tagged antigens lS ~ ~or antibodies, the preferred radioactive material, any radioi~otope generally used for tagging or tracing antigens or antibodies in radioimmunoassay procedure~ may be utilized.
labeled iodine is preferred because of its long sixty tay half life. Other radioisotopes that can be utilized 20 ~ include~I131 havlng an eight day half life, p32 having a fourteen day half life, as well as other radioisotopes such as Tritium. Mixtures of the above can also be employed.
- As noted above, the process of the present invention utilizes a direct~radioimmunoassay technique for the determination of various antigens and their antibodies, especially hepatitis associated antigen or its antibody.
:lOS9901 Furthermore, if a quantitative determination of hepatitis associate~d antigen i8 desired, a standardized curve directly showing the relationship between counts per minute and the amount of hepatitis assod atet antigen may be utilized.
The foregoing methods and the apparatus of the present invention will now be illustrated by thc following specific example which is for the purpose of illustration only and is not to be taken as limiting. In the following .
example all parts and percentages are by weight~and all temperatures in degrees centigrade.
EXAMPLE I
The tube as shown in the drawing which is molded from polystyrene or polypropylene is coated with a purified :
h-patltls associated antibody. This coating is applied by m ~ ~ ; 8ub3ecting the surface of the tube to a diluted solution ~ ~ of hepatitis associated antibody in 0.01 molar Tris-HCl , .
at a pH of 7.1 and 0.02% by weight sodium azide and the coat~e~d tube is incubated at room temperature for one day.
m ~20 The tube is then washed with aliquots of 0.01 molar Tri~-HCl plus 0.02% by weight sodium azide. These tubes may be stored at 4C. until use. Three 100 microliter plasma samples are placed one each in three separate coated tubes, : :
; each tube being coated with hepatitis associated antibody.
One of these plasma samples is the unknown, the other two are negative for hepatitis associated antigen. Each step in the procedure i8 applied to each o the three samples, The two negative samples provide the background radiation against which the unknown sample is ultimatPly compared.
These samples are then ~et aside and incubated for 18 hours at room temperature. At the end of this time, the coated t~bes are wa~hed with aliquots of the incubation buffer mixture or water. At this time, 2.5 nanograms of purLfied I125 labeled hepatitis associated antibody in 0.1 ml. volume are placed into each coated tube. The tubes are again set aside and incubated for two hours after which time the tube is washed again with water or aliquots of the incubation buffer mixture. Each of the negative plasma samples is counted utilizing a conventional radiation counter having a well and capable of detecting gamma radiation. The negative samples are counted for 1 minute and the average number of counts per minute is determined;
in this case, 200 count~ per minute. The unknown sample is then counted in the same manner and compared with the average value of counts per minute of the negative plasma samples plus a correction factor equal to 50% of the counts per minute of the negative sample. The unknown plasma in this ca~e has a count rate of 400 counts per minute which i~ above the 300, i.e., 200 plus 50% of 200 = 300, which is the maximum for a negative test.
EXAMPLE II
The following is an example of a test procedure employing a short incubation time and described with reference to the detection of hepatitis assaciated antigen.
0.1 ml. of serum to be tested is added to the bottom of an antibody coated tube, ensuring that the serum is evenly distributed therein. The tube is placed in a water bath maintained at about 45C. to incubate for a period of two hours. The contents of the tube are then aspirated to remove the serum and washed with d~ tilled or deionized water or with an aliquot of a prepared rinse solution comprising trlmethamine buffer diluted with deionized or distilled water. The washing process is repeated four additional times. 0.1 ml. I125 labeled hepatitis associated antibody solution is then added to the bottom of each tube, again ensuring even distribution of the labeled antibody. The tube is then incubated at about 45C. for one hour. Thereafter, the contents are aspirated in order to remove any unbound labeled antibody and the tu~e is rinsed five times as previously described.
The tube is ~ubsequently placed in a suitable well-type gamma scintillation detector and the net counts per minute determined. To obtain the net counts per minute, any machine or background count is deducted from the total counts per minute obtained for the sample. The actual ~ ` ~
1059~01 radioactivi~y of the sample is thus obtained which is a direct measure of the antigen present in the sample.
