1. Special RIA buffer*, 50ml (4 x concentrate)
2. Standard Protein 5 ng, 1ml
3. Rabbit antiserum specific for the RELM-Beta (Mouse) , 13ml (lyophilized powder)
4. ¹²⁵I-RELM-Beta (Mouse) , 1.5µCi (lyophilized powder)
5. Goat Anti-Rabbit IgG Serum (GAR), 13 ml (lyophilized powder)
6. Normal Rabbit Serum (NRS), 13ml (lyophilized powder)
7. Positive control, 1ml (lyophilized powder)
8. Instructions, 1 booklet

Note:* A special RELM-Beta (Mouse) RIA Buffer is different with other Phoenix's peptide RIA buffer. Phoenix Pharmaceuticals guarantees that its products conform to the information contained in this publication. The purchaser must determine the suitability of the product for its particular use.

Extraction of plasma or serum is not required in RELM-Beta (Mouse) (Human) RIA Assay

1. Dilute the RIA buffer (concentrate) with 150 ml of distilled water. This buffer will be used to reconstitute all of the other compounds in this kit and should be used for dilution of samples if needed.
2. Reconstitute the standard RELM-Beta (Mouse) with 1 ml of RIA buffer, mix well and store on ice.
   Note: Before adding buffer, carefully examine the eppendorf tube containing the standard. During shipping, part or the entire lyophilized RELM-Beta (Mouse) may have come loose from the bottom of the tube and sticking to the cap or walls of the tube. Gently tap or centrifuge the tube to dislodge powder from the cap or walls. Carefully open the tube and add buffer.
   
   After adding the RIA buffer, vortex for approximately 2 minutes until ALL the RELM-Beta (Mouse) powder is completely dissolved. For hydrophobic and hard to dissolve RELM-Beta (Mouse) s, longer vortexing may required.
3. Reconstitute the rabbit anti-RELM-Beta (Mouse) serum with 13ml of RIA buffer, mix well and store on ice.
   Note: The remaining reagents are not required at this time and should be stored in their lyophilized state until needed.
4. Reconstitute samples with RIA buffer (we cannot assure success with other buffers since they have not been tested).
5. Prepare RELM-Beta (Mouse) standard solutions as follows:
   

   Tube	Sample	RIA Buffer	Concentration of Standard RELM-Beta (Mouse) in RIA reaction
   A (Stock)	1000µl		5000 pg/ml
   B	300µl A	300µl	2500 pg/ml
   C	300µl B	300µl	1250 pg/ml
   D	300µl C	300µl	625 pg/ml
   E	300µl D	300µl	312.5 pg/ml
   F	300µl E	300µl	156.25 pg/ml
   G	300µl F	300µl	78.125 pg/ml
   H	300µl G	300µl	39.06 pg/ml

6. Set up initial RIA reactions (see diagram on page 4) in 12 x 75 mm polystyrene tubes.
   1. Number tubes TC-1, TC-2, NSB-1, NSB-2, TB-1, TB-2 and #7-#22 for the standards.
   2. Number tubes #23 & 24 for positive control
   3. Number tubes #25 up to #125 for the unknown samples .
   4. Pipette 200 ul of RIA buffer into each NSB tube.
   5. Pipette 100 ul of the RIA buffer into each TB tube.
   6. Pipette 100 ul of the standards H through A into duplicate tubes #7-#22.
      Note: Reverse the order of preparation so that the concentration increases as the number of the tube increases. For example, pipette 100 ul of standard H into tubes #7 & #8.

