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Human anti-murine antibodies interfere with CPR assays performed with commercial kits
Diabetes Research and Clinical Practice 48 (2000) 105 – 111 www.elsevier.com/locate/diabres
Human anti-murine antibodies interfere with CPR assays performed with commercial kits Fujiko Sasakuma *, Takao Shimizu, Hiroko Wada, Takurou Morii, Akira Sasaki, Manabu Ehara Department of Clinical Laboratory Medicine, Osaka Medical Center for Cancer and Cardio6ascular Diseases, 1 -3 -3 Nakamichi, Higashinari-ku, Osaka 537 -8511, Japan Received 17 May 1999; received in revised form 26 November 1999; accepted 26 November 1999
Abstract Quantitation of C-peptide is important for the assessment of insulin secretion, in particular in patients receiving insulin therapy. Since the CPR levels become much higher than the concentration of C-peptide for several reasons, such as the high concentration of proinsulin, CPR values sometimes need to be assessed carefully. We have had two diabetic patients whose CPR values were abnormally high when determined with a Daiichi C-peptide kit III (method 1). CPR values determined by other methods were from two to ten times lower, indicating considerable interference when method 1 was used. Since method 1 uses mouse monoclonal antibodies (mmab) for detection antibodies, we suspected that human anti-murine antibodies (HAMA) were responsible for the interference. HAMA were detected in serum from both patients (45 and 460 ng/ml in case 1 and case 2 (at peak), respectively). Removal of HAMA from serum eliminated the interference. Modification of method 1 to exclude mmab from the assay system removed all interference. HAMA were, therefore, considered to be the cause of the interference. In case 2, the peak concentration of HAMA was recorded 16 months earlier than the maximum of interference. Further analysis revealed that HAMA with high affinities were responsible for the interference. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: C-peptide; HAMA; Immunoassay; Interference
1. Introduction Quantitation of C-peptide is important for assessment of insulin secretion from islets [1]. CPR, the sum of concentrations of C-peptide and proinsulin (including metabolites of proinsulin), is usually determined for quantitation of C-peptide. CPR values become substantially higher than the * Corresponding author. Tel.: +81-6-69721-11181.
concentration of C-peptide when the concentration of proinsulin is high (e.g. in patients with insulinoma). The presence of anti-insulin antibodies in the serum also increases the CPR value since antibody-bound proinsulin is accumulated. Therefore, abnormally high CPR values need to be assessed carefully. We have had two diabetic patients whose CPR values, determined with Daiichi C-peptide kit III (Daiichi, Tokyo, Japan; method 1), were abnor-
0168-8227/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 8 2 2 7 ( 9 9 ) 0 0 1 4 2 - 4
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mally high. This kit uses mouse monoclonal antibodies (mmab), and human anti-murine antibodies (HAMA) interfered with the assay. By contrast concentrations of HAMA determined with an Immustrip® kit (Immunomedics, Warren, NJ) were not correlated with the extent of interference. Analysis of the characteristics of HAMA showed that HAMA with high affinities were important in the interference with the assay using the Daiichi kit.
2. Cases Case 1 was a 3-year-old girl with IDDM. She had mild diabetes and was followed without insulin therapy. During observation, her blood glucose level increased gradually. Before starting insulin therapy, she was found to have a high CPR value (3.0 ng/ml by method 1), as compared with a low IRI value (2 mU/ml). The concentration of proinsulin was not elevated (9.0 pmol/1). A sample collected before insulin therapy was sent to our laboratory for further analysis. Case 2 was a 74-year-old woman with NIDDM. She had had cancer of the caudal pancreas, which had been curatively removed. At surgery, she received 600 ml blood transfusion. Her glucose tolerance had been monitored regularly since the operation. Interference with the C-peptide assay (method 1) was first noticed 6 months after surgery. At that time, concentrations of IRI, CPR (method 1) and proinsulin were 4 mU/ml, 4.9 ng/ml and 15.0 pmol/1, respectively. She did not receive any blood, fresh frozen plasma or immunoglobulin transfusion after surgery. No specific symptom was observed when her CPR values became abnormally high. Two years and 3 months after surgery, the patient became diabetic. Her diabetes has been controlled with glibenclamide until the present time. Neither patient had a history of known exposure to murine immunoglobulins or had antibodies against C-peptide or insulin. RA was negative in both cases. Samples of serum from glucose tolerance tests of normal controls were also studied. Such serum contained less than 4 ng/ml HAMA.