As is evident, the above-noted tes~ procedures provide a simple yes - no test for determining~the presence S or ab8ence of hepatitis associated antigeh ih an unknown ~ample of blood or plasma. Although some correction factor iæ required, the test is quite conclusive and reprotucible and has a high degree of accuracy.
While the process of the present invention has ~ been illustrated by way of the foregoing specific example, ; the process of the present invention should be in no way : ~ :
limited thereto but should be construed as broadly as any and all equivalents in the appended claims.
- ~ ~
I
. ` .
.
This application is related to Canadian Patent Application No. 158,644, filed December 12, 1972.
Backg~ound of the Invention This invention relates to a diagnostic method for the radioimmunoassay of antigens and their antibodies and a method for coating apparatus useful in the above determination. More particularly, this invention relates - to a direct method for determining hepatitis associated antigens or their antibodies and is also directed toward a method for preparing diagnostic apparatus suitable for use in the same.
Although there have been methods for determining the presence of antigenically active macromolecules such as intact viruses, virus capsids, virus subunits, bacteria, membranes, cell walls, hormones, etc., there has been a lack - of a simple, yet sensitive, test method and apparatus for - 15 determining the presence of these materials. Viral hepatitis, including so-called serum hepatitis, which is a relatively common disease, has not been heretofore easily detected by a sensitive test which is both specific and reproducible for quickly determining whether or not the sera from a patient or a donor contains hepatitis associated antigens or antibodies.
Furthermore, radioimmunoassay techniques have been developed in the past for various antigen-antibody materials;
however, these radioimmunoassay techniques such as disclosed in articles by Kevin Catt et al in the Journal of BiochemistrY, 1966, Volume 100, pages 31c and 33c and in Science, ~ 59 9 ~
Volume 158, page 1570, 1967, are an indirect radioim~unoassay technique wherein the amount of antigen present in roughly inversely proportional to the amount of radiation emitted by the tracer material. These procedures required the use of correlation tables and other materials which generally rendered the resu~ts less than reproducible and exact.
Wide, Karolinska Simposia, 1st Simposia, September 23 to 25, 1969, University Hospital, Upsila, Sweden, pages 207 to 214 discloses a radioimmunoassay wherein an allergen is absorbed on a plastic polymer and reagin is bound immuno-chemically to the solid phase allergen and then labeled anti-reagen is bound to the reagln.
Briefly, it has been discovered that the above-noted difficulties, i.e., lack of reproducibility and exactness, have been overcome by utilizing the method of the precent invention. Briefly, in one application, the method of the present invention comprises contacting an unknown serum sample with an antibody coated implement, incubating the test implement and serum for a period of from 0.5 to 42 hours, aspirating and washing, contacting an Il 5 labeled antibody with the serum and coated the test apparatus and incubating for from 1 to 6 hours, aspirating and washing, and counting the tube for I gamma radiation.
It is therefore the principal object of the present invention to provide a novel method for the direct determination of antigens and their antibodies.
' `1059901 It is a further object of the present invention to provide a method for coating diagnostic apparatus for use in radioimmunoassay determinations.
It is a still further object of the present invention to provide a method for quickly and accurately determining the presence of hepatitis associated antigens or antibodies in sera.
Still further objects and advantages of the diagnostic method for direct radioimmunoassay and method for coa~ing test implements useful in the same will become more apparent from the following more detailed description thereof and the following attached drawing wherein:
The drawing is a cut-away view of a coated test implement useful in the method of the present invention.
The drawing shows a test tube shaped test implement 1 with a coated portion 2. Coated portion 2 has a coating of an antigen or its antibody preferably located as shown, i.e., in the bottom of the tube. Although the drawing shows one embodiment of apparatus suitable for use in performing the method of the present invention, the method of this invention should not be limited thereto. Other solid state devices may be employed such as inserts as disclosed in copending Canadian Patent Application No. 158,644, filed December 12, 1972g discs, spherical beads or particles, and the like, all of which may be coated with an antigen or antibody.
