VetLine Babesia ELISA 产品说明书

VetLine Babesia

ELISA

For Research Use Only

RUO

English .......................................................................................................................................................................... 2

Bibliography / Literatur / Bibliographie / Bibliografia / Bibliografía/ Blibiografia ............................................................ 6

Abbreviations / Abkürzungen / Abréviations / Abbreviazioni / Abreviaciónes / Abreviaturas ....................................... 6

Symbols Key / Symbolschlüssel / Explication des Symboles / Legenda / Símbolos / Tabela de símbolos ................. 7

Summary of Test Procedure / Kurzanleitung Testdurchführung / Résumé de la procedure de test / Schema della

procedura / Resumen de la técnica / Resumo do Procedimento de Teste .................................................................. 8

Product Number:

BABVT0890 (96 Determinations)

ENGLISH

1. INTRODUCTION

2. INTENDED USE

3. PRINCIPLE OF THE ASSAY

The qualitative immunoenzymatic determination of specific antibodies is based on the ELISA (Enzyme-linked Immunosorbent

Assay) technique.

Microtiterplates are coated with specific antigens to bind corresponding antibodies of the sample. After washing the wells to

remove all unbound sample material a horseradish peroxidase (HRP) labelled conjugate is added. This conjugate binds to the

captured antibodies. In a second washing step unbound conjugate is removed. The immune complex formed by the bound

conjugate is visualized by adding Tetramethylbenzidine (TMB) substrate which gives a blue reaction product.

The intensity of this product is proportional to the amount of specific antibodies in the sample. Sulphuric acid is added to stop

the reaction. This produces a yellow endpoint colour. Absorbance at 450/620 nm is read using an ELISA Microtiterplate reader.

4. MATERIALS

4.1.

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Reagents supplied

Microtiterplate: 12 breakapart 8-well snap-off strips coated with Babesia antigens; in resealable aluminium foil.

Sample Dilution Buffer: 1 bottle containing 100 mL of phosphate buffer (10 mM) for sample dilution; pH 7.2 ± 0.2;

coloured yellow; ready to use; white cap; ≤ 0.0015 % (v/v) CMIT/MIT (3:1).

Stop Solution: 1 bottle containing 15 mL sulphuric acid, 0.2 mol/L; ready to use; red cap.

Washing Buffer (20x conc.): 1 bottle containing 50 mL of a 20-fold concentrated phosphate buffer (0.2 M),

pH .2 ± 0.2, for washing the wells; white cap.

Conjugate: 1 bottle containing 20 mL of peroxidase labelled Protein A/G; coloured yellow, ready to use; white cap;

≤ 0.02 % (v/v) MIT.

TMB Substrate Solution: 1 bottle containing 15 mL 3,3',5,5'-tetramethylbenzidine (TMB), < 0.1 %; ready to use;

yellow cap.

Positive Control: 1 vial containing 2 mL control; coloured yellow; ready to use; red cap; ≤ 0.02 % (v/v) MIT.

Cut-off Control: 1 vial containing 3 mL control; coloured yellow; ready to use; green cap; ≤ 0.02 % (v/v) MIT.

Negative Control: 1 vial containing 2 mL control; coloured yellow; ready to use; blue cap; ≤ 0.0015 % (v/v) CMIT/MIT

(3:1).

For hazard and precautionary statements see 12.1

For potential hazardous substances please check the safety data sheet.

4.2.

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Materials supplied

1 Cover foil

1 Instruction for use (IFU)

1 Plate layout

4.3.

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Materials and Equipment needed

ELISA Microtiterplate reader, equipped for the measurement of absorbance at 450/620 nm

Incubator 37 °C

Manual or automatic equipment for rinsing Microtiterplate wells

Pipettes to deliver volumes between 10 and 1000 µL

Vortex tube mixer

Distilled water

Disposable tubes

5. STABILITY AND STORAGE

Store the kit 8 °C. The opened reagents are stable up to the expiry date stated on the label when stored 8 °C.

6. REAGENT PREPARATION

It is very important to bring all reagents and samples to room temperature (20…25 °C) and mix them before

starting the test run!

6.1. Microtiterplate

The break-apart snap-off strips are coated with Babesia antigens. Immediately after removal of the strips, the remaining strips

should be resealed in the aluminium foil along with the desiccant supplied and stored 8 °C.

