Ab136942 – LVV Hemorphin 7 ELISA Kit

ab136942 – LVV Hemorphin 7 ELISA Kit

Instructions for Use

For the quantitative measurement of LVV Hemorphin 7 concentrations in serum and tissue homogenate samples.

This product is for research use only and is not intended for diagnostic use. 1 Table of Contents

1. Introduction 3

2. Principle of the Assay 4

3. Assay Summary 5

4. Kit Contents 6

5. Storage and Handling 7

6. Additional Materials Required 7

7. Protocol 8

8. Calculation of Results 15

9. Performance Characteristics 17

10. Troubleshooting 22

2 1. Introduction ab136942 is a complete kit for the quantitative determination of LVV Hemorphin 7 in serum and tissue homogenates, with results in three hours. Please read the entire kit insert before performing this assay. This kit is not intended for use with plasma samples.

Hemorphins are opioid peptides derived by proteolysis from hemoglobin. Their sequences are identical in several mammalian species including human, sheep and bovine.

LVV‐hemorphin 7 (LVVYPWTQRF) binds strongly to the Angiotensin IV (AT4) receptors in the brain. The AT4 receptor is an integral membrane aminopeptidase also known as IRAP (insulin‐ regulated membrane aminopeptidase). LVV‐hemorphin 7 and AT4 are not substrates but rather inhibitors of the AT4 (IRAP) receptor. Both promote learning and memory and reverse amnesia in animal models. Elevated serum levels of LVV Hemorphin 7 have also been documented in patients with some forms of breast cancers that are associated with an increased expression of Cathespins B and D.

3 2. Principle of the Assay

1. Standards and samples are added to wells coated with a GxR IgG antibody. A blue solution of LVV Hemorphin 7 conjugated to biotin is added, followed by a yellow solution of rabbit polyclonal antibody to LVV Hemorphin 7.

2. During a simultaneous incubation at room temperature the antibody binds, in a competitive manner, the LVV Hemorphin 7 in the sample or conjugate. The plate is washed, leaving only bound LVV Hemorphin 7.

3. A solution of Streptavidin conjugated to horseradish Peroxidase is added to each well, to bind the biotinylated LVV Hemorphin 7. The plate is again incubated.

4. The plate is washed to remove excess HRP conjugate. TMB substrate solution is added. An HRP‐catalyzed reaction generates a blue color in the solution.

5. Stop solution is added to stop the substrate reaction. The resulting yellow color is read at 450 nm. The amount of signal is inversely proportional to the level of LVV Hemorphin 7 in the sample.

4 3. Assay Summary

Biotinylated antigen, sample antigen and primary antibody are added to the pre-coated well coated with affinity purified antibody

Incubate

Wash

Add Streptavidin-HRP

Incubate

Wash

Add TMB Substrate

Incubate

Add Stop Solution

Read Plate (measure at 450 nm)

5 4. Kit Contents

Item Description Quantity

Goat anti‐Rabbit IgG A clear plate of 1x 96 wells Microtiter Plate break‐apart strips coated with a goat anti‐rabbit polyclonal antibody. Assay Buffer 16 Tris buffer containing 30 ml proteins and preservative. LVV Hemorphin 7 Two vials containing 2 vials Standard 40 ng lyophilized LVV Hemorphin 7. LVV Hemorphin 7 A yellow solution of 6 ml Antibody polyclonal antibody to LVV Hemorphin 7. LVV Hemorphin 7 A blue solution of 6 ml Conjugate biotinylated LVV Hemorphin 7. Streptavidin‐HRP One vial containing 1 vial 12.5 µg of lyophilized streptavidin conjugated to horseradish peroxidase. Wash Buffer Concentrate Tris buffered saline 27 ml containing detergents. TMB Substrate A solution of 3,3’5,5’ 2 x 10 ml tetramethylbenzidine (TMB) and hydrogen peroxide. Stop Solution 2 A 1N solution of 10 ml hydrochloric acid in water. Plate Sealer 2 units

6 5. Storage and Handling

All components of this kit, except the Standard, should be stored at 4°C upon receipt. The Standard should be stored at ‐20°C.

6. Additional Materials Required

 Deionized or distilled water.

 Precision pipets for volumes between 5 μl and 1000 μl.

 Repeater pipets for dispensing 50 μl and 200 μl.

 Disposable beaker for diluting buffer concentrates.

 Graduated cylinders.

 A microplate shaker.

 Lint‐free paper toweling for blotting.

 Microplate reader capable of reading at 450 nm.

7 7. Protocol

A. Sample Handling

The assay is suitable for the measurement of LVV Hemorphin 7 in serum and tissue homogenates. This kit is not species specific. However, samples containing rabbit IgG will interfere in the assay due to the GxR IgG coated plate. Prior to assay, frozen samples should be brought to 4°C and centrifuged, if necessary, to isolate residual debris.