3. Materials and methods
3.1. Methods for quantitation of C-peptide C-peptide was quantitated with three commercial kits that are sold in Japan. For method 1, we used a kit ‘Daiichi C-peptide kit III’ (Daiichi). This kit is the most popular assay kit and accounts for 50% of assays in Japan. The kit is used for a two-antibody competitive RIA. The first antibodies are goat polyclonal antibodies against human C-peptide. The second antibodies are mmab, which are bound to a polystyrene bead. We modified method 1 by replacing the second antibodies by rabbit polyclonal antibodies against goat IgG that were bound to magnetic particles (CF130; Japan Paint, Tokyo, Japan). The modification was designed to exclude mmab from the assay reagents. Magnetic particle-bound antibodies were prepared as reported previously [2]. Antigen–antibody complexes were isolated with a magnetic separator. All other procedures and reagents were the same as in method 1. Method 2 involves a one-antibody competitive RIA (Shionogi C-peptide RIA; Shionogi, Osaka, Japan) using rabbit polyclonal antibodies. Method 3 is a two-site immunometric assay (Tosoh II C-peptide; Tosoh, Tokyo, Japan) that uses mmab for the detection antibodies. The capture antibodies are rabbit polyclonal antibodies.
3.2. Treatment of serum with polyethylene glycol (PEG) Serum was mixed with an equal volume of 25% PEG to remove immunoglobulin. After incubation at 0°C for 10 min, the sample was centrifuged at 2200 ×g at 4°C. The concentration of C-peptide in the supernatant was determined by method 1.
3.3. Treatment of serum with mouse IgG We assayed C-peptide by method 1 after adding mouse IgG (up to 2000 mg/ml) to the assay buffer. We postulated that if HAMA were the cause of interference, interference should be attenuated by coexisting mouse IgG.
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We removed HAMA from serum using magnetic particles with bound mouse IgG. Magnetic particles with bound IgG, suspended in phosphate-buffered saline (pH 7.4) plus 0.1% bovine serum albumin (mouse IgG, 600 mg/ml; magnetic particles, ca. 1%, w/v), were prepared as reported previously [2]. An aliquot of the suspension of magnetic particles (0 – 120 mg IgG equivalent) was placed in a tube and the supernatant was removed with a magnetic separator. Then 200 ml of serum was added. After incubation for 3 h at 25°C, the supernatant was prepared using the magnetic separator. The concentration of C-peptide in the supernatant was determined by method 1.
represents the ratio of mouse IgG that bound HAMA to mouse IgG that did not bind HAMA. The concentration of mouse IgG with bound HAMA was calculated by multiplying the total concentration of mouse IgG by the ratio of B to T. HAMA bound mouse IgG was plotted versus B/F. From data of binding curves, we calculated the apparent affinities and apparent titers of HAMA using the Scatchard plot [3]in Berson’s modification.
3.4. Quantitation of HAMA
When C-peptide was assayed by method 1, both our cases gave abnormally high CPR values as compared with low IRI values. CPR values obtained by the other two methods were low and similar to each other (Table 1), indicating that the high values obtained by method 1 were due to interference. We calculated ratios of CPR values as indices of the extent of interference since CPR values assayed by the three methods were almost proportional to each other. In case 2, interference appeared first 6 months after surgery and was maximal 22 months after surgery. The concentration of HAMA in case 1 was 45 ng/ml (it was less than 4 ng/ml in normal controls). In case 2, the concentration of HAMA increased above the limit for normal controls 6 months after surgery. Changes in the concentration of HAMA were apparently associated with increases in interference, but the peak level of HAMA (460 ng/ml) was recorded earlier (at 6 months after surgery) than the peak interference.
