Clin. Biochem. 11 (3) 97-100 (1978)

• A Convenient Method for the Determination of Non. Specific Binding In Commercially Available Solid Phase

Iodine Labeled Radioimmunoassay Kits PAUL CARTER

The Radioassay Office, Delaware Division, The Wilmington Medical Center, Wilmington, Delaware 19899, USA

(Accepted September 26, 1977)

CLBIA, 11, (3) 97-100 (1978) Clin. Biochem.

Carter, Paul

The Radioassay Office, Delaware Division, The Wilmington Medical Center, Wilmington, Delaware, 19899, U.S.A.

A CONVENIENT METHOD FOR THE DETER- MINATION OF NON-SPECIFIC BINDING IN COMMERCIALLY AVAILABLE SOLID PHASE IODINE LABELED RADIOI,MMUNOASSAY KITS

57Co-cyanocobalamin is substituted for iodine labeled antigen in three commercially available solid phase radioimmunoassay kits to evaluate the extent of non-specific binding by determination of the supernatant and residual fraction of unbound ligand and thus obtain a value for corrected binding throughout the range of the calibrated standard curve. In the present state of technology correction appears to have a trifl ing effect on the overall accuracy of the assay; however, the method as de- scribed allows the laboratorian to confirm for him- self when testing a new product.

MOST OF THE OUTPUT FROM THE BURGEONING NEW ART

of radioassay is being accomplished through com- mercial ki ts because the prepara t ion and isolation of highly pur i f ied tagged ant igens and the i r correlat ive ant ibodies is not a prof i table venture for a modest clinic to undertake unless the labora to t ry production volume is excessively large. About 25% of all k i t manufac tu re r s offered a solid phase methodology as revealed in a recent survey of the ten most frequently performed tes t s in April , 1976. This f igure had in- creased to about 28% when a second census was taken six months later. Of the more troublesome aspects in immunoassay non-specific binding remains par- amount ' " . Var ia t ion is a t t r i bu ted to a number of causes such as contaminat ion by extraneous radio- active substances, coupling ins tabi l i ty of ant ibody to insoluble part icle polymers or inadequate r ins ing of the part icles to which ant ibody is conjugated. In adsorpt ion techniques manufac ture rs advise ki t users to per form "correct ions" by subs t i tu t ing buf fe r for an t ibody; however, in some solid phase methodology the s ta t ionery inclusion of the antibody to the react ion vessel i tself precludes this approach and the extent of non-specific b inding is ignored. This communication allows the labora tor ian to judge for himself by a

*This communication was delivered in par t at the Annual Meeting Canadian Society of Clinical Chemists, Van- couver, June 1977

simple a l ternat ive where sTCo-cyanocobalamin which is unbound to the solid phase is subst i tu ted through the course of the reaction to determine the super- na tan t and residual f ract ion of free l igand and obtain a corrected es t imate in order to monitor the severi ty of non-specific binding throughout the concentration range of the assay. The present s ta te of the tech- nology is revealed through an evaluation of three commercial products.

MATERIALS AND METHODS

Chemicals unless otherwise specified were obtained from Fisher Scientific, King of Prussia, Pa. 19406. All measurements were performed on model 1185 series auto- matic gamma counting system, Searle Analytic, Inc., Des Plaines, Ill., 60018.

(1) IMMUNOPHASE T M DIGOXIN 125I Radioimmuno- assay (RIA) test kit was obtained from Corning Medical Diagnostics, Medfield, Ma, 02052; (2) GAMMACOAT CORTISOL ~ I RIA test kit was obtained from Clinical Assays, Inc., Cambridge, Ma, 02142; (3) SPAC T M DIG- OXIN 1~I RIA kit was obtained from Mallinckrodt Nuclear Pharmaceuticals, Hazlewood, Mo, 63042 (4) aqueous solution of radioactively labeled 57Co-cyanocobal- amin containing less than 1 /~Ci was obtained from Bio- Rad Laboratories, Richmond, Ca, 94804 (5) phosphate buffer was prepared by mixing 0.4176 g K.+HP04 -b 0.083 g Na I'I2PO4 + 0.877 g NaC1 + 0.25 g bovine serum albumin (;BSA) Fraction V, Sigma Chemical Co., St. Louis, Mo., 63178 and diluting to 100 ml to yield 7.1 pH (6) TRIS buffer was prepared by mixing 0.120 g Tris (hydroxymethyl) aminomethane + 0.877 g NaC1 in water and diluting to 100 ml to yield 7.4 pH (7) working sTCo tracer for IMMUNOPHASE dixogin assay was prepared by adding 200 ~l 57Co-cyanocobalamin to 1.0 ml phosphate buffer (8) working ~TCo tracer for GAMMACOAT eor- tisol assay was prepared by adding 200 ~1 57Co-cyano- cobalamin to 2.0 ml TRIS buffer (9) working STCo tracer for SPAC digoxin assay was prepared by adding 200 ~1 sTCo-cyanocobalamin to 20 ml phosphate buffer (10) RIA control serum I and II was obtained from Ortho Diagnos- tics, Raritan, N.J. 08869 (11) RIA Control serum II was obtained from Lederle Diagnostics, Pearl River, NY, 10965; (12) TRI-RAC T M I RIA control serum was obtained from Dade, Div. American Hospital Supply Corp,. Miami, FA, 33152.

