Diagnostic device > Other diagnostic sets
DIAGNOSTIC SETS 2 x 25 tests Code: SO 301
ITEST ASO
The kit for titration of antistreptolysin O in serum.
Significance and application:
ITEST ASO kit is designed for the titration of antistreptolysin O (ASO) in sera. ASO is an antibody against streptolysin O (SLO) which is an extracellular toxin of group A streptococci (1). An experience of group A Streptococcus infection is manifested by the development or increase of the ASO titre. These infections are serious especially due to their consequences - acute rheumatic fever and acute glomerulonephritis. Therefore the determination of ASO titre as an evidence of previous Streptococcus infection is an inevitable complement of the diagnosis of the named diseases. ASO titration can be fairly well standardised and the antibodies can be quantitatively determined (2).
With regard to the enormous frequency of Streptococcus infection the ASO titration can be also used as a "screening method" for evaluation of unsufficient immune system response.
The kit contains all reagents needed for the test execution except rabbit erythrocytes. These can be ordered separately (ITEST plus s.r.o., catalogue No. SO 306).
Principle of the test:
The principle of the test is SLO neutralization with antibody(ASO) present in the serum. Residual SLO is proved with hemolysis of the added erythrocytes. For passing the hemolysis, SLO must be in reduced form.
Kit contents:
1. Streptolysin O (catalogue No. 302).
Purified and freeze-dried filtrate from a strain of group A Streptococcus producing SLO. The bottle contains the number of the International Unit equivalents shown on the bottle label.
2. Reducing agent (catalogue No. 303).
Reducing agent – Sodium dithionite. 20 mg in the bottle.
3. Concentrated buffered saline with bovine albumin (catalogue No. 304).
The bottle contains 8 ml of 15x concentrated saline with phosphate buffer and bovine albumin, pH 6.5. The solution is preserved with 0.1% sodium azide.
4. Antistreptolysin O standard (catalogue No. 305).
Purified and freeze-dried human IgG tested for presence of HIV, HBsAg and HCV with negative results. The bottle contains the number of International Unit equivalents shown on the bottle label.
Procedure:
1. Preparation of buffered saline with bovine albumin (BSA).
Dilute the bottle content concentrated BSA with 112 ml of distilled water. You will obtain 120 ml of the working solution, pH approximately 6.5. Store the solution at +4oC and use it within seven days.
2. Preparation of rabbit erythrocyte suspension.
Wash rabbit erythrocytes three times in BSA and centrifuge them at 1000 g for 10 minutes. Prepare 1% suspension in BSA from the sedimented erythrocytes after last centrifugation.
3. Preparation of reduced streptolysin.
Put 1 ml of BSA into each bottle with SLO and Reducing agent.
After the complete reconstitution transfer quantitatively the contents of both bottles into ml of BSA. The SLO solution prepared this way contains 1 International Unit equivalent in 0.5 ml. Use the solution the same day.
4. Serum dilution.
Prepare two basic solutions of the examined, inactivated (56oC for 30 minutes) serum in the test tubes:
a) 1:25 (50 µl serum + 1.2 ml BSA)
b) 1:30 (0.5 ml of serum diluted 1:25 + 0.1 ml BSA)
5. Preparation of the microtitration plates (with U-shaped wells) and the test execution.
Place the 96-wells plate in such a way that the H1 well would be at the top left. Titrate each serum in two rows of the plate, i.e. in 16 wells (six sera on one plate). Put into first two wells 50 µl BSA in to each for each serum examined. Put 25 µl BSA into the remaining 14 wells.
Transfer 50 µl of serum diluted 1:25 into the first well and after a proper stirring transfer 75 µl by micropipette into the third well. In the same way continue in the serum dilution in all odd wells till the fifteenth well. Remove 75 µl of diluted serum from the 15th well.
Transfer 50 µl of serum diluted 1:30 into the second well and after the proper stirring transfer 75 µl by micropipette into the fourth well. Continue in the same way in the serum dilution in all even wells till the sixteenth well. Remove 75 µl from the 16th well.
Add by the dropping pipette 25 µl BSA into each of the 16th wells and stirr it properly (on the shaking machine or by tapping on the wall of the plate) - in each well is the final amount of the diluted serum 50 µl.
Add 25 µl of reduced SLO solution into each well. Incubate, after proper stirring, in the moisture chamber at 37oC for 15 minutes. After the incubation add 25 µl of 1% suspension of rabbit erythrocytes (stirr properly before each suspension application). Mix the well content properly and incubate under the same condition for 45 minutes.
6. Reading of the results.
Substract the last well without hemolysis: reciprocal value of pertinent diluted serum gives ASO titre in International Unit equivalents (I.U). We introduce the reciprocal values of serum dilution (ASO titre) in the separate well for the survey:
Well No. 1 2 3 4 5 6 7 8
I.U. 100 120 133 160 178 213 237 284
Well No. 9 10 11 12 13 14 15 16
I.U. 316 379 421 505 562 673 749 897
7. Control.
7.1. Serum with known titre.
For the ASO titration in sera, the titration of serum with known titre, which is ASO standard delivered in the kit, is necessary. Reconstitute freeze-dried ASO standard in 1 ml of BSA (basic dilution 1:25). Further continue in the way as given in the point 4.b - 5. Titre of ASO standard diluted in this way, is 421 I.U. (i.e. the last well without hemolysis is No. 11).
Carry out ASO standard titration in 14 wells only, well No. 15 and 16 use for the erythrocyte suspension control and SLO control.
7.2. Control of erythrocyte suspension.
Put 75 µl BSA into the fifteenth well (in the row, where the ASO standard is titred) + 25 µl of rabbit erythrocytes. When substracting the results, 100% hemolysis must not occur.
7.3. SLO control.
Put 50 µl BSA + 25 µl SLO + 25 µl suspension of rabbit erythrocytes into sixteenth well (in the row, where the ASO standard is titred). When substracting the results, 100% hemolysis must occur.
Evaluation:
"Normal" ASO values in serum are highly variable and depend on the age of the patient, geographgical area, epidemiological situation, season, etc. The titre of 200 I.U. is usually considered as the upper limit of "normal values" of ASO titre (3). Finding of a significant increase of ASO titre in the patient`s serum is more reliable for the evidence of previous Streptococcus infection. It is the difference between titration of two samples of sera bigger than one well. Increase of ASO titre after Streptococcus infection reaches usually maximum between the 3th and 5th week after its development. This is the reason why we recommend titration of two samples of serum at least, taken in a particular time interval. ASO titre increase occurs after Streptococcus infection approximately in 80% of cases. Titration of some other Streptococcus antibodies, e.g. antideoxyribonuclease B is necessary for finding higher number of Streptococcus infections. Nonspecifically increased ASO titre can occur also in the presence of free cholesterol in serum or in contaminated serum.
References:
1. Hostetler C.L., Sawyer K.P., Nachamkin I. (1988): Comparison of three rapid methods for detection antibodies to streptolysin O and DNase B. J. Clin. Microbiol. 26: 1406.
2. Wannamaker L.W., Ayoub E.M. (1960): Antibody titres in acute rheumatic fever. Circulation 21: 598.
3. Rotta J., Facklam R.D. (1980): Determination of streptolysin O. In: Manual of microbiological diagnostic methods for Streptococcus infection and their sequelae. WHO, p. 39.
3. Bícová R. (1997): Stanovení protilátky proti streptolysinu O mikrometodou. Epidemiol.Microbiol.Imunol., 46, č. 4, 140-144.
4. Johnson D.R., Kaplan E.L. et al. (1996): Laboratory diagnosis of group A streptococcal infections. WHO, Geneva.
Caution:
1. The kit is determined for in vitro diagnostic use only!
2. Store the kit at +4oC and use it till the expiration date given on the wrapping.