~ ` 1059901 Coated portion 2 is coated with either an antigen or an antibody depending on the material to be tested.
The antibody or antigen can be affixed to the test apparatus by contact coating from solution or any other means, the ~ manner of affixation not being critical. Since the method i5 similar with regard to almost all antigens and antibodies, the process for coating these tubes will be described with reference to a particular hepatitis associated antibody, i.e., anti-Australia antigen. A solution of anti-Australia antigen having a concentration of from about 1 to about 100 micrograms of protein per ml. is prepared from an antl-Australia antigen serum in from about 0.005 to about 0.02 molar Tris-HCl, i.e., 2-amino-2-hydroxymethyl-1,3-propanedîol-HCl. The Tris-HCl buffers the solution to a pH of from about 7.1 to about 9.5 together with from about 0.01% to about 0.05% sodium azide. This anti-Australia antigen solution is then coated on the tube surfaces and incubated at room temperature for from 6 to 72 hours and preferably for from 12 to 48 hours. These coated tubes are then washed with from about 0.005 to about 0.02 molar Tris-HCl at a pH of 6.9 to 8.4 plus from about 0.01% to about 0.05% sodium azide. Following this washing and rinsing step, the test implements may be stored at 4C. until necessary for use for radioimmunoassay. For additional details for methods of preparing antigen or antibody or the affixing of such to tubes or other apparatus, reference can be made to the ~~
publication by ~. M~ Ling and L. R~ ~verby, ~Preval~nce of Hepatitis B Antigen as Revealed by Direct Radioimmunoassay with I125 Antibody", the Journal of Immunolo~y, Volume 109, ~o. 4, October 1972.
It is preferred to utilize an 0~01 molar solution of Tris-HCl and 0.02% sodium azide buffered at a pH bf 7~1 for both the incubation medium and the washing medium.
The amount of antibody or antigen coated in the tubes i9 not critical since the test is run each time in comparison with at least one blank test~ No standard curves or charts are necessary for the test of the present invention; therefore, no specific amount of antibody or antigen in the coating is required as long as two similar tubes are used.
~ Although the coating method has been described with reference to a coated tube, the coating method may be utilized to prepare coated lnserts, beads, or any apparatus for use with wells, etc. by dipping the inserts in the antigen or antibody solution and following the remaining procedure. Likewise, while generally described in terms of coating of a tube, the antibody or antigen can be affixed to the test apparatus to provide a layer of the antibody or antigen, using any suitable methQd for achieving bonding or attachment thereto. The time required for incubating the affixed antigen or antibody to the test apparatus is not critical and may be as low as about ten minutes. However, where a coated tube is used, production processes dictate that a period of 6 to 72 hours is preferred.
Antigens and antibodies which may be determined by mean~ of the method of the present invention include:
various intact viruses, virus capsids, virus subunits, bacteria, membranes, cell walls, various hormones, gamma globulins, etc. The only requirement with regard to the above materials i8 that the materials havè a ~inimum of two antigenically active sites. Furthermore, antigens and antibodies having multiple combining sites are detectable even in the presence of their re3pective antibodies and antigens, provided a minimum of two free combining sites remain available. Although the radioimmunoassay method of the present invention is useful for ~etecting the above class of materials, it is especially well adapted, and this is a preferred embodiment of the present invention, for the determination of the presence of hepatitis associated antigens and antibodies, such as Australia antigen and anti-Australia antigen.
While the radioimmunoassay method of the present invention has been briefly described above, the method will now be described with reference to the specific materials and steps necessary for conducting the direct radioimmunoassay technique for determining the presence of the hepatitis associated antigen, Australia antigen.
-lOS9901 FirstJ a measure sample of plasma or blood to be tested for hepatitis associated antigen is placed in an anti-Australia antigen or antibody coated tube. The material is incubated for a period of from 0.5 to 42 hours at room temperature and preferably for from 12 to 24 hours. The coated material is washed with the buffer mixture, i.e., Tris-HCl and sodium azide or with distilled or deionized water. A measured amount of purifLed I125 labeled anti-Australia antigen is then added to the tube or test receptacle well and the tube or insert in contact therewith is incubated for an additional 1 to 24 hours at room temperature and preferably for from 1.5 to 6 hours.
Following thls incubation, the contents are washed utilizing the buffer mixture or distilled or deionized water and the tube is placed in the well of a counter which is capable of counting gamma radiation. Background controls in duplicate are run simultaneously utilizing a normal plasma in place of the tested plasma and tested iD a similar manner. If the unknown plasma has a higher count rate than the backgroundJ
it is considered hepatitis associated antigen positive.
Generally, it is preferred to utilize a counting time of one minuteJ howeverJ if a sample is quite close to the upper limit of the controlJ a longer counting time up to ten minutes may be utilized in order to obtain exact .
~ 25 counting results.
, ~ .
~ - 8 -~05990~
As noted above, the incubations are generally conducted at room temperature although slight warming up to about 35 C. may be utilized to shorten the incubation time. Where a short incubation time is desirable as a ; 5 means of reducing the total time required to conduct the test procedure, the incubation time can be greatly reduced ; by increasing the reaction temperature to from about 35 to 55C., especially from 38 to 50C. J with about 45C.
~; being most preferred. Use of the foregoing higher temper-atures can effectively reduce the incubation time to about l/6 to 3 hour~, especlally about 1 to about 2 hdurs. For example, in the procedure described above, after the plasma or blood to be te8ted is placed in the coated tube, or other apparatus, the incubation time can be greatly reduced to about two hours by incubating at about 45C. rather than at room tenperature. After incubation, the plasma or blood sample is removed from the test apparatus and rinsed with distilled , or deionized water, the washing procedure being preferably repeated. Labeled antibody is then added to the test apparatus and the apparatus incubated at about 45C. for about one hour. At the end of the hourJ the antibody~solution is removed from the test apparatus which is then rinsed with distilled or deionized water as previously described. The test apparatus is subsequently placed in a suitable well-type gamma scintillation counter and the count rate deter-mined. The presence or absence of antigen in the plasma or blood sample is then determined by relating net counts ~' _ g _ ~ ~059901 per minute of the unknown sample to net counts per minute of a negative control sample.
To determine the presence of an antibody in a sample, the procedure is reversed by instead affixing to the test apparatus a suitable antLgen. As described, the sample is then placed in contact with the apparatus having an antigen layer whereby any antibody in the sample will bond to the antigen. After washing, a suitably labeled antigen is then placed in contact with the apparatus to bond the labeled antigen to the antibody being measured.
~: .
The radioactivity is then measured as previously described ; to determine the presence or absence of antibody.
While an incubation temperature of from about 35 to 55C. can be employed as a means of reducing the incubation time, incubation temperatures greater than 55& .
and especially 60C. are undesirable 9ince activity drops off rapitly because of the destrùction or denaturing of the reagents which is believed to occur at these temperatures, i.e., the layer of antigen or antibody becoming disassociated from the test apparatus, as well as denaturing of the labeled - antiboty or antigen.
Short incubation times are desirable since rapid completion of the test procedure is permitted. In the collection and use of human blood for example, two to three hour incubation periods permit the collection, testing and use of blood, all within the same day. As used herein, the term "deionized" water is taken to mean water where the ions of any impurities have been removed by any conventional means.
~059901 Generally, the buffer medium ~ontains from about 0.005 to about 0.02 molar Tris-HCl and from about 0.01 to 0.05% by weight sodium azide at a pH of 6.9 to 8.4. The preferred buffer comprises 0.01 mo~ar Tris-HCl and 0.02X by weight sodium azide.
Generally, tne tests are run using undiluted blood serum or plasma, however, if samples are limited, a suitable dilution of the sample in normal serum or plasma, such a~ bovine serum albumen or in a buffer mixeure, such as a mixture of Tris-NCl and sodium azide, a mixture of Tris-HCl, sodium azide and 1% bovine serum albumen, etc.
may be used.
Also, although the method of the present invention has been-described with reference to I125 tagged antigens lS ~ ~or antibodies, the preferred radioactive material, any radioi~otope generally used for tagging or tracing antigens or antibodies in radioimmunoassay procedure~ may be utilized.
labeled iodine is preferred because of its long sixty tay half life. Other radioisotopes that can be utilized 20 ~ include~I131 havlng an eight day half life, p32 having a fourteen day half life, as well as other radioisotopes such as Tritium. Mixtures of the above can also be employed.
- As noted above, the process of the present invention utilizes a direct~radioimmunoassay technique for the determination of various antigens and their antibodies, especially hepatitis associated antigen or its antibody.
:lOS9901 Furthermore, if a quantitative determination of hepatitis associate~d antigen i8 desired, a standardized curve directly showing the relationship between counts per minute and the amount of hepatitis assod atet antigen may be utilized.
The foregoing methods and the apparatus of the present invention will now be illustrated by thc following specific example which is for the purpose of illustration only and is not to be taken as limiting. In the following .
example all parts and percentages are by weight~and all temperatures in degrees centigrade.
EXAMPLE I
The tube as shown in the drawing which is molded from polystyrene or polypropylene is coated with a purified :
h-patltls associated antibody. This coating is applied by m ~ ~ ; 8ub3ecting the surface of the tube to a diluted solution ~ ~ of hepatitis associated antibody in 0.01 molar Tris-HCl , .
at a pH of 7.1 and 0.02% by weight sodium azide and the coat~e~d tube is incubated at room temperature for one day.
m ~20 The tube is then washed with aliquots of 0.01 molar Tri~-HCl plus 0.02% by weight sodium azide. These tubes may be stored at 4C. until use. Three 100 microliter plasma samples are placed one each in three separate coated tubes, : :
; each tube being coated with hepatitis associated antibody.
One of these plasma samples is the unknown, the other two are negative for hepatitis associated antigen. Each step in the procedure i8 applied to each o the three samples, The two negative samples provide the background radiation against which the unknown sample is ultimatPly compared.
These samples are then ~et aside and incubated for 18 hours at room temperature. At the end of this time, the coated t~bes are wa~hed with aliquots of the incubation buffer mixture or water. At this time, 2.5 nanograms of purLfied I125 labeled hepatitis associated antibody in 0.1 ml. volume are placed into each coated tube. The tubes are again set aside and incubated for two hours after which time the tube is washed again with water or aliquots of the incubation buffer mixture. Each of the negative plasma samples is counted utilizing a conventional radiation counter having a well and capable of detecting gamma radiation. The negative samples are counted for 1 minute and the average number of counts per minute is determined;
in this case, 200 count~ per minute. The unknown sample is then counted in the same manner and compared with the average value of counts per minute of the negative plasma samples plus a correction factor equal to 50% of the counts per minute of the negative sample. The unknown plasma in this ca~e has a count rate of 400 counts per minute which i~ above the 300, i.e., 200 plus 50% of 200 = 300, which is the maximum for a negative test.
EXAMPLE II
The following is an example of a test procedure employing a short incubation time and described with reference to the detection of hepatitis assaciated antigen.
0.1 ml. of serum to be tested is added to the bottom of an antibody coated tube, ensuring that the serum is evenly distributed therein. The tube is placed in a water bath maintained at about 45C. to incubate for a period of two hours. The contents of the tube are then aspirated to remove the serum and washed with d~ tilled or deionized water or with an aliquot of a prepared rinse solution comprising trlmethamine buffer diluted with deionized or distilled water. The washing process is repeated four additional times. 0.1 ml. I125 labeled hepatitis associated antibody solution is then added to the bottom of each tube, again ensuring even distribution of the labeled antibody. The tube is then incubated at about 45C. for one hour. Thereafter, the contents are aspirated in order to remove any unbound labeled antibody and the tu~e is rinsed five times as previously described.
The tube is ~ubsequently placed in a suitable well-type gamma scintillation detector and the net counts per minute determined. To obtain the net counts per minute, any machine or background count is deducted from the total counts per minute obtained for the sample. The actual ~ ` ~
1059~01 radioactivi~y of the sample is thus obtained which is a direct measure of the antigen present in the sample.
As is evident, the above-noted tes~ procedures provide a simple yes - no test for determining~the presence S or ab8ence of hepatitis associated antigeh ih an unknown ~ample of blood or plasma. Although some correction factor iæ required, the test is quite conclusive and reprotucible and has a high degree of accuracy.
While the process of the present invention has ~ been illustrated by way of the foregoing specific example, ; the process of the present invention should be in no way : ~ :
limited thereto but should be construed as broadly as any and all equivalents in the appended claims.
- ~ ~
I
. ` .
.
Claims (9)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for determining the presence of an antigen or an antibody capable of being bound to two associated antigens or antibodies respectively in an unknown sample utilizing direct radioimmunoassay comprising:
(a) forming a solution of said antigen or antibody in a buffer mixture or deionized water;
(b) affixing the antigen or antibody contained in said solution to a test apparatus;
(c) incubating said affixed test apparatus;
(d) washing said incubated affixed test apparatus with a buffer mixture or deionized water;
(e) placing said unknown sample in contact with said incubated and washed test apparatus;
(f) incubating said unknown sample while in con-tact with said coated test apparatus for from 0.5 to 42 hours at from 38 to 50°C. to bond any of said antigen or antibody present in said unknown sample to said incubated test apparatus;
(g) washing said incubated test apparatus with a buffered solution or deionized water to remove said unknown sample leaving said bonded antigen or antibody on the affixed portion of said test apparatus;
(h) contacting said washed test apparatus with a purified form of said antigen or antibody labeled with a radioactive isotope selected from at least one of the group consisting of I125 I131 p32 and Tritium;
(i) incubating said washed test apparatus at from 38 to 50°C. while in contact with said purified form of said antigen or antibody labeled with a radioactive isotope so as to bond said purified form to said antigen or antibody bonded on said test apparatus and thereby produce a radioactively traced incubated coating;
(j) washing said radioactively traced incubated coating with a buffered solution or deionized water to remove any unbonded purified form;
(k) counting radiation emitted from said radio-actively traced incubated coating; and (l) comparing the number of counts from said coating with the number of counts from a control sample pre-pared by steps (a) to (k).
(a) forming a solution of said antigen or antibody in a buffer mixture or deionized water;
(b) affixing the antigen or antibody contained in said solution to a test apparatus;
(c) incubating said affixed test apparatus;
(d) washing said incubated affixed test apparatus with a buffer mixture or deionized water;
(e) placing said unknown sample in contact with said incubated and washed test apparatus;
(f) incubating said unknown sample while in con-tact with said coated test apparatus for from 0.5 to 42 hours at from 38 to 50°C. to bond any of said antigen or antibody present in said unknown sample to said incubated test apparatus;
(g) washing said incubated test apparatus with a buffered solution or deionized water to remove said unknown sample leaving said bonded antigen or antibody on the affixed portion of said test apparatus;
(h) contacting said washed test apparatus with a purified form of said antigen or antibody labeled with a radioactive isotope selected from at least one of the group consisting of I125 I131 p32 and Tritium;
(i) incubating said washed test apparatus at from 38 to 50°C. while in contact with said purified form of said antigen or antibody labeled with a radioactive isotope so as to bond said purified form to said antigen or antibody bonded on said test apparatus and thereby produce a radioactively traced incubated coating;
(j) washing said radioactively traced incubated coating with a buffered solution or deionized water to remove any unbonded purified form;
(k) counting radiation emitted from said radio-actively traced incubated coating; and (l) comparing the number of counts from said coating with the number of counts from a control sample pre-pared by steps (a) to (k).
2. The method of claim 1 wherein the incubation of steps (f) and (i) is conducted at a temperature of about 45°C.
3. The method of claim 1 wherein said antigen or its antibody is a hepatitis associated antigen or its antibody.
4. The method of claim 1 wherein the radioactive isotope is I125.
5. The method of claim 1 wherein the incubation of steps (f) and (i) is conducted at from 0.5 to 3 hours.
6. The method of claim 5 wherein the incubation time is from 1 to 2 hours.
7. The method of claim 1 in which step (b) is con-ducted by contacting said test apparatus with the solution of step (a) so as to form a coating.
8. The method of claim 1 wherein the washing is con-ducted with deionized water.
9. A method for determining the presence of a hepatitis associated antigen or its antibody in an unknown sample utilizing direct radioimmunoassay comprising:
(a) contacting said unknown sample with a hepatitis associated antigen or its antibody affixed to a test apparatus to provide a layer of said antigen or its antibody;
(b) incubating said unknown sample a first time for a period of about one to two hours at a temperature of about 38° to 50°C while in contact with said affixed layer;
(c) washing said incubated layer;
(d) contacting said washed layer with a radio-actively traced purified material selected from said hepatitis associated antigen or its antibody with the proviso that if said affixed layer is said antigen, said radioactively traced material is also said antigen, and if said affixed layer is said antibody said radioactively traced material is also said antibody;
(e) incubating said washed layer a second time for a period of about one to two hours at a temperature of about 38°
to 50° while in contact with said radioactively traced material;
(f) washing said radioactively traced incubated layer;
(g) counting radiation emitted from said radio-actively traced layer; and (h) comparing the number of counts from said layer with the number of counts from a negative sample prepared by steps (a) to (g).
(a) contacting said unknown sample with a hepatitis associated antigen or its antibody affixed to a test apparatus to provide a layer of said antigen or its antibody;
(b) incubating said unknown sample a first time for a period of about one to two hours at a temperature of about 38° to 50°C while in contact with said affixed layer;
(c) washing said incubated layer;
(d) contacting said washed layer with a radio-actively traced purified material selected from said hepatitis associated antigen or its antibody with the proviso that if said affixed layer is said antigen, said radioactively traced material is also said antigen, and if said affixed layer is said antibody said radioactively traced material is also said antibody;
(e) incubating said washed layer a second time for a period of about one to two hours at a temperature of about 38°
to 50° while in contact with said radioactively traced material;
(f) washing said radioactively traced incubated layer;
(g) counting radiation emitted from said radio-actively traced layer; and (h) comparing the number of counts from said layer with the number of counts from a negative sample prepared by steps (a) to (g).
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US05/480,180 US4012494A (en) | 1971-12-21 | 1974-06-17 | Direct radioimmunoassay for antigens and their antibodies |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1059901A true CA1059901A (en) | 1979-08-07 |
Family
ID=23906963
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA225,016A Expired CA1059901A (en) | 1974-06-17 | 1975-04-10 | Direct radioimmunoassay for antigens and their antibodies |
Country Status (3)
| Country | Link |
|---|---|
| CA (1) | CA1059901A (en) |
| DE (1) | DE2526800A1 (en) |
| FR (1) | FR2274687A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2418457A1 (en) * | 1978-02-22 | 1979-09-21 | Commissariat Energie Atomique | Device for sepg. e.g. antibodies in radioimmunology - having extended surface of material adsorbing or covalently bonding with antibodies and fixable in tube above end |
| FR2495326B1 (en) * | 1980-12-03 | 1986-03-28 | Georges Desmonts | AGGLUTINATION TYPE IMMUNOLOGICAL ASSAY METHOD FOR THE DETECTION OF IMMUNOGLOBULIN TYPE ANTIBODIES |
| CA1172561A (en) * | 1981-01-28 | 1984-08-14 | Richard H. Decker | Igm detection method |
-
1975
- 1975-04-10 CA CA225,016A patent/CA1059901A/en not_active Expired
- 1975-06-16 FR FR7518785A patent/FR2274687A1/en active Granted
- 1975-06-16 DE DE19752526800 patent/DE2526800A1/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| FR2274687A1 (en) | 1976-01-09 |
| FR2274687B1 (en) | 1980-05-16 |
| DE2526800A1 (en) | 1976-01-02 |
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