Assay for Non-Specific Binding

(a) for CORNING IMMUNOPHASE products:

Label duplicate antibody tubes for standard concen- tration as provided by the manufacturer, and pipet the appropriate standard volume into the correlatively marked antibody tube. Substitute the identical volume of the appropriate working ~rCo-cyanocobalamin solution for "iodinated tracer" to each tube. Incubate and centrifuge as described then carefully decant the supernatant from each tube into a 12 x 75 mn polyethylene test tube

98 CARTER

TABLE 1

DETERMINATION OF NSB DIGOXIN IMMUNOPHASE TM

Total Counts = 9740 cpm

NSB Bcorr NSB bap (cpm) % T

conc. ng/ml 102x bap (cpm) (cpm) ~ T Post-Wash Post-Wash

0 5.48 6706 6531 1.8 6543 0.1 0.5 5.46 6471 6282 1.9 6317 0.4 1.0 6.37 5235 4928 3.1 5008 0.8 2.5 6.15 3751 2826 4.4 3064 2.4 5.0 6.65 2101 1557 5.6 1931 3.8

TABLE 2

DETERMINATION OF NSB DIGOXIN SPAC TM

Total Counts = 2026

NSB Bcorr NSB bap (cpm) % T

conc. ng/ml 10~x bap (cpm) (cpm) % T Post-Wash Post-Wash

0 3.81 12217 11895 1.5 11930 0.2 0.5 3.17 10414 10079 1.6 10130 0.2 1.0 3.51 8405 7961 2.2 8057 0.5 2.5 3.75 5380 4789 2.9 5083 1.4 5.0 3.59 3578 2944 3.1 3384 2.1

(Falcon Plastics, Los Angeles, Ca.) and count for 1 minute. Measure the residual activity" in the reaction vessel by counting for 10 minutes.

(b) /or GAMMACOAT CORTISOL ml RIA:

Pipet 1.0 ml TRIS buffer followed by 100 ~l of the appropr ia te denatured s tandard to each of duplicate labeled antibody tubes. Vortex and incubate the specimens for 10 min. and add 100 ~1 of appropr ia te working 57Co- cyanocobalamin t racer to all tubes. Af t e r la tera l shaking for 45 min. decant the superna tan t to a 12 x 75 mn poly- ethylene test tube, wash with TRIS, adding the washings to the supernatant . Count the washing and residual vessel act ivi ty for 1 rain. and 10 min. respectively.

(c) for SPAC DIGOXIN ~ I RIA: Pipet 1.0 ml TRIS buffer followed by 100 ~1 of the

appropr ia te s tandard in each tube. Add 1.0 ml of appro- pr ia te sTCo-cyanocobalamin t racer to all tubes. Mix and incubate for 1 hour a t 37°C and decant the superna tan t to a 12 x 75 mm polyethylene tube counting the super- na t an t and residual act ivi ty for 1 min. and 10 min. respectively

Simultaneous to all assays for non-specific blanking perform standardizat ion for each kit assay exactly as described by the manufacturer .

RESULTS

Non-spec i f i c b ind ing is de f ined he re a r b i t r a r i l y as t h a t po r t ion of f r ee labeled a n t i g e n which f i n d s i t s e l f m e a s u r e d wi th the bound f r a c t i o n due to i m p e r f e c t p rocedura l s e p a r a t i o n of the bound and f r ee f r ac t ion . I t m a y a r i s e because of (1) incomple te a d s o r p t i o n me thodo logy (2) sol id phase i n t e r s t i t i a l occlusion (3) i m p r o p e r d e c a n t a t i o n of the aqueous phase (4) i m p r o p e r wash ing . Obvious ly as the to ta l concen t r a - t ion of f r ee a n t i g e n in the sys t em inc reases as is the case when h i g h levels of unknown a r e measu red , a p~'iori the non-spec i f i c b ind ing , i.e. the f r ee labeled a n t i g e n t r a p p e d in the bound phase , m u s t p r o p o r t i o n - a t e ly inc rease as well. Th is v a r i a t i o n can be de te r -