The concentration of HAMA was determined with Immustrip® (Immunomedics). The assay is based on the ability of HAMA to bridge two mmab (i.e. to bridge a capture antibody and a detection antibody).
3.5. Analysis of the kinetics of binding of HAMA to a bead We analyzed the kinetics of binding of HAMA to a bead in method 1. In this experiment, we assumed that the affinity of HAMA for a bead was the same as that for mouse IgG. Serum from patients was treated with charcoal (final concentration, 1.25%, w/v) to remove C-peptide. Then 75 ml of a solution of mouse IgG at various concentrations (0 – 200 mg/ml) were added to a mixture of 25 ml of charcoal-treated serum, 100 ml of a solution of goat antibodies against C-peptide, 100 ml of a solution of 125I-C-peptide, and a polystyrene bead to which mmab against goat IgG had been bound (reagent in method 1). After incubation at 4°C for 24 h, the radioactivity bound to the bead was determined (F). Total radioactivity (T) was also determined under same conditions except without mouse IgG and serum. When F is the radioactivity of a bead without bound HAMA, the difference (B) between T and F represents radioactivity of a bead with bound HAMA. Thus, the ratio of B to F
4. Results
4.1. CPR 6alues determined with three assay kits and concentrations of HAMA
4.2. Pretreatment of serum with PEG Pretreatment of serum with polyethylene glycol (PEG) eliminated interference in method 1. After PEG treatment, CPR values in the supernatant, as determined by method 1, were similar to those determined by method 2. The relationship between the level of CPR determined by method 1 after PEG treatment and by method 2 was: CPR (method 2)=1.0× CPR (method 1, + PEG)−
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0.1, r= 0.95 (n= 30, data from both cases were used to generate the equation).
4.3. Effects of mouse IgG Addition of mouse IgG to the assay buffer in method 1 decreased interference (Fig. 1). Interference was totally eliminated in serum that showed weak interference (case 1, and case 2 at 43 months after surgery). Interference was increased, paradoxically, in a sample from case 2 at 6 months after surgery. In the serum that showed strong interference (i.e. 6, 22 and 30 months after surgery), interference remained even after addition of mouse IgG at 2000 mg/ml. Complete elimination of interference was achieved by pretreating serum with magnetic particle-bound mouse IgG (Fig. 1). As little as 150 mg/ml IgG was enough to eliminate interference in a sample of serum that showed the strongest interference.
4.4. Modification of method 1 Method 1 was modified to exclude mmab from the assay system, and modified method 1 was free from interference. The relationship between CPR (modified method 1) and CPR (method 2) was: CPR (method 2)=1.1 ×CPR (modified method 1) + 0.1, r= 0.99 (n =30, data from both cases were used to generate the equation).
4.5. Characteristics of HAMA and interference Kinetic analysis revealed that HAMA consisted of populations with at least two different affinities (Fig. 2). Assuming that HAMA consist of two different affinities, we calculated their affinities and titers. As shown in Fig. 3, the extent of interference by HAMA was apparently related to HAMA with high affinity. Either total HAMA or HAMA with low affinity seemed to reflect the concentration of HAMA determined with Immustrip®.
