CA1227130A - Identification of infected states - Google Patents
Identification of infected statesInfo
- Publication number
- CA1227130A CA1227130A CA000453199A CA453199A CA1227130A CA 1227130 A CA1227130 A CA 1227130A CA 000453199 A CA000453199 A CA 000453199A CA 453199 A CA453199 A CA 453199A CA 1227130 A CA1227130 A CA 1227130A
- Authority
- CA
- Canada
- Prior art keywords
- immune complex
- complex
- samples
- antibody
- bound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
ABSTRACT
A method of diagnosis using an attached material for binding to an immune complex, by treating the bound complex with a series of different reagents or mixtures thereof and detec-ting the presence or absence of reaction in each case, this method being based on immune complexes produced in the body during infection and allowing much earlier detection and diagnosis of infection thereby providing the facility for treatment to reduce the damage caused to the body by the formation of complex.
A method of diagnosis using an attached material for binding to an immune complex, by treating the bound complex with a series of different reagents or mixtures thereof and detec-ting the presence or absence of reaction in each case, this method being based on immune complexes produced in the body during infection and allowing much earlier detection and diagnosis of infection thereby providing the facility for treatment to reduce the damage caused to the body by the formation of complex.
Description
1227~30 Identification of Infected States This invention relates to the identification of an infected state in a living body.
When a patient is infected by a micro-organism, or antigen, the entry of the micro-organism into the patient's blood-stream causes the production of an antibody by the patient's body and the antibody combines with an antigen from the 10 micro-organism and with "complement", which is contained in the blood, to form an immune complex in the patient's body.
Recently immune complexes have been implicated in the etiology of some human diseases, in particular as a cause 15 of tissue damage, and thus the accurate measurement of complexes is of growing importance to medical practitioners. Many of the procedures currently used for their measurement rely upon interaction of complex with the complement system, for instance by virtue of the decreases 20 in serum complement levels so caused. Alternatively, complex may be detected by measuring differences in their physico-chemical characteristics relative to uncombined antibody. These techniques, however, are generally insensitive, inaccurate, difficult to use and give little 25 information about the antigen and antibody components of the complex. Other techniques make use of living cells or human reagents and as such are unsuitable for application on the extensive scale required by hospital laboratories
When a patient is infected by a micro-organism, or antigen, the entry of the micro-organism into the patient's blood-stream causes the production of an antibody by the patient's body and the antibody combines with an antigen from the 10 micro-organism and with "complement", which is contained in the blood, to form an immune complex in the patient's body.
Recently immune complexes have been implicated in the etiology of some human diseases, in particular as a cause 15 of tissue damage, and thus the accurate measurement of complexes is of growing importance to medical practitioners. Many of the procedures currently used for their measurement rely upon interaction of complex with the complement system, for instance by virtue of the decreases 20 in serum complement levels so caused. Alternatively, complex may be detected by measuring differences in their physico-chemical characteristics relative to uncombined antibody. These techniques, however, are generally insensitive, inaccurate, difficult to use and give little 25 information about the antigen and antibody components of the complex. Other techniques make use of living cells or human reagents and as such are unsuitable for application on the extensive scale required by hospital laboratories
- 2 - lZ27~30 During the initial stages of infection the patient's body contains an excess of infecting micro-organisms over antibody but as the infection proceeds the body produces more antibody until there is an excess of antibody. In the early stages only a small amount of complex is formed in view of the small amounts of antibody produced by the body, and this does not activate the complement, so little damage is caused to the body and no symptoms are detected. As the production of antibody increases however the amount of complex increases 10 correspondingly, and as the stage approaches where the anti-gun and antibody are in equal proportions in the body the complex is present in substantial amounts. Moreover, it has been found that the complex produced at this stage has a half-life of about a week, so its effect is felt over a pro-15 longed period. On passing the antigen/antibody balance point the antibody is produced in excess and it has been found that the half-life of the complex produced then is reduced to a few hours. Thereafter the antibody excess increases until the antigen and the complex are no longer 20 present.
In general, symptoms appear in a patient only during the period of antibody excess, and this may be due to the high concentration of complex present, both of long half-life 25 from before the antigen/antibody balance point and of short half-life from during and after the balance point. By the time that the symptoms appear, therefore, the infection is well advanced and treatment may have to be drastic.
Further, the symptonls often appear after the infectious 30 stage has been passed so that the infection may have been already passed on to others before detection.
It has previously been proposed to determine the amount of immune complex in a sample by means of an assay using 35 conglutinin, as this material has the property of combining
In general, symptoms appear in a patient only during the period of antibody excess, and this may be due to the high concentration of complex present, both of long half-life 25 from before the antigen/antibody balance point and of short half-life from during and after the balance point. By the time that the symptoms appear, therefore, the infection is well advanced and treatment may have to be drastic.
Further, the symptonls often appear after the infectious 30 stage has been passed so that the infection may have been already passed on to others before detection.
It has previously been proposed to determine the amount of immune complex in a sample by means of an assay using 35 conglutinin, as this material has the property of combining
3 issue with an immune complex. The use of conglutinin has however been restricted to the assay.
According to the present invention there is provided a method of identifying the presence or absence of an infected state in a living body before the appearance of symptoms of the infection, comprising obtaining a biological fluid sample from the living body not displaying symptoms of the particular infection, adding to the sample a material which 10 is known to bind with immune complex characteristic of said particular infection, adding to the resulting bound immune complex a labeled reagent which is known to react specifically with the antibody or antigen portion of said characteristic immune complex, separating excess reagent 15 and detecting the presence or absence of said label in the residual mixture.
If required, the presence or absence of immune complex in the sample can be detected after treatment of the body 20 extract with the attachment material.
