GB2098322A - Determination of antistreptolysine antibody - Google Patents

Abstract

Antistreptolysin antibody content of blood or serum samples is determined by contacting a solution of known concentration of oxidised O- streptolysin with a diluted blood or serum sample, allowing the mixture obtained to stand at 15 to 75 DEG C, adding a solution of a reducing agent, waiting for red blood cell sedimentation, checking if haemolysis has occurred, and checking the condition of a control blank. If a serum sample is used, it is necessary to add red blood cells thereto prior to the treatment with the reducing agent. It is possible to use the method for the determination of the antibody threshold, and to continue the method if the preselected threshold has been exceeded. A diagnostic kit containing the reagents necessary for the method is also described.

Description

SPECIFICATION Determination of antistreptolysine antibody This invention relates to an improved and integrated method for determining the presence and/or the quantity of antistreptolysin antibody in blood, and relates also to means for carrying out such determinations.

As is known, O-streptolysin is a toxin having a proteinic nature, secreted by certain Streptococci of the A Group. The toxin is lethal to test animals, it destroys cell cultures, and it lysates the red blood cells and the white blood cells of a number of animal species.

It is also known that some oxidising agents may be able to inhibit the lytic power of the toxin and, if the oxidation is mild, the reaction is reversible.

This means that, under the action of an appropriate reducing agent, the toxin acquires its haemolytic power again.

The determination of antistreptolysin antibody has long been suggested as an appropriate method for determining the status and seriousness of a pathological condition associated with the presence of microorganisms which produce O-streptolysin. One procedure used for this purpose is that described by Todd in J. Exp.

Med., 55, 267-280 (1932). This author ascertained that antistreptolysin antibody was present in the serum of patients affected by infections due to Streptococci. The relative diagnostic test was carried out by neutralizing 0streptolysin by means of different quantities of the serum being tested, which serum had previously been deactivated. An excess of non-neutralized 0streptolysin was evidenced by haemolysis of red blood cells added to the system.

Rantz and Randall (Proc. Soc. Exp. Biol. Med., 59,2225 (1945)), have suggested the use, for determining the antistreptolysin antibody content of serum, concentrated solutions of 0-streptolysin which have been reduced totally prior to carrying out the diagnostic test.

We are the patentees of British Patent No.

1,531,530 which describes and claims a method of measuring the anti-0-streptolysin antibody content of blood using a plurality of solutions containing known and differing amounts of oxidised 0-streptolysin, which method comprises mixing a known quantity of blood, diluted with a non-haemolytic diluent, with each of said plurality of solutions and incubating the resulting bloodcontaining solutions, mixing each of the incubated solutions with a known quantity of a reducing agent to reduce the oxidised O-streptolysin and incubating the resulting solutions for a time sufficient for sedimentation of red blood cells to occur, observing those solutions in which no haemolysis has occurred, and determining therefrom the anti-0-streptolysin antibody content of the blood.

None of the above methods or any other method, has, however, suggested the carrying out, either simultaneously or sequentially according to the desire or the necessity of the person carrying out the method, a quantitative evaluation of the presence or the absence of antistreptolysin antibody and/or an accurate determination of the antistreptolysin antibody content.

The method as described in Patent No.

1,531,530, even though it has reliable advantages deriving from the use of O-streptolysin which has directly been oxidised during progress of the analysis and the use of full blood, is, however, open to criticism, inevitably, whenever it is desired to change from an accurate determination of the antistreptolysin antibody content directly to a purely qualitative estimate of the mere presence of the antibody or of the value of the antibody threshold of a given sample. As a matter of fact, the use of a plurality of solutions having different known concentrations of oxidised O-streptolysin has many advantages when it is desired to determine the O-streptolysin antibody content, but loses a part of its operational utility when a single determination of the antibody threshold is desired.

We have now found, in accordance with the present invention, a solution which permits one to exploit the advantages of the use of oxidised 0streptolysin both in the case of the determination of the exact antibody content and in the case in which it is desired to determine the antibody threshold only.

Thus the present invention provides a composition which is adapted to achieve both of the above purposes, and which comprises a known quantity of oxidised O-streptolysin, a reducing agent for restoring the haemolytic properties of O-streptolysin, a diluent for the sample to be tested, and a control blank. The components may be contained in different containers, or, still separate, they can make up a single diagnostic kit.

In both cases, the components may be in a ready-for-use form, or in the form of a concentrated solution, or in solid form. Also, the oxidised O-streptolysin can be present as a series of solutions of preselected concentrations.

Thus, by way of a nonlimiting example, a composition for the determination of the antibody may comprise three discrete portions, one of which contains oxidised O-streptolysin, the other the reducing agent and the third the diluent.

