67

Clinica Chimica Acta, @ Elsevier/North-Holland

86 (1978)

67-72

Biomedical

Press

CCA 9382

A RAPID RADIOIMMUNOASSAY

W.E. BLANK-LISS,

FOR HUMAN CY-FETOPROTEIN

P.C.W. LAI, D.M. HAY a and F.L. LORSCHEIDER

Divisions of Medical Physiology and a Obstetrics University of Calgary, and the Alberta Children’s Alberta T2N lN4 (Canada)

(Received

December

and Gynaecology, Hospital Research

*

Faculty Centre,

of Medicine, Calgary,

5th, 1977)

Summary Our previous double antibody radioimmunoassay for human AFP was run at 4°C and required 48 h for completion. A method is described which permits the assay to be performed within 5 h due to an increased incubation temperature and the use of polyethylene glycol to precipitate the antigen-antibody complex. The modified radioimmunoassay was standardized against an international reference preparation of human AFP. Characteristics of this assay are discussed.

Introduction Alpha-fetoprotein (AFP) is considered to be a useful diagnostic marker in hepatic malignancy and pathological pregnancy [ 11. In our studies on maternal serum AFP in normal and abnormal pregnancies [2,3] the AFP levels were determined by radioimmunoassay (RIA) as previously described [ 41. However, expansion of our clinical screening program necessitated that we modify our RIA in order to shorten the assay time. The former assay, which was based on a double antibody system, could be completed within 48 h. Another objective was to standardize our assay against the recommended international reference preparation for human AFP [ 5,6]. Materials and methods (i) Reagents Rabbit anti-human AFP and a standard human AFP were supplied by Behring Diagnostics (Montreal, Quebec). The international reference preparation (IARC standard No. 72/225) of human AFP was donated by the Intema* To

whom

correspondence

should

be addressed.

68

tional Agency for Research on Cancer (Lyon, France). Pooled cord serum, which was calibrated against both the IARC and Behring standards, was used as our secondary standard of human AFP. Other reagents used were carrier-free ‘*‘I (Amersham Searle Corp., Oakville, Ont.; activity 100 mCi/ml), bovine serum albumin (BSA) and bovine gamma globulin (BSG) (Calbiochem, San Diego, Ca.) and polyethylene glycol (PEG; mol. wt. 6000-7500) (Baker Chemical Co., Phillipsburg, Pa.). Purification of human AFP from cord serum was carried out in two steps; first by affinity chromatography [4] and secondly by prep~ative polyacrylamide gel electrophoresis (71. Details of iodination of purified AFP are as described earlier [ 41.

Standard human AFP was diluted with normal human serum. The working range of the standard curves was constructed with seven points between 5 and 224 ng/O.l ml. The 1251-labelled AFP was diluted with assay buffer containing 0.05 M sodium phosphate, pH 7.6, 0.5% BSA and 0.01% NaN3 to a concentration of approximately 13000 d.p.m./O.l ml. Labelled AFP could be used for 3 months. Rabbit anti-human AFP was diluted 1 : 1600 with the assay buffer (a final concentration during incubation in each assay tube of 1 : 6400) which was the dilution read from an antiserum titre curve which gave optimal assay precision. Table I lists the contents of each assay tube (10 X 75 mm) in order of addition. After incubation at room temperature for 5 h, 1 mg of BSG in 0.1 ml of the phosphate/BSA buffer and 0.5 ml of 16% PEG solution were added to each assay tube to precipitate the antigen-antibody complex. Each tube was stirred on a mixer and centrifuged at 3000 X g for 30 min at 4°C. The average initial radioactive counts were determined in 10 tubes selected at random and the supernatant was removed by aspiration from all assay tubes. The precipitates

TABLE

I

CONTENTS

(IN

ml)

OF

Reagent

EACH

RADIOIMMUNOASSAY

RIA

Binding

standards

12SI-AFP

Assay

buffer

0.1 -

human

DURING

INCUBATION Determination

Test samples in

nonspecific

the absence

Maternal

Diluted

of unlabelled

serum

amniotic

AFP

Normal

of

TUBE

*

0.1 0.1

or cord

**

precipitation

fluid serum 0.2

0.1 -I

0.1

0.1

serum Standard with

AFP

fdil.