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6.2. Washing Buffer (20x conc.)

Dilute Washing Buffer 1 + 19; e. g. 10 mL Washing Buffer + 190 mL distilled water. The diluted buffer is stable for 5 days at

room temperature (20…25 °C). In case crystals appear in the concentrate, warm up the solution to 37 ° in a water bath.

Mix well before dilution.

6.3. TMB Substrate Solution

The reagent is ready to use and has to be stored 8 °C, away from the light. The solution should be colourless or could

have a slight blue tinge. If the substrate turns into blue, it may have become contaminated and should be thrown away.

7. SAMPLE COLLECTION AND PREPARATION

Use veterinary serum samples with this assay. If the assay is performed within 5 days after sample collection, the samples

should be kept 8 °C; otherwise they should be aliquoted and stored deep-frozen (-70…-20 °C). If samples are stored

frozen, mix thawed samples well before testing. Avoid repeated freezing and thawing.

Heat inactivation of samples is not recommended.

7.1. Sample Dilution

Before assaying, all samples should be diluted 1+100 with Sample Dilution Buffer. Dispense 10 µL sample and 1 mL Sample

Dilution Buffer into tubes to obtain a 1+100 dilution and thoroughly mix with a Vortex.

8. ASSAY PROCEDURE

Please read the instruction for use carefully before performing the assay. Result reliability depends on strict adherence to the

instruction for use as described. The following test procedure is only validated for manual procedure. If performing the test on

ELISA automatic systems we recommend increasing the washing steps from three up to five and the volume of Washing Buffer

from 300 µL to 350 µL to avoid washing effects. Pay attention to chapter 12. Prior to commencing the assay, the distribution and

identification plan for all samples and standards/controls (duplicates recommended) should be carefully established on the plate

layout supplied in the kit. Select the required number of microtiter strips or wells and insert them into the holder.

Perform all assay steps in the order given and without any delays.

A clean, disposable tip should be used for dispensing each standard/control and sample.

Adjust the incubator to 37 ± 1 °C.

1. Dispense 100 µL standards/controls and diluted samples into their respective wells. Leave well A1 for the Substrate

Blank.

2. Cover wells with the foil supplied in the kit.

3. Incubate for 1 hour ± 5 min at 37 ± 1 °C.

4. When incubation has been completed, remove the foil, aspirate the content of the wells and wash each well three times

with 300 µL of Washing Buffer. Avoid overflows from the reaction wells. The interval between washing and aspiration

should be > 5 sec. At the end carefully remove remaining fluid by tapping strips on tissue paper prior to the next step!

Note: Washing is important! Insufficient washing results in poor precision and false results.

5. Dispense 100 µL Conjugate into all wells except for the Substrate Blank well A1.

6. Incubate for 30 min at room temperature (20...25 °C). Do not expose to direct sunlight.

7. Repeat step 4.

8. Dispense 100 µL TMB Substrate Solution into all wells.

9. Incubate for exactly 15 min at room temperature (20...25 °C) in the dark. A blue colour occurs due to an enzymatic

reaction.

10. Dispense 100 µL Stop Solution into all wells in the same order and at the same rate as for the TMB Substrate Solution,

thereby a colour change from blue to yellow occurs.

11. Measure the absorbance at 450/620 nm within 30 min after addition of the Stop Solution.

8.1. Measurement

Adjust the ELISA Microtiterplate reader to zero using the Substrate Blank.

If - due to technical reasons - the ELISA Microtiterplate reader cannot be adjusted to zero using the Substrate Blank, subtract its

absorbance value from all other absorbance values measured in order to obtain reliable results!

Measure the absorbance of all wells at 450 nm and record the absorbance values for each standard/control and sample in the

plate layout.

Bichromatic measurement using a reference wavelength of 620 nm is recommended.

Where applicable calculate the mean absorbance values of all duplicates.

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9. RESULTS

9.1. Run Validation Criteria

In order for an assay to be considered valid, the following criteria must be met:

▪ Substrate Blank: Absorbance value < 0.100

▪ Negative Control: Absorbance value < 0.200 and < Cut-off

▪ Cut-off Control: Absorbance value 0.150 – 1.300

▪ Positive Control: Absorbance value > Cut-off

If these criteria are not met, the test is not valid and must be repeated.

9.2. Calculation of Results

The Cut-off is the mean absorbance value of the Cut-off Control determinations.