5. Discussion We have had two diabetic patients with high CPR values (as determined by method 1) and low IRI values. They are our first two cases we noticed as having high CPR (method 1)/IRI ratio with normal proinsulin. At first, we suspected that the level of proinsulin might be high but such was not the case. No insulin-specific antibodies were detected either. When three assay kits (methods 1–3) were compared, only method 1 yielded high values, indicating that interference has occurred in method 1. We treated serum with PEG to examine the cause of interference. The supernatant after PEG treatment did not show any interference in
Table 1 Concentrations of CPR and HAMA in cases 1 and 2 Case 2 (months after surgery) Case 1 Fasting CPR (ng/ml) Method 1 Method 2 Method 3 HAMA (ng/ml)
3.0 0.7 0.6 45
Ratio of CPR 6alues a Method 1/method 2 Method 3/method 2
4.19 0.1 (3) 0.9 (1)
1
1.1 1.1 1.0 3 1.090.1 (5) 0.9 (1)
6
22
30
43
2.8 1.0 0.9 460
9.5 0.7 0.7 287
4.9 0.7 0.7 65
2.8 0.9 0.9 12
2.09 0.6 (5) 0.9 (1)
10.49 2.7 (5) 0.99 0.1 (5)
5.09 1.2 (5) 1.0 9 0.0 (5)
2.3 90.4 (5) 1.0 90.0 (5)
Means 9S.D. (number) are shown. The relationship between CPR (method 1) and CPR (method 2) in normal controls was: CPR (method 2) = 1.0×CPR (method 1)−0.1, r = 0.95 (n= 30). The relationship between CPR (method 1) and CPR (method 3) in normal controls was: CPR (method 3) = 0.95 CPR (method 1)+0.12, r =0.95 (n = 30). a
F. Sasakuma et al. / Diabetes Research and Clinical Practice 48 (2000) 105–111
Fig. 1. Effects of mouse IgG. (a) Effects of the addition of mouse IgG to the assay buffer in method 1. (b) Pretreatment of serum with mouse IgG that was bound to magnetic particles. The ratio of the CPR value obtained by method 1 to that obtained by method 2 was calculated as an index of interference. Half-filled squares with a dotted line denote case 1. Closed circles, open circles, open triangles, and open squares denote samples from case 2 collected 6, 22, 30 and 43 months after surgery, respectively.
Fig. 2. Analysis of kinetics of binding of MAMA. Closed circles, open circles, and open triangles refer to case 2 at 6, 22 and 30 months after surgery, respectively. HAMA with high affinities were most abundant in serum 22 months after surgery. B/F, bound/free.
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method 1. The material responsible for interference was recovered in the precipitate (data not shown). Antibodies were, therefore, suspected as a cause of interference. Since HAMA are well known as substance that interferes with immunoassays that use mmab [4], we postulated that HAMA were the most probable cause of interference. The incidence of interference in immunoassays due to HAMA was reported to be 74% in patients receiving mmab, and 9–40% in the normal population [5,6]. In 503 healthy Japanese individuals, false positive rates with CEA measurement were reported as high as 29.0% in one HAMA-sensitive assay method, and 5.4 and 5.8% in two commercial kits [7]. Method 3, which also uses mmab, might be resistant to this kind of interference. Therefore, we quantitated HAMA and found that levels of HAMA were above normal limits in both cases. Although the observation that the peak of interference did not coincide with that in the level of HAMA in case 2 appears not to support such proposed inference, two other observations do favor this scenario. Removal of HAMA from serum (using magnetic particle-bound mouse IgG) eliminated interference completely. Furthermore, modified method 1 was no longer susceptible to interference. HAMA were, therefore, considered to be the cause of interference in method 1. This is the first report of HAMA interference of C-peptide assay. Little is known about etiology of HAMA in normal subjects. It was proposed that HAMA may merely reflect a facet of the normal immune system [5]. Case 2 was apparently exposed to antigen in her daily life. We do not consider that interference was related to pancreatectomy since other 80 patients with pancreatectomy did not show such abnormal CPR values. This is the first report of natural course of HAMA in a normal subject. As was apparent in case 2, the extent of interference did not reflect the concentration of HAMA directly. To elucidate the relationship between the concentration of HAMA and interference, we studied the characteristics of HAMA in case 2. Analysis of kinetics showed that HAMA consisted of antibodies with different affinities. Levels of HAMA with high affinities, and not