0.1

-

normal

serum) Assayed

specimen

-

-

0.1

0.1

-

Labelled

AFP

0.1

0.1

0.1

0.1

0.1 0.1

0.1 0.1

0.1 -

Anti-human * For

AFP initial

* * Diluted

point

in assay

on standard buffer.

curve

only.

of

69

were counted calculator.

on a Searle

Gamma

Counter

type

1197 equipped

with an RIA

(iii) Recovery unalysis A cord serum specimen was calibrated for AFP concentration by radialimmunodiffusion using standard human AFP and an M-Partigen immunodiffusion plate (Behring Diagnostics). Varied amounts of the calibrated cord serum (8-265 ng AFPj5 ~1) were added to 0.1 ml of 10 different matem~ serum samples of known AFP concentration as well as to 0.1 ml of 4 amniotic fluid samples of known AFP concentration diluted with phosphate/BSA buffer. The samples were analyzed for AFP by RIA. (iv) Goat anti-human AFP Production and confirmation of specificity of goat anti-human AFP was carried out as we have previously described for goat anti-rat AFP f7] as an alternative to the commercially available rabbit anti-human AFP. Results (i) ~ec~pitution of antibody-boned AFP by PEG The precipitation of antigen-antibody complex as a function of PEG concentration revealed that 8% (w/v) PEG in final concentration was sufficient to insure complete precipitation of antibody-bound AFP. The nonspecific precipitation of labelled AFP was approximately 5%. BSG added in varied amounts from 0 to 5 mg/ml of total assay tube volume did not produce any noticeable differences in efficiency of precipitation of the antigen-antibody complex. (ii) Time and temperature of incubation The time and temperature effects on formation of the antigen-antibody complex were determined within a range from 3 to 28 h at 4,21 and 37°C. Formation of the complex was completed after 5 h of incubation at 21°C as indicated by percent binding of labelled AFP in the absence of unlabelled AFP. Nonspecific precipitation of labelled AFP did not change significantly with either time or temperature of incubation, (iii) Precision The average coefficient of variation for 5 different maternal serum samples containing AFP in the range of 8-100 ngf0.1 ml and assayed in replicate (n = 9) on the same standard curve was 3.7% {range: 2.2-4.8%). For 5 amniotic fluid samples diluted in a range of 15-150 ng/O.l ml and assayed in replicate (n = 9) on the same standard curve, the average coefficient of variation was 3.9% (range: 2.6-6.0%). The inter-assay variability was determined by measuring the same 5 serum and 5 amniotic fluid samples as above in triplicate on 5 different standard curves. The average inter-assay coefficient of variation for and for amniotic fluid 7.5% maternal serum was 4.5% (range: 1.3~8.0%) (range: 3.4-9.8%). (iv) Recovery analysis The average analytical

recovery

was 96.8% (range: 85-114%)

for 7 maternal

70

serum and 3 diluted amniotic fluid samples to which known amounts of AFP were added (7-170 ng AFP). Samples with concentrations of AFP above 250 ng/O.l ml had a higher recovery variability with a tendency for recovery to be as much as 140%. (u) Proportionality of results as a function of dilution Five to seven dilutions of cord serum, maternal serum, amniotic fluid and hepatoblastoma serum, each in a range from IO-400 ng AFP/O.l ml were assayed by RIA. Under actual assay conditions, deviation of AFP values from linearity of dilutions was observed for AFP concentrations above 180 ng/O.l ml in hepatoblastoma serum and above 250 ng/O.l ml in cord serum and amniotic fluid. (vi) Correlation between RIA results with PEG vs. secondary antibody Twenty different maternal serum samples and ten amniotic fluid samples

I

100

200

ng AFP/tube

(0) 1

I

67

134

m/tube(i) 1

1

I:107 dilution

I:53 (x)

Fig. 1. Radioimmunoassay standard curve comparison Values are corrected for nonspecific precipitation. lA, I.A.R.C. reference standard. SeNm; o--13,

of three preparations containing human AFP. standard cord Behring standard; X-X,

71

were assayed by a double antibody method [4] and the modified RIA employing PEG. Varied concentrations of AFP were chosen from 5-135 ng/O.l ml. Concentrations of AFP measured by both methods were highly correlated (r = 0.98, p < 0.0001). (vii) Standardization

