CA1057194A - C1q in immunochemical determination - Google Patents
C1q in immunochemical determinationInfo
- Publication number
- CA1057194A CA1057194A CA310,401A CA310401A CA1057194A CA 1057194 A CA1057194 A CA 1057194A CA 310401 A CA310401 A CA 310401A CA 1057194 A CA1057194 A CA 1057194A
- Authority
- CA
- Canada
- Prior art keywords
- clq
- complex
- amount
- agglutination
- particles
- 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
Abstract
ABSTRACT OF DISCLOSURE
Fluid Samples such as serum are assayed for Ab:Ag complex by adding to the sample a solution of Clq as well as a material which is caused to agglutinate on contact with Clq by determin-ing the extent of agglutination or non-agglutination of the material.
Fluid Samples such as serum are assayed for Ab:Ag complex by adding to the sample a solution of Clq as well as a material which is caused to agglutinate on contact with Clq by determin-ing the extent of agglutination or non-agglutination of the material.
Description
lOS'7~94 This invention is concerned with the analysis of fluids, such as urine or serum, for the determination of the presence, amount and/or nature of antibodie~, antigens and antibody:antigen complexes. tFor ~implicity hereinafter the symbols "Ab", "Ag" and "Ab:Ag" are used for "antibody", "antigen", and "antibody-antigen complex", respectively.) As iq well known, it is important to be able to analyse biological fluids for Ab, Ag and Ab:Ag complexesO
For example many diseases are characterised by the presence in the circulation of Ab:Ag complexes. The Ag may be any of a wide variety of proteins including those due to the presence of bacteria or viruses or those released from human tissues or cancer cells. The Ab are, of cour~e~
specific to the particular Ag and are predominantly immuno-globulins of the IgG class synthe~ised by the ~ub~ect's lymphoid system. The detection of Ab:Ag complexe~ in blood, and their ~eparation and characterisation, provide infor-mation of value and can be used, for example, in the diagnosis of disease.
There nre a number of techniques known for detecting and quantifying Ag, Ab and Ab:Ag complexe~ and particularly for determining the nature and amount of Ag present. These quantification techniques are called "immunoassay" procedures.
It has been known for some time that the naturally occurring substance Clq ha~ the property of combining with Ab:Ag complexes but not with either free Ag or free Ab.
Whilst there has been a prior propo~al (Agnello et al, J. Exp. Med., 134, 228, 1971) to use this property in one ~057194 particular way for the detection (but not the quantitative assay or absolute determination) of Ab:Ag complexe~, it has never previously been realised that Clq is potentially an extremely useful reagent in the analysis of Ab, Ag and Ab:Ag complexes.
We have now devised a method of immunoassRy using Clq.
According to the invention, there is provided a method of assaying a fluid sample for Ab:Ag complex, which comprises adding to the sample a solution of Clq, charac-terised in that there is also added a material which is caused to agglutinate on contact with Clq, and wherein the extent of the agglutination or non-agglutination of the material is detected.
]5 Clq is a natural circulating protein and method for its separation and purification are known. It is usually obtained from human, rabbit or bovine serum by a technique known as "euglobulin precipitation" which is des-cribed in, for example, J. Immunol., 106, 304-413 (1~71).
The Ab:Ag complex to be assayed may be formed by adding to a fluid containing an Ab or Ag, the corresponding Ag or Ab, respectivelyO In this way it is posslble to determine the amount of an Ab or Ag in a fluid.
In the method of the invention, the sald m~terial which is caused to agglutinate can vary widely. Thu~, Clq causes agglutination of red blood cells. Also, it wlll cause agglutination of materialq which, for example, com-prise immunoglobulins, e.g. have a coating or outer ~urface of immunoglobulins, such as polystyrene particles coated with immunoglobulins. Such coatings can be formed by immunological reactions between Ab and membrane antigens, or by physical adsorption or chemical reaction. Poly-styrene particles coated with immunoglobulini are commer-cially available but can, in any event, easily be prepared.
When such coated particles or red blood cells contact Clq, agglutination begins to occur, but if there is also present in solution an Ab:Ag complex, thi~ will react with the Clq relatively quicker and the Clq will become bound to the Ab:Ag complex in solution and~ a~ a result, no agglutination of the coated particles (or red blood cells) will occur. Thus, the presence of Ab:Ag complexes in serum, for example, can be detected by con-tacting the ~erum with Clq and with particles coated with immunoglobulins. If agglutination is observed, the ~erum does not contain any Ab:Ag complexes.
This is a very simple and accurate te~t, and is applioable to the detection of all Ab:Ag complexes. An example of the procedure is as follows:
50 ~1 of serum are added to 50 ~1 of a ~olution of soluble Clq, and the mixture i~ combined with 50 ~1 oP a ~u~pen~ion oP poly~tyrene particle~
coated with immunoglobulin~. Any re~ulting agglutination of the particles can readil~ be observed.
The above test can also be used for detectlng the presence of a particular Ag or Ab in serum, For example, when testing for a particular antigen, Ag', there i~ added to the serum the appropriate specific antibody Ab', and then the test is made for the presence of an Ab:Ag complex, 3o namely Ab':Ag'. If the fferum originally contain~ other Ab:Ag con~plexes, tllcse must first be removed (for example using insolubili~ed RF or Clq as described in our co-C~
pending~application no. 227,481, filed May 21, 1975).
The method of the invention may also be effected quantitatively. Eor examplc, the sample under te~t 18 mixed with a solution of Clq and with a known amount of latex particles coated with IgG, whereby Clq becomes bound to any Ab:Ag complex present and the remainder of the Clq causes agglutination of the latex particles. The amount of the said Clq remainder can be assayed by counting the agglutinated or non-agglutinated latex particles and com-paring the results with standard results. From the result, the amount of Ab:Ag complex can be determined.
In~order that the invention may be more fully understood, the following Examples are given by way of illustration only~
EXA~LE 1 Determination of IgE
0.1 ml of the sample containing IgE wa~ pip~tted int,o a 5 ml tube and 1.0 ml of rabbit anti-human IgE anti-serum (diluted 1:128) was added. The mixture was incubated for 1 hour at 37C and 1 ml of a solution containing 60000 latex particles of o.8 micron diameter which had previously been incubated in a solution of physiological saline con-taining 1 mg of IgG/ml, wa~ added. The mixture,was shaken and 0.1 ml of a solution containing 600 nanograms of Clq was added.
'The-mixture was incubated overnight and then -counted in a particle counter capable of rejecting partlcles `~ 1057194 groater than 1 micron in diameter. The concentrRtion o~
IgE was inver~ely proportional to the particle count and could be determined from a pr~viouYly prepared cAlibrAtion curve obtained with -qolutionq containing known ~oncentration~
of IgE.
Automatic analysis for C~1 foetal proteln ~ _ .
A flow of 0.1 ml/minute of the sample to be te~ted wa~ pumped into an automatic analyser of the con~lnuou~ flow type, together with the same flow Gf-a ~olution contAlning - 60000 particles of latex per ml. The latex particle~ were .o.8 micron in diameter and the solu~ion had previou~ly been incubated with an antiserum to ~ foetal protei~ diluted 64 with physiological saline~ The mixed streamn ~ere qegmented with air pumped into the system at 0.32 ml/~inute.
Following the introduction of the air, a flow of 0.4 ml~
minute of A solution of Clq was introduced.
The segmented stream wa~ heated to 37C for 10 -minutes and then further heated to 63C for 2 minutec to ~0 Adestroy the Clq and hence release nll latex par~icle~ bound by Clq, but not tho~e bound by Ag:Ab coupling.
The stream was then passed through a pArticle counter which is capable of rejecting particles $reator than 1 micron in diameter. The concentration ofa~1 ~oetnl protein ~was inversely proportional to the particle count and c4uld be -~determined from a previously prepared calibration cur~e obt~ined with solutions containing known concentrntionJ of ~oetal protein~
_This-application is a division of copending Canadian _application Serial No. 227,493, filed May 21, 1975.
For example many diseases are characterised by the presence in the circulation of Ab:Ag complexes. The Ag may be any of a wide variety of proteins including those due to the presence of bacteria or viruses or those released from human tissues or cancer cells. The Ab are, of cour~e~
specific to the particular Ag and are predominantly immuno-globulins of the IgG class synthe~ised by the ~ub~ect's lymphoid system. The detection of Ab:Ag complexe~ in blood, and their ~eparation and characterisation, provide infor-mation of value and can be used, for example, in the diagnosis of disease.
