PLATELIA™ M. pneuMoniae IgG

96 72780SEmI-quAnTITATIvE dETEcTIon of AnTI-MycoplasMa pneuMoniae IgG In humAn SErum by EnzymE ImmunoASSAy

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1- CLINICAL INTERESTMycoplasma pneumoniae is one of the major etiological agents of atypical pneumonias in children and young adults, with a predominance at school age. The biological diagnosis is most often based on serology. The presence of IgM class antibodies, which appear and disappear before IgG class antibodies, allows the diagnosis of acute infection directly on the first serum sample. The appearance of, or a significant increase in the titer of the IgG between two samples collected at an interval of about 2 weeks allows the diagnosis of a recent or developing infection.

2- PRINCIPLE OF THE TESTThe principle of PlateliaTM M. pneumoniae IgG, a test kit for semi-quantitative detection of anti-Mycoplasma pneumoniae IgG in human serum, is based on a solid-phase enzyme immunoassay referred to as an indirect EIA. Test sera and calibrators are diluted and placed into the M. pneumoniae antigen coated wells of the microplate. During the first incubation period, M. pneumoniae IgG antibodies present in the sample bind to the M. pneumoniae antigens. After incubation, non-specific immunoglobulins and serum proteins are removed by washings. Then, the conjugate, a peroxidase-labeled monoclonal antibody specific for human gamma chains, is added to the microplate wells and incubated. During the second incubation period, the labeled monoclonal antibody binds to the M. pneumoniae IgG antibody- M. pneumoniae antigen complexes. After incubation, unbound-labeled antibody is removed by washings. The addition of a solution containing a peroxidase substrate and a chromogen initiates a colour development reaction. The enzymatic reaction is stopped by the addition of an acid. The optical density reading, obtained with a spectrophotometer set at 450 / 620 nm, is proportional to the amount of M. pneumoniae IgG antibodies present in the test sample and is converted into AU/ml (Arbitrary Unit) using a standard curve.

3- PRODUCT INFORMATIONFor storage conditions and expiration date refer to the indications mentioned on the box.

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Labeling Reagents Presentation

R1 Microplate Microplate : 12 strips with 8 wells each, sensibilized with M. pneumoniae Ag

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R2 Concentrated Washing

Solution (20x)

Concentrated Washing Solution (20x): TRIS-NaCl buffer (pH 7.4), 2% Tween® 20Preservative : < 1.5% ProClin™ 300

1 x 70 ml

R3 Calibrator 0 Calibrator 0 : TRIS-NaCl buffer (pH 8 ± 0.1), bovine serum albumin, glycerol, E102 and E122.Preservative: < 1.5% ProClin™ 300

1 x 1 ml

R4a Calibrator 10 Calibrator 10 AU/ml: TRIS-NaCl buffer (pH 8 ± 0.1), human serum reactive against M. pneumoniae Ag, bovine serum albumin, glycerol, E102 and E122.Preservative: < 1.5% ProClin™ 300

1 x 1 ml

R4b Calibrator 50 Calibrator 50 AU/ml: TRIS-NaCl buffer (pH 8 ± 0.1), human serum reactive against M. pneumoniae Ag, bovine serum albumin, glycerol, E102 and E122.Preservative: < 1.5% ProClin™ 300

1 x 1 ml

R4c Calibrator 100 Calibrator 100 AU/ml: TRIS-NaCl buffer (pH 8 ± 0.1), human serum reactive against M. pneumoniae Ag, bovine serum albumin, glycerol, E102 and E122.Preservative: < 1.5% ProClin™ 300

1 x 1 ml

R6a Conjugate Conjugate (50X) : Murine monoclonal antibody to human gamma-chains coupled with horseradish Peroxidase.Preservative: < 1.5% ProClin™ 300

1 x 0.3 ml

R6b Conjugate Diluent

Conjugate Diluent : Preservative: < 1.5% ProClin™ 300

1 x 12 ml

R7 Sample Diluent

Sample diluent : TRIS-NaCl buffer (pH 7.7 ± 0.1), bovine serum albumin, 0.1% Tween® 20 and phenol red.Preservative: < 1.5% ProClin™ 300

2 x 120 ml

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4- WARNINGS AND PRECAUTIONS The reliability of the results depends on correct implementation of the following Good Laboratory Practices: • Donotuseexpiredreagents.• Donotmixorassociatewithinagivenrunreagentsfromdifferentlots.