As an alternative, the three portions can be assembled, though separated, in a single kit. The oxidised O-streptolysin, in its turn, may be present in different portions, separated from each other, in quantities which can be equal to or different from each other.

Oxidised O-streptolysin can be present in the solid state, or diluted in an appropriate medium, as a powder, or in any other possible form whatsoever, such as in tablet form or freeze-dried form.

The present invention also provides a method for the analysis of antistreptolysin antibody, the method comprising contacting oxidised 0streptolysin with the sample to be tested, subsequent treatment with a reducing agent to restore the haemolytic power of the 0 streptolysin, and comparison with a control blank.

More particularly, the present invention provides an integrated method for the analysis of antistreptolysin antibody, the method comprising the steps of a preliminary determination of the antibody threshold value and subsequent accurate measurement of the content of antistreptolysin antibody. The second step can be dispensed with if the preliminarily obtained value is below the threshold iimit.

The reactions involved in the method of the invention are explained in Patent No. 1,531,530, more particularly with reference to Figures 1 to 5 thereof.

One of the advantages of the method of the invention is the possibility of using either full blood or serum, as such and not deactivated.

Detailing now the individual steps, the integrated method for the analysis of antistreptolysin antibody according to the present invention comprises the steps of preparing a solution of oxidised O-streptolysin of known concentration, preparing one or more solutions of an appropriate reducing agent, diluting the sample with a non-haemolytic medium, contacting the oxidised O-streptolysin with a preselected quantity of the sample, allowing the mixture thus obtained to stand at a temperature of from 15 to 750C, adding the reducing agent, waiting for sedimentation of the red blood cells, controlling the haemolysis or the non-haemolysis condition, and checking the status of the control blank.

The sequence of the above steps has, for its objective, the determination of the antistreptolysin antibody content, which will be determined as from the last testing step in which no red blood cell haemolysis has been detected. Such a result can be obtained by using solutions of oxidised 0streptolysin having a constant content thereof, and by adding thereto different quantities of the sample to be tested, or, as an alternative, using a set of solutions of oxidised O-streptolysin having known oxidised O-streptolysin contents which are gradually increased, and adding to ali of them the sample to be tested in the same amount.

In its turn, a single test run carried out by adding a solution of the sample being tested to a solution having a known concentration of oxidised O-streptolysin and subsequently introducing the reducing agent, permits one to ascertain, upon comparison with a control blank, the presence, of any, of antistreptolysin antibody in the sample and to calculate, on the basis of the concentration of O-streptolysin, the quantity corresponding to such a concentration. If the concentration is selected properly, this single test run clearly indicates the value of the antibody threshold.

As suggested hereinbefore, these alternative steps have been made possible by the very nature of the composition, which comprises a number of components, the latter being separate from each other in the form of a single diagnostic kit or, if preferred, being held each in a separate container.

Reducing agents for use in the invention include thiols (e.g. mercaptoethanol, dithiothreitol, cysteine, and N-acetyl cysteine), sodium metabisulphite and sodium sulphite.

As outlined above, it is possible to employ serum rather than full blood. If so, red blood cells must be added after the addition of the reducing agent, to check whether or not haemolysis takes place.

The invention will now be illustrated by the following Example.

EXAMPLE For the analysis of antistreptolysin antibody in a set of 80 blood samples (or serum samples, in which case it is necessary to add, at the appropriate time, red blood cells to the individual samples), and assuming that the antibody threshold value is selected a 200 l.U./ml (International Units per millilitre), a value below which the essay is considered to be negative while the presence of antistreptolysin antibody in an amount above such a value is regarded, conversely, as a positive essay, there were prepared 80 containers in each of which oxidised O-streptolysin was present in such an amount as to correspond to the value of the antibody threshold. These containers were the containers for the threshold test.

The 80 blood samples were diluted with an appropriate diluent and analysed (according to the procedure outlined above) using the containers for the threshold test. On completion of the analyses, after having carried out a comparison with the respective control blanks, the samples were divided into two sets, namely: (1) Samples in which haemolysis in the containers had occurred, i.e. samples in which the quantity of O-streptolysin antibody was lower than the quantity regarded as the antibody threshold.In the case of these samples, the analysis can be considered as having been completed, and the final result is expressed in terms of an antistreptolysin antibody content below 200 l.U./ml; (2) Samples in which haemolysis in the containers had not occurred, i.e. samples in which the quantity of O-streptolysin antibody present was equal to or higher than the quantity regarded as being the antibody threshold. In the case of these samples, the analysis cannot be regarded as having been completed since it is necessary to determine the exact antistreptolysin antibody content.

