Peviva M65 EpiRat® ELISA

The Peviva M65 EpiRat™ ELISA is a one-step in vitro immunoassay for the quantitative determination of soluble keratin 18 in rat serum and plasma. The Peviva M65 EpiRat™ ELISA is based on the M5 and M6 antibodies, which bind to the biomarker Keratin-18 (K18). K18 is an intermediate filament protein found in epithelial cells. The concentration of K18 reflects the amount of overall cell death, both apoptosis and necrosis, in K18 positive cells. Levels of K18 are commonly elevated when the liver endures damage, through disease or toxicological effects.

Peviva M65 EpiRat™ ELISA is suitable for use in research, pre-clinical trials, and toxicological studies.

The Peviva M65 EpiRat™ ELISA is optimized as a rat specific assay and represents the final piece of the puzzle to complete our portfolio K18 assays, making the Peviva product line available and valuable in all stages of drug development.

Peviva M65 EpiRat™ ELISA’s unique characteristics

• Rat specific measurement of total cell death in K18 positive cells
• Quantification of K18 in rat serum and plasma samples.
• Convenient ready to use format and can be split up for use at several occasions.

Materials provided for 96 determinations

M6 Coated Microstrips	One microplate, 12 strips with 8 wells each, 96 dry wells in total. The wells are coated with mouse monoclonal K18 antibody M6. The microplate is sealed in an aluminum bag, which contains a desiccating device. If not all the strips are used, reseal the bag and keep the desiccating device inside. Ready for use!
M65 HRP Conjugate	Concentrate (24 × conc). One vial containing 0.4ml of mouse monoclonal M5 antibody (anti-rat-K18) conjugated with horseradish peroxidase (HRP) in phosphate buffer with protein stabilizers. Preservative added. Should be diluted with M65 Conjugate Dilution Buffer. Note! Do not expose to light!
M65 Conjugate Dilution Buffer	One vial containing 11ml of phosphate buffer with protein stabilizers for dilution of the M65 HRP Conjugate. Preservative added. Blue colored. Ready for use!
M65 Standard A– E	Standard A containing 2ml of phosphate buffer with foetal calf serum (FCS). Standard B – E, 0.5ml each, containing standard material in phosphate buffer with FCS. The values of Standard A–E are 0, 250, 500, 1000, 2000 U/L, respectively. Preservative added. Yellow colored. Ready for use!
Wash Tablet	One tablet for 500ml of prepared wash solution. Dissolve the Wash Tablet in 500ml of fresh deionized water.
TMB Substrate	One bottle containing 22ml of TMB (3,3’,5,5’-Tetramethyl- benzidine) Solution. Note! Do not expose to light! Ready for use!
Stop Solution	One vial containing 7ml of 1.0M sulphuric acid. Ready for use!
Sealing Tape	One (1) sheet.
Instructions for Use
Certificate of Analysis

Materials required but not provided

• Microplate reader (wavelength: 450nm; linear 0–3OD)
• Microplate shaker (oscillation: 600rpm, orbit: 1.5–4mm)
• 96-well microtiter plate washer or multichannel pipette (volume 250μl)
• Vortex mixer
• Precision pipettes: 25, 50, 75 and 200μl
• Cylinder (500ml)
• Deionized water

Research

Toxicology
The Peviva M65 EpiRat™ ELISA can be used in toxicological studies. Rodent species are commonly used in traditional toxicology testing guidelines to predict human health toxicity outcomes. Rodents have significant conventional knowledge, historical availability, and ease of care and breeding, making them ideal test subjects for the assessment of possible adverse health effects and sensitivities from chemical exposures. As rodent species are commonly used globally in traditional toxicology testing guidelines to predict human health toxicity outcomes, the Peviva M65 EpiRat™ will help by quantifying the amount of cell death in hepatocytes to assess liver toxicity. This in turn saves Pharmaceutical companies a lot of money where they can discontinue projects where the tested compound has too high toxicological effect on the liver.

Hepatology & Oncology
The Peviva M65 EpiRat™ ELISA detects total amount of K18, which quantifies the total amount of cell death in epithelial cells. This allows for the use of the assay in different research fields such as in hepatology and oncology. In pre-clinical studies, the Peviva M65 EpiRat™ ELISA can be used to analyze the effect of drugs on treatment effect and disease progression for example for new compounds in Non Alcoholic Fatty Liver (NAFL) and Non Alcoholic Steatohepatitis (NASH).

The kit can also be applicatory in veterinary research regarding different liver diseases.

Assess Preclinical Hepatotoxicity in Drug Development

• Increase liver specificity
• Determine DILI at earlier time points
• Provide mechanistic information
• Defend the safety of your new drug
• Reduce risk of unnecessary costs
• Prevent unnecessary withdrawal or clinical hold for promising new drug candidates
• Prevent delay of new medications to the market

Assay Principle and Procedure

Principle of the Method
The Peviva M65 EpiRat™ ELISA is a solid-phase sandwich enzyme immunoassay. Standards, controls and samples react with a solid phase capture antibody M6 directed against K18 and the HRP-(horseradish peroxidase) conjugated M5 antibody directed against a different epitope. Unbound conjugate is removed by a washing step. TMB Substrate is added. The color development is stopped and the absorbance is read. The resulting color is directly proportional to the concentration of the analyte.

By plotting a standard curve from known concentrations versus measured absorbance, the amount of antigen in the sample can be calculated. The concentration of the antigen is expressed as units per liter (U/L).

Assay Protocol
Warnings and Precautions

1. Peviva M65 EpiRat™ ELISA kit is intended for Research Use Only.
2. Do not mix reagents from different kit lots.
3. All sample specimens should be regarded as contagious and handled and disposed of according to appropriate regulations.
4. Do not use samples that are contaminated.
5. The Stop Solution contains 1.0M sulphuric acid, which will cause irritation of the skin and is harmful to the eyes. In case of contact, flush with plenty of water and seek medical advice

Component Preparation
Dilution of M65 Conjugate
Dilute the M65 HRP Conjugate with M65 Conjugate Dilution Buffer. The M65 HRP Conjugate vial contains exactly 0.4ml. Add 9.2ml of the M65 Conjugate Dilution Buffer directly to the M65 Conjugate vial and mix.

Dissolving of Wash Tablet
Dissolve one Wash Tablet in 500ml of fresh deionized water.

Storage and Shelf Life
Store reagents in their original containers at 2–8°C.

The TMB Substrate and the M65 Conjugate are sensitive to light and metal ions and should be stored in the original amber bottles at 2–8°C at all times. TMB Substrate cannot be used after exposure to light.

Assay Procedure
The Peviva M65 EpiRat™ ELISA should be performed at room temperature (24 ± 3 °C).

1. Allow all reagents to reach room temperature before performing the assay. Vortex all reagents prior to use.
2. Dissolve the Wash Tablet in fresh deionized water (see “Component Preparation”).
3. Dilute the M65 HRP Conjugate with M65 Conjugate Dilution Buffer (see “Component Preparation”) and mix.
4. Pipette 25μl of M65 Standard (A–E) or sample per well (duplicates are recommended).
5. Add 75μl of the diluted M65 Conjugate solution to each well.

Note! Steps 4 and 5 should be performed sequentially without interruption within 20 minutes.

6. Cover the wells with sealing tape or a microtiter plate lid.
7. Incubate on shaker for four (4) hours. Speed setting: 600rpm.
8. Wash the plate in a plate washer five (5) times with 300–400μl/well of Wash Tablet solution (overflow wash) or Wash the plate manually, discarding the incubation solution and washing the wells five (5) times with 250μl of Wash Tablet solution. Avoid contamination between wells.
9. Add 200μl of TMB Substrate to each well. Incubate in darkness at room temperature for 20 ± 1 minute.
10. Add 50μl of Stop Solution to each well. To ensure complete mixing of the TMB Substrate and the Stop Solution, shake the microplate for 5–10 seconds. Leave the microplate for 5 minutes before reading the absorbance.
11. Determine the absorbance at 450nm in a microplate reader within 30 minutes and record the results.
12. Calculate the results as described in section “Calculation of Analytical Results”.

Flow Chart

Dilute M65 HRP Conjugate and dissolve Wash Tablet.
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Pipette 25μl of Standard, Control or sample per well.
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Add 75μl of diluted M65 HRP Conjugate per well.
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Incubate for 4 hours on plate shaker (600rpm).
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Wash in plate washer with 5 × 300–400μl (overflow), or manually with 5 × 250μl.
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Add 200μl of TMB Substrate per well. Incubate in darkness 20 ± 1min.
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Add 50μl of Stop Solution per well. Shake 5–10sec. Read absorbance after 5–30min.

Calculation of Analytical Results

The Peviva M65 EpiRat™ ELISA results are calculated using computer-assisted methods. Evaluate the values of controls and samples using a suitable program for handling ELISA-type data. Fitting algorithm: Cubic Spline. x-axis: concentration (U/L); y-axis: absorbance at 450nm (A450).

Note! If samples have been diluted, the observed concentration must be multiplied by the dilution factor.

Shipping and Storage

The Peviva M65 EpiRat™ ELISA is shipped in cooled conditions and should be stored at 2-8°C. Note! Do not freeze!