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Automation of IDEXX Milk Pregnancy ELISA

Crocodile ELISA miniWorkstation - Application Note 2016/01

Here we describe the successful automation of IDEXX Milk Pregnancy ELISA with the Crocodile ELISA miniWorkstation. The IDEXX test is an enzyme-linked immunoassay for the detection of Pregnancy-Associated Glycoproteins (PAGs) in bovine milk as a marker.

Introduction

Accurate and timely detection of pregnancy in dairy cows is an essential component of today's reproductive management programs, as a high reproductive efficiency is a prerequisite for high life-time production from dairy animals. Laboratory methods for pregnancy determination using milk as starting sample have numerous advantages over traditional methods, such as rectal palpation or transrectal ultrasound, as they are safer, equally or less expensive, and do not require trained personnel or special equipment on-farm. Pregnancy-associated glycoproteins (PAGs) constitute a large family of glycoproteins expressed in the outer epithelial cell layer (chorion/trophectoderm) of the placenta of cows and other eutherian species. PAGs can be detected in milk of pregnant cows, thus providing a convenient, specific and sensitive method for pregnancy diagnosis.

The IDEXX Milk Pregnancy Test, for use in bovine milk samples, is an enzyme-linked immunoassay for the detection of PAGs in bovine milk as a marker for pregnancy from ≥ 35 days post-breeding. The test can be used ≥ 60 days post calving.

Crocodile ELISA miniWorkstation

Materials

  • Crocodile ELISA MiniWorkstation (Berthold Technologies GmbH & Co. KG)
  • Milk Pregnancy Test (Part number 99-41209, IDEXX Laboratories Inc.)
  • Adhesive plate covers
  • Precision micropipettes or multi-dispensing micropipettes, with suitable disposable tips
  • Distilled or deionized water

 

 

Methods

All reagents were brought up to room temperature for 30 minutes prior to use. Wash Solution was prepared following the manufacturer's instructions.

Controls and samples were pipetted according to the manufacturer’s instructions. 3 samples known to be positive, and 3 samples known to be negative, were used. All controls and samples were run in triplicate.

The plate should be tightly sealed with adhesive cover during incubations to avoid evaporation. Manual steps were included in the program of the Crocodile to allow the user to put or remove the sealing, as needed. In order to reduce the amount of user intervention and improve automation, the assay was tested covering the plate only in the first incubation (2 h at 37°C) and leaving the plate uncovered in the other incubation steps (20-30 minutes at room temperature); no significant evaporation was observed, and the assay performed as expected.

The Crocodile ELISA miniWorkstation was programmed with the steps summarized in Table 1 (see below).

 

 

Results

For the assay to be valid, the Positive Control mean minus the Negative Control mean must be greater than or equal to 0.500, and the Negative Control mean must be less than or equal to 0.200. Both criteria were fulfilled running the assay in the Crocodile ELISA miniWorkstation.

Pregnant or open (not pregnant) status is determined by the corrected OD values (S-N: OD of the Sample minus OD of the Negative Control) for each sample: if the S-N value is less than 0.100, the animal is considered not pregnant (open); if it is equal to or greater than 0.250, the animal is considered pregnant; and if is less than 0.250 but greater than or equal to 0.100, the animal should be re-checked to confirm pregnancy status. All negative samples were determined as negative, and all positive samples were determined as positive. There were no false positives or false negatives.

Results are summarized in Figure 1.

Milk Pregnancy ELISA

Figure 1. Left: Table displaying the results for controls and samples; OD values correspond to OD (450 nm) - OD (620 nm). Data shown are averages of triplicate measurements. Right: screenshot of data reduction software (MikroWin).

Summary

The assay fulfilled both validation conditions and all positive and negative samples were correctly determined. The assay procedure is simple and involves only the addition of controls and samples, while the instrument is processing all necessary dispense, wash, incubation and reading steps. The addition of manual steps in the Crocodile Control Software allows the user to remove the cover when required, thus providing an easy and convenient integration of manual and automated steps. In consequence, the Crocodile ELISA miniWorkstation, in combination with the MikroWin data reduction software, provides a convenient and easy-to-use method to automate the IDEXX Milk Pregnancy Test.

 

 

Acknowledgements

Samples kindly provided by Franziska Breitenwieser (Milchprüfring Baden-Württemberg).

Special thanks to IDEXX Laboratories Inc. for their support.

Table 1

Summary of steps programmed in the Crocodile Control Software

# Step name Description and parameters
1 Incubator ON Incubation
Incubator On, Temperature: 37°C
2 Incubator heat up Manual
“Insert plate when the incubator reaches 37°C and press Continue”, Duration: 00:10:00, Mode: User Continue, Position: Insert Position
3 Sample Incubation Shaking
For 02:00:00, at Incubator, with 1 mm Amplitude at 5 Hz
4 Incubator OFF Incubation
Incubator Off
5 Remove adhesive cover Manual
“Please remove cover”, Duration: 00:02:00, Mode: user continue, Position: Insert position, Alarm Notification: sound of choice, 0 %
6 Wash Solution priming Washing
Method: Prime Washer, Wash Solution Inlet: 1, Cycles: 3, Volume: 800 µL
7 Wash Washing
Method: Standard, Wash Solution Inlet: 1, Cycles: 4, Volume: 300 µL, Delay: 1 s, Wait: 200 ms, Dispenser Depth: 1300 (Plate Offset: -50), Aspiration Depth: 2910* (Plate Offset: 20), Sweep: 4 mm @ 2 mm/s
8 Aspiration Washing
Method: Aspirate Only, Cycles: 1, Delay: 1s, Wait: 500 ms, Dispenser Depth: 1300 (Plate Offset: -50), Aspiration Depth: 2920* (Plate Offset: 20), Sweep: 4 mm @ 2 mm/s
9 Detector priming Dispensing
Volume: 850 µL, Inlet: 1, Method: Priming
10 Detector distribution Dispensing
Volume: 100 µL, Inlet: 1, Method: Standard
11 Mix Shaking
For 00:00:10 at Shaker Position with 1 mm Amplitude at 5 Hz
12 Detector incubation Manual
Message: “Incubating at Room Temperature”, Duration: 00:30:00, Mode: Auto Continue, Position: Insert Position
13 Wash Washing
Method: Standard, Wash Solution Inlet: 1, Cycles: 4, Volume: 300 µL, Delay: 1 s, Wait: 200 ms, Dispenser Depth: 1300 (Plate Offset: -50), Aspiration Depth: 2910* (Plate Offset: 20), Sweep: 4 mm @ 2 mm/s
14 Aspiration Washing
Method: Aspirate Only, Cycles: 1, Delay: 1s, Wait: 500 ms, Dispenser Depth: 1300 (Plate Offset: -50), Aspiration Depth: 2920* (Plate Offset: 20), Sweep: 4 mm @ 2 mm/s
15 Conjugate priming Dispensing
Volume: 850 µL, Inlet: 2, Method: Priming
16 Conjugate distribution Dispensing
Volume: 100 µL, Inlet: 2, Method: Standard
17 Mix Shaking
For 00:00:10 at Shaker Position with 1 mm Amplitude at 5 Hz
18 Conjugate incubation Manual
Message: “Incubating at Room Temperature”, Duration: 00:30:00, Mode: Auto Continue, Position: Insert Position
19 Wash Washing
Method: Standard, Wash Solution Inlet: 1, Cycles: 4, Volume: 300 µL, Delay: 1 s, Wait: 200 ms, Dispenser Depth: 1300 (Plate Offset: -50), Aspiration Depth: 2910* (Plate Offset: 20), Sweep: 4 mm @ 2 mm/s
20 Aspiration Washing
Method: Aspirate Only, Cycles: 1, Delay: 1s, Wait: 500 ms, Dispenser Depth: 1300, Aspiration Depth: 2920*, Sweep: 4 mm @ 2 mm/s
21 Substrate priming Dispensing
Volume: 850 µL, Inlet: 3, Method: Priming
22 Substrate distribution Dispensing
Volume: 100 µL, Inlet: 3, Method: Standard
23 Mix Shaking
For 00:00:10 at Shaker Position with 1 mm Amplitude at 5 Hz
24 Substrate incubation Manual
Message: “Incubating at Room Temperature”, Duration: 00:20:00, Mode: Auto Continue, Position: Insert Position
25 Stop solution priming Dispensing
Volume: 850 µL, Inlet: 4, Method: Priming
26 Stop solution distribution Dispensing
Volume: 100 µL, Inlet: 4, Method: Standard
27 Mix well Shaking
For 00:00:10 at Shaker Position with 1 mm Amplitude at 5 Hz
28 Measure Reading
Reference Measurement, Filter 1: 450 nm, Filter 2: 620 nm

 

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