Dicas Técnicas

Os testes IDEXX são fabricados em lotes com padrões de qualidade rigorosos. A aprovação do lote pelas autoridades reguladoras, como o USDA, baseia-se nos resultados dos testes internos de controle de qualidade para cada um. O certificado de análise (C de A) para um teste documenta o desempenho do teste no momento do teste de liberação. Os dados de desempenho incluem os valores de densidade óptica (OD) dos controles de teste e os resultados calculados e / ou interpretação qualitativa de um grupo selecionado de amostras utilizadas durante a fabricação. Todos esses valores devem estar dentro de um intervalo especificado para o lote de teste a ser aprovado para venda.

Os valores de OD dos controles de teste não precisam permanecer dentro dos intervalos de C de A durante o tempo de vida do teste. Os valores da densidade óptica podem mudar ao longo do tempo, sem comprometer a qualidade do teste.

Os testes diagnósticos são compostos de componentes biológicos que nem sempre são estáveis ​​quando removidos do ambiente natural. Apesar dos estabilizadores utilizados para controlar a vida útil, a maioria dos componentes biológicos perdem alguma atividade com o tempo. Quando isso ocorre, os valores de OD podem diminuir. A diminuição nos valores de OD dos controles de teste é paralela à diminuição dos valores de DO nas amostras testadas, de modo que os resultados calculados permanecem os mesmos. Os valores de DO inferiores aos do C de A são aceitáveis ​​desde que as especificações de validade na inserção de teste sejam atendidas para o ensaio.

Os critérios de validade na inserção diferem dos valores observados no C de A. Os critérios de validade na inserção geralmente especificam um limite em um valor de OD de controle ou uma proporção de valores de OD de controle. Se uma placa de teste funcionar dentro das especificações de validade da inserção, o teste está funcionando corretamente e os resultados do teste podem ser usados.

A vida útil de um teste de diagnóstico IDEXX pode variar de 9 meses a 2 anos, dependendo do produto. O teste é projetado para dar resultados válidos ao longo de sua vida útil.

Com qualquer ELISA, a reprodutibilidade e a confiabilidade dependem da técnica adequada, do equipamento, dos fatores ambientais e da atenção aos detalhes. O C de A serve como guia para o desempenho do teste antecipado.

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O ELISA é dividido em três formatos principais: indireto, bloqueador (competitivo) e captura de antígenos (direto).

Formato Indireto
No formato indireto, o anticorpo da amostra é intercalado entre o antígeno revestido na placa e um conjugado de globulina antiespécie marcado com enzima. A adição de um reagente de enzima-reagente cromogênio faz com que a cor se desenvolva. Esta cor é diretamente proporcional à quantidade de anticorpo de amostra ligada. Quanto mais anticorpo presente na amostra, maior o desenvolvimento de cor no teste bem. Este formato é adequado para determinar o nível total de anticorpos em amostras (Johne, etc.).

Formato Bloqueador (Competitivo)
Neste formato, os anticorpos específicos da amostra competem ou bloqueiam o anticorpo específico marcado com enzima no conjugado. A adição de um reagente de enzima-reagente cromogênio faz com que a cor se desenvolva. Esta cor é inversamente proporcional à quantidade de anticorpo de amostra ligada. Quanto mais anticorpos presentes na amostra, menor desenvolvimento de cor no poço de teste (CAV, etc.).

Formato de Captura de Antigenos (Direto)
O antígeno na amostra é intercalado entre anticorpos revestidos na placa e um conjugado marcado com enzima. O conjugado de anticorpo pode ser monoclonal ou policlonal. A adição de um reagente de enzima-reagente cromogênio faz com que a cor se desenvolva. Esta cor é diretamente proporcional à quantidade do antígeno alvo presente nas amostras (LLAg, etc.).

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Sample quality and handling

• The assay can be run on whole or skim milk samples and samples that have been exposed to heat treatment (during milk component analysis).
• Bronopol or a similar preservative may be used to maintain sample quality—this will not affect test results.
• Samples should be mixed gently prior to pipetting.
• Poor quality samples may compromise the accuracy of test results. Prior to testing, milk samples should be checked to ensure that they are not soured or separated and are free from contamination.
• Care should be taken to minimize the likelihood of milk carryover from cow to cow during sample collection, particularly when using samples collected for routine herd recording.