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with low affinities, changed in parallel with the extent of interference. This observation indicates that HAMA with high affinities were important for interference. The Immustrip® kit detects HAMA that bind two mouse antibodies. What we observed as interference in method 1 was the ability of HAMA to disturb the binding of mmab to its antigen (goat antibodies). All HAMA that were detected by Immustrip® kit do not necessarily disturb this binding of mmab. A difference in affinities probably explain, the different effects of each particular subgroup of HAMA on the assay in case 2. With respect to methods of quantitating HAMA, significant differences in results have been reported [8]. As for epitope, most HAMA are specific for the Fc region, and some HAMA may recognize both the F(ab) and the Fc region [6, 9]. Addition of mouse IgG to the assay mixture did not eliminate interference completely, and a paradoxical increase in interference was observed in the serum at 6 months after surgery in case 2. The limited capacity of co-existing mouse immunoglobulin to eliminate interference has been reported [8, 9]. Pretreatment with magnetic particle-bound mouse IgG proved to be an excellent and straightforward method for elimination of interference. According to the manual for Immustrip® HAMA IgG, serum with HAMA above 40 ng/ml is judged HAMA-positive. We observed interfer-
ence in a sample with as little as 12 ng/ml HAMA. The reference value in the manual is very strict. The concentration of HAMA in normal serum that showed no interference was 2.79 0.6 ng/ml in our hands. When compared three assay kits, each assay kit has its advantage and disadvantage. Method 2 requires manual centrifugation step, and might be vulnerable to the interference by anti-rabbit immunoglobulin antibodies. Method 3 is best for handling, but most expensive. Method 3 may not detect C-peptide fragments in old sample (urine and serum sample preserved without anti-proteolytic reagent). All three methods are similar in respect of sensitivity. We noticed interference in our two cases because CPR values were too high when compared with IRI values. If differences from anticipated values are not striking, interference might be overlooked. This is an important issue in the assessment of insulin-secreting ability in insulinopenic patients who receive insulin therapy. Above consideration also applies to other immunoassays for other hormones, tumor makers, etc. Sera from case 2 interfered the thyroglobulin assay kit (Daiichi Thyroglobulin IRMA Pasteur (Daiichi)) that uses mmab. Thyroglobulin level was 18, 1300, and 51 ng/ml at 1, 6 and 43 months after surgery, respectively (reference value B 40 ng/ml). Medical doctors should always keep in mind the possibilities of interference by heterophilic antibodies in immunoassays. It is also noteworthy that not all immunoassays that use mmab are vulnerable to interference by HAMA.
References
Fig. 3. Time course of changes in the HAMA titer and interference in case 2. The ratio of the CPR value obtained by method 1 to that obtained by method 2 is shown by open circles. Closed circles and closed triangles denote titers of HAMA with high affinities and with low affinities, respectively.
[1] D.L. Horwitz, H. Kuzuya, A.H. Rubenstein, Circulating serum C-peptide: a brief review of diagnostic implications, New Engl. J. Med. 295 (1976) 207 – 209. [2] F. Sasakuma, T. Shimizu, O. Ishikawa, et al., An improved method for determination of C-peptide levels in serum: pretreatment of test samples with anti-insulin antibody insolubilized with magnetic particles, Rinsho Byori 42 (1994) 183 – 188. [3] S.A. Berson, R.S. Yalow, Quantitative aspects of the reaction between insulin and insulin binding antibodies, J. Clin. Invest. 38 (1959) 1996 – 2016.
F. Sasakuma et al. / Diabetes Research and Clinical Practice 48 (2000) 105–111 [4] L.J. Kricka, P.D. Schmerfeld, M. Senior, D.B. Goodman, P. Kaladas, Interference by human anti-mouse antibody in two-site immunoassays, Clin. Chem. 36 (1990) 892– 894. [5] R.J. Thompson, A.P. Jackson, N. Langlois, Circulating antibodies to mouse monoclonal immunoglobulins in normal subjects — incidence, species specificity, and effects on a two-site assay for creatine kinase-MB isoenzyme, Clin. Chem. 32 (1986) 476 –481. [6] J.M. Boscato, M.C. Stuart, Heterophilic antibodies: a problem for all immunoassays, Clin. Chem. 34 (1988) 27– 33.
