An automated turbidimetric rate method for immunoglobulin assays

523

Clinica Chimica Acta, 88 (1978) 523-530 @ ElsevierlNorth-Holland Biomedical Press

CGA 9648

AN AUTOMATED TURBIDIMETRIC IMMUNOGLOBULIN ASSAYS

H. MALKUS Department (Received

*, P. BUSCHBAUM of Pathology,

RATE METHOD

FOR

and A. CASTRO

University of Miami, Miami, Florida 33152

(U.S.A.)

April 6th, 1978)

summary A turbidimetric rate method for the determination of immunoglobulins IgG, IgA, and IgM has been adapted to an automatic kinetic rate analyzer. The procedure can be run on mildly lipemic sera without correction for sample light scatter. We report correlations with results by an immunodiffusion method and a manual laser nephelometric technique. The automated rate procedure described provides a rapid, accurate, precise and sensitive way to measure immunoglobulins.

Introduction During the past few years there has been considerable clinical interest in developing convenient automated analyses for specific immunoglobulins [ 1,2]. Although the precipitin reaction was first identified by Krauss in 1897 [3] and the manual nephelometric quantitative analysis of specific proteins was described by Libby in 1938 [ 4,5], it has been the recent commercial availability of high quality monospecific antiserum that has led to the development of automated nephelometric and turbidimetric analyses. The automated equilibrium techniques developed on continuous flow instrumentation [6,7] and centrifugal analyzers [8,9] all require sample blanks due to nonspecific light scatter. In an attempt to correct for sample induced light scatter a reaction rate analyzer was adapted using an early post mixing reading [lo]. A rate method of detecting antibody immunoglobulin complexes was developed on a centrifugal analyzer modified with a laser for light scatter measurements [ 11-141. We have adapted the rate technique which offers the advantage of automatic blank correction to the Perkin Elmer KA-150 kinetic analyzer. The method described here gives excellent sensitivity and a large working range for immunoglobylins IgG, IgA and IgM. * To

whom correspondence should be addressed.

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Materials and methods Instrumentation We used a Perkin Elmer KA-150 Kinetic Analyzer equipped with the Auxilliary Control Panel (Perkin-Elmer Corp., Norwalk, CT 06856). The KA-150 has been designed to perform enzyme tests on serum utilizing the classical tworeagent methods. A lo-~.11aliquot of sample is diluted with 90 ~1 of water into cups carried in small magazines. Reagent pipettes deliver 50 ,ul of reagents 1 and 2 to the reaction cups. The pipettes operate on a displacement principle so that only the replaceable tips come in contact with liquid. All pipette tips are wiped both before and after delivery, The reaction mixture is stirred mechanically during the 24-s (second) interval between addition of reagent 2 and transfer to the photometer cell through a thermoelectrically controlled heat exchanger. The instrument we used was factory adjusted to perform analyses at 30°C. The diluted sample is preincubated with reagent 1 for 6 min prior to addition of reagent 2. The absorbance is observed continuously for 9 s. During this interval the average rate of absorbance change and the curvature is measured simult~eously. With the auxilliary panel a substrate mode can be used which will allow a 17% negative deviation from linearity expected for first order reactions. Error messages include exceeding the preset limits of expected curvature and checks against high and low absorbance thresholds. We used the following auxiliary panel settings: 340 nm; high absorbance threshold, 1.5; low absorbance threshold, 0; incubation, 24 s; direction, up; substrate; sensitivity, low; preincubator, off; scale factor, 4000. A Laser Nephelometer “PDQ” (Hyland Laboratories, Inc., Costa Mesa, Calif. 92626) was used according to manufacturer’s instructions. Reagents Antiserum. Antiserum to immunoglobulins G (lot 204J190), M (105 205HOSO) and A (105 2025060) were obtained from Kallestad Laboratories, Chaska, Minn. 55318. LAS-R kits for immunoglobulins IgG, IgA and IgM were purchased from Hyland Laboratories, Inc. Immunoglobin standards. The immunoglobin standards were obtained from Kallestad Laboratories (lot R055KlOO) and from Hyland (Iots 8554P002A, 8555P002A, 8556P002A, 8557P002A, 8558P002A and 8559P002A). Buffer solution Phosphate buffered saline (PBS, pH 7.2, 20 mmol/l in phosphate, 0.15 molfl in NaCl) was prepared by dissolving 9.0 g NaCI, 2.36 g NaZHP04 and 0.446 g NaH2P04 in 1 liter of deionized water. The solution was filtered through a 0.2 pm (av. pore size) filter (Gelman Filtration Products, Ann Arbor, Mich. 48106). Polyethylene glycol (PEG) diluents The solution used as reagent 1 on the KA-150 and as the antibody diluent used to make reagent 2 was prepared by dissolving 40 g PEG (mol. wt. 60007500) and 0.5 g NaN, in one liter of the buffer solution. The final reagent was filtered through a 0.2‘1.irn (av. pore size) filter. The diluent was stable at room temperature for at least two months.