Incubation

• Use adhesive covers as specified during sample, detector and conjugate incubation steps. Adhesive plate covers validated for use with this test can be purchased from IDEXX. Ask your local sales or customer service representative for details.
• Use of a plate shaker incubator is needed. Please reference the test insert for rotation and temperature specifications or ask your local sales representative for more details.

Washing

• The assay requires 4–5 washes at each wash step during the assay protocol.
• Washing with good quality water and a clean and maintained washer is crucial.
• IDEXX can provide specific wash settings to be used for the IDEXX Milk Pregnancy Test. Please ask your LPD Technical Services representative for details.

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• The IDEXX Porcine Reproductive and Respiratory Syndrome (PRRS) Oral Fluids Ab Test is easy to run; however, please keep the following in mind regarding sample collection and handling.
• Samples of oral fluids can be collected from individual pigs or from groups of pigs in pens using ropes that are hung at pig shoulder height.
• Ropes should remain in the pens for 20–30 minutes to ensure adequate exposure.
• Insert the wet end of the rope into a clean plastic boot or bag.
• Strip the rope so the fluid accumulates in the corner of the bag or flows into the tube, if the tube is connected to the bag.
• Cut a corner of the plastic bag and drain fluids into the collection tube. If using a bag with a connected tube, then disconnect the tube.
• A minimum sample of 2.5 mL is recommended. Make sure that collection tubes do not contain additives such as EDTA or heparin. If using glass blood collection tubes, use only red-top tubes (without additives).
• Discard all ropes after collection. Never reuse ropes, plastic bags or collection tubes. Never leave ropes in the pens after sampling is finished. Do not pool oral fluids samples from ropes in different pens.
• Be sure all tubes are tightly sealed and packaged with ice before shipping. Overnight delivery is preferred, as it ensures timely arrival of chilled samples for best results.
• If the oral fluid sample contains particulate from the mouths of the animals, the sample can be centrifuged at 2500g for 5–10 minutes before use.

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IDEXX ELISA test kits are designed to work as a set of reagents. However, generic components, such as substrate, wash, and stop solutions can be mixed within kits having the same part number. Other reagents such as kit controls, conjugates and plates have been optimized to work together for that kit lot/batch. Should you have any questions on generic components or what can be used across kits, please contact your LPD Technical Services representative.

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It is good laboratory practice to periodically test the calibration of your microplate reader. IDEXX checks the calibration of its readers using calibration test plates, which can be purchased from most reader manufacturers or from laboratory equipment providers.

Important: The calibration plate must be calibrated for the wavelength you normally use for testing (e.g., 630 nm, 650 nm, 450 nm). The calibration can be performed by the manufacturer of the calibration plate.

Use the following steps to run a calibration test plate and to print the results:

1. Start the xChekPlus® software and log on.
2. Turn on the plate reader, and place the calibration plate in the holder.
3. From the File menu, select Template. Create a template for an assay you normally use, with a case name of your choosing and a count of 96. Save the template.
4. From the File menu, select Read.
5. Choose the assay and template you just created.
6. After the data is displayed, you may save it (using the save icon), and/or print it (using the print icon).
7. Using the printout, compare the optical density (OD) values for specific wells to the OD values on the calibration plate test card for the wavelength used.

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Mixing samples before plating is a very important step when running an ELISA test. Samples that are not mixed well will produce variable results.

Frozen samples can be thawed at room temperature or in a refrigerator. All thawed samples need to be thoroughly mixed prior to dilution to ensure that the proteins are dispersed throughout the sample. Mix by gentle vortexing or inverting at least five times. Frothing or overmixing of samples will cause denaturation of serum proteins.

Prepared dilutions, especially the 1:500 in poultry kits, also require mixing with gentle vortexing or a pipette set at a volume of 100 µl or greater prior to plating. Mixing the diluted sample with a micropipette, such as the one utilized for transferring the 1 µl of sample, is not appropriate as the volume exchanged is not enough to thoroughly mix the sample.

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Careful pipetting is crucial in obtaining accurate test results when performing any ELISA test. Sometimes air, resulting in bubbles, can be drawn into the pipette or dispensed into the wells. If this happens, bubbles can influence optical density values and results. To minimize or eliminate this problem, reverse pipetting is recommended for the addition of reagents to the ELISA plate.

Reverse pipetting with a multichannel pipette:

1. Put new tips on the pipette, ensuring they are on tight and straight.
2. Press the plunger past the first stop and halfway to the second stop.
3. Draw the liquid in a slow motion, being careful that no air bubbles are drawn into the tip. Check for consistency of volume in the tips.
4. Touch the tips to the edge of the reagent reservoir to remove excess liquid on the outside of the tips.
5. If the wells on your plate are empty, position the tips into the lower corner of the wells.
6. If the wells on your plate contain liquid, position the tips above the liquid.
7. Slowly dispense the liquid into the wells by depressing the plunger to the first stop. Be careful not to splash liquid out of the wells, and make sure there are no drops left on the tips.
8. To repeat, hold the plunger at the first stop and continue with step 3.
9. Eject the tips into an appropriate waste container.

Note: Reverse pipetting uses more reagent/volume (="dead volume").

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The plate washer plays a key role in achieving accurate, reproducible test results. Make sure your plate washer is adjusted properly. Change settings as needed so the washer meets these requirements:

• Buffer is dispensed gently, at approximately 200 μL/sec, with the tops of the aspiration pins level with the tops of the wells. Avoid bottom wash or similar modes. In bottom wash, the buffer is dispensed closer to the bottom of the wells, which increases the pressure on the well. There should be just enough pressure to clean the well.
• During aspiration, liquid is pulled from the surface of the wells, not from below or above. Synchronize the aspiration rate and the movement of the pins so that the pins always remain in contact with the liquid's surface as the level goes down. Use a high aspiration rate; the actual rate will vary depending on the washer. The aspiration pins should never be lower than 1 mm above the bottom of the wells.

Contact your local LPD Technical Services representative for any washer programming information available or any questions you may have.

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The Total Counts Report provides the number of negative, positive, suspect, and total number of samples for each case selected. It also gives you a grand total for each category for all the cases selected. When creating the report, you can select the assay as well as the date range. The Total Counts Report allows a variety of uses. You may want to use it to determine a percentage of positive samples when testing presumed negative flocks or herds. You can also use the report to determine monthly or quarterly sample tallies for testing in your lab.

To run a total counts report from xChekPlus:

1. Choose Reports > Total Counts from the menu bar.
2. In the Total Counts dialog box, choose the assay and the data range (or leave the date range open to select all cases).

Note: You can also select the spreadsheet option if you want to save the data as a Microsoft® Excel spreadsheet.

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IDEXX ELISA screening assays are used to screen populations representing herds or flocks. Some negative populations may present with a higher number than expected rate of positives. Unexpected positives generated using any screening assay should be confirmed using an alternate confirmatory method appropriate for the test, such as Hemagglutination Inhibition (HI), quantitative polymerase chain reaction (qPCR), virus isolation (VI), etc.

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Many IDEXX ELISA test kits require the use of a diluted wash buffer between incubation steps. To maintain a high level of ELISA test kit performance, keep these guidelines in mind when using wash solutions:

• Use high-quality distilled or deionized water to mix with the specified volume of a wash buffer concentrate.
• Consult the test kit’s insert to check the amount of wash buffer required to run each plate. The wash volume needed may vary depending upon the washer. Automated washers tend to use more wash buffer than manual washers because they need to be primed and then flushed after use.

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When running ELISAs, a good measure of test performance is the reproducibility of the positive and negative control. xChekPlus software offers a report that displays not only data on the control replicates, but also control tracking over time.

It is important to review control data often to ensure that values are not trending or exhibiting excess variability. Control values for a specific lot should be similar on a run-to-run basis.

To run a control tracking report from xChekPlus:

1. Click on Reports > Control Tracking Report.
2. Highlight the test, specify a date, choose graphing options and check by well (% diff) > OK.
3. A control tracking report will be displayed that can be saved and/or printed and then evaluated.

Note: We recommend that the difference between control replicates be no greater than 20%.

A key component of the control tracking report is that it can display the lot number of the kit being used and represented in the control tracking report. This is useful information when evaluating quality control data. If you currently do not track the kit lot, it can be entered into the software in two ways: either through database > assays, or in plate view at the time that you are reading your plate.

If you are seeing poor control reproducibility, it may be an indication that your pipettes are not functioning properly, your wash system is malfunctioning, or you have poor distribution of antibodies in your samples.

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