By using the method of this invention it is possible to test individual patients for the presence or absence of complex and to identify the complex, at an early stage of complex 25 production in the body and before the formation of damaging long-half-life complex. This can be done in circumstances, for example, where an infection has been detected by symptoms in an individual and it is desired to prevent the infection spreading to others. Thus those individuals who 30 have been in contact with the infected patient can be tested by the present method before symptoms appear, and isolated and treated if the diagnosis shows that they have the infection.
12Z7~30 In an alternative example, regular diagnosis by the present method can be carried out in environments where infections could spread rapidly and with great effect, for example in schools, old peoples' homes and other places where large numbers of people come together regularly. In this example the individuals can be "screened" periodically to detect and diagnose any infections before they would otherwise become apparent and before they have reached a stage where they can /
, 1ZZ7~30 be transmitted to others.
The present method also allows infections to be treated early and can reduce thereby the amount of suffering of a patient.
As the infection may be detected and diagnosed before the damaging long-half-life complex is produced, antibody specie lie to the offending antigen can be administered in excess so that the antigen/antibody balance point is rapidly reached and passed, and only a small amount of complex having a loo 10 half-life is produced. The patient therefore would export-once considerably less discomfort as the symptoms would be reduced in intensity.
The material for attachment to the complex is preferably 15 conglutinin as this is the most convenient material avail-able at this time; however, other materials may be used if appropriate, for example immunoconglutinin or rheumatoid factor. Immunoconglutinins are naturally occurring anti-bodies formed against complement components in an antigen/
20 antibody/complement complex, and conveniently react specie focally with complement which has been altered by inter-action with complex. Immunoconglutinins may be obtained from the sofa of most animals, e.g. sheep or rabbits which have previously been challenged with suitable complexes.
The conglutinin may be prepared by treating conglutinin-containing serum with an absorbent for conglutinin, washing the treated serum to separate the absorbed conglutinin, and separating the conglutinin from the absorbent.
Preferably, the absorbent is zymosan, which may be obtained by treating baker's yeast with elm 2-mercaptoethanol for 2 hours and alkylating the product with 0.02M iodacetamide in phosphate-buffered saline solution.
6 12z7~30 Preferably also the conglutinin is separated from the absorb bent by centrifuging and dialyzing. Purification can be by chromatography, for example by passage through Sephadex G-20n. (Sephadex is a Registered Trade Mark).
s Conglutinin advantageously reacts only with a Cub component of complement which has been formed after interaction with an antigen/antibody complex. Conglutinin may be obtained from the sofa of ruminant animals, preferably from a lo euglobulin fraction of bovine serum, although it may also be obtained from the serum of other ruminants e.g. African buffalo, water buck, Uganda knob etc. In a particularly preferred method, conglutinin is extracted from bovine serum by use of zymosan, a yeast cell wall preparation. Kingly-15 tenon may also be separated from other serum constituents bagel filtration, for instance through Sephadex G-2~û in the presence of mercaptoethanol and ETA, as described by Lachman (Advances in Immunology 1967, 6, 479). Conglutinin rich preparations may be obtained by the combination of these 20 procedures in any sequence to achieve the standard of purity desired.
The material, especially when it is conglutinin, is prefer-ably attached to a solid substrate prior to treatment with 25 the immune complex, and it has been found to be very effect live to use as the substrate balls of plastics material such for example as those sold by Euro-Matic Limited, which offer an extensive surface area over which the material can attach itself. Alternatively the solid substrate can be in the 3û form of a receptacle such as a dish or a test tube, or can be coated on the surface of such a receptacle.
Conglutinin and immunoconglutinin can be linked to the solid substrate by any suitable means. The conglutinin and 35 immunoconglutinin are preferably in a purified state, though l;~Z7~30 impure or partially purified forms may be used such as the complete euglobulin fraction of bovine serum. The method used to link the conglutinin or immunoconglutinin to the solid substrate may include use of a coupling reagent and/or 5 appropriate preparation of the solid substrate. For example it has been found that conglutinin may be linked to Sephadex particles which have been activated with cyanogen bromide.
When the presence of immune complex has been detected, but 10 its identity is not known, reagents can be used which are active against either the antigen or the antibody. An effective method is to prepare mixtures of known reagents and to test samples of the immune complex against these mixtures. If a mixture is found which reacts with the 15 component of the complex, either further mixtures or individual ingredients of that mixture can then be tested until the active ingredient is recognized, thus allowing identification of the component of the complex. Reagents active against the antigen component, known as anti-20 antigens, are preferably employed.
The presence or absence of reaction can for example be detected by labeling the reagent with a radioactive label, and after treatment of the immune complex testing for the 25 presence of radioactivity. In an alternative method the reagent may be enzyme labeled, and reaction detected by adding an enzyme substrate and determining a color change, for example by spectrophotometry.
30 An embodiment of the method of this invention will now be described by means of the following Examples, of which Example 1 describes the detection and identification of infection and Example 2 describes such a procedure in use for early diagnosis in accordance with the invention.
Preparation of Conglutinin A conglutinin-rich bovine serum is selected by a red cell 8 ~.ZZ7~30 clumping test, as described on pages 172 to 175 of "The Serology of Conglutination and its Relation to Disease" by Combs et at (Bleakly, 1961). 1 lithe of high title bovine serum (1 in 64n) is mixed with 1509 of zymosan for 2 hours at 4C. The zymosan product is then washed with Vernal buffered saline solution and eluded with O.OlM PBS-ETA for 10 minutes at room temperature. 0.5 ml/ml of yeast suspension is used. The product is then centrifuged and the supernatent liquid containing the conglutinin is 10 dialyzed against 0.5M Nail overnight at 4C.
The liquid then has its pi adjusted to 3.0 using N Hal and is dialyzed against O.OOlN Hal and 0.5M Nail for 2 hours.
15 Pepsin is added to the resulting liquid in an amount of 0.5 my per ml in O.OlN Hal to a final concentration of owe.
The solution is allowed to stand at 4C for 18 hours, and a buffer solution of elm Nope an Own Noah is added until a pi of 7 2 is achieved. The solution is then dialyzed 20 against û.02M PBS-EDTA and then passed through a Sephadex G-200 chromatography column to separate out the conglutinin which is identified by the red cell test.