The minimum recommended dilution to remove matrix interference from serum samples and tissue homogenates is 1:16. Due to differences in samples, users must determine the optimal sample dilution for their particular experiments.

B. Protocol for Tissue Homogenate

1. Collect tissue and store in liquid nitrogen.

2. Using a homogenizer, homogenize 4 grams of tissue in 20 ml of 10% acetic acid.

3. Centrifuge the homogenate at 1500 x g for 15 min. Place supernatant into a clean tube.

4. The supernatant may be divided into aliquots and stored at or below ‐20°C, or used immediately in the assay.

8 5. Avoid repeated freeze‐thaw cycles.

C. Protocol for Serum

1. Collect whole blood in appropriate serum tubes.

2. Incubate upright at room temperature for 30 - 45 minutes to allow clotting to occur.

3. Centrifuge at 1000 x g for 15 minutes at 4°C. Do not use brake.

4. Without disturbing the cell layer, place supernatant into a clean tube.

5. The supernatant may be divided into aliquots and stored at or below -20°C, or used immediately in the assay.

6. Avoid repeated freeze-thaw cycles.

D. Reagent Preparation

1. Wash Buffer

Prepare the wash buffer by diluting 5 ml of the supplied Wash Buffer Concentrate with 95 ml of deionized water. This can be stored at room temperature for 3 months.

9 2. LVV Hemorphin 7 Standard

Reconstitute one vial of LVV Hemorphin 7 standard with 1 ml of the assay buffer. Vortex to ensure the entire cake is dissolved. Label six 12 x 75mm tubes #1 through #6. Pipet 750 μl of the assay buffer into tubes #1 through #6, remove 250 μl from the reconstituted vial and add to tube #1, this is standard #1. Vortex thoroughly. Add 250 μl from tube #1 to tube #2, vortex thoroughly. Continue this for tubes #3 through #6.

Diluted standards should be used within 60 minutes of preparation. The concentrations of LVV Hemorphin 7 in the tubes are specified below:

Tube 1: 10000 pg/ml; Tube 2: 2500 pg/ml; Tube 3:625 pg/ml; Tube 4: 156 pg/ml; Tube 5: 39.1 pg/ml; Tube 6: 9.8 pg/ml.

3. Streptavidin-HRP

Reconstitute one vial of Streptavidin-HRP with 250 μl of deionized water and vortex thoroughly. Store at 4ºC for up to 3 months. For prolonged storage, aliquot and freeze at -20°C. Avoid repeated freeze/thaw cycles. Prepare the working concentration by diluting stock 1:1000 in the assay buffer. Do not store diluted Streptavidin-HRP.

10 E. Assay Procedure

Refer to the Assay Layout Sheet to determine the number of wells to be used. Remove the wells not needed for the assay and return them, with the desiccant, to the mylar bag and seal. Store unused wells at 4°C.

1. Pipet 150 μl of the assay buffer into the NSB (non‐specific binding) wells.

2. Pipet 100 μl of the assay buffer into the Bo (0 ng/ml standard) wells.

3. Pipet 100 μl of Standards #1 through #6 to the bottom of the appropriate wells.

4. Pipet 100 μl of the samples to the bottom of the appropriate wells.

5. Pipet 50 μl of the conjugate into each well except the Blank.

6. Pipet 50 μl of the antibody into each well except the Blank, and NSB wells.

7. Seal the plate. Incubate for 2 hour on a plate shaker (~500 rpm*) at room temperature.

11 8. Empty the contents of the wells and wash by adding 400 μl of wash buffer to every well. Repeat 3 more times for a total of 4 washes. After the final wash, empty or aspirate the wells and firmly tap the plate on a lint free paper towel to remove any remaining wash buffer.

9. Pipet 200 μl of Streptavidin-HRP to each well except the Blank.

10. Seal the plate. Incubate for 30 minutes on a plate shaker (~500 rpm*) at room temperature.

11. Wash as above (Step 8).

12. Add 200 μl of the TMB substrate solution into each well.

13. Incubate for 30 minutes at room temperature without shaking.

14. Pipet 50 μl of stop solution into each well.

15. After blanking the plate reader against the substrate blank, read optical density at 450 nm. If plate reader is not capable of adjusting for the blank, manually subtract the mean OD of the substrate blank from all readings.

12 *The optimal speed for each shaker will vary and may range from 120‐700 rpm. The speed must be set to ensure adequate mixing of the wells, but not so vigorously that the contents of the wells splash out and contaminate other wells.

13 Assay Layout Sheet:

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 Blank Std 3 S0

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 Blank Std 3 S0

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 NSB Std 4

D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 NSB Std 4

E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 Std 1 Std 5

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 Std 1 Std 5

G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 Std 2 Std 6

H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 Std 2 Std 6

14 8. Calculation of Results

Several options are available for the calculation of the concentration of LVV Hemorphin 7 in the samples. We recommend that the data be handled by an immunoassay software package utilizing a 4 parameter logistic (4PL) curve fitting program.

Samples with concentrations outside of the standard curve range will need to be re‐ analyzed using a different dilution.

Typical Results

The results shown below are for illustration only and should not be used to calculate results. Average Net LVV Hemorphin Sample Percent Bound OD 7 (pg/ml)

Blank (mean) (0.040) - -

NSB 0.039 0% -

Bo 0.553 100% 0

S1 0.027 4.7% 10000

S2 0.074 13.1% 2500

S3 0.205 35.9% 625

S4 0.387 69.2% 156.3

S5 0.508 91.2% 39.1

S6 0.539 97.5% 9.8

15 120 0.600

100 0.500

80 0.400

60 0.300 B/Bo (%) Optical Density

40 0.200

20 0.100

B/Bo (%) OD

0 0.000 1 10 100 1000 10000

LVV Hemorphin 7 (pg/mL)

16 9. Performance Characteristics

A. Specificity

The cross reactivities for a number of related compounds were determined by diluting the cross reactants to concentrations in the range of 0.1 pM to 500 nM. These samples were then measured in the assay.

Analyte Sequence Percent cross reactivities in the range of 0.1 pM ‐ 500 nM

LVV Hemorphin 7 LVVYPWTQRF 100

Leu-Valorphin-Arg VVYPWTQR <0.003 (LVV Hemorphin-6)

Valorphin VVYPWTQ <0.003

Ang(1-12) DRVYIHPFHLVI <0.003

Ang I DRVYIHPFHL <0.003

Ang(1-9) DRVYIHPFH <0.003

Ang II DRVYIHPF <0.003

Ang(1-7) DRVYIHP <0.003

Ang A ARVYIHPF <0.003

Ang III RVYIHPF <0.003

Ang IV VYIHPF <0.003

17 B. Sensitivity

The sensitivity, defined as 2 standard deviations from the mean signal at zero, was determined from 6 independent standard curves. The standard deviation was determined from 12 zero standard replicates. The sensitivity of the assay was determined to be 6.1 pg/ml.

C. Dilutional Linearity

Human samples containing LVV Hemorphin 7 were serially diluted 1:4 in the kit assay buffer and measured in the assay. The results are shown in the table below.

% of Expected Dilution Serum Tissue Homogenate

1:16 98% 93%

1:64 100% 88%

1:256 - 100%

1:1024 - -

18 D. Parallelism

Dose‐response curves from human plasma and serum diluted into assay buffer were compared to the LVV Hemorphin 7 standard curve. The parallel response indicates the standard effectively mimics the native protein.

100,000

10,000

1,000

(pg/mL) 100

1 1:1 1:4 1:16 1:64 1:256 1:1024 Dilution Factor

LVV Hemorphin 7 Standard Rat Heart Homogenate Human Serum

19 E. Precision

Intra‐assay precision was determined by assaying 20 replicates of three buffer controls containing LVV Hemorphin 7 in a single assay.

pg/ml %CV

708.1 4.1

305.5 8.8

71.5 20.9

Inter-assay precision was determined by measuring buffer controls of varying LVV Hemorphin 7 concentrations in multiple assays over several days.

pg/ml %CV

707.0 9.2

334.1 9.5

72.3 16.5

20 F. Sample Recoveries

Recombinant LVV Hemorphin 7 was spiked at high, medium and low concentrations to samples that had been diluted sufficiently to eliminate matrix interference. Endogenous LVV Hemorphin 7 was subtracted from the spiked values and the average recovery in the spiked matrices was compared to the recovery of identical spikes in the assay buffer. The mean and the range percent recovery at the three concentrations are indicated below for each matrix.

Sample Matrix Dilution Spike Recovery of Concentration Spike (Range) (ng/ml) Human Serum 1:16 5000 89% (73-101%) (n=5) 1500 103% (97‐ 107%) 100 90% (63‐112%)

Rat Heart 1:16 5000 96% Homogenate 1500 99%

100 91%

21 10. Troubleshooting

Weak Color Development It is possible that Stop Solution was How long was the substrate added to the plate without allowing the incubation? full substrate incubation. If a plate is left to incubate on a cold lab bench or under a drafty area during What were the conditions of ambient incubations, signal values (e.g. the substrate incubation? optical density) may be lower than expected. It is important to ensure that all reagents are brought to room temperature prior to use, or as mentioned in the product specific instruction manual. Usually leaving the kit out on the bench top at ambient temperature for about half an Were reagents brought to hour prior to setting up the assay will be room temperature prior to sufficient, when the reagents can be use? stored at 4°C. Frozen volumes take a little more time to come to room temperature. Do not thaw frozen reagents in a water bath. If a different standard/sample diluent is used (such as culture media) this must also be warmed.

22 If the incubation times and temperatures are not observed, this can lead to lower What were the conditions of than expected signal values (e.g. optical the incubations? density). Pay attention that in air- conditioned rooms the temperature does not drop below 21°C. If customers do not have a plate shaker, they will often use an orbital flask shaker or some other piece of equipment. This is not a problem as long as the liquid is vigorously displaced about 3/4 of the How was the plate shaken way up the sides of the wells without during incubations (if coming out. It is very important that the required)? plate is secured into place. If the plate is not shaken and it is required in the procedure, a longer incubation may be necessary to bring the reagents to equilibrium. The plate needs to be read at the How long after the addition of correct wavelength as soon as possible Stop Solution was the plate after the addition of the Stop Solution. read? We generally recommend that the plate be read within 10 minutes.

23 High Background It is important that the plate is washed thoroughly. If plate washing is troublesome, a squirt bottle can be filled with diluted Wash Buffer and all of the wells completely filled from this. The plate contents can be dumped into the sink and shaken to remove excess buffer. This should be repeated for the How was the plate washed? number of times recommended in the instruction manual. It is important to remember that adding too little Wash Buffer can result in high background, while adding too much is not a problem. The contents of the wells should be aspirated and the plate tapped dry on lint-free paper towels. If the plate was incubated for too long or What were the incubation at a higher than recommended times and temperatures? temperature, high background could result.

24 Drift If the reagents are not at a constant temperature prior to their addition into Were reagents brought to the wells, the results from one side of room temperature prior to the plate to the other can differ use? depending on the temperatures at addition. If the assay is interrupted at any point during the addition of reagents, it is possible that differing results will be Was the set-up of the assay seen before the interruption versus after. interrupted? The wells that had reagents added before the interruption will have been incubating for longer than those after.

25 Poor Precision All wells receive the same treatment during the wash step. If some are washed less than others, this can translate to poor precision. It is important that the plate is washed thoroughly. If plate washing is troublesome, a squirt bottle can be filled with diluted Wash Buffer and all of the wells completely filled from this. The Were the wells washed plate contents can be dumped into the properly? sink and shaken to remove excess buffer. This should be repeated for the number of times recommended in the instruction manual. It is important to remember that adding too little Wash Buffer can result in high background, while adding too much is not a problem. The contents of the wells should be aspirated and the plate tapped dry on lint-free paper towels.

26 It is very important that as little Wash Buffer as possible remains in the wells after aspiration. Residual buffer can Were the wells aspirated cause dilution of subsequent reagents. sufficiently after the wash After the last wash step, it is a good idea steps? to hit the plate several times over a piece of paper toweling to remove excess buffer. In order to eliminate precision error, customers need to remember to pre- rinse all pipet tips used in the assay. We usually recommend that the customer draw up the liquid into the tip and aspirate it three times prior to addition How were reagents pipetted into the well. Regular pipet calibration into wells? and maintenance is also essential to ensure that the tips fit properly and that the correct volumes are dispensed. Be sure reagents are not splashed between wells or outside of the wells during pipeting (especially if using repeater pipets).

27 Poor Standard Curve Diluents other than the supplied assay What was used as the buffer may contain interfering standard diluent? substances that can affect the standard curve. If the %CV values for the standard curve signal values (e.g. optical density) are consistently above 5%, it may be a good idea to pay particular attention to How was the precision of the pipetting technique. If the standard standard curve? curve signal values were acceptable but the sample precision was not, the problem relates to the sample. Also, see recommendations under "Poor Precision". If the net signal values (e.g. optical density) are not used, the signal values Were the Blank and NSB will appear higher than those presented values subtracted out? in the sample data in the instruction manual.

28 It is important that test tubes of an appropriate size and material are used. Standard dilutions must be properly mixed (e.g. vortexed) while preparing the serial dilutions. It is also crucial that the standard dilutions be prepared and used within the time specified in the product specific instruction manual. How were the standard Never store unused standard dilutions dilutions prepared? for a later use.

29 Edge Effects Often times the conditions for ambient incubations can be less than ideal. If Where was the plate there is a draft in the area or the plate is incubated? incubated on a cold lab bench, this can lead to uneven color development. Multiple plates should only be incubated If multiple plates were run, in a single layer. This will assure that no were they stacked on top of area of the plate is at a different each other during incubation? temperature than any other. Making sure that the plate sealer is If a non-ambient incubation tightly covering all of the wells will help was required, was the plate to discourage uneven evaporation of the properly sealed? well contents, or condensation for colder incubation conditions.

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