Fig. 1 shows that Behring standard AFP, our secondary standard of pooled cord serum and the IARC reference standard gave virtually identical standard curves when used in the modified RIA. The AFP concentration of the IARC standard is designated as 50 000 I.U. per ml [6] and the Behring preparation of standard human AFP, which has hitherto been used in our RIA, contains 150 I.U./O.l ml for the point equivalent to 224 ng/O.l ml on the standard curve. Our secondary standard of cord serum which is to be introduced into the RIA is thus calculated to contain 71400 I.U./ml (or 106.6 I-18AFP/ml according to the Behring standard). (viii) Goat

anti-human

AFP

The optimal RIA titer for our goat anti-human AFP was found to be 1 : 12 800 as compared to 1 : 1600 for the commercial rabbit anti-human AFP. The results of using both rabbit and goat antiserum under actual radioimmunoassay conditions were compared in 23 maternal sera and 18 amniotic fluid specimens. AFP values obtained with either antiserum were highly correlated (maternal serum, r = 0.97, p < 0.0001; amniotic fluid, r = 0.99, p < 0.0001). Discussion Use of PEG to separate free and antibody-bound antigen has been employed for radioimmunoassays of peptide hormones [8] as well as for human [9] and rat [ 71 AFP. When this modification is incorporated into our previous RIA for human AFP [ 41, the incubation time is reduced from 2 days to 5 h and there is a tolerable increase in nonspecific precipitation from 3% to 5%. Addition of all reagents, including PEG, in one stage as recommended by Vince et al. [9] could not be used without significantly reducing the slope of the standard curve. The present assay can be run at room temperature whereas at 4°C the 5-h incubation time is insufficient and the assay is not appreciably shortened by increasing the incubation temperature from 21 to 37°C. The advantages of PEG at room temperature permit one technician to complete the assay of one hundred serum specimens within one working day. The precision of the present assay is acceptable for maternal serum samples with an intra-assay coefficient of variation of 3.7% and an inter-assay variability of 4.5%. A higher inter-assay coefficient of variation of 7.5% for amniotic fluid samples can be explained by the necessity of additional dilution steps preliminary to assay. The working range of 5-224 ng/O.l ml on the standard curve was suitable for measuring concentrations of AFP found in maternal serum. Analytical recovery, reproducibility, and proportionality of results were optimal within this range. In assaying other specimens containing higher levels of AFP, such as amniotic fluid, cord serum and hepatoblastoma serum, it was necessary to

72

dilute samples to the range of the standard curve. Since the total protein concentration was reduced in the diluted specimens, thereby reducing the accuracy of the assay, 0.1 ml normal human serum was added to such samples after dilution to make the protein level comparable to that of serum. This procedure insured a relatively constant value for nonspecific precipitation and maintained the proportionality of assay values in diluted specimens. The correlation between 25 assay results determined manually and by computer using the Searle RIA calculator was 0.996 (p < 0.0001). Standardization of human AFP standard against the recommended IARC standard 72/225 will permit comparison of data between laboratories. Our published clinical results [2,3] can be converted to the international system by dividing our previous values by a factor of 1.49, which is the relationship of our AFP weight unit to the proposed international unit. Moreover, our large scale production of goat anti-human AFP has resulted in a higher titered antibody than any previously reported. Our antibody has proven useful in both the assay and isolation of human AFP. Acknowledgements

This investigation was supported by the Medical Research Council of Canada, Grant No, MA 5292 and a research grant from the Alberta Children’s Hospital Foundation. The authors are indebted to Dr. P. Sizaret, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69008, Lyon, France for providing the international reference standard for AFP. P.C.W.L. is the recipient of an M.R.C. Studentship. References Alpert,

E. (1976)

Hay.

D.M.,

Progr.

Forrester,

Liver P.I..

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Hancock,

R.L.

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Lorscheider.

F.L.

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GynawoI.

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D.M.,

Forrester,

Forrester, P.I.,

P.I.,

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Lai,

P.C.W.

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Lorscheider,

D.M.,

Lai,

P.C.W.

F.L.

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Lomheider.

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