Assay and Diagnosis I" polystyrene balls obtained from Euro-Matic Limited, are incubated in a solution of conglutinin in a carbonate buffer in an approximate concentration of 100 gel at pi 9.6.
The balls, with the conglutinin on their surface, are then 30 washed 3 times in vernal buffered saline/Tween*solution ova t and placed in test tubes.
A standard of aggregated human Gig is prepared by aggregate in Gig at 63C for 30 minutes, and a stock of solution at a 35 concentration of 1000 gel is diluted in 350 I volumes to *Trade Mark 122~30 a concentration of 1 yg/ml. To each dilution it added 50 I
of fresh normal human serum, and the mixture is incubated at 37UC for 30 minutes to allow complement fixation. The total 400 I volumes of the standard and of the Test sample are added to the coated polystyrene balls in separate test tubes and incubated for 3 hours at room temperature. The balls in each tube are then washed 3 times with the vernal buff freed saline/Tween solution at room temperature. 0.4 ml of radiolabelled anti-human immunoglobulin is added to each 10 tube and incubated for 3 hours at room temperature, then being washed 3 times with the vernal buffered saline/Tween solution. The radioactivity of the bound immunoglobulin in the standard and test samples is then measured, the standard providing a datum for determining the presence of immune 15 complex quantitatively in the test sample.
Having measured the level of immune complex, further ides-tidal test samples are added to conglutinin-coated balls, incubated and washed as described above. Thereafter dip-20 fervent selected mixtures of radioactive-labelled anti-antigens, which are known antibodies against bacteria, fungi or viruses, are added to the test samples, incubated and washed as described above, and the coated balls are then tested for radioactivity using an ION Gamiest 500 counter.
When a mixture of anti-antigens is found to have bound to the immune complex, the components of that mixture are tested individually or in further mixtures against the immune complex in the same manner until the anti-antigen 30 which has bound to the complex is identified.
In this way not only the quantity of immune complex present in the sample but also the nature of the antigen in the complex can be determined, thus providing a quantitative and 35 qualitative test for immune complex.
1;~27130 The invention as described in this Example therefore pro-vises a diagnostic test for identifying infection in a test sample.
This Example shows that early detection and identification of immune complexes can be made, before symptoms are apt parent. Routine samples of blood serum was obtained from 10 two individuals, both young, apparently healthy, adult males. The samples were each subjected to assay as in Example 1, and then diagnosis was carried out by adding to the conglutinin-coated balls with the complex attached a series c-f non-radiolabelled anti-zntigens, namely:
rabbit anti-influenza B viruses rabbit anti-respiratory syncytial viruses rabbit anti-adeno viruses and rabbit anti-influenza A viruses The balls were then treated with radiolabelled sheep anti-20 rabbit Gig, polyvzlent reagent, and then tested for radio-activity after washing. The belts treated with anti-influenza B produced a positive result.
The antibodies used were prepared in viva but it is equally 25 effective to use monoclonal antibodies.
The day after sampling, both individuals developed flu-like symptoms which became worse throughout that day arid into the next. By the third day they were both improving and 30 on the fourth were able to return to work where blood swamp-lies were taken. Further blood samples were obtained 16 days later and conventional serological analysis of these (i.e. by antibody detection) confirmed that both had been infected with a strain of Influenza B virus.
1227~30 The results obtained with an immune complex assay are summarized in Tables 1 and 2. The assays were carried out as described in the Example 1. Plastic balls were used to carry the conglutinin, radiolabelled sheep anti-human Gig 5 detected the immune complexes and quantitated them, and radiolabelled rabbit anti-influenza B antibody was the anti-antigen which identified the antigen.
Table 1. Results of conglutinin radio immunoassay for immune complex Sample Corrected count_ug/ml equivalents Reeducate viny immune complexes cam 15 Standard (1)23010 600 (2) 13590 300 (3) 8690 150
According to the present invention there is provided a method of identifying the presence or absence of an infected state in a living body before the appearance of symptoms of the infection, comprising obtaining a biological fluid sample from the living body not displaying symptoms of the particular infection, adding to the sample a material which 10 is known to bind with immune complex characteristic of said particular infection, adding to the resulting bound immune complex a labeled reagent which is known to react specifically with the antibody or antigen portion of said characteristic immune complex, separating excess reagent 15 and detecting the presence or absence of said label in the residual mixture.
If required, the presence or absence of immune complex in the sample can be detected after treatment of the body 20 extract with the attachment material.
By using the method of this invention it is possible to test individual patients for the presence or absence of complex and to identify the complex, at an early stage of complex 25 production in the body and before the formation of damaging long-half-life complex. This can be done in circumstances, for example, where an infection has been detected by symptoms in an individual and it is desired to prevent the infection spreading to others. Thus those individuals who 30 have been in contact with the infected patient can be tested by the present method before symptoms appear, and isolated and treated if the diagnosis shows that they have the infection.
12Z7~30 In an alternative example, regular diagnosis by the present method can be carried out in environments where infections could spread rapidly and with great effect, for example in schools, old peoples' homes and other places where large numbers of people come together regularly. In this example the individuals can be "screened" periodically to detect and diagnose any infections before they would otherwise become apparent and before they have reached a stage where they can /
, 1ZZ7~30 be transmitted to others.
The present method also allows infections to be treated early and can reduce thereby the amount of suffering of a patient.
As the infection may be detected and diagnosed before the damaging long-half-life complex is produced, antibody specie lie to the offending antigen can be administered in excess so that the antigen/antibody balance point is rapidly reached and passed, and only a small amount of complex having a loo 10 half-life is produced. The patient therefore would export-once considerably less discomfort as the symptoms would be reduced in intensity.
