I’m attached to endocrine laboratory and from the word ‘endocrine’, you all should know what I am studying about. And ya, its hormone! My lab actually collaborates with the Singapore zoo to conduct projects on the animals. The purpose is to determine the hormone levels of the animals using their feces, urine or plasma/serum. For example, the cortisol level will allows us to know whether the animal is in stress or progesterone level to determine the ovulation cycle for reproduction.
So for the past 2 weeks, I was handling with white tigers’ urine. There are three white tigers in the Singapore zoo and they are Omar (the male tiger), Winnie and Jippie (female tigers). I have to determine their cortisol levels and the technique that I used is a modified method of radioimmunoassay (RIA), called RIA-SPA (scintillation proximity assay).
Radioimmunoassay, sound familiar? Ya, we have learnt the theory before but not practical. I am taught from basic what RIA is about and started to do the assay. RIA is a technique used to determine the concentration of an unknown sample. It employs the use of radioisotopes to label antigens and these labeled antigens (known concentration) will compete with the non-labeled antigens (the sample or standard) for the limited number of antibody binding sites. There are 5 steps in RIA: Preparation, purification, competition, separation and computation. The principle of RIA-SPA is the same as that of RIA, the only difference is that RIA-SPA eliminates the need for a separation step and the addition of scintillation fluid.
Separation is needed in conventional RIA to separate the bound fraction from the free fraction. Bound fraction includes bound labeled antigens and bound non-labeled antigens. Liquid scintillant is then added to the bound fraction. Only the labeled antigens will emit radiation which causes the scintillant to fluoresce. If there is no separation of the fractions, the scintillant will also bind to the free-labeled antigens, leading to inaccuracy of results. The fluorescence will be measured in a beta-scintillation counter (when beta-emitting isotope is used) or gamma-scintillation counter (when gamma-emitting isotope is used). 3H is a weak β-emitter in an aqueous environment thus it is brought close to a scintillant in order to fluoresce and be measured in β-scintillation counter. In the absence of scintillant, the radiation energy of the radioactive substance is dissipated and lost in the solvent.
In RIA-SPA, SPA reagent is used. It contains second antibody bound to fluomicrosphere. Fluomicrosphere is a bead that contains scintillant. The bound antigens (both labeled and non-labeled) will be immobilized on the fluomicrospheres and because of the close proximity between the bound labeled antigens and fluomicrospheres, the radiation produced by the labeled antigens will stimulate the embedded scintillant to emit light. The bound non-labeled will not cause the scintillant to emit light as there is no radiation produced. For free labeled antigens, the radiation energy is absorbed by millions of surrounded solvent molecules and is dissipated as heat. This does not affect the fluorescent process thus separation of bound and free fractions is unnecessary.
Cortisol test
Preparation of reagents
1. Sample: Urine sample was extracted using dichloromethane. The purpose of extraction was to remove steroid-binding protein that will interfere with the measurement of hormones.
2. Standard: The standard, which was provided in a bottle, was diluted into varying concentrations. Standard is a known concentration of non-labeled antigens (cortisol).
3. Labeled radioactive antigen: ³H Cortisol was used in this assay. A fixed concentration of labeled antigen was used and the radioactivity was set around 10,000cpm.
4. Antibody: Known concentration of rabbit antibodies was used.
5. SPA reagent: It contains second antibody bound to fluomicrosphere (embedded with scintillant).
Competition
The assay was first performed using the standard, antibodies and labeled antigens to determine the dose-response curve. The standard, with varying concentrations, was added to the respective assay tubes (in duplicates). It was followed by the addition of labeled antigens (³H Cortisol), antibodies and SPA reagent to all the tubes. The tubes were incubated overnight.
Computation
After incubation, the tubes were counted in a beta-scintillation counter. This counter would measure the radioactivity of bound labeled antigens. The results obtained were plotted into a dose-response curve.
This is an example of a dose-response curve. Dose refers to the concentration of non-labeled antigens and response refers to the amount of bound labeled antigens. Because of the competition binding to the antibodies, the amount of bound labeled antigen is inversely proportional to the amount of non-labeled antigen. Higher amount of non-labeled antigens will displace more of the labeled antigens from the antibody thus resulting in less bound labeled antigens (lower response).
Assay was then continued on the samples. All the reagents and steps performed were the same as mentioned above (sample was used instead of standard). After measuring the radioactivity of an unknown sample, the result can be compared and interpolated from the standard curve and the cortisol concentration in the urine smaples can be determined.
Beta-scintillation counter
Retrieved on June 06, 2009 from website http://www.gla.ac.uk/media/media_61475_en.jpg
The end! =D
Zhang’e
0704086H
TG02
This comment has been removed by the author.
ReplyDeleteHello Zhang E,
ReplyDeleteso u visited the zoo? thanks for sharing their names. hahaha.
anyway. i'm curious. how do you collect their urine?
and erm
"Preparation of reagents
1. Sample: Urine sample was extracted using dichloromethane. "
do you mind explaining this?
so RIA and RIA-SPA. is there a reason why RIA-SPA instead of RIA is conducted?
Yanhong
tg01 / 0703979E
HELLO ZHANG`E! omg, u get to play with white tigers? tats awesomeeeee!
ReplyDeletehmm. i'm quite confused, the RIA and RIA-SPA both uses the scintillant right? den whats the difference ah?
Joanna Yeo!
0702054H
Reply to yanhong
ReplyDeleteHi yanhong! Sorry for the late reply! Anyway, I didn’t visit the zoo. It will be fun if I have. Haha.. Anyway, the urine was collected by the zoo keeper and sent to our lab. Although the white tigers are kept together, I heard that they are trained and they have their own “toilet”. Therefore, the zoo keepers are able to collect the urine.
Qn1
Dichloromethane is a solvent that is used frequently to extract hormones.
In the urine, the hormones are bound to the proteins (steroid-binding proteins). When the hormone is bound to the protein, we are unable to measure the level accurately. It has been researched that the proteins will somehow interfere with the assay. Therefore, we use DCM to extract (pick up/absorb) the hormones and separate the proteins from the hormones. This will yield a more accurate analysis of hormones.
Steps in urine extraction
1. 200ul of the urine samples were added to the tubes
2. 1ml of DCM was added to the tubes, vortex and centrifuged for 20 minutes
Vortex: The purpose was to mix the urine and DCM together and DCM will extract the hormones out.
Centrifuge: The purpose was to separate the hormones and proteins
3. The aqueous phase was aspirated and discarded
After centrifuge, aqueous phase was found on top of the organic phase. Aqueous phase contained all the proteins and was discarded
4. The organic phase was placed in the water bath at 60°C and blew dried using the blower (nitrogen)
Organic phase is a clear layer which contained the hormones and DCM. It has been researched that solvent will also eventually interfere with the hormones thus we dry the solvent
5. The dried tubes were reconstituted with 400μl of Buffer ‘S’
Qn2
RIA-SPA, as explained above, it eliminates separation step and the addition of scintillation fluid.
In normal RIA, we have to do separation to separate the bound fraction and free fractions (explained in para 3). There are different types of separation techniques used but each technique involves a series of steps.
In RIA-SPA, a SPA reagent is used. Separation technique is not needed (explained in para4). Hence, there are less manual steps in the method and this reduces human error (eg, add wrong reagent or the amount of reagent added is inaccurate). RIA-SPA is also faster and less tedious.
Reply to Joanna
Whoo! Hello Joanna! Haha.. Nope, of course I didn’t get to play with the tigers. I don’t wish to get bitten by them! =X
Back to your question. Ya, both uses scintillant but RIA-SPA is a reagent that is bought commercially and it already contains the scintillant. For normal RIA, we have to add the scintillant ourselves. It involves one more manual step. But the major difference between the two methods is the need for a separation step in normal RIA. Normal RIA requires separation and the addition of scintillant (in total, there is a series of steps to be performed), however, in RIA-SPA, only a SPA reagent is needed (just one step). The SPA reagent act like a “separation reagent” (as explained in para 3 and 4 of the blog entry). Thus, using RIA-SPA, it is more convenient, faster, less tedious and prevent human error.
I had explained to yanhong about this too (you can read what I had replied to her) =D
Zhang'e
0704086H
TG02 =D
Hi, may I know more in detail about what is a scintillant used for? I'm not really clear about its function.
ReplyDeletethanks.
Muna
0703791D
Hello Muna,
ReplyDeleteScintillant actually means spark or a flash. It is actually about physics. Its function is to amplify the radioactivity of the beta-isotopes as the radiation that they emit is very low and hard to detect. Therfore, scintillant is used to amplify the radiation (produced by the beta isotopes)so that it can be detected.
Zhang'e
0704086H
TG02