There nre a number of techniques known for detecting and quantifying Ag, Ab and Ab:Ag complexe~ and particularly for determining the nature and amount of Ag present. These quantification techniques are called "immunoassay" procedures.
It has been known for some time that the naturally occurring substance Clq ha~ the property of combining with Ab:Ag complexes but not with either free Ag or free Ab.
Whilst there has been a prior propo~al (Agnello et al, J. Exp. Med., 134, 228, 1971) to use this property in one ~057194 particular way for the detection (but not the quantitative assay or absolute determination) of Ab:Ag complexe~, it has never previously been realised that Clq is potentially an extremely useful reagent in the analysis of Ab, Ag and Ab:Ag complexes.
We have now devised a method of immunoassRy using Clq.
According to the invention, there is provided a method of assaying a fluid sample for Ab:Ag complex, which comprises adding to the sample a solution of Clq, charac-terised in that there is also added a material which is caused to agglutinate on contact with Clq, and wherein the extent of the agglutination or non-agglutination of the material is detected.
]5 Clq is a natural circulating protein and method for its separation and purification are known. It is usually obtained from human, rabbit or bovine serum by a technique known as "euglobulin precipitation" which is des-cribed in, for example, J. Immunol., 106, 304-413 (1~71).
The Ab:Ag complex to be assayed may be formed by adding to a fluid containing an Ab or Ag, the corresponding Ag or Ab, respectivelyO In this way it is posslble to determine the amount of an Ab or Ag in a fluid.
In the method of the invention, the sald m~terial which is caused to agglutinate can vary widely. Thu~, Clq causes agglutination of red blood cells. Also, it wlll cause agglutination of materialq which, for example, com-prise immunoglobulins, e.g. have a coating or outer ~urface of immunoglobulins, such as polystyrene particles coated with immunoglobulins. Such coatings can be formed by immunological reactions between Ab and membrane antigens, or by physical adsorption or chemical reaction. Poly-styrene particles coated with immunoglobulini are commer-cially available but can, in any event, easily be prepared.
When such coated particles or red blood cells contact Clq, agglutination begins to occur, but if there is also present in solution an Ab:Ag complex, thi~ will react with the Clq relatively quicker and the Clq will become bound to the Ab:Ag complex in solution and~ a~ a result, no agglutination of the coated particles (or red blood cells) will occur. Thus, the presence of Ab:Ag complexes in serum, for example, can be detected by con-tacting the ~erum with Clq and with particles coated with immunoglobulins. If agglutination is observed, the ~erum does not contain any Ab:Ag complexes.
This is a very simple and accurate te~t, and is applioable to the detection of all Ab:Ag complexes. An example of the procedure is as follows:
50 ~1 of serum are added to 50 ~1 of a ~olution of soluble Clq, and the mixture i~ combined with 50 ~1 oP a ~u~pen~ion oP poly~tyrene particle~
coated with immunoglobulin~. Any re~ulting agglutination of the particles can readil~ be observed.
The above test can also be used for detectlng the presence of a particular Ag or Ab in serum, For example, when testing for a particular antigen, Ag', there i~ added to the serum the appropriate specific antibody Ab', and then the test is made for the presence of an Ab:Ag complex, 3o namely Ab':Ag'. If the fferum originally contain~ other Ab:Ag con~plexes, tllcse must first be removed (for example using insolubili~ed RF or Clq as described in our co-C~
pending~application no. 227,481, filed May 21, 1975).
The method of the invention may also be effected quantitatively. Eor examplc, the sample under te~t 18 mixed with a solution of Clq and with a known amount of latex particles coated with IgG, whereby Clq becomes bound to any Ab:Ag complex present and the remainder of the Clq causes agglutination of the latex particles. The amount of the said Clq remainder can be assayed by counting the agglutinated or non-agglutinated latex particles and com-paring the results with standard results. From the result, the amount of Ab:Ag complex can be determined.