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KA-150 diluent The diluent used on the KA-150 was prepared by dissolving 44.44 g of PEG, 10.0 g NaCl and 0.1 ml of Triton-X100 to 1 liter of deionized water. TritonXl00 is most conveniently added as a 10% solution prepared by diluting 100 ml to 1 liter with deionized water. The KA-150 diluent was filtered through a 0.2 pm (av. pore size) filter. The KA-150 diluent was stable for at least two months when stored at room temperature. Antisera dilutions Antisera were diluted 1 : 26 in polyethylene glycol diluent. The resulting solution was filtered through a 0.2 pm (av. pore size) filter. Although the diluted antisera solutions were prepared fresh each time they were found to be stable for several days at room temperature. Procedure Duplicate dilutions of 1 : 41 and 1 : 81 for IgA and IgG and 1 : 21 and 1 : 41 for IgM in buffer solution were prepared using a high speed automatic dilutor (Micromedic Systems, Inc., Horsham, PA 19044). The diluted samples, references and controls were loaded onto the sample tray and assayed using the PEG KA-150 diluent in place of the manufacturer’s diluent, polyethylene glycol diluent as reagent 1 and the appropriate diluted antibody solution as reagent 2. Samples with low concentrations of IgA and IgM such as for newborns were routinely run at dilutions of 1 : 6 and 1 : 11. A series of six Hyland standards or six serial dilutions of the Kallestad reference standard in 50 g/l human serum albumin, 0.15 M NaCl were included in each run. Results were obtained from the calibration curves and were reported in WHO mg/l units. Results Standard curves Figs..l, 2, and 3 show the standard curves for IgA, IgG and IgM, respectively. These curves were obtained using the Hyland “LAS-R” reference standards at

lg A (mg/liter)

Ig G (mg/liter)

Fig. 1. Calibration curve for the IgA assay on the KA-1 50 at a 1

: 41

serum dilution.

Fig. 2. Calibration curve for the IgG assay on the KA-150 at a 1

: 41

serum dilution.

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lg A tmg/liter)

Ig M (mg/litarl Fi$. 3. Calibration curve for the IgM assay on the KA-150 Fig. 4. Antigen excess curve for IgA at a 1

: 41

at a 1

: 21

serum d&&ion.

serum dilution.

1 :41 dilution for IgA and IgG and a 1 : 21 dilution for IgM._The Kallestad reference serum gave similar results. Detection limits of these curves are 500 mg/l, 300 mg/l, and 150 mg/l for IgA, IgG, and IgM, respectively. Antigen excess was determined for each of the immunoglobulins by assaying serial dilutions of Kallestad reference serum. The change in turbidity is plotted against the assayed values of the dilutions corresponding to a 1 : 41 dilution of sample as shown in Fig. 4 for IgA. With the antibody lots used, antigen excess occurred at 10 000 mg/l and 30 000 mgfl at 1 : 41 dilution and at 20 000 mg/l, and 60 000 mgfl at 1 : 81 dilution for IgA and IgG and at 8000 mg/l at 1 : 21 and 16 000 mg/l at 1 : 41 for IgM. Run-to-run repeatability, determined by assaying 20 to 22 samples in duplicate runs about 2 h apart, is shown in Table I. Within-run precision was estimated by performing replicate analyses on two pooled serum samples of differing immunoglobulin levels (Table I). Day-to-day precision was estimated for TABLE I SUMMARY

OF PRECISION STUDIES FOR IM~UNOGLOBWLIN

ASSAYS

ON KA-150

@A

IgG

&M

Run-to-run precision

Y = 0.967% + 79 r = 0.999, n = 21

Y = 0.982x + 52 P = 0.9888. n = 20

Y = 0.999x + 57 r = 0.994, R = 22

Within run we&ion

?? n x R

= = = =

;E n x n

= 6290, C.V. = 3.9% =23 = 10400. C.V. = 3.2% =22

x n x R

= 10600, C.V. = 5.5% -20 = 1880.1, C.V. = 3.9% =22

Day-to-day precision

x II x n

= 748, C.V. = 8.8% =12 = 1730, C.V. = 4.2% = 12

x n x n

= 6280, C.V. = 3.5% =12 = 10400, C.V. = 3.8% =12

x n x n

= 1110, =12 = 2030, =12

791, C.V. = 9.6% 21 1810. C.V. = 3.1% 24

C.V. = 4.5% C.V. = 3.9%

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lg M (mg/Htsr)

lg A hg/lHa)

Fig. 5. Effect of PEG concentration upon the IgM calibration curve at a 1

: 21 serum dilution.

Fig. 6. Effect of antiserum concentration upon the IgA calibration curve at a 1

: 41 serum dilution.

two frozen serum pools (Table I). All results summarized in Table I are at the 1 : 41 dilution for IgA and IgG, and 1 : 21 for IgM.

The effect of PEG on the reaction rate of fgM (Fig. 5) was investigated by adjusting the PEG concentration in reagent 1 and reagent 2 to give a final concentration of 35 g/l, 37.5 g/l and 40 g/l. The addition of albumin, 5 g/l, to the sample diluent had no effect on the reaction kinetics as observed by the KA-150. The effect of changing antibody concentration on the kinetic rate was inves-

0

mo

2cmo

LASER ~~EL~ETER

b#@ed

RADiAL

IMMU~IFFU~ON

Fig. 7. Comparison of results for &A with the KA-150 vs. the laser nephelometer. Fig. 8. Comparison of results for IgA with the KA-150 vs. radial immunodiffusion

agate)

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LASER NEPHELOMETERfmg/litwf

RADIAL IMM~~FUSION

(tm#iiterf

Fig. 9. Comparison of results for IgG with the KA-150 vs. the laser nephelometer, Fig. 10. Comparison of results for IgG with the KA-150 vs. radial immunodiffusion.

tigated by changing the antibody dilution for the IgA reaction, Fig. 6. No interference from gross lipemia, hemolysis or bihrubin was observed the assays of the three immunoglobulins.

for

~omp~son methods used were the Hyland Laser Nephelometer end point procedure and the Hyland Immunoplate radial immunodiffusion method with use of manufacturer’s instrumentation and reagents. The scale factor of 4000 used on the KA-150 is empirical and has been used for all assays to obtain a suitable range of relative changes in turbidity. The results of the correlation studies are shown in Figs. 7-12.

LASER NE~EL~TER

hgJltter)

RADIAL ~MMUNO~F~~

Fii. 11. Comparison of results for IgM with the KA-150 vs. the laser nepbefometer. Fig. 12. Comparison of results for IgM with the KA-150 vs. radial bnmunodiffwion.

bq’liter~

529

Discussion The calibration curves obtained by this method for the kinetic immunoprecipitation assay of IgA and IgM are sigmoid in shape. This is due in part to the average fixed time rate measurements of the formation of antibody-antigen complexes. The inflection points at a 1 : 41 dilution of standards occurring at 2000 mg/l for IgA and 100 mg/l for IgM represent the antigen concentration at which the maximum rate is achieved within 25-34 s after antibody addition. The calibration curve for IgG exhibits two inflection points at 2000 mg/l and 24 000 mg/l. This biphasic response is probably due to antigen induced aggregation of the initial antibody-antigen complexes. The sensitivity of the assay is dependent upon the rate of the reaction. Variables that affect the rate of complex formation as measured by the KA-150 include PEG concentration, antibody dilution and ionic strength of the reaction mixture. It was found necessary to maintain a phosphate ion concentration of 10 mmol, a sodium chloride concentration of 0.15 M and a pH of 7.2 in the final reaction mixture in order to remove interference from varying protein concentrations in the calibrating standards. Calibrating curves obtained from 1 : 41 dilutions of the Hyland standards in both water and 5% albumin in 0.15 M NaCl were virtually superimposable. Thus undiluted sera could be assayed with detection limits of 12 mg/l, 7 mg/l and 8 mg/l for IgA, IgM and IgG, respectively. This is especially of importance in measuring IgA and IgM levels in newborns. PEG increases the rate of turbidity formation and thus increases sensitivity of the assay. The optimum PEG concentration in the final reaction mixture is 40 g/l (Fig. 5). Higher concentrations of PEG may cause nonspecific precipitation of proteins in some samples exhibited negative IgA results on the Laser Nephelometer. This was due to incomplete blanking of the nonspecific PEGsample interaction as measured on the Laser Nephelometer and could be corrected by substituting a PEG sample blank for the water-buffer blank in the manufacturer’s directions, Several of these samples were examined on the KA150 substituting PEG diluent for the antibody solution as reagent 2. No nonspecific rate reaction could be detected. It was necessary to include PEG in the KA-150 diluent as attempts to achieve a final PEG concentration of 40 g/l by increasing the PEG concentration in reagent 1 to 12 g/l failed. The higher viscosity of the 12 g/l PEG solution did not give complete mixing with the diluted sample and caused considerable protein precipitation at the interface. This precipitate which did not redissolve before the read cycle gave falsely low rate measurements resulting in negative protein values on many low-level IgA and IgM samples. An antibody dilution of 1 : 26 with serum dilutions of 1 : 41 and 1 : 81 for IgG and IgA and 1 : 21 and 1 : 41 for IgM gave sufficiently high antigen excess for these immunoglobulins to allow for the assay of most samples without additional dilutions. Under these conditions, the usable range of antigen concentration is up to 20 000 mg/l, 60 000 mg/l and 16 000 mg/l for IgA, IgG and IgM, respectively. In the substrate mode the computation algorithm of the KA-150 approximates the conditions of a first order reaction and allows for a 17% negative

530

deviation from linearity. As the immunoprecipitation reaction fluctuates between first and second order, error messages due to ascending or descending curvature (AC or DC) are to be expected in certain ranges of immunoglobulin concentration. Therefore, to detect random errors duplicate determinations are necessary. The correlation studies show good agreement with both radial immunodiffusion and a manual laser nephelometric technique. Grossly lipemic samples (up to 50 g/l can be assayed without prior filtration. The method is both more sensitive and as precise as either of the comp~son procedures and requires considerably less technologist time. One technologist can produce up to 35 patient results per hour. If performed singly at two dilutions up to 75 patient results per hour can be obtained. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14

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