A Rapid, Simple, Low-Cost Radioreceptor Assay
for Serotonin with FemtoMolar Sensitivity

Background

A schematic representation of Serotonin

This assay was conceived and initially developed by Dr. Bruce E. Morton and his students in the early 1990's at the Dept. of Biochemistry and Biophysics, University of Hawaii School Of Medicine, Honolulu, Hawaii. In May of 1997 I started a series of developmental experiments in the laboratory of Dr. Morton to further refine and make more efficient the assay. This writing reports upon the refinements of the Assay to September of 1997. I refined the assay from 4 hours and results overnight to 1 and 1/4 hours and results in 3 hours. I also discovered that Tris-HCl buffer at 50 mM concentration gave substantial pH drift towards alkalinity with a lowering of temperature. Consequently, I changed the setup and filtration temperatures from on ice to room temperature (r.t.). Buffers employed were made and kept at r.t. They are used quickly and do not spoil. Resuspended brain membranes were viable, kept in the refrigerator for as long as three weeks, without substantial loss of membrane functionality. This assay detects serotonin in any biological fluid without any prior preparation other than dilution, i.e., 1/10 for urine and 1/5 for saliva.

Over 80 % of the 5-HT found in the body is synthesized and stored by the enterochromaffin cells in the gastrointestinal system and most circulating serotonin arises from enterochromaffin cells triggered by parasympathetic drive where it is used to regulate intestinal movements. Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine found in the central nervous system, gastrointestinal tract, and blood with broad physiological functions in neurotransmission, gastric motility, rheostasis, and cardiovascular integrity. 5-HT1A expression in mammals is strongest in CNS limbic pathways that control emotion, including tissue of the hippocampus, the dorsal raphé, and the septum.

Serotonin that is secreted from the enterochromaffin cells eventually finds its way out of gut tissues into the blood. There, it is actively taken up by blood platelets and stored. When platelets then bind to a clot, they release serotonin, that serves as a local vasoconstrictor and thus helps to regulate rheostasis and blood clotting.

Materials:

Brandel 24 Place cell harvester

Filters

• 1. Whatman GF/B from Brandel

  Catalogue # FP-100, 100 sheets

  Biomedical Research and Development laboratories, 8561 Atlas Drive, Gaithersburg, MD 20877 Telephone: 301-869-5570

• 2. Schlecher and Schuell #32

  Catalogue # 46690, 100 sheets

  Schlecher and Schuell, P.O. Box 2012, Keene, NH 03431 Telephone: 603-352-3810

Scintillation Counter

• Scintillation Vials- 3 ml. size, polyethylene

• Scintillation Fluid- Fisher Scientific, Scintisafe Econo 1

• Scintillation Standard- Tritium, usually comes with the machine

Dilution Buffer

• 50 mM Tris-HCl, pH 7.4 at r.t., needed to wash filters in Brandel

• 50 mM Tris-HCl, pH 7.4 at 4 degrees Celsius, needed to make brain homogenate

• store at make-up temperature

Incubation Buffer

• Components for 100 ml:

  1. 50 mM Tris- 605 mg

  2. 4 mM Calcium Chloride- 44.4 mg

  3. 10 microlitres Pargyline- use 10 ml of a 100 microlitres Pargyline solution(19.6 mg/ml)

• Make-up

  1. dilute additions to 90 ml with ultrapure water

  2. adjust pH with HCl to 7.4 at r.t.

  3. add ultrapure water to 100 ml

  4. Store at r.t.

Unlabeled Serotonin Standards

• Serotonin, MW 387.4 complexed with creatine sulfate

• sensitive to light

• water soluble, aqueous solutions stable at pH 2.6-4.0 with 0.01 M aq. solution at pH 3.6

• Sigma Chemical Co., catalogue # H- 7752

Stock Serotonin (E-3 M) in Citrate (E-3 M), pH 3.0, 100 ml

• serotonin-38.74 mg

• citrate-citric acid monohydrate, 21.0 mg

• dilute additions to 95 ml with ultrapure water

• adjust pH with HCl to 3.0

• add ultrapure water to 100 ml

• store in refrigerator

Citrate Dilution Buffer

• 1 mM Citrate, pH 3.0, 1 litre

• add 210 mg of citric acid monohydrate to 900 ml ultrapure water

• adjust pH to 3.0 with HCl

• bring volume to 1 litre

• refrigerate

Preparation

• use stock (E-3 M) serotonin

• make dilutions with Citrate Dilution Buffer

• Make E-4 M through E-11 M standards

• refrigerate

Tritiated-Serotonin Stock Solution (20 nM)

• Tritiated-Serotonin complexed with creatine sulfate

• Source is New England Nuclear, Catalogue # NET-498

• To minimize autodegredation, do not freeze

• 1 mCi provided in 1 ml ethanol-water

• Add 100 microlitre of original commercial preparation to 210 microlitre of 1 mM Citrate Buffer, pH 3.0

• refrigerate

• Final concentration should yield up to 2000 cpm at the lowest serotonin standard of E-12 M and about 350 cpm for the highest standard of E-5 M, complexed with brain homogenate

Protein Assay For Total Protein in Brain Homogenate

• BioRad Protein Assay

• Coomassie Brilliant Blue G-250 by a procedure developed by Bradford, Anal. Biochem 72, 248 (1976)

• BioRad One Step Protein Assay Kit

• Catalogue # 500-0001

• follow the directions with the kit

• read absorbency at 595 nM between the 5 and 60 minute window

Standards

• Human Serum Albumin (HSA), crystalline, Sigma Chemical Co.

• HSA primary standard is 1.4 mg/ml in ultrapure water (28 mg/20 ml)

• HSA secondary standards

• 1.4 mg/ml use 5.0 ml straight

• 1.1 mg/ml use 4 ml 1.4 std to 1.10 ml ultrapure water

• 0.8 mg/ml add 3 ml 1.4 std to 2.25 ml ultrapure water

• 0.5 mg/ml add 2 ml 1.4 std to 3.60 ml ultrapure water

• 0.2 mg/ml add 1 ml 1.4 std to 6.00 ml ultrapure water

Brain Homogenate Preparation

• Remove brains from source (pig, rat, guinea pig, etc.) and remove cerebellum, brain stem and any adhering arachnoid/pia matter

• Wash cerebrum(s) in 0.9% w/v NaCl

• Blot dry and store in freezer bags at -70 degrees Celsius or lower (label bag with pertinent data) if not used immediately

• Weigh out approximately 50 gms wet weight of cerebrum which will yield between 40 and 50 tubes of homogenate

• Thaw frozen cerebrum in Dilution Buffer and weigh after thawed and blotted

• Dilution Buffer should be pH 7.4 at 4 degrees Celsius and made in a 4 degree Celsius cold room for this specific purpose (50 mM Tris-HCl)

• Disperse by cutting up cerebrum in a dish on ice into small pieces (the smaller the better)

• Further disperse by subjecting to two up-down passes in a Potter-Elvehjem (P-E) tissue homogenizer on ice (Arthur B. Thomas, glass-teflon, size C)

• Homogenize dispersant with a Brinkman Polytron in the glass half of the P-E, set at #5, for 20 seconds

• Add dilution Buffer to make up liquid volume to be 1.4 litre for the 50 gms wet weight

• Spin homogenate at 33,000 g for 15 minutes at 2-4 degrees Celsius

• Re-suspend pellets in Dilution Buffer made at r.t. and pH 7.4

• Pre-incubate homogenate to remove any endogenous serotonin for 15 minutes at 37 degrees Celsius

• Spin pre-incubated homogenate at 33,000 g for 15 minutes at r.t.

• Discard supernatant and re-suspend with Incubation Buffer made at r.t. and pH 7.4 if using immediately

• If freezing aliqouts, re-suspend (see below) in Dilution Buffer made at 4 degrees Celsius and pH 7.4

• Cover tubes with caps or parafilm and freeze at -70 degrees Celsius or lower

Preparation of Frozen/Fresh Aliqouts of Homogenate

• The target protein concentration in the assay is 0.5 mg/tube in the 1 ml final volume

• This requires for a 50 tube assay a 25 mgs of protein membrane concentration per frozen aliquot tube (a double incubation assay is 48 tubes plus two tubes extra for easier pipetting) as the Brandel is a 24 tube device

• Determine the protein concentration per ml

• Determine what volume of buffer/homogenate mix will yield 25 mgs of protein per aliquot tube and pipette into the tubes

• Spin tubes at 33,000 g for 15 minutes and discard supernatant

• Cap or use parafilm and freeze at -70 degrees Celsius or lower

Methods:

Fresh

• Measure aliquot to determine volume

• Add Dilution Buffer made at r.t. and pH 7.4 to make final volume 40 ml

• Make one up-down-up pass in the P-E, size C

• Put into 125 Erhlenmeyer flask and add a magnetic stirrer flea

• stir w/o frothing to keep suspended

Frozen

• Obtain tube from freezer and warm in hand so as to melt frost on outside

• Use tubes wide enough to allow P-E size A pestle to go to the bottom of the tube

• Stir with some of the 40 ml at 1725 RPM to re-suspend pellet

• Add the rest of the 40 ml, make one up-down-up pass in P-E size C

• Put into 125 ml Erhlenmeyer flask and add a magnetic stirrer flea

• Stir w/o frothing to keep suspended

Incubation of Homogenate/Standards/Unknowns

• Obtain two test tube racks which will allow the Brandel 24 place Dispersion/Collection Head to easily fit into the 24 test tubes

• Fill 2 test tube racks(Rack A & Rack B) with 1 cm X 10 cm Wasserman test tubes

• To Rack A add known standards: E-11 M, E-10 M, E-9 M, E-8 M, E-7 M, E-6 M, E-5 M in triplicate plus one unknown, 100 microlitre each

• To Rack B add known stds E-8 M and E-7 M and six unknowns in triplicate, 100 microlitres each

• This is to allow variation between Rack A and Rack B incubation and filtration runs to be taken into account during analysis of the experimental data

• You may wish to determine your own protocol as you gain experience with the assay in your own lab

• Next, add 800 microlitre of homogenate to each tube in Rack A & Rack B, vortex briefly

• Incubate tubes for 15 minutes at 37 degrees Celsius in a shaker water bath, otherwise agitate racks every 3 minutes

Pre-Filtration

• Prepare Brandel 24 place apparatus for filtration

• Place inlet tube into Dilution Buffer made at r.t. and pH 7.4

• Turn on suction motor and allow air bubble to exit Dispersion/Collection Head

• Place filter over the 24 hole supports and wet with ultrapure water

• close block and tighten clamps

Filtration

• 1. At the appointed time, remove Rack A from the shaker bath and place in a plastic dishpan next to the Brandel

  2. Place the head ports into the 24 test tubes

  3. Turn on Dispersion motor and fill tubes 1/2 full

  4. Turn on suction Vacuum and empty tubes

  5. Refill tubes 1/2 full and then allow suction vacuum to empty tubes

  6. Turn off suction, open block, remove filter

  Repeat steps 2-6 for Rack B

Drying Filter Strip For Counting

Place filter strip on slide warmer at 55 degrees Celsius

Allow 20-30 minutes to dry

Preparation of Filters/Vials for Counting

• Remove filter disks from filter strip with tweezers and place into 3 ml vials in the order taken from the strip

  Fill vials with 3 ml of Scintisafe Econo 1 and cap

  Place vials in order into counter, close cover

  Let vials sit for 2 hours to allow for wetting of disks (gives 80%-90% of overnight soaking counts)

  Count vials for 1 minute

Analysis Of Data

• Mark off triplicates and average each set of counts

  Subtract value for the E-6 m counts from each previous value to set to zero (saturation with E-6 M 5-HT)

  Value of the E-12 M is the total specific binding for the run of Rack A & Rack B

  Using the E-12 M value as 100%, calculate the % binding for the remaining values

  Inflection of the sigmoid curve should be at 3 X E-9 M 5-HT as 50% value on semilog graphpaper with y(linear)=% and x(log)=E-? M

  Calculate the unknown values according to the known curve values as percentages of the E-12 M value on the semilog plot of the Standard curve

  Take into account any dilution factors

  If the difference between the E-9 M and E-8 M values of Rack A and Rack B are greater than 100 counts, use ratio and proportion to bring Rack B values into the Rack A scale

  If any of the triplicates is separate from the other two values by more than 200 counts, discard count and average the two remaining values

Discussion:

This assay as described is rapid, simple, easy, low-cost and yields femtoMolar sensitivity of 5-HT. The membranes are sensitive to shifts in pH towards alkalinity with a decrease in binding activity. Buffers made at r.t., pH 7.4 and then cooled to 4 degrees Celsius yield a pH of 8.0 or so in the concentration used (50 mM Tris-HCl) and thus the protocol was changed to eliminate this effect.

High salt and protein concentration interfere with the assay and thus dilution of certain biological fluids is necessary, apart from the high serotonin content. Ascorbic acid was shown to interfere with the assay and should definitely NOT be added to any of the solutions.

With this assay, it is possible to easily follow 5-HT content in urine, saliva, blood, cerebral spinal fluid, etc. over time. The applications are numerous: post-prandial effects, drug effects, pre/post parturition effects, stressor effects, etc.