   7. Pipette 100 ul of positive control into duplicate tubes: tube #23 and 24.
   8. Pipette 100 ul of unknown sample into duplicate tubes: tube #25 and up.
   9. Pipette 100 ul of primary antibody (rabbit anti-RELM-Beta (Mouse) serum) into TB-1, TB-2 tubes and up. DO NOT ADD TO TC AND NSB TUBES.
   10. Vortex the contents of each tube.
   11. Cover and incubate all tubes for 16-24 hours at 4°C.
7. Reconstitute the 125-I-RELM-Beta (Mouse) with 13 ml of RIA buffer and mix well to make tracer solution. Please check the concentration of this tracer solution and adjust it with RIA buffer until the concentration is 8,000~10,000 cpm/100 ul.
8. Add 100 ul of the tracer solution to each tube.
9. Vortex the contents in each tube.
10. Cover and incubate all tubes for 16-24 hours at 4°C.

    Tube	Contents	RIA Buffer	STD or Samples	Primary Antibody	125I RELM-Beta (Mouse)
    TC-1&2	Total Counts				100µl
    NSB-1&2	Non-specific Binding	200µl			100µl
    TB-1&2	Total Binding	100µl		100µl	100µl
    7,8	H Standard		100µl	100µl	100µl
    9,10	G Standard		100µl	100µl	100µl
    11,12	F Standard		100µl	100µl	100µl
    13,14	E Standard		100µl	100µl	100µl
    15,16	D Standard		100µl	100µl	100µl
    17,18	C Standard		100µl	100µl	100µl
    19,20	B Standard		100µl	100µl	100µl
    21,22	A Standard		100µl	100µl	100µl
    23,24	Positive control		100µl	100µl	100µl
    25,26	Sample 1		100µl	100µl	100µl
    27,28	Sample 2		100µl	100µl	100µl
    etc.	etc.		100µl	100µl	100µl
    Contents Before Incubation

11. Reconstitute the Goat Anti-Rabbit IgG Serum (GAR) with 13.0ml of RIA buffer.
12. Reconstitute the Normal Rabbit Serum (NRS) with 13.0ml of RIA buffer.
13. Add 100µl of GAR to each tube except the TC tubes.
14. Add 100µl of NRS to each tube except the TC tubes.
15. Vortex the contents of each tube. Incubate all tubes at room temperature for 90 minutes.
16. Add 500µl of RIA buffer to each tube except the TC tubes and vortex the contents in each tube.
17. Centrifuge all tubes (except the TC tubes) at 3,000rpm (approx. 1700 x g) for 20 minutes at 4°C.
18. Carefully aspirate off ALL the supernatant (without touching the pellet) immediately follow centrifugation (do not decant since the pellet might be lost or excess liquid could be left). DO NOT ASPIRATE THE TC TUBES.
    Note: For best results, the supernatant should be immediately aspirated after centrifugation. If the pellet is allowed to sit for more than 15-30min, it may become detached and make aspiration difficult. Do not aspirate any solid.
19. Use a Gamma-counter to count the CPM of the pellet.

1. Using cpm, calculate the average NSB and label this as average NSB.
2. Using cpm, calculate the average TB and label this as average TB.
3. To find Bo use the following equation:
   
   Bo = averageTB - average NSB
4. To determine the B/Bo (%) for paired standards and unknown samples use the following calculation:
   1. Example for Standard H:
      
   2. Standards G through A (tubes #9-#22), positive control (tube#23, 24) and the unknown samples (tubes #25 up to #125) are handled as shown above for standard H.

5. Examples of tabulated data:
   

   Tube	Samples	RELM-Beta (Mouse)	Average cpm	B/B0(%)
   TC-1&2			9,000
   NSB-1&2			150
   TB-1&2		0 ng/ml	4,000	100
   7,8	H Standard	39.06 pg/ml	3,741	93.3
   9,10	G Standard	78.12 pg/ml	2,287	55.5
   21,22	A Standard	5000 pg/ml	420	7.0
   23,24	positive control		1650	41.3
   25,26	Sample 2	?	976	21.5
   27,28	Sample 3	?	1,383	32.0
   Tabulated Data After Calculation

   
   Total Count [total activity] (cpm/100µl)=9,000cpm
   NSB=150cpm
   TB=4,000cpm
   B₀=4,000cpm-150cpm=3,850cpm
6. On semilog graph paper, plot B/B₀(%) (in decimal scale) versus the standard RELM-Beta (Mouse) concentrations (in long scale).
   • Label the concentrations of standard H through A (39.06 - 5000 pg/ml) on the X-axis (log scale).
   • Label B/Bo (%) (0 to 100%) on the Y-axis (decimal scale).
   • Plot B/Bo (%) for each standard concentration directly above its X-axis designation.
   • Draw the "Best fit" curve.
7. Determination of the concentrations of RELM-Beta (Mouse) in unknown samples.
   • Using B/Bo (%) calculated for each unknown sample read, across the graph to the point of intersection with the "Best fit" curve.
   • The corresponding X-axis coordinate is equivalent to the concentration of RELM-Beta (Mouse) (pg/ml) in the assayed sample.
   • To calculate the amount of RELM-Beta (Mouse) in the original sample, multiply the concentration of the assayed sample by any dilution factor used to prepare the sample.

1. Add sample of standard and antibody.
2. Vortex and incubate 16-24 hours at 4°C.
3. Add ¹²⁵I-RELM-Beta (Mouse) .
4. Vortex and incubate 16-24 hours at 4°C.
5. Add GAR and NRS.
6. Vortex and incubate at room temperature for 90 minutes.
7. Add RIA buffer.
8. Vortex and centrifuge for 20 minutes at 1,700 x g
9. Aspirate of the supernatant (except TC tubes)
10. Count assay tubes
11. Calculation of results

SUGGESTED METHOD:
Blood Withdrawal:
Collect blood samples into the Lavender Vacutaner tubes (#VT6450) which contain EDTA and can collect 7ml blood/tube. Gently rock the Lavender Vacutaner tubes several times immediately after collection of blood for anti-coagulation. Transfer the blood from the Lavender Vacutaner tubes to centrifuge tubes containing aprotinin (0.6TIU/ml of blood) and gently rock for several times to inhibit the activity of proteinase. Centrifuge the blood at 1,600 x g for 15 minutes at 4°C and collect the plasma. Plasma kept at -70°C may be stable for one month.

1. Berson, S.A. and Yalow, R.S. Kinetics of reaction between insulin and insulin binding antibody. J. Clin Invest 36: 873, 1957.
2. Patrono, C. and Peskar, B.A. (eds) Radioimmunoassay in basic and clinical pharmacology. Heidelberg, Springer-Verlag, 1987
3. Reuter, A., Vrindts-Gevaerts, Y., Meuleman-Gathy, R., Joris, J., Chretien, M. and Franchimont, P. A radioimmunoassay for beta-endorphins. (BETA-END) and (BETA-LPH) in plasma. Horm Res 25: 236, 1987.
4. Dwenger, A. Radioimmunoassay: an overview. J Clin Biochem 22:883, 1984.
5. Wang, Y.N., Chou J., Chang, D., Chang, J.K., Avila, C. and Romero, R. Endothelin-1 in human plasma and amniotic fluid. In Endothelin-derived contracting factors, edited by G. Rubanyi and P. Vanchoutte, Karger, Basel, pp. 143, 1990.

Precautions in Handling Radioactive Material:
The user should store the by-product material, until used, in the original shipping container or in a container providing equivalent radiation protection. There should be no drinking, eating or smoking while radioactive material is being handled. Hands should be covered with gloves during, and thoroughly washed after the handling of radioactive material. When handling radioactive material do not pipette by mouth. Spills must be quickly and thoroughly cleaned up and the surfaces involved shall be washed with an alkali detergent (alconox or the equivalent). Persons under 18 should not be permitted to handle radioactive material or enter radioactive areas.

Disposal:
Used radioactive test solutions must be disposed of by flushing down a laboratory sink drain with copious amounts of water. Radioactive waste should be disposed of in compliance with Federal, State, and Local Government regulations.