Assuming that the samples of the second group were 10 in number, there were prepared 10 sets of containers which contain gradually increasing and predetermined quantities of oxidised 0streptolysin (the number of the containers of each set and the quantities of oxidised O-streptolysin were selected consistently with the range of antistreptolysin antibody content one desires to assay).

These samples were then analysed (according to the procedure as described hereinbefore), using one of the sets of containers in each case.

The final result is defined by the quantity of 0 streptolysin which is present in the container in which no red blood cell haemolysis was detected and which immediately precedes the first of the containers in which red blood cell haemolysis was detected.

Claims (17)

1. A method of determining whether or not the antistreptolysin antibody content of a sample suspected of containing such antibody is below a given threshold value, using a solution containing a known amount of oxidised O-streptolysin, which method comprises mixing the sample with said solution and incubating the resulting solution, mixing the incubated solution with a quantity of a reducing agent to reduce the oxidised 0streptolysin and incubating the resulting solution for a time sufficient for sedimentation of red blood cells to occur (red blood cells being added to the solution in the event that such cells are not present in the solution), observing whether or not haemolysis has occurred, and determining therefrom whether or not the antistreptolysin antibody content of the sample is below said threshold value; the amount of oxidised 0streptolysin in the solution thereof being such that haemolysis does odcur when the antistreptolysin antibody content is below said threshold value but does not occur when the antistreptolysin antibody content is not below said threshold value.

2. A method of analysing a plurality of samples suspected of containing antistreptolysin antibody, which method comprises (A) determining whether or not the antistreptolysin antibody content of each sample is below a given threshold value, by a method according to claim 1; and (B) in respect of the or at least one sample whose antistreptolysin antibody content is not below the given threshold value, measuring the antistreptolysin antibody content thereof by a method according to claim 1, using a plurality of solutions containing known and differing amounts of oxidised O-streptolysin.

3. A method according to claim 1 or 2, wherein the or each sample is blood or blood serum.

4. A method according to claim 3, wherein the or each sample is diluted with a non-haemolytic diluent.

5. A method according to claim 1 or 2, substantially as described herein.

6. A composition suitable for the analysis of antistreptolysin antibody, comprising oxidised 0streptolysin in a known amount, a reducing agent for restoring the haemolytic power of 0streptolysin, a diluent for the sample to be tested, and a control blank.

7. A composition according to claim 6, wherein the oxidised O-streptolysin in known amount is present in a subdivided set of samples in preselected concentrations.

8. A composition according to claim 6 or 7, wherein oxidised O-streptolysin is present in the solid state, or in solution at known concentration, or in the freeze-dried state.

9. A composition according to any of claims 6 to 8, wherein the reducing agent is mercaptoethanol, dithiothreitol, cysteine, Nacetylcysteine, sodium metabisulphite or sodium sulphite.

10. A diagnostic kit for the analysis of antistreptolysin antibody, comprising, in separate containers, oxidised O-streptolysin in known amount, a reducing agent for restoring the haemolytic properties of O-streptolysin, and a diluent for the sample to be tested.

11. A diagnostic kit according to claim 10, wherein the oxidised O-streptolysin in known amount is present in a set of separate containers each containing a fraction having a preselected concentration.

12. A diagnostic kit according to claim 10 or 11, wherein the oxidised O-streptolysin is present in the solid state, in solutions of known concentrations, or in the freeze-dried state.

1 3. A diagnostic kit according to any of claims 10 to 12, wherein the reducing agent is mercaptoethanol, dithiothreitol, cysteine, Nacetylcysteine, sodium metabisulphite or sodium sulphite.

14. An integrated method for the analysis of antistreptolysin antibody, comprising the steps of preliminarily determining the threshold value of the antibody content and subsequently possibly accurately measuring the antistreptolysin antibody content.

1 5. A method according to claim 14, wherein the first step is carried out by contacting a known quantity of oxidised O-streptolysin with the sample concerned, subsequently treating what is obtained with a reducing agent, waiting for red blood cell sedimentation, checking possible haemolysis of the red cells, and checking the status of a control blank.

1 6. A method according to claim 14, comprising the steps of preparing one or more solutions of known concentrations of oxidised 0streptolysin, preparing a solution of an appropriate reducing agent, diluting the sample to be tested with a non-haemolytic medium, contacting the oxidised O-streptolysin with a preselected amount of the sample to be tested, allowing the mixture thus obtained to stand at a temperature of from 1 5 to 750C, adding a reducing agent, waiting for red blood cell sedimentation, checking haemolysis, and checking the condition of a control blank.

17. A method according to any of claims 14 to 16, wherein the sample to be tested is full blood or serum as such.

1 8. A method according to claim 17, wherein the sample to be tested is serum as such and the restoration of the haemolytic properties of 0streptolysin is preceded by an appropriate addition of red blood cells.