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[7] M. Kuroki, Y. Matsumoto, F. Arakawa, et al., Reducing interference from heterophilic antibodies in a two-site immunoassay for carcinoembryonic antigen (CEA) by using a human/mouse chimeric antibody to CEA as the tracer, J. Immunol. Methods 180 (1995) 81 – 91. [8] HAMA-Survey-Group, Survey of methods for measuring human anti-mouse antibodies, Clin Chim Acta 215 (1993) 153 – 163. [9] H.C. Vaidya, B.G. Beatty, Eliminating interference from heterophilic antibodies in a two-site immunoassay for creatine kinase MB by using F(ab%)2 conjugate and polyclonal mouse IgG, Clin. Chem. 38 (1992) 1737 – 1742.
Human anti-murine antibodies interfere with CPR assays performed with commercial kits Fujiko Sasakuma *, Takao Shimizu, Hiroko Wada, Takurou Morii, Akira Sasaki, Manabu Ehara Department of Clinical Laboratory Medicine, Osaka Medical Center for Cancer and Cardio6ascular Diseases, 1 -3 -3 Nakamichi, Higashinari-ku, Osaka 537 -8511, Japan Received 17 May 1999; received in revised form 26 November 1999; accepted 26 November 1999
Abstract Quantitation of C-peptide is important for the assessment of insulin secretion, in particular in patients receiving insulin therapy. Since the CPR levels become much higher than the concentration of C-peptide for several reasons, such as the high concentration of proinsulin, CPR values sometimes need to be assessed carefully. We have had two diabetic patients whose CPR values were abnormally high when determined with a Daiichi C-peptide kit III (method 1). CPR values determined by other methods were from two to ten times lower, indicating considerable interference when method 1 was used. Since method 1 uses mouse monoclonal antibodies (mmab) for detection antibodies, we suspected that human anti-murine antibodies (HAMA) were responsible for the interference. HAMA were detected in serum from both patients (45 and 460 ng/ml in case 1 and case 2 (at peak), respectively). Removal of HAMA from serum eliminated the interference. Modification of method 1 to exclude mmab from the assay system removed all interference. HAMA were, therefore, considered to be the cause of the interference. In case 2, the peak concentration of HAMA was recorded 16 months earlier than the maximum of interference. Further analysis revealed that HAMA with high affinities were responsible for the interference. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: C-peptide; HAMA; Immunoassay; Interference
1. Introduction Quantitation of C-peptide is important for assessment of insulin secretion from islets [1]. CPR, the sum of concentrations of C-peptide and proinsulin (including metabolites of proinsulin), is usually determined for quantitation of C-peptide. CPR values become substantially higher than the * Corresponding author. Tel.: +81-6-69721-11181.
concentration of C-peptide when the concentration of proinsulin is high (e.g. in patients with insulinoma). The presence of anti-insulin antibodies in the serum also increases the CPR value since antibody-bound proinsulin is accumulated. Therefore, abnormally high CPR values need to be assessed carefully. We have had two diabetic patients whose CPR values, determined with Daiichi C-peptide kit III (Daiichi, Tokyo, Japan; method 1), were abnor-
0168-8227/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 8 2 2 7 ( 9 9 ) 0 0 1 4 2 - 4
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mally high. This kit uses mouse monoclonal antibodies (mmab), and human anti-murine antibodies (HAMA) interfered with the assay. By contrast concentrations of HAMA determined with an Immustrip® kit (Immunomedics, Warren, NJ) were not correlated with the extent of interference. Analysis of the characteristics of HAMA showed that HAMA with high affinities were important in the interference with the assay using the Daiichi kit.