mined r ead i l y by s u b s t i t u t i n g into t he s t a n d a r d i z a t i o n p rocedu re a f o r e i g n t r a c e r which r e m a i n s a lways f r ee t h r o u g h o u t the course of the reac t ion . A l t h o u g h 6°Co has been s u g g e s t e d as a vo lume m a r k e r in some RIA, r ecen t s tud ies s u g g e s t t h a t some heavy me ta l s t end to b ind se rum p r o t e i n s l ead ing to an e r roneous ly h igh pe r cen t age o f r a d i o a c t i v i t y r e m a i n i n g in the bound phase '2~. F o r th i s r eason and because of i t s ava i l - a b i l i t y in V i t a m i n B12 assay , wh ich is p e r f o r m e d rou t i ne ly in m a n y serv ice l abo ra to r i e s , 57Co-cyanoco- b a l a m i n has been se lec ted as an a p p r o p r i a t e a l t e rna t i ve .

The co r rec t ed labe led concen t r a t i on in the bound phase can be exp res sed a s :

(1) Bcor r ---- bap -- Fx , w h e r e bap is the a p p a r e n t labeled concen t r a t i on in the bound phase as measu red , F is the f r ee labe led an t igen , x is the d i s t r i b u t e d f r a c t i o n of f o r e i g n t r a c e r be tween the bound and f r ee phase , and consequen t ly F x is the non-spec i f i c b ind ing . S u b s t i t u t i n g ( T - B c o r r ) ---- F, w h e r e T is the cpm of to ta l r a d i o l i g a n d in the sy s t em and solving.

(2) Bcon" = bap - Tx 1 - x

Since x can be exp res sed as x~/(x, + xl) w h e r e x~ and x, a r e cpm of t a g in t he solid and l iqu id phase respec t ive ly .

(3) Bcorr = bap(x, +x,) - Tx. X!

R a d i o a c t i v i t y non-spec i f i ca l ly bound a t any concen t r a - t ion can be o b t a i n e d f r o m E q u a t i o n (1) and i t is o f t en exp re s sed as a p e r c e n t a g e of t o t a l counts in the sys tem.

Fx bap - Bcorr (5) ~ X 100 = T X 100

C O N V E N I E N T M E T H O D F O R T H E D E T E R M I N A T I O N OF N O N - S P E C I F I C B I N D I N G 99

U

T/B

so- / /

/ . /

¢ s - / • /

/

4.0- / " /

/ * I # / .

/ * I

2.0

/ /

F i g . i - - C a l i b r a ~ n c h a r t f o r d i g o z i n a s s a y : u n c o r r e c t e d ( e ) f o r n o n spec i f i c b ind ing r = 0.99,~5, a f t e r w a s h i n g ( - - ) ~" --- 0.9935, correc ted ( - ) r - - 0.9965.

L 0 r . - ~ - i i i I I 1.0 2.0 3.O 49

ng/ml Digoxin

Table 1 illustrates the mathematical parameters required for the determination of non-specific bind- ing in the digoxin assay where IMMUNOPHASE TM

technology is utilized. An additional procedural step has been added to show the effect of washing. After activity in the reaction vessels was determined, test tubes were immediately removed from the counter and washed with 2.0 ml of phosphate buffer, recentrifuged, redecanted and recounted. The carry over loss from rinse is expressed as the % free in bound post wash.

TABLE 3

DETERMINATION OF NSB GAMMACOAT

Total Counts -- 15216

NSB conc. (ng/ml) 10Sx bap (cpm) Bcorr cpm % T

0 0.46 5923 5879 0.2 1 0.48 4797 4749 0.3 3 0.47 3705 3651 0.4

10 0.45 2185 2128 0.4 25 0.46 1525 1462 0.4 60 0.48 944 875 0.5

Table 2 and Table 3 illustrate the variation when SPAC TM and GAMMACOAT technologies are eval- uated for the same parameters. Rinsing the solid phase is a part of the GAMMACOAT procedure therefore no measurements were made of activity of the bound fraction before the last addition of TRIS buffer. By including a single wash in the IMMUNO- PHASE TM and SPAC TM methodologies the correction for non-specific binding could be reduced considerably.

About 6% free tracer remains in the test tube after decantation to separate the bound from the free phase in IMMUNOPHASE TM methodology as illustrated in the second column "102x '' in Table 1. Correlative values for SPAC TM and GAMMACOAT are estimated at 3.56 and 0.47 mean % respectively. This distribu- tion of free tracer between the solid and liquid phases can be used as a monitor of the reproducibility of decantation technique. It is comparable in the IM- MUNOPHASE TM and SPAC TM methodology and yields a variation coefficient of 8.87% and 6.97% respec- tively.

Variation of non-specific binding throughout the ranges of the standard curve is differentiated by a

100 CARTER

TABLE 4

ASSAYED SERUM BEFORE AND AFTER CORRECTION FOR NON SPECIFIC BINDING

Post MFG Uncorrected Wash Corrected C.V. Intra-Assay

1. 0.50 0.56 0.48 9.7 ca. 8.6 2. 3.60 3.20 4.00 11.0 ca. 8.6

IMMUNOPHASE~

SPACrM

GAMMACOAT~

1. 0.62 0.73 0.70 8.2 ca. 6.3 2. 4.7 4.5 4.30 6.2 ca. 4.5

1. 103 92 5.1 ca. 7.8 2. 446 396 6.0 ca. 4.0

comparison of corrected plots as illustrated in Figure 1. The theoretical implication can be conveniently shown through linearized reciprocal bound plots. I f antibody binds homogeneously and the antigen con- centration has not been minimized then there should be considerable linear response over wide ranges of concentration. In practice however, there is always some underestimation as the antigen concentration tends to increase leading to slight curvature. While the concentrations listed here have not been extended far enough to exacerbate the situation still there is already evidence of slight improvement in the linear- ity correlation coefficient after correction for non- specific binding has been made.

Commercially available sera which had been pre- assayed at low and high antigen value were assayed as described by the manufacturers and as described in this communication in which standards and un- knowns were treated identically. Results of Table 4 indicate that variation is not appreciable since values lie close to the accepted precision claimed by the manufacturer. It is felt therefore that in the present state of technology correction is unnecessary although this is a conclusion which the laboratorian will want to confirm for himself when testing a new product. Provided adequate decantation technique has been observed the correction for non-specific binding appears to have little effect on the overall accuracy and precision of the assay.

DIscussioN

Each of the commercial products selected for this study is in its way typical of the present state of solid phase technology. In GAMMACOAT methodology a thin layer of antibody is applied within a plastic tube whereas in the SPAC T M methodology effort is made to increase tube to tube antibody uniformity by immersing the reaction portion of the vessel in a protective coating in order that the antibody can be covalently bound to the tube surface wall ̀ 3). IMMUNO- PHASE T M methodology utilizes vials as reaction tubes to which immobilized antibody succinylated on glass particles and buffer have been predispensed c~" 5). Non-

specific binding in the IMMUNOPHASE T M and SPAC T M technology is almost identical although a slightly lower value is obtained via GAMMACOAT. This is not surprising in light of the large surface area generated by the f i rs t two techniques giving rise to increased interstitial occlusion, which is that re- sidual adsorption remaining after correction for washing. In any case the values are comparable to those obtained in this laboratory when IMMUNO- BEAD T M reagent (Bio-Rad Laboratories), a goat anti- rabbit gamma globulin immobilized on a polymeric buffered resin bead was dispensed in the absence of antiserum for the assay of thyroid stimulating hormone. These values range from 0.2% to 5.6% of the total activity in the system depending on whether a washing step has or has not been included in the protocol. Published methods which employ antibody coated tubes generally advise one washing step (''*), though it is becoming increasingly popular to avoid this task in commercially available kits by the justifi- cation that if the supernatant from standards and un- knowns is decanted with identical technique then the residual errors ought to cancel out. To a point this is t rue; however recently when a dual label was applied to a dual counting system equipped to measure both levels simultaneously and reduce the data after cor- rection for residual unbound antigen, workers ob- tained values with up to 50% improvement in precision (8).

REFERENCES

1. Pizarro, M. A., and Kalonowski, J. (1972). J. S~eroid Biochem. 3, 349-356.

2. Peters, T. L., and Vantrappen, G. R., (1977). Clin. Chem. 23, 1052-1053.

3. Cart, K. J., (1969). Aeta~ Endoc~rinol. (Supp) 142, 222-230.

4. Weetal, H., (1970). Biochem. J. 117, 257-61. 5. Rushing, J. F., Luttrell, G. H., and Papariello, G. J.

(1976) Anal. Chem. 48, 1211-1215. 6. Cawley, L. P., Goodwin, W., Moeder, M. and Dibbern,

P., (1974) Clin. Chem. 20, 376-382. 7. Nicklin, M. G., Hewitt, J. V., and Watson, D., (1976),

J. Clin. Path. 29, 561-564. 8. Miller, W. W. (Abstract) (1975) Clin. Chem. 21, 1012.