Herbert H. Kohl' and W. 8. Wheatley Adourn Unwers ty Auburn. A.abama 36830
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A Laboratory Introduction to Competitive Protein Binding and Radioimmunoassay
We describe in this paper a simple inexpensive experiment designed to measure the levels of cortisol in a serum or plasma sample. This experiment can be efficiently performed in less than 3 hours. The experiment is designed to acquaint the undergraduate chemistrv student with the theorv and technique'hf hoth mdia,imkunoassay, RIAJ and competitive rotei in bindinr! analvsis. CI'HA.'h number of review articles B Aalready appeared (1-3). concerning RIA ~ ~ ~ - C Phave Although a great many experiments can be designed for many radioimmunoassays, the considerable expense involved in purchasing labile antibodies and other reagents often precludes their use in laboratory teaching situations involving large numbers of students. The 24,48, or 72 hours often required to reach equilibrium in the competition reaction between labelled and non-labelled hormone for antibody is also a disadvantage in many laboratory teaching situations involving limited time and where the entire experiment must be completed in one laboratory session. In contrast, equilibrium in this experiment is attained in well under 30 min and the cost of plasma derived binding protein is minimal. College level chemistry students today should he familiar with CPBA and RIA because of their extensive analytical capacity and widespread use in chemistry. These techniques are virtually revolutionizing the field of clinical chemistry today. A recent bibliography of RIA literature lists over 1700 publications ). -.( 4neory I
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RIA and CPBA are relatively new techniques enabling substances to be measured in biological fluids a t levels smaller than the nanogram range. These techniques utilize radioactive substances which can be quantitated with both great sensitivity and accuracy. RIA makes use of the specificity inherent in antigen antibody binding while CPBA utilizes non-imare readilv available protein (7). T h e above binding. proteins . in serum or plasma derived frnm \rarious human and anunal sourcrs, whilt: srwcific antil~ndirsmust often be purchdsed from commercial sources. RIA has several advantages over CPBA in that it is both more sensitive and specific although CPBA is more economical for a limited number of analyses. CPBA also requires sample deproteinization (see discussion) while RIA does not. The principles of RIA and CPBA can be summarized by the comoetine reactions shown below. The reactions describe the competitron of both radioactively labelled and native hormone, or other antigen, for a limited number of binding sites on a specific reactor, either antibody or binding globulin. Basic Reaction Representing Radioimmunoassay + Ab Ab-F* + Ab-F F* + F (Antibody) (Antibody bound (Unbound or free radioactive and nonradioactive and nonradioactive ligands) radioactive ligands) Basic Reaction Representing Competitive Protein Binding + BG BG-F* BG-F F* + F (Binding (Binding globulinbound (Unbound or free radioactive and nonradioactive and nonglobulin) radioactive ligands) radioactive ligands) Both RIA and CPBA exploit the ability of unlabeled hor-
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612 / Journal of Chemical Education
mones to compete with radioactively labeled hormone for limited binding sites on an antibody or binding globulin. T h e binding of the labeled hormone is thus reduced because of displacement. RIA and CPBA are considered to be forms of disnlacement analvsis ( 8 ) or saturation analvsis ( 9 ) . Both ti& of antigen (l~hcledand unlabeled) comp&e for binding sites on a specific reactor (antilwdv or hindinr alohulin, in accordance-with the law of mass action. As a&ult of this competition the ratio of antibody-bound radioactivity to total radioactivity (denoted BIT), is diminished as the concentration of unlabeled hormone is increased. The hormone concentration in a n unknown sample is obtained by comparing the observed (BIT) with that produced by standard solutions containine known amounts of hormone. One of the essential requirements of these types of assays is the ability to separate bound from free hormone. Another essential requirement in only CPBA is the removal of any binding proteins present in individual samples. In the present experiment the bTlnding proteins which a;e present in unknown serum samples are removed by alcohol precipitation. This is essential sinck variable amounts of binding proteins are contained in differing samples. This would in itself produce changed levels of bindine. An aliauot of the alcohol s u ~ e r n a t a ncontainine t the extracted cortisol is then taken 1 0 dr)~ncisand a constant amuunt of suluhlt~I~indinrrlobulin containinr! radioarri\,e cortisol (CBG, see methoisj is added. Equilibration of the comoetition reaction takes d a c e in an ice bath and is complete within 30 min. The protein-bound and unbound fractions are separated by means of absorbing the unbound (or free) frac~ ~~ 't~ ~i h~ t ~ of i btracer ~ t i ~ ~ tion to insoluble ~ ~ ~l l ~particles. cortisol is then determined by counting supernatant samples containing radioactive cortisol bound to cortisol binding globulin (CBG). ~h~ steroid supplied from the unknown samDle disDlaces a oo*ion of the tracerbound to CBG and the ment and steroid concentratibn is then established by a standard curve. Methods and Materials Tritioted Cortisol: This reagent was purchased from the New England Nuclear Corporation as (Hydrocortisone lA3H(N)40 Cil mmole). We diluted 250 &i to 50 ml in 5:l-benzene:ethanol. This solution was stored refrigerated in a plastic vial. Standard Cortisol Solution: A stock standard of 100 urlml was oreoared bv weiebine 10me of cortisol (~aliforniaBiocbe&als) into
working standard concentrationis 0.1 rglml and was used to prepare standard curves. Radiooetiue Cortisol Binding Globulin Solution (CRG): Onemilliliter aliquots of tritiated cortisol solution (5 #Ci) are added to individual glass test tubes and evaporated with air in a 37'C water All correspondence to this author. %Theabbreviations used are: RIA, radioimmunoassay; CPBA, competitive protein binding analysis; *F, radioactive free ligand; F, non-radioactive free ligand; Ab, antibody; BG, binding globulin; BG-F, binding globulin bound ligand; BG-P*,binding globulin bound radioactive ligand; Ab-F, antibody bound ligand; AB-F*, antibody bound radioactive ligand; BIT, ratio of bound aver total radioactivity; CBG, cortisol binding globulin; CV, coefficientof variation;
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