The material for attachment to the complex is preferably 15 conglutinin as this is the most convenient material avail-able at this time; however, other materials may be used if appropriate, for example immunoconglutinin or rheumatoid factor. Immunoconglutinins are naturally occurring anti-bodies formed against complement components in an antigen/
20 antibody/complement complex, and conveniently react specie focally with complement which has been altered by inter-action with complex. Immunoconglutinins may be obtained from the sofa of most animals, e.g. sheep or rabbits which have previously been challenged with suitable complexes.
The conglutinin may be prepared by treating conglutinin-containing serum with an absorbent for conglutinin, washing the treated serum to separate the absorbed conglutinin, and separating the conglutinin from the absorbent.
Preferably, the absorbent is zymosan, which may be obtained by treating baker's yeast with elm 2-mercaptoethanol for 2 hours and alkylating the product with 0.02M iodacetamide in phosphate-buffered saline solution.
6 12z7~30 Preferably also the conglutinin is separated from the absorb bent by centrifuging and dialyzing. Purification can be by chromatography, for example by passage through Sephadex G-20n. (Sephadex is a Registered Trade Mark).
s Conglutinin advantageously reacts only with a Cub component of complement which has been formed after interaction with an antigen/antibody complex. Conglutinin may be obtained from the sofa of ruminant animals, preferably from a lo euglobulin fraction of bovine serum, although it may also be obtained from the serum of other ruminants e.g. African buffalo, water buck, Uganda knob etc. In a particularly preferred method, conglutinin is extracted from bovine serum by use of zymosan, a yeast cell wall preparation. Kingly-15 tenon may also be separated from other serum constituents bagel filtration, for instance through Sephadex G-2~û in the presence of mercaptoethanol and ETA, as described by Lachman (Advances in Immunology 1967, 6, 479). Conglutinin rich preparations may be obtained by the combination of these 20 procedures in any sequence to achieve the standard of purity desired.
The material, especially when it is conglutinin, is prefer-ably attached to a solid substrate prior to treatment with 25 the immune complex, and it has been found to be very effect live to use as the substrate balls of plastics material such for example as those sold by Euro-Matic Limited, which offer an extensive surface area over which the material can attach itself. Alternatively the solid substrate can be in the 3û form of a receptacle such as a dish or a test tube, or can be coated on the surface of such a receptacle.
Conglutinin and immunoconglutinin can be linked to the solid substrate by any suitable means. The conglutinin and 35 immunoconglutinin are preferably in a purified state, though l;~Z7~30 impure or partially purified forms may be used such as the complete euglobulin fraction of bovine serum. The method used to link the conglutinin or immunoconglutinin to the solid substrate may include use of a coupling reagent and/or 5 appropriate preparation of the solid substrate. For example it has been found that conglutinin may be linked to Sephadex particles which have been activated with cyanogen bromide.
When the presence of immune complex has been detected, but 10 its identity is not known, reagents can be used which are active against either the antigen or the antibody. An effective method is to prepare mixtures of known reagents and to test samples of the immune complex against these mixtures. If a mixture is found which reacts with the 15 component of the complex, either further mixtures or individual ingredients of that mixture can then be tested until the active ingredient is recognized, thus allowing identification of the component of the complex. Reagents active against the antigen component, known as anti-20 antigens, are preferably employed.
The presence or absence of reaction can for example be detected by labeling the reagent with a radioactive label, and after treatment of the immune complex testing for the 25 presence of radioactivity. In an alternative method the reagent may be enzyme labeled, and reaction detected by adding an enzyme substrate and determining a color change, for example by spectrophotometry.
30 An embodiment of the method of this invention will now be described by means of the following Examples, of which Example 1 describes the detection and identification of infection and Example 2 describes such a procedure in use for early diagnosis in accordance with the invention.
Preparation of Conglutinin A conglutinin-rich bovine serum is selected by a red cell 8 ~.ZZ7~30 clumping test, as described on pages 172 to 175 of "The Serology of Conglutination and its Relation to Disease" by Combs et at (Bleakly, 1961). 1 lithe of high title bovine serum (1 in 64n) is mixed with 1509 of zymosan for 2 hours at 4C. The zymosan product is then washed with Vernal buffered saline solution and eluded with O.OlM PBS-ETA for 10 minutes at room temperature. 0.5 ml/ml of yeast suspension is used. The product is then centrifuged and the supernatent liquid containing the conglutinin is 10 dialyzed against 0.5M Nail overnight at 4C.
The liquid then has its pi adjusted to 3.0 using N Hal and is dialyzed against O.OOlN Hal and 0.5M Nail for 2 hours.
15 Pepsin is added to the resulting liquid in an amount of 0.5 my per ml in O.OlN Hal to a final concentration of owe.
The solution is allowed to stand at 4C for 18 hours, and a buffer solution of elm Nope an Own Noah is added until a pi of 7 2 is achieved. The solution is then dialyzed 20 against û.02M PBS-EDTA and then passed through a Sephadex G-200 chromatography column to separate out the conglutinin which is identified by the red cell test.
Assay and Diagnosis I" polystyrene balls obtained from Euro-Matic Limited, are incubated in a solution of conglutinin in a carbonate buffer in an approximate concentration of 100 gel at pi 9.6.
The balls, with the conglutinin on their surface, are then 30 washed 3 times in vernal buffered saline/Tween*solution ova t and placed in test tubes.
A standard of aggregated human Gig is prepared by aggregate in Gig at 63C for 30 minutes, and a stock of solution at a 35 concentration of 1000 gel is diluted in 350 I volumes to *Trade Mark 122~30 a concentration of 1 yg/ml. To each dilution it added 50 I
of fresh normal human serum, and the mixture is incubated at 37UC for 30 minutes to allow complement fixation. The total 400 I volumes of the standard and of the Test sample are added to the coated polystyrene balls in separate test tubes and incubated for 3 hours at room temperature. The balls in each tube are then washed 3 times with the vernal buff freed saline/Tween solution at room temperature. 0.4 ml of radiolabelled anti-human immunoglobulin is added to each 10 tube and incubated for 3 hours at room temperature, then being washed 3 times with the vernal buffered saline/Tween solution. The radioactivity of the bound immunoglobulin in the standard and test samples is then measured, the standard providing a datum for determining the presence of immune 15 complex quantitatively in the test sample.