In~order that the invention may be more fully understood, the following Examples are given by way of illustration only~
EXA~LE 1 Determination of IgE
0.1 ml of the sample containing IgE wa~ pip~tted int,o a 5 ml tube and 1.0 ml of rabbit anti-human IgE anti-serum (diluted 1:128) was added. The mixture was incubated for 1 hour at 37C and 1 ml of a solution containing 60000 latex particles of o.8 micron diameter which had previously been incubated in a solution of physiological saline con-taining 1 mg of IgG/ml, wa~ added. The mixture,was shaken and 0.1 ml of a solution containing 600 nanograms of Clq was added.
'The-mixture was incubated overnight and then -counted in a particle counter capable of rejecting partlcles `~ 1057194 groater than 1 micron in diameter. The concentrRtion o~
IgE was inver~ely proportional to the particle count and could be determined from a pr~viouYly prepared cAlibrAtion curve obtained with -qolutionq containing known ~oncentration~
of IgE.
Automatic analysis for C~1 foetal proteln ~ _ .
A flow of 0.1 ml/minute of the sample to be te~ted wa~ pumped into an automatic analyser of the con~lnuou~ flow type, together with the same flow Gf-a ~olution contAlning - 60000 particles of latex per ml. The latex particle~ were .o.8 micron in diameter and the solu~ion had previou~ly been incubated with an antiserum to ~ foetal protei~ diluted 64 with physiological saline~ The mixed streamn ~ere qegmented with air pumped into the system at 0.32 ml/~inute.
Following the introduction of the air, a flow of 0.4 ml~
minute of A solution of Clq was introduced.
The segmented stream wa~ heated to 37C for 10 -minutes and then further heated to 63C for 2 minutec to ~0 Adestroy the Clq and hence release nll latex par~icle~ bound by Clq, but not tho~e bound by Ag:Ab coupling.
The stream was then passed through a pArticle counter which is capable of rejecting particles $reator than 1 micron in diameter. The concentration ofa~1 ~oetnl protein ~was inversely proportional to the particle count and c4uld be -~determined from a previously prepared calibration cur~e obt~ined with solutions containing known concentrntionJ of ~oetal protein~
_This-application is a division of copending Canadian _application Serial No. 227,493, filed May 21, 1975.
Claims (8)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method of assaying a fluid sample for Ab:Ag complex, which comprises adding to the sample a solution of Clq, characterised in that there is also added a material which is caused to agglutinate on contact with Clq, and wherein the extent of the agglutination or non-agglutination of the material is detected.
2. A method according to claim 1, characterised in that the material comprises an immunoglobulin.
3. A method according to claim 2, characterised in that the material comprises an immunoglobulin coating on inert carrier particles.
4. A method according to claim 3, characterised in that the said particles are polystyrene.
5. A method according to claim 1, 2, or 3 charac-terised in that the assay is quantative and that said mat-erial is a known amount of latex particles coated with IgG, whereby Clq becomes bound to any antibody:antigen complex present and the remainder of the Clq causes agglut-ination of the latex particles; and determining the amount of said remainder of Clq (and therefrom the amount of antibody: antigen complex) by counting the agglutinated or non-agglutinated latex particles and comparing the result with standard results.
6. A method according to claim 1, 2 or 3, character-ised in that the said complex is formed by adding to a fluid, originally containing an Ab or Ag, the corres-ponding Ag or Ab respectively, whereby the amount of said Ab or Ag present originally in the fluid can be determined.
7. A method according to claim 1, 2 or 3.
characterised in that the assay is quantitative.
characterised in that the assay is quantitative.
8. A method according to claim 1, 2 or 3 characterised in that it is effected in a continuous flow manner.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA310,401A CA1057194A (en) | 1978-08-31 | 1978-08-31 | C1q in immunochemical determination |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA310,401A CA1057194A (en) | 1978-08-31 | 1978-08-31 | C1q in immunochemical determination |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1057194A true CA1057194A (en) | 1979-06-26 |
Family
ID=4112247
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA310,401A Expired CA1057194A (en) | 1978-08-31 | 1978-08-31 | C1q in immunochemical determination |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1057194A (en) |
-
1978
- 1978-08-31 CA CA310,401A patent/CA1057194A/en not_active Expired
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