NOTE: For Washing Solution (R2, label identification: 20x colored green), Chromogen (R9, label identification: TMB colored turquoise) and Stopping Solution (R10, label identification: 1N colored red), it is possible to use other lots than those contained in the kit, provided these reagents are strictly equivalent and the same lot is used within a given test run.

NOTE: The washing solution (R2 identified* in green as 20x) may not be mixed with the Washing Solution (R2 identified* in blue as 10x) provided in Bio-Rad reagent kits.* on the vial label• Before use, wait for 30 minutes to allow reagents to reach room

temperature (+18-30°C).• Carefullyreconstituteordilutethereagentsavoidinganycontamination.• Do not carry out the test in the presence of reactive vapors (acid,

alkaline, aldehyde vapors) or dust that could alter the enzyme activity of the conjugate.

• Useglassware thoroughlywashedandrinsedwithdeionizedwateror,preferably disposable material.

• Washing the microplate is a critical step in the procedure: follow therecommended number of washings cycles and make sure that all wells are completely filled and then completely emptied. Incorrect washings may lead to inaccurate results.

• Do not allow the microplate to dry between the end of the washingsoperation and the reagent distribution.

• Never use the same container to distribute the conjugate and thedevelopment solution.

Labeling Reagents Presentation

R9 Chromogen TMB

Chromogen (Ready-to-use): 3.3’.5.5’ tetramethylbenzidine (< 0.1%), H2O2 (<1%)

1 x 28 ml

R10 Stopping Solution

Stopping Solution (Ready to use) : 1N sulfuric acid solution

1 x 28 ml

Plate Sealers 4

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• The enzymatic reaction is very sensitive to metal or metal ions.Consequently, do not allow any metal element to come into contact with the various solutions containing the conjugate or the chromogen.

• Chromogensolution(R9)shouldbecolorless.Theappearanceofabluecolor indicates that the reagent cannot be used and must be replaced.

• Useanewpipettetipforeachsample.• Check the pipettes and other equipments for accuracy and correct

operations.

HeALTH AnD SAfeTy inSTRUCTionS• Weardisposablegloveswhenhandlingreagents.• Donotpipettebymouth.• Humanoriginmaterialusedinthepreparationofthereagentshasbeen

testedandfoundnon-reactiveforhepatitisBsurfaceantigen(HBsAg),antibodies to hepatitis C virus (anti-HCV) and to human immunodeficiency virus (anti-HIV1 and anti-HIV2). Because no method can absolutelyguarantee the absence of infectious agents, handle reagents of human origin and patient samples as if capable of transmitting infectious disease.

• Anymaterial,includingwashingssolution,thatcomesdirectlyincontactwith samples and reagents containing materials of human origin, should be considered as if capable of transmitting infectious disease.

• Avoid spilling, but if in the event of a spill with an acid, it must beneutralized with sodium bicarbonate, then cleaned with bleach at the concentration of 12° diluted to 1/10 and dried absorbent paper. The material used for cleaning must be discarded in a contaminated residue container.

• Patient samples, reagents containing human origin material, as wellas contaminated material and products should be discarded after decontamination only.

• Do not introduce solutions containing sodium hypochlorite into theautoclave.

CAUTION : Some of the reagents contain ProClin™ 300 < 1.5%

For risks and security recommendations refer to the table at the end of the package insert.

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5- SPECIMEN1. Serum collected on dry tubes is the recommended sample type. 2. Observe the following recommendations for the handling, processing,

and storing serum samples:- Collect all serum samples observing routine precautions.- Allow samples to clot completely before centrifugation. - Keep tubes stoppered at all times.- After centrifugation separate the serum and store it in a tightly stoppered

storage tube.- The specimens can be stored at +2-8°C if screening is performed

within 24 hours. If the assay will not be completed within 24 hours, or for shipment of samples, freeze at –20°C, or colder.

- Preferably, thaw samples one time only. Previously frozen specimens should be thoroughly mixed after thawing prior to testing.

3. Interference’s due to high levels of albumin or bilirubin are unknown. Do not use lipemic or hemolyzed samples.

4. Do not heat the samples.

6- ASSAY PROCEDURE6 .1 - MATeRiALS ReQUiReD noT PRoViDeD- Vortex mixer.- Microplate reader equipped with 450 nm and 620 nm filters*.- Manual, semi-automatic or automatic microplate washer*.- Container for biohazard waste.- Sodium hypochlorite (bleach) and sodium bicarbonate.- Germ-free distilled or deionized water.- Graduated cylinders of 25, 50, 100 and 1000ml capacity. - Disposable latex gloves.- Goggles or safety glasses.- Absorbent paper.- Automatic or semi-automatic, adjustable or preset, pipettes or multipipettes

to measure and dispense 10 to 1000µl and 1, 2 and 10 ml.- Disposable tubes.(*) Consult our technical department for detailed information about the recommended equipment

6.2 - ReAGenTS ReConSTiTUTionR2 : Dilute 1/20 the washing solution R2 in distilled water: for example 50 ml of R2 and 950 ml of distilled water to get the ready-to-use washing solution. Prepare 350 ml of diluted washing solution for one plate of 12 strips if washing manually

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R6a: the conjugate R6a is concentrated 50x. For one strip, dilute 20 µl of R6a in 1 ml of R6b. Multiply these volumes by 10 for one microplate.

6.3 - SToRAGe of oPeneD AnD/oR ReConSTiTUTeD ReAGenTSR1 : once the vacuum-sealed bag has been opened, the strips remain stable for up to eight weeks provided they are stored at +2-8°C in the same carefully closed bag.R2 : Once diluted, the Washing Solution can be kept for 2 weeks at +2-30°C. Once opened, the concentrated Washing Solution stored at +2-30°C, in absence of contamination, is stable until the expiration date indicated on the label.R6 : once diluted, the conjugate should be used withtin 6 hours of preparation.R9 : Once opened and without any contamination, the reagent stored at +2-8°C is stable for up to 8 weeks.R3, R4a, R4b, R4c, R6a, R6b, R7 and R10 : once opened, reagents stored at +2-8°C in absence of contamination, are stable until the expiry date indicated on the label.

6.4 - PRoCeDUReStrictly follow the assay procedure. Use the calibrators with each run to validate the assay results.Beforeuse,allowreagentstoreachroomtemperature(+18-30°C)1) Document the sample distribution and identification plan2) Prepare the dilution of the wash solution (R2). (Refer to section 6.2).3) Take the carrier tray and the strips (R1) out of the protective pouch.4)Wash microplate strips once with diluted R2. Invert microplate and

gently tap on absorbent paper to remove remaining liquid.5) Dilute calibrators R3, R4a, R4b, R4c and test sample in R7 to give a

1/201 dilution: 10 µl sample + 2 ml of R7. Vortex diluted samples.6) For manual performance of the assay, add 100 µl of diluted calibrators

and diluted test samples to the wells as suggested below:

1 2 3 4 5 6 7 8 9 10 11 12A R3 S5 B R4a S6 C R4b S7 D R4c S8 E S1 S9 F S2 S10 G S3 S11 H S4 S12

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7) Cover the microplate with an adhesive plate sealer; pressing firmly onto the plate to ensure a tight seal. Incubate the microplate for 1 hour at room temperature (+18-30°C).

8) At the end of first incubation period, remove the adhesive plate sealer. Aspirate the contents of all wells into a container for biohazard waste (containing sodium hypochlorite). Wash microplate 3 times. Invertmicroplate and gently tap on absorbent paper to remove remaining liquid.

9)PrepareWorkingConjugate(R6)(refer to section 6.2). Add 100 µl of the conjugate solution (R6) to all wells. The solution must be shaken gently before use.

10) Cover the microplate with adhesive plate sealer, pressing firmly onto the plate to ensure a tight seal. Incubate the microplate incubator for 1 hour at room temperature (+18-30°C).

11) At the end of the second incubation period, remove the adhesive plate sealer. Aspirate the content of all wells into a container for biohazard waste(containingsodiumhypochloride).Washmicroplate4timeswith350µloftheWashingSolution(R2).Invertthemicroplateandgentlytapon adsorbent paper to remove remaining liquid.

12) Quickly distribute into each well and away from light 200 µl of Chromogen solution (R9). Allow the reaction to develop in the dark for 30 ± 5 minutes at room temperature (+18-30°C). Do no use adhesive plate sealer during this incubation.