Having measured the level of immune complex, further ides-tidal test samples are added to conglutinin-coated balls, incubated and washed as described above. Thereafter dip-20 fervent selected mixtures of radioactive-labelled anti-antigens, which are known antibodies against bacteria, fungi or viruses, are added to the test samples, incubated and washed as described above, and the coated balls are then tested for radioactivity using an ION Gamiest 500 counter.
When a mixture of anti-antigens is found to have bound to the immune complex, the components of that mixture are tested individually or in further mixtures against the immune complex in the same manner until the anti-antigen 30 which has bound to the complex is identified.
In this way not only the quantity of immune complex present in the sample but also the nature of the antigen in the complex can be determined, thus providing a quantitative and 35 qualitative test for immune complex.
1;~27130 The invention as described in this Example therefore pro-vises a diagnostic test for identifying infection in a test sample.
This Example shows that early detection and identification of immune complexes can be made, before symptoms are apt parent. Routine samples of blood serum was obtained from 10 two individuals, both young, apparently healthy, adult males. The samples were each subjected to assay as in Example 1, and then diagnosis was carried out by adding to the conglutinin-coated balls with the complex attached a series c-f non-radiolabelled anti-zntigens, namely:
rabbit anti-influenza B viruses rabbit anti-respiratory syncytial viruses rabbit anti-adeno viruses and rabbit anti-influenza A viruses The balls were then treated with radiolabelled sheep anti-20 rabbit Gig, polyvzlent reagent, and then tested for radio-activity after washing. The belts treated with anti-influenza B produced a positive result.
The antibodies used were prepared in viva but it is equally 25 effective to use monoclonal antibodies.
The day after sampling, both individuals developed flu-like symptoms which became worse throughout that day arid into the next. By the third day they were both improving and 30 on the fourth were able to return to work where blood swamp-lies were taken. Further blood samples were obtained 16 days later and conventional serological analysis of these (i.e. by antibody detection) confirmed that both had been infected with a strain of Influenza B virus.
1227~30 The results obtained with an immune complex assay are summarized in Tables 1 and 2. The assays were carried out as described in the Example 1. Plastic balls were used to carry the conglutinin, radiolabelled sheep anti-human Gig 5 detected the immune complexes and quantitated them, and radiolabelled rabbit anti-influenza B antibody was the anti-antigen which identified the antigen.
Table 1. Results of conglutinin radio immunoassay for immune complex Sample Corrected count_ug/ml equivalents Reeducate viny immune complexes cam 15 Standard (1)23010 600 (2) 13590 300 (3) 8690 150
(4) 3750 75
(5) 3670 37 20 First Patient Day 1 15450* 350*
Day 4 8710 190 Day 20 480 ~75 Second Patient 25 Day 1 22460* 540*
Day 4 7900 170 Day 20 600 ~75 Normal sofa (1) 82 ~75 30 (2) 306 C75 (3) 285 ~75 _ *Denotes exceptionally high values The assay quantitates satisfactorily between 75 and 600 ug/ml. Normal fall in the range 0-75~ug/ml 12 12Z7~30 able 2. Detection of influenza B antigen in immune complexes Sample Radioactivity counts/min.
Normal (1) 136û
(2) 1718 First Patient 10 Day 1 2221*
Day 4 1474 Day 20 1780 Second Patient 15 Day 1 2444*
Day 4 1683 Day 20 1746 20 It should be noted that the assay can be made to quantitate satisfactorily from 0-75 yg/ml, but for screening purposes, when looking for abnormal values, this is not essential.
From these results it is clear that on Day 1 - i.e. one day 25 before symptoms appeared- high levels of immune complexes were present in the blood of both individuals. The level was lower by Day 4 by which time they were recovering and by Day 20 both were normal.
30 The examples show that infection, even simple respiratory virus infection could be detected by the finding of abnormal levels of immune complex in the blood.
Table 2 gives the results obtained with an anti-antigen (i.e.
35 anti-influenza B virus antibody). As can be seen, it 12Z7~30 satisfactorily identified influenza virus in the appropriate samples.
Note that antigen was only detected in the "early" phase of infection, not during recovery; this is to be expected because after recovery begins antigen clearance from the blood will be rapid.
EXAMPLE _ 10 Trypanosomiasis (sleeping sickness) in rabbits New Zealand White rabbits were infected on Thea February with a clone preparation of Trypanosoma Bruce Bruce (WIG-lo). Daily blood samples were taken to establish whether 15 the animals had parasites in their blood (by Leishman-stained films and microscopy), and for immune complexes by the method described in Example l. The weight of the animals was recorded each day and a careful watch was kept for signs of illness. A summary of the results from one 20 representative experiment is shown in the accompanying Figure l.
It can be seen that after injection a progressive increase in immune complex levels occurred, reaching a peak between 25 the lath and Thea March. The abnormality of the animal was evident in raised immune complex levels prior to this time, and it was only later that weight reduction was seen.
Symptoms appeared only about Thea March, and throughout the whole experiment trypanosomes were not observed in the blood 30 films by microscopy. Hence the immune complexes were a very sensitive indicator of the infective process which was going on in this animal. The complex detection was with a radiolabelled sheep anti-rabbit Gig, which favors antibody excess immune complexes.
1227~30 The present inventor has discovered that production of immune complex takes place in the body at an early stage of infection and long before any symptoms appear, and this immune complex can be detected and identified at a stage where action can be taken to treat the body with drugs before the patient is aware that he or she is ill; the infection may possibly be cured without the patient experiencing any symptoms at all. His or her day-to-day activities may therefore not be affected by the infection in cases where otherwise a prolonged period of discomfort through the onset of symptoms would be experienced.