2. Cases Case 1 was a 3-year-old girl with IDDM. She had mild diabetes and was followed without insulin therapy. During observation, her blood glucose level increased gradually. Before starting insulin therapy, she was found to have a high CPR value (3.0 ng/ml by method 1), as compared with a low IRI value (2 mU/ml). The concentration of proinsulin was not elevated (9.0 pmol/1). A sample collected before insulin therapy was sent to our laboratory for further analysis. Case 2 was a 74-year-old woman with NIDDM. She had had cancer of the caudal pancreas, which had been curatively removed. At surgery, she received 600 ml blood transfusion. Her glucose tolerance had been monitored regularly since the operation. Interference with the C-peptide assay (method 1) was first noticed 6 months after surgery. At that time, concentrations of IRI, CPR (method 1) and proinsulin were 4 mU/ml, 4.9 ng/ml and 15.0 pmol/1, respectively. She did not receive any blood, fresh frozen plasma or immunoglobulin transfusion after surgery. No specific symptom was observed when her CPR values became abnormally high. Two years and 3 months after surgery, the patient became diabetic. Her diabetes has been controlled with glibenclamide until the present time. Neither patient had a history of known exposure to murine immunoglobulins or had antibodies against C-peptide or insulin. RA was negative in both cases. Samples of serum from glucose tolerance tests of normal controls were also studied. Such serum contained less than 4 ng/ml HAMA.
3. Materials and methods
3.1. Methods for quantitation of C-peptide C-peptide was quantitated with three commercial kits that are sold in Japan. For method 1, we used a kit ‘Daiichi C-peptide kit III’ (Daiichi). This kit is the most popular assay kit and accounts for 50% of assays in Japan. The kit is used for a two-antibody competitive RIA. The first antibodies are goat polyclonal antibodies against human C-peptide. The second antibodies are mmab, which are bound to a polystyrene bead. We modified method 1 by replacing the second antibodies by rabbit polyclonal antibodies against goat IgG that were bound to magnetic particles (CF130; Japan Paint, Tokyo, Japan). The modification was designed to exclude mmab from the assay reagents. Magnetic particle-bound antibodies were prepared as reported previously [2]. Antigen–antibody complexes were isolated with a magnetic separator. All other procedures and reagents were the same as in method 1. Method 2 involves a one-antibody competitive RIA (Shionogi C-peptide RIA; Shionogi, Osaka, Japan) using rabbit polyclonal antibodies. Method 3 is a two-site immunometric assay (Tosoh II C-peptide; Tosoh, Tokyo, Japan) that uses mmab for the detection antibodies. The capture antibodies are rabbit polyclonal antibodies.
3.2. Treatment of serum with polyethylene glycol (PEG) Serum was mixed with an equal volume of 25% PEG to remove immunoglobulin. After incubation at 0°C for 10 min, the sample was centrifuged at 2200 ×g at 4°C. The concentration of C-peptide in the supernatant was determined by method 1.
3.3. Treatment of serum with mouse IgG We assayed C-peptide by method 1 after adding mouse IgG (up to 2000 mg/ml) to the assay buffer. We postulated that if HAMA were the cause of interference, interference should be attenuated by coexisting mouse IgG.
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We removed HAMA from serum using magnetic particles with bound mouse IgG. Magnetic particles with bound IgG, suspended in phosphate-buffered saline (pH 7.4) plus 0.1% bovine serum albumin (mouse IgG, 600 mg/ml; magnetic particles, ca. 1%, w/v), were prepared as reported previously [2]. An aliquot of the suspension of magnetic particles (0 – 120 mg IgG equivalent) was placed in a tube and the supernatant was removed with a magnetic separator. Then 200 ml of serum was added. After incubation for 3 h at 25°C, the supernatant was prepared using the magnetic separator. The concentration of C-peptide in the supernatant was determined by method 1.
represents the ratio of mouse IgG that bound HAMA to mouse IgG that did not bind HAMA. The concentration of mouse IgG with bound HAMA was calculated by multiplying the total concentration of mouse IgG by the ratio of B to T. HAMA bound mouse IgG was plotted versus B/F. From data of binding curves, we calculated the apparent affinities and apparent titers of HAMA using the Scatchard plot [3]in Berson’s modification.
3.4. Quantitation of HAMA
When C-peptide was assayed by method 1, both our cases gave abnormally high CPR values as compared with low IRