Mouse RELM-b RIA

ntended Use:

The Resistin molecule, expressed in adipose tissue, has been implicated in insulin resistance of obesity and diabetes and as a possible link between obesity and diabetes.1-4 In addition to mouse resistin, two other resistin-like molecules, RELM-a and RELM-b with 29 and 37 percent sequence homology at the protein level are expressed in white adipose tissue and the gut, respectively5. The secretory form of a consensus of RELMs is 85-94 amino acid residues with a variable N-terminal sequence of about 35 amino acid residues and a highly conserved C-terminal domain with 10 cysteines of an unique spacing motif 3,4,5.. LINCO’s Mouse RELM-β is the first available assay to specifically measure RELM- β in mouse serum, plasma, or tissue culture media. Measurement of mouse RELM-β levels in circulation under different patho-physiological, nutritional and genetic manipulations should result now in a better understanding of the etiology of type 2 diabetes, obesity and inflammatory diseases. This kit is for research purposes only.

Principles of Procedure

In radioimmunoassay, a fixed concentration of labeled tracer antigen is incubated with a constant dilution of antiserum such that the concentration of antigen binding sites on the antibody is limited, for example, only 50% of the total tracer concentration may be bound by antibody 6. If unlabeled antigen is added to this system, there is competition between labeled tracer and unlabeled antigen for the limited and constant number of binding sites on the antibody. Thus, the amount of tracer bound to antibody will decrease as the concentration of unlabeled antigen increases. This can be measured after separating antibody-bound from free tracer and counting one or the other, or both fractions. A calibration or standard curve is set up with increasing concentrations of standard unlabeled antigen and from this curve the amount of antigen in unknown samples can be calculated 7. Thus, the four basic necessities for a radioimmunoassay system include a specific antiserum to the antigen to be measured, the availability of a radioactive labeled form of the antigen, a method whereby antibody-bound tracer can be separated from the unbound tracer, and finally, an instrument to count radioactivity 8.

The LINCO Research Mouse RELM-β RIA assay utilizes 125I-labeled Murine RELM-β and a Mouse RELM-β Guinea pig antiserum to determine the level of mouse RELM-β in serum, plasma or tissue culture media by the double antibody/PEG technique.

Reagents Supplied

Each kit is sufficient to run 250 tubes and contains the following reagents.

  1. Assay buffer
    0.05M Phosphosaline containing 0.025M EDTA, 0.08% Sodium Azide, 0.1 % BSA and protease inhibitors.
    Quantity: 50 ml/vial
    Preparation: Ready to use
  2. Mouse RELM-b Antibody
    Guinea pig anti-Mouse RELM-b Antibody
    Quantity: 13 ml/vial
    Preparation: Ready to use
  3. 125I-Mouse RELM-b
    125I-RELM-beta Label (<1.5 µCi, <56kBq)
    Lyophilized for stability. Freshly iodinated label contains < 1.5 µCi, (<56 kBq) calibrated to the 1st Monday of each month.
    Quantity: 13.5 ml/vial upon hydration
    Preparation: Contents Lyophilized. Hydrate with 13.5 ml of Assay Buffer. Allow to sit at room temperature for 10-15 minutes, with occasional gentle mixing.
  4. Mouse RELM-b Standard
    Recombinant Mouse RELM-b, 5000 pg
    Lyophilized for stability.
    Quantity: 1 ml upon hydration (5000 pg/ml).
    Preparation: Contents Lyophilized. Hydrate with 1 ml distilled or deionized water.
  5. Quality Controls 1 and 2
    Recombinant Mouse RELM-b
    Lyophilized for stability.
    Quantity: 1 ml/vial upon hydration.
    Preparation: Contents Lyophilized. Hydrate with 1 ml distilled or deionized water.
  6. Guinea pig Carrier
    Assay buffer containing normal guinea pig IgG as a carrier.
    Quantity: 2 ml/vial
    Ready to use
  7. Precipitating Reagent
    Goat anti-Guinea pig IgG Serum, 3% PEG and 0.05% Triton X-100 in 0.05M Phosphosaline, 0.025M EDTA, 0.08% Sodium Azide
    Quantity: 130 ml/vial
    Preparation: Ready to use; chill to 4

Storage and Stability

For short-term storage, refrigerate all reagents between 2 and 8°C. For prolonged storage (>2 weeks), freeze at £  -20°C. Avoid multiple freeze/thaw cycles. Refer to date on bottle for expiration when stored at £  -20°C. Do not mix reagents from different kits unless they have the
same lot number. Store remaining hydrated Standard, Quality Controls and Tracer at -20°C.

Radioactive Materials

This radioactive material may be received, acquired, possessed and used only by research personnel or clinical laboratories for in vitro research tests not involving internal or external administration of the material, or the radiation therefrom, to Mouse beings or animals. Its receipt, acquisition, possession, use and transfer are subject to the regulations of the U. S. Nuclear Regulatory Commission (NRC) or of a State with which the Commission has entered into an agreement for the exercise of regulatory authority.

The following are suggested general rules for the safe use of radioactive material. The customer’s Radiation Safety Officer (RSO) is ultimately responsible for the safe handling and use of radioactive material.

  1. Wear appropriate personnel devices at all times while in areas where radioactive materials are used or stored.
  2. Wear laboratory coats, disposable gloves, and other protective clothing at all times.
  3. Monitor hands, shoes, and clothing and immediate area surrounding the work station for contamination after each procedure and before leaving the area.
  4. Do not eat, drink, or smoke in any area where radioactive materials are stored or used.
  5. Never pipette radioactive material by mouth.
  6. Dispose of radioactive waste in accordance with NRC rules and regulations.
  7. Avoid contaminating objects such as telephones, light switches, doorknobs, etc.
  8. Use absorbent pads for containing and easily disposing of small amounts of contamination.
  9. Wipe up all spills immediately and thoroughly and dispose of the contaminated materials as radioactive waste. Inform Radiation Safety Officer.

    Sodium Azide

    Sodium Azide has been added to all reagents as a preservative at a concentration of 0.08%. Although it is at a minimum concentration, sodium azide may react with lead and copper plumbing to form highly explosive metal azides. On disposal, flush with a large volume of water to prevent azide build up.

Materials Required but not provided

  1. Borosilicate glass tubes, 12 x 75 mm. (NOTE: Polypropylene or polystyrene tubes may be used if the investigator finds that the pellet formation is acceptably stable in their system.)
  2. Borosilicate glass tubes, 12 x 100 mm, or equivalent for sample and standard dilutions
  3. 10, 20, 100 and 1000 µl pipettes with disposable tips
  4. 100 µl & 1.0 ml repeating dispenser
  5. Refrigerated swing bucket centrifuge capable of developing 2,000 - 3,000 xg. (Use of fixed-angle buckets is not recommended.)
  6. Absorbent paper
  7. Vortex mixer
  8. Refrigerator
  9. Gamma Counter

Specimen Collection and Storage

  1. A maximum of 100 µl per assay tube of serum or plasma can be used, although, 50 µl per assay tube is adequate for most applications. Tissue culture and other media may also be used.
  2. Since RELM-b levels in tissue culture medium depend on incubation conditions and the cell density, it is advisable to pilot test for appropriate volume and/or dilution before assaying all the samples. Use unconditioned tissue culture medium to determine the background or basal level.
  3. When analyzing different volumes of the samples, it is important to adjust the final result by the appropriate factor to compensate for the different volume.
  4. Avoid using samples with gross hemolysis or lipemia.

Assay Procedure

For optimal results, accurate pipetting and adherence to the protocol are recommended.

Mouse RELM-b Standard Preparation

  1. Use care in opening the lyophilized Standard vial. Using a pipette, reconstitute the Mouse RELM-b Standard with 1 ml distilled or deionized water into the glass vial to give a 5000 pg/ml concentration of Standard. Mix well.
  2. 2. Label 8 glass tubes 2500, 1250, 625, 312.5, 156, 78, 39 and 19.5 pg/ml. Add 0.5 ml Assay Buffer to each of the 8 tubes. Prepare serial dilutions by adding 0.5 ml of the 5000 pg/ml reconstituted Standard to the 2500 pg/ml tube, mix well and transfer 0.5 ml of the 2500 pg/ml Standard to the 1250 pg/ml tube, mix well. Repeat the serial dilution until you reach 19.5 pg/ml.
    Note: Do not use a Repeater pipette. Change tip for every dilution. Wet tip with Standard before dispensing. Unused portions of standard should be stored at £  -20°C. Avoid multiple freeze/thaw cycles.

Standard Concentration
pg/ml

Volume of Deionized
water to Add

Volume of Standard
to Add

5000

1 ml

0


 

Standard Concentration
pg/ml

Volume of Assay Buffer to Add

Volume of Standard to Add

2500

0.5 ml

0.5 ml of 5000 pg/ml

1250

0.5 ml

0.5 ml of 2500 pg/ml

625

0.5 ml

0.5 ml of 1250 pg/ml

312.5

0.5 ml

0.5 ml of 625 pg/ml

156

0.5 ml

0.5 ml of 312.5 pg/ml

78

0.5 ml

0.5 ml of 156 pg/ml

39

0.5 ml

0.5 ml of 78 pg/ml

19.5

0.5 ml

0.5 ml of 39 pg/ml


Mouse RELM-b Quality Control 1 and 2 Preparation

Use care in opening the lyophilized Quality Control vials. Using a pipette, reconstitute each of the Mouse RELM-b Quality Control 1 and Quality Control 2 with 1 ml distilled or deionized water into the glass vials. Mix well. Unused portions of Quality Controls should be stored at £  -20°C. Avoid multiple freeze/thaw cycles.

Assay Set-Up, Day One

  1. Pipet 310 µl of Assay Buffer to the Non-Specific Binding (NSB) tubes (3-4), 210 µl to the Reference (Bo) tubes (5-6), and 110 µl to standard and control tubes (7-28) and 200 µl to sample tubes 29-through the end of the assay.
  2. Pipet 100 µl of Standards and Quality Controls in duplicate (see flow chart). For preparation, see Section VIII, Part A and B.
  3. Pipet 10 µl of each Sample in duplicate. Refer to Section IX for calculation modification for different volume of the samples.
  4. Pipet 100 µl of RELM-b antibody to all tubes except Total Count tubes (1-2) and NSB tubes (3-4).
  5. Vortex, cover, and incubate overnight (20-24 hours) at 4°C.

Day Two

  1. Pipet 100 µl of 125 I-RELM-b (Important: For preparation, see Section III, Part C ) to all tubes. Vortex, cover, and incubate overnight (20-24 hours) at 4°C.
     

Day Three

  1. 10 µl of Guinea pig Carrier to all tubes except Total Count tubes (1-2).
  2. Add 1.0 ml of cold (4°C) Precipitating Reagent to all tubes except Total Count tubes (1-2).
  3. Vortex and incubate 20 minutes at 4°C.
  4. Centrifuge at 4°C, all tubes [except Total Count tubes (1-2)] for 20 minutes at 2,000-3,000 xg. NOTE: If less than 2,000 xg is used or if slipped pellets have been observed in previous runs, the time of centrifugation must be increased to obtain a firm pellet (e.g., 40 minutes). Multiple centrifuge runs within an assay must be consistent.
    Conversion of rpm to xg:
    xg = (1.12 x 10-5) (r) (rpm)2
    r = radial distance in cm (from axis of rotation to the bottom of the tube)
    rpm = rotational velocity of the rotor
  5. Immediately decant the supernate of all tubes except Total Count tubes (1-2), drain tubes for at least 15-60 seconds (be consistent between racks), and blot excess liquid from tip of tubes.
  6. Count all tubes in a gamma counter for 1 minute. Calculate the pg/ml of RELM-b in unknown samples using automated data reduction procedures (see Section IX).