Such diagnosis of infection prior to the appearance of symptoms has great significance in other areas also. For example, many infections such as trypanosomiasis, schistosomiasis and others caused by parasites are very difficult to diagnose and identify. The symptoms may appear in a number of people, but many more can be carriers of the infection without displaying any symptoms whatever.
The method of this invention allows such people to be tested to determine whether infection is present in their bodies, and the infection can be identified and treated. This prevents any further transfer of the infection from these people unwittingly to others, and the spread of the 25 infection can therefore be arrested.
Hitherto such carriers of infection could not be identified as they appeared to be healthy, and treatment was confined to those who displayed symptoms and could thereby be 30 recognized.
A further area in which this invention is extremely important is in the identification of infections which cause no clear overt symptoms or malaise. Such infections are 35 brucellosis, hepatitis B, leprosy and post-viral depression, lo 12~L30 and in each case the patient may feel depressed or slightly below strength without displaying any clear symptoms of illness; this can continue for many years if untreated and can result in considerable long-term suffering to the patient.
The method of this invention allows a serum sample from a patient to be taken and the infection diagnosed and treated.
Hitherto positive diagnosis could not be made in this way as 10 the antibodies are not free, being locked into immune complexes in the body.
The invention can also be used in areas where infection has broken out and rapid spread is likely, as in epidemics.
15 Here the infection can be detected and diagnosed in people who have not yet displayed symptoms but are nevertheless infectious to others. Measles, typhoid and many other infections are passed on as a result of early diagnosis being unavailable, as the most infectious period occurs 20 before any symptoms occur. Now, however, the method of the invention can be applied to large numbers of people in an area of infection and those who are infected can be isolated and treated before the onset of their symptoms. This is especially important in institutions where many people are 25 living in the same place and infection can spread very rapidly, for example old people's homes, children's homes, and hospital wards especially geriatric and cancer units.
Day 4 8710 190 Day 20 480 ~75 Second Patient 25 Day 1 22460* 540*
Day 4 7900 170 Day 20 600 ~75 Normal sofa (1) 82 ~75 30 (2) 306 C75 (3) 285 ~75 _ *Denotes exceptionally high values The assay quantitates satisfactorily between 75 and 600 ug/ml. Normal fall in the range 0-75~ug/ml 12 12Z7~30 able 2. Detection of influenza B antigen in immune complexes Sample Radioactivity counts/min.
Normal (1) 136û
(2) 1718 First Patient 10 Day 1 2221*
Day 4 1474 Day 20 1780 Second Patient 15 Day 1 2444*
Day 4 1683 Day 20 1746 20 It should be noted that the assay can be made to quantitate satisfactorily from 0-75 yg/ml, but for screening purposes, when looking for abnormal values, this is not essential.
From these results it is clear that on Day 1 - i.e. one day 25 before symptoms appeared- high levels of immune complexes were present in the blood of both individuals. The level was lower by Day 4 by which time they were recovering and by Day 20 both were normal.
30 The examples show that infection, even simple respiratory virus infection could be detected by the finding of abnormal levels of immune complex in the blood.
Table 2 gives the results obtained with an anti-antigen (i.e.
35 anti-influenza B virus antibody). As can be seen, it 12Z7~30 satisfactorily identified influenza virus in the appropriate samples.
Note that antigen was only detected in the "early" phase of infection, not during recovery; this is to be expected because after recovery begins antigen clearance from the blood will be rapid.
EXAMPLE _ 10 Trypanosomiasis (sleeping sickness) in rabbits New Zealand White rabbits were infected on Thea February with a clone preparation of Trypanosoma Bruce Bruce (WIG-lo). Daily blood samples were taken to establish whether 15 the animals had parasites in their blood (by Leishman-stained films and microscopy), and for immune complexes by the method described in Example l. The weight of the animals was recorded each day and a careful watch was kept for signs of illness. A summary of the results from one 20 representative experiment is shown in the accompanying Figure l.
It can be seen that after injection a progressive increase in immune complex levels occurred, reaching a peak between 25 the lath and Thea March. The abnormality of the animal was evident in raised immune complex levels prior to this time, and it was only later that weight reduction was seen.
Symptoms appeared only about Thea March, and throughout the whole experiment trypanosomes were not observed in the blood 30 films by microscopy. Hence the immune complexes were a very sensitive indicator of the infective process which was going on in this animal. The complex detection was with a radiolabelled sheep anti-rabbit Gig, which favors antibody excess immune complexes.
1227~30 The present inventor has discovered that production of immune complex takes place in the body at an early stage of infection and long before any symptoms appear, and this immune complex can be detected and identified at a stage where action can be taken to treat the body with drugs before the patient is aware that he or she is ill; the infection may possibly be cured without the patient experiencing any symptoms at all. His or her day-to-day activities may therefore not be affected by the infection in cases where otherwise a prolonged period of discomfort through the onset of symptoms would be experienced.
Such diagnosis of infection prior to the appearance of symptoms has great significance in other areas also. For example, many infections such as trypanosomiasis, schistosomiasis and others caused by parasites are very difficult to diagnose and identify. The symptoms may appear in a number of people, but many more can be carriers of the infection without displaying any symptoms whatever.
The method of this invention allows such people to be tested to determine whether infection is present in their bodies, and the infection can be identified and treated. This prevents any further transfer of the infection from these people unwittingly to others, and the spread of the 25 infection can therefore be arrested.
Hitherto such carriers of infection could not be identified as they appeared to be healthy, and treatment was confined to those who displayed symptoms and could thereby be 30 recognized.
A further area in which this invention is extremely important is in the identification of infections which cause no clear overt symptoms or malaise. Such infections are 35 brucellosis, hepatitis B, leprosy and post-viral depression, lo 12~L30 and in each case the patient may feel depressed or slightly below strength without displaying any clear symptoms of illness; this can continue for many years if untreated and can result in considerable long-term suffering to the patient.