Example: Absorbance value Cut-off Control 0.44 + absorbance value Cut-off control 0.42 = 0.86 / 2 = 0.43

Cut-off = 0.43

9.2.1. Results in Units [NTU]

Sample (mean) absorbance value x 10 = [NovaTec Units = NTU]

Cut-off

Example: 1.591 x 10 = 37 NTU

0.43

9.3. Interpretation of Results

10 NTU

> 11 NTU

9 – 11 NTU

< 9 NTU

Cut-off

Positive

Equivocal

Negative

10. SPECIFIC PERFORMANCE CHARACTERISTICS

For further information about the specific performance characteristics please contact NovaTec Immundiagnostica GmbH.

10.1. Precision

10.2. Diagnostic Specificity

10.3. Diagnostic Sensitivity

10.4. Interferences

Interferences with hemolytic, lipemic or icteric samples are not observed up to a concentration of 10 mg/mL hemoglobin,

5 mg/mL triglycerides and 0.5 mg/mL bilirubin.

10.5. Cross Reactivity

11. LIMITATIONS OF THE PROCEDURE

Bacterial contamination or repeated freeze-thaw cycles of the sample may affect the absorbance values.

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12. PRECAUTIONS AND WARNINGS

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For research use only!

All materials of human or animal origin should be regarded and handled as potentially infectious.

All components of human origin used for the production of these reagents have been tested for anti-HIV antibodies, anti-

HCV antibodies and HBsAg and have been found to be non-reactive.

Do not interchange reagents or strips of different production lots.

No reagents of other manufacturers should be used along with reagents of this test kit.

Do not use reagents after expiry date stated on the label.

Use only clean pipette tips, dispensers, and lab ware.

Do not interchange screw caps of reagent vials to avoid cross-contamination.

Close reagent vials tightly immediately after use to avoid evaporation and microbial contamination.

After first opening and subsequent storage check conjugate and standard/control vials for microbial contamination prior to

further use.

To avoid cross-contamination and falsely elevated results pipette samples and dispense reagents without splashing

accurately into the wells.

The ELISA is only designed for qualified personnel who are familiar with good laboratory practice.

12.1. Safety note for reagents containing hazardous substances

Reagents may contain CMIT/MIT (3:1) or MIT (refer to 4.1)

Therefore, the following hazard and precautionary statements apply.

Warning H317 May cause an allergic skin reaction.

P261 Avoid breathing spray

P280 Wear protective gloves/protective clothing.

P302+P352 IF ON SKIN: Wash with plenty of soap and water.

P333+P313 If skin irritation or rash occurs: Get medical advice/attention.

P362+P364 Take off contaminated and Wash it before reuse.

Further information can be found in the safety data sheet.

12.2. Disposal Considerations

Residues of chemicals and preparations are generally considered as hazardous waste. The disposal of this kind of waste is

regulated through national and regional laws and regulations. Contact your local authorities or waste management companies

which will give advice on how to dispose hazardous waste.

13. ORDERING INFORMATION

Prod. No.: BABVT0890 VetLine Babesia ELISA (96 Determinations)

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BIBLIOGRAPHY / LITERATUR / BIBLIOGRAPHIE / BIBLIOGRAFIA / BIBLIOGRAFÍA/

BLIBIOGRAFIA

ABBREVIATIONS / ABKÜRZUNGEN / ABRÉVIATIONS / ABBREVIAZIONI / ABREVIACIÓNES /

ABREVIATURAS

CMIT

MIT

5-chloro-2-methyl-4-isothiazolin-3-one

2-methyl-2H-isothiazol-3-one

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SYMBOLS KEY / SYMBOLSCHLÜSSEL / EXPLICATION DES SYMBOLES / LEGENDA /

SIMBOLOS / TABELA DE SIMBOLOS

Manufactured by / Hergestellt von / Fabriqué par / Prodotto da / Fabricado por /

Fabricado por

Lot Number / Chargenbezeichnung / Numéro de lot / Lotto / Número de lote / Número

de lote

Expiration Date / Verfallsdatum / Date de péremption / Scadenza / Fecha de

caducidad / Data de Validade

Storage Temperature / Lagertemperatur / Température de conservation / Temperatura

di conservazione / Temperatura de almacenamiento / Temperatura de

Armazenamento

For research use only/ Nur für Forschungszwecke / Destiné à la recherche

uniquement/

Solo per scopi di ricerca/ Uso exclusivo en investigación / Apenas para

fins de pesquisa

Catalogue Number / Katalog Nummer / Référence du catalogue / Numero di codice /

Número de Catálogo / Número de Catálogo

Consult Instructions for Use / Arbeitsanleitung beachten / Consulter la notice

d’utilisation / Consultare le istruzioni per l’uso / Consulte las Instrucciones de Uso /

Consultar as Instruções de Utilização

Microtiterplate / Mikrotiterplatte / Plaque de Microtitrage / Piastre di Microtitolazione /

Placa de Microtitulación / Placa de Microtitulação

Conjugate / Konjugat / Conjugué / Coniugato / Conjugado / Conjugado

Negative Control / Negativkontrolle / Contrôle Négatif / Controllo Negativo / Control

Negativo / Controle Negativo

Positive Control / Positivkontrolle / Contrôle Positif / Controllo Positivo /

Control Positivo / Controle Positivo

Cut-off Control / Cut-off Kontrolle / Contrôle Cut-off / Controllo Cut-off /

Control Cut-off / Controle Cut-off

Sample Dilution Buffer / Probenverdünnungspuffer / Tampon de Dilution d’Échantillon /

Tampone di Diluizione del Campione / Tampón de Dilución de Muestras / Tampão de

Diluição de Amostra

Stop Solution / Stopplösung / Solution d’Arrêt / Soluzione Bloccante / Solución de

Parada /Solução de Bloqueio

TMB Substrate Solution / TMB-Substratlösung / Solution de Substrat TMB / Soluzione

Substrato TMB / Solución de Sustrato de TMB / Solução Substrato TMB

Washing Buffer 20x concentrated / Waschpuffer 20x konzentriert / Tampon de Lavage

concentré 20 x / Tampone di Lavaggio concentrazione x20 / Tampón de Lavado

concentrado x20 / Tampão de Lavagem concentrada 20x

Contains sufficient for “n” tests / Ausreichend für “n” Tests / Contenu suffisant pour “n”

tests / Contenuto sufficiente per “n” saggi / Contenido suficiente para ”n” tests /

Conteúdo suficiente para “n” testes

LOT

RUO

REF

MTP

CONJL

CONTROL│-

CONTROL│+

CUT OFF

DIL

SOLN│STOP

SUB│TMB

WASH│BUF│20x

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SUMMARY OF TEST PROCEDURE / KURZANLEITUNG TESTDURCHFÜHRUNG / RÉSUMÉ DE LA

PROCEDURE DE TEST / SCHEMA DELLA PROCEDURA / RESUMEN DE LA TÉCNICA / RESUMO

DO PROCEDIMENTO DE TESTE

SCHEME OF THE ASSAY

VetLine Babesia ELISA

Test Preparation

Prepare reagents and samples as described.

Establish the distribution and identification plan for all samples and standards/controls on the plate

layout supplied in the kit.

Select the required number of microtiter strips or wells and insert them into the holder.

Assay Procedure

Negative Control

Cut-off Control

Positive Control

(diluted 1+100)

Substrate Blank

(A1)

Negative

Control

100 µL

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Cut-off

Control

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100 µL

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Positive

Control

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100 µL

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Sample

(diluted 1+100)

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100 µL

Sample

Cover wells with foil supplied in the kit

Incubate for 1 h at 37±1 °C

Wash each well three times with 300 µl of Washing Buffer

Conjugate - 100 µL 100 µL 100 µL 100 µL

Incubate for 30 min at room temperature (20...25 °C)

Do not expose to direct sunlight

Wash each well three times with 300 µL of Washing Buffer

TMB Substrate

Solution

100 µL 100 µL 100 µL 100 µL 100 µL

Incubate for exactly 15 min at room temperature (20...25 °C) in the dark

Stop Solution 100 µL 100 µL 100 µL 100 µL 100 µL

Photometric measurement at 450 nm

(reference wavelength: 620 nm)

NovaTec Immundiagnostica GmbH

Waldstraße 23 A6

63128 Dietzenbach, Germany

Tel.:

Email:

Internet:

+49 (0) 6074-48760

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Fax: +49 (0) 6074-487629

BABVT0890-engl-29042020-TL-RUO

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