13) Stop the enzymatic reaction by adding 100 µl of Stopping Solution (R10) in each well. Use the same sequence and rate of distribution as for the development solution.

14) Carefully wipe the plate bottom. Read the optical density at 450/620 nm using a plate reader within 30 minutes after stopping the reaction. The strips must always be kept away from light before reading.

15)Beforereportingresults,checkforagreementbetweenthereadingandthe distribution plan of plate and samples.

7- INTERPRETATION OF RESULTS

7.1 – QUALiTy ConTRoLInclude all the calibrators for each test run. For the assay to be considered valid, the following criteria must be met. • Opticaldensityvalues:

- OD R4c > 1.000 • Ratio:

- OD R4a / OD R3 > 2.0If the quality control criteria are not met, the test run should be repeated.

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7.2 – eSTABLiSHinG THe STAnDARD CURVeThe optical density reading is proportional to the amount of M. pneumoniae IgG antibodies present in the test sample and is converted into AU/ml using a standard curve.

a) Drawing the standard curveFor manual data reduction, use linear paper and plot the OD readings of R3, R4a, R4b, and R4c on the vertical (y) axis. Plot the corresponding concentration in AU/ml on the horizontal (x) axis. Draw the point-to-point curve through the four points. For automated data reduction, use the point-to-point function to construct a curve from the OD readings obtained for the calibrators.

b) Calculation of the concentration in AU/mlIf the OD reading of the test sample is in the interval of OD R4a and OD R4c, find the value corresponding to the OD reading of the test sample on the y-axis and draw a horizontal line to the standard curve. At the point of intersection with the standard curve, draw a vertical line to the x-axis. Read the concentration in AU/ml at the point of intersection with the x-axis.Note : If the OD reading of a test sample is > OD R4c, this test sample should be diluted 1/2010 in R7 and re-run in the assay. The calculated AU/ml values must then be multiplied by a factor of 10.

7.3 - inTeRPReTATion of THe ReSULTS

Titer interpretation and recommendations

> 40AU/ml High titerHigh antibody rate, compatible with a current or recent M. pneumoniae infection

≤ 40 AU/ml > 20 AU/ml

Moderate titerInterpretation of the test can only be given after a further serum sample been collected 8 to 15 days later, the two samples being tested in a same serie.

≤ 20 AU/ml ≥ 10 AU/ml

Low titer

< 10 AU/ml Non significant titer

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Interpretation after a second sample taken from the same patient:

7.4 – TRoUBLe SHooTinG GUiDeNon validated or non repeatable reactions are often caused by:• Inadequatemicroplatewashings,• Contamination of negative samples by serum or plasma with a high

antibody titer,• Contaminationofthedevelopmentsolutionbyoxidizingagents(bleach,

metal ions...),• Contaminationofthestoppingsolution.

8- LIMITATIONS OF THE ASSAYDiagnosis of a recent infection can only be established on the basis of a combination of clinical observations and serological data. The result of a single serum sample does not constitute sufficient proof for diagnosis of recent infection.

X = Titer of the 1st serum

y = Titer of the 2nd serum

interpretation and recommendations

X ≤ 40 AU/ml X ≥ 10 AU/ml

Y > 2X ou Y > 40 AU/ml

High antibody rate, compatible with a current or recent M. pneumoniae infection

X < Y < 2X

Non significant increase in antibody titer, which does not allow us to interpret that there exists an active M. pneumoniae infection. Collecting a 3rd sample one week later is recommended to exclude an infection at its early stage

Y = X Y < X

Low to moderate antibody rate, compatible with past M. pneumoniae infection.

Y < 10 AU/ml No argument in favor of M.pneumoniae infection

X < 10 AU/ml10 AU/ml < Y < 20AU/ml

Non significant increase in antibody titer, which does not allow us to interpret that there exists an active M. pneumoniae infection. Collecting a 3rd sample one week later is recommended to exclude an infection at its early stage

Y > 20 AU/mlSeroconversion compatible with an evolutive M. pneumoniae infection

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9- PREVALENCEThe prevalence of positive / negative results with PlateliaTM M. pneumoniae IgG OPD (72776) was determined from 396 samples obtained from blood donors:

10- PERFORMANCES

10.1- SenSiTiViTy AnD SPeCifiCiTyThe results of the correlation studies obtained with PlateliaTM

M. pneumoniae IgG (72780) confirm that the modifications made on this kit did not affect the performances announced below obtained with the test PlateliaTM M. pneumoniae IgG OPD (72776).

• Specificity57 samples were tested in Complement Fixation Reaction (CFR). One discrepant result was found: this sample highly positive with PlateliaTM M. pneumoniae IgG, but lowly positive in CFR (16) was confirmed highly positive in immunofluorescence (>64). Specificity established on 47 newborn samples was 100% (47/47).

• Sensitivity

1. Comparative studies

The sensitivity of PlateliaTM M. pneumoniae IgG determined on 3 evaluation sites. 50 out of 59 serum specimens were found positive, the sensitivity being then of 84.7%, with 3 specimens with a titer below 10 AU /ml, the other specimens with low to moderate titers.

60

50

40

30

20

10

0

FR

EQ

UE

NC

Y

Prevalence study on Ab igG anti M. pneumoniae

on a population of blood donnors (n=396)

< 10 11 - 20 21 - 40 > 40

53.79%

TITER AU/ml

26.52%

13.89%

5.81%

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2. Seroconversions

114 serum specimens from 48 patients were tested:• In 1 case, the second sample was found non-positive with PlateliaTM

M. pneumoniae IgG.• In8cases,PlateliaTM M. pneumoniae IgG allowed the detection of the

seroconversion earlier than with CFR (previous samples).

10.2 - PReCiSion

Intra-assay repeatability:

Intra-assay variations demonstrate CV ≤ 10% on non significative titers and CV ≤ 8% on low to high titers.

Inter-assay reproducibility:

Inter-assay variations demonstrate CV ≤ 30% on non significative titers and CV ≤ 20% on cut-off titers and on low to high titers.

10.3 - CRoSS ReACTiViTy189 specimens with characteristics which could potentially result in non-specific reactions, such as specimens from patients with respiratory infections and specimens positive for rheumatoid factors and autoantibodies were tested with PlateliaTM M. pneumoniae IgG OPD (72776). The 6 samples, which were found positive with PlateliaTM M. pneumoniae IgG, were also confirmed positive by complement fixation reaction (CFR) or by other techniques (immunofluorescence; agglutination test).

Samples negative Positive 1 Positive 2 Positive 3

Titer

Mean 1,69 16.26 35.15 51.83

SD 0.16 1.31 1.39 1.95

CV % 9.4 8.0 3.9 3.8

Samples negative Positive 1 Positive 2 Positive 3

Titer

Mean 2.71 9.30 20.92 52.44

SD 0.74 1.68 3.97 6.55

CV % 27.16 18.05 18.96 12.48

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11- QUALITY CONTROL OF THE MANUFACTURERAll manufactured reagents are prepared according to our Quality System, starting from reception of raw material to the final commercialization of the product. Each lot is submitted to quality control assessments and is only released to the market, after conforming to pre-defined acceptance criteria. The records relating to production and control of each single lot arekeptwithinBio-Rad.

12- REFERENCES1.ABRAMOVITZ,P.,SCHVARTZMAN,P.,HAREL,D.,LIS, I.,andNAOT,

Y. Direct invasion of the central nervous System by Mycoplasma pneumoniae: a report of two cases. The Journal of Infectious diseases, Vol. 1545, N° 3 March 1987.

2.BUSOLO.F.,MELONI,G.A.SerodiagnosisofM. pneumoniae infections by enzyme-linked immuno-sorbent assay (ELISA). The Yale Journal of BiologyandMedicine56(1983),517-521.

3.CLYDE, W.A., Jr Mycoplasma pneumoniae infections of man. The Mycoplasmas, Vol II, 1979 by Academic Press, Inc ; 275-302.

4. KENNY, G.E., GRAYSON, J.T. Eaton pleuropneumoniae-like organism (Mycoplasma pneumoniae) complement-fixing antigen : extraction with organic solvents. The Journal of lmmunology, Vol. 95, N°1 (1965).

5. RAISANEN, S.M., SUNI, J.I., LEINIKKI, P.O. Serological diagnosis of Mycoplasma pneumoniae infection by enzyme immunoassay. J. Clin. Pathol. 1980 ; 33 : 836-840.

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