The method of this invention allows a serum sample from a patient to be taken and the infection diagnosed and treated.
Hitherto positive diagnosis could not be made in this way as 10 the antibodies are not free, being locked into immune complexes in the body.
The invention can also be used in areas where infection has broken out and rapid spread is likely, as in epidemics.
15 Here the infection can be detected and diagnosed in people who have not yet displayed symptoms but are nevertheless infectious to others. Measles, typhoid and many other infections are passed on as a result of early diagnosis being unavailable, as the most infectious period occurs 20 before any symptoms occur. Now, however, the method of the invention can be applied to large numbers of people in an area of infection and those who are infected can be isolated and treated before the onset of their symptoms. This is especially important in institutions where many people are 25 living in the same place and infection can spread very rapidly, for example old people's homes, children's homes, and hospital wards especially geriatric and cancer units.
Claims (21)
1. A method of identifying the presence or absence of an infected state in a living body before the appearance of symptoms of the infection, comprising obtaining a biological fluid sample from the living body not displaying symptoms of the particular infection, adding to the sample a material which is known to bind with immune complex characteristic of said particular infection, adding to the resulting bound immune complex a labeled reagent which is known to react specifically with the antibody or antigen portion of said characteristic immune complex, separating excess reagent and detecting the presence or absence of said label in the residual mixture.
2. A method according to Claim 1, wherein the biological fluid sample is serum.
3. A method according to Claim 1 or 2, wherein the material which is known to bind with said characteristic immune complex is selected from conglutinin, immunocon-glutinin and rheumatoid factor.
4. A method according to Claim 1, wherein the material which is known to bind with said characteristic immune complex is carried by an inert solid substrate.
5. A method according to Claim 4, wherein the substrate is in the form of plastics balls whose surfaces have a coating comprising said material.
6. A method according to Claim 1, wherein prior to addition of the labeled reagent the presence of immune complex is detected.
7. A method according to Claim 6, wherein the presence of immune complex is detected by treating the bound complex with radiolabelled immunoglobulin.
8. A method according to Claim 1 or 6, wherein the bound immune complex is identified by treating samples of the bound complex with different labelled antibody mixtures, detecting the mixture which has reacted with the bound complex, treating the samples of bound complex with individual labelled components of the mixture which was found to react with the bound complex, and detecting the component which has reacted with the bound complex.
9. A method according to Claim 1 or 6, wherein the bound immune complex is identified by treating samples of the bound complex with individual labelled antibodies, and detecting the antibody which has reacted with the bound complex.
10. A method according to Claim 1 or 6, wherein the bound immune complex is identified by treating samples of the bound complex with different unlabelled antibody mixtures, treating the resulting products with a polyvalent labelled reagent thereby to label reacted antibody mixture, detecting the antibody mixture which has reacted with the bound complex, treating the samples of bound complex with individual unlabelled components of the antibody mixture which reacted with the bound complex, treating the resulting products with polyvalent labelled reagent thereby to label reacted component, and detecting the component which has reacted with the bound complex.
11. A method according to Claim 1, wherein the label is a radioactive or enzyme label.
12. A method according to Claim 1, wherein the labeled reagent is known to react with the antigenic portion of said immune complex.
13. A method of identifying the presence or absence of an infected state in a living body, comprising:
(a) obtaining biological fluid from a living body;
(b) contacting the fluid with binding material selected from conglutinin, immunoconglutinin or rheumatoid factor to bind any immune complex in the fluid to the bind material;
(c) adding to discrete samples of the resulting mixture a series of labelled reagents each of which is known to react specifically with a different antigen portion of immune complex;
(d) separating excess of the reagents from the samples;
and (e) detecting the presence or absence of said label in each of the samples thereby to identify therein the nature of any reacted antigen portion of immune complex.
(a) obtaining biological fluid from a living body;
(b) contacting the fluid with binding material selected from conglutinin, immunoconglutinin or rheumatoid factor to bind any immune complex in the fluid to the bind material;
(c) adding to discrete samples of the resulting mixture a series of labelled reagents each of which is known to react specifically with a different antigen portion of immune complex;
(d) separating excess of the reagents from the samples;
and (e) detecting the presence or absence of said label in each of the samples thereby to identify therein the nature of any reacted antigen portion of immune complex.
14. A method of identifying the presence or absence of an infection in a living body before the appearance of symptoms of said infection, comprising:
(a) obtaining biological fluid from a living body not displaying symptoms of said infection;
(b) contacting the fluid with binding material selected from conglutinin, immunoconglutinin and rheumatoid factor;
(c) adding to discrete samples of the resulting mixture a series of labelled reagents each of which is known to react specifically with a different antigen portion of immune complex;
(d) separating excess of the reagents from the samples;
and (e) detecting the presence or absence of said label in each of the samples thereby to identify therein the nature of any reacted antigen portion of immune complex characteris-tic of said infection.
(a) obtaining biological fluid from a living body not displaying symptoms of said infection;
(b) contacting the fluid with binding material selected from conglutinin, immunoconglutinin and rheumatoid factor;
(c) adding to discrete samples of the resulting mixture a series of labelled reagents each of which is known to react specifically with a different antigen portion of immune complex;
(d) separating excess of the reagents from the samples;
and (e) detecting the presence or absence of said label in each of the samples thereby to identify therein the nature of any reacted antigen portion of immune complex characteris-tic of said infection.
15. A method of identifying the presence or absence of an infected state in a living body, comprising:
(a) obtaining biological fluid from a living body;
(b) contacting the fluid with conglutinin to bind any immune complex in the fluid to the conglutinin;
(c) adding to discrete samples of the resulting mixture a series of labelled reagents each of which is known to react specifically with a different antigen portion of immune complex;
(d) separating excess of the reagents from the samples;
and (e) detecting the presence or absence of said label in each of the samples thereby to identify therein the nature of any reacted antigen portion of immune complex.
(a) obtaining biological fluid from a living body;
(b) contacting the fluid with conglutinin to bind any immune complex in the fluid to the conglutinin;
(c) adding to discrete samples of the resulting mixture a series of labelled reagents each of which is known to react specifically with a different antigen portion of immune complex;
(d) separating excess of the reagents from the samples;
and (e) detecting the presence or absence of said label in each of the samples thereby to identify therein the nature of any reacted antigen portion of immune complex.
16. A method according to Claim 13, 14 or 15, wherein a plurality of different labelled antibody mixtures are added one to each of the samples of said resulting mixture con-taining bound immune complex, excess of each of said labelled antibody mixtures is separated, the presence or absence of said label is detected in each sample to identify which of the mixtures has reacted with the immune complex, individual labelled antibodies of the mixture which reacted with the immune complex are then added to further samples of said resulting material, excess antibody is separated in each case, and the presence or absence of said label is detected in each sample to identify the antibody which has reacted with the immune complex.
17. A method according to Claim 13, 14 or 15, wherein a plurality of different antibody mixtures are added one to each of the samples of said resulting mixture containing bound immune complex, excess antibody mixture is separated in each case, a polyvalent labelled reagent known to react with the antibody is then added to each of the reaction mix-tures obtained, excess polyvalent reagent is separated in each case, the presence or absence of said label in each of the reaction mixtures is then detected to identify which of the mixtures has reacted with the immune complex, individual antibodies of the mixture which reacted with the immune complex are added to further samples of said resulting mix-ture containing bound immune complex, excess antibody is separated in each case, polyvalent labelled reagent known to react with the antibody is then added to each of the reaction mixtures, excess polyvalent reagent is separated in each case, and the presence or absence of said label is detected in each sample to identify which of the antibodies has reacted with the immune complex.
18. A method according to Claim 15, wherein the con-glutinin is coated on an inert solid substrate.
19. A method according to Claim 18, wherein the inert solid substrate is in the form of plastics balls whose sur-faces are coated with the conglutinin.
20. A method according to Claim 13, 14 or 15 wherein prior to the addition of the labelled reagents radiolabelled immunoglobulin is added to the samples of said resulting mixture for reaction with any immune complex present, excess radiolabelled immunoglobulin is separated and the presence or absence of said label is detected in the remaining reac-tion mixture.
21. A method according to Claim 13, 14 or 15 wherein the label is a radiolabel or an enzyme label.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000453199A CA1227130A (en) | 1984-04-30 | 1984-04-30 | Identification of infected states |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000453199A CA1227130A (en) | 1984-04-30 | 1984-04-30 | Identification of infected states |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1227130A true CA1227130A (en) | 1987-09-22 |
Family
ID=4127759
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000453199A Expired CA1227130A (en) | 1984-04-30 | 1984-04-30 | Identification of infected states |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1227130A (en) |
-
1984
- 1984-04-30 CA CA000453199A patent/CA1227130A/en not_active Expired
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4548909A (en) | Method of diagnosis | |
| Allan et al. | A quantitative comparison of the sensitivity of serological tests for bovine brucellosis to different antibody classes | |
| Shackelford et al. | Spectrum of IgG2 subclass deficiency in children with recurrent infections: prospective study | |
| Shulman | Hepatitis-associated antigen | |
| Aotsuka et al. | Measurement of anti-double-stranded DNA antibodies in major immunoglobulin classes | |
| US4353982A (en) | Immunochemical assay for creatine kinase-MB isoenzyme | |
| Ruddy et al. | Hereditary deficiency of the second component of complement (C2) in man: correlation of C2 haemolytic activity with immunochemical measurements of C2 protein | |
| SINGER et al. | The presence of anti-gamma globulin factors in sera of patients with active pulmonary tuberculosis | |
| Greenwood et al. | Rheumatoid factor in Nigerian sera | |
| Calder et al. | Cytotoxic lymphocytes in Hashimoto thyroiditis: an in vitro assay system using 51Cr-labelled chicken red blood cells coated with thyroglobulin | |
| Chaplin et al. | Quantitation of red blood cell‐bound C3d in normal subjects and random hospitalized patients | |
| Viljanen et al. | Radioimmunoassay of class-specific antibodies (RIACA): chicken antibodies to bovine serum albumin | |
| Schmidt et al. | Complement fixation and immunodiffusion tests for assay of hepatitis-associated “Australia” antigen and antibodies | |
| Schinski et al. | Enzyme-and 125I-labeled anti-immunoglobulin assays in the immunodiagnosis of schistosomiasis | |
| US4329331A (en) | Diagnostic method for detection of systemic lupus erythematosus | |
| CA1168580A (en) | Immunological reagent for the detection of tubes of the rheumatoid factor in a biological specimen and process for preparing this novel reagent | |
| Cupps et al. | Activation of human peripheral blood B cells following immunization with hepatitis B surface antigen vaccine | |
| McDuffie et al. | The behavior in the Coombs test of anti-A and anti-B produced by immunization with various blood group A and B substances and by heterospecific pregnancy | |
| Lind et al. | Observations on blood glucose and insulin determinations | |
| Aukrust et al. | Demonstration of distinct allergens by means of immunological methods: Comparison of crossed radioimmunoelectrophoresis, radioallergosorbent test and in vivo passive transfer test | |
| Edrissian et al. | Application of enzyme-linked immunosorbent assay (ELISA) to identification of Anopheles mosquito bloodmeals | |
| CA1227130A (en) | Identification of infected states | |
| US4609630A (en) | Method for improving the specificity of immunoassays | |
| Enerbäck et al. | Binding to galactose alpha 1----4galactose beta-containing receptors as potential diagnostic tool in urinary tract infection | |
| Sarsfield et al. | A modified radioallergosorbent test for the in vitro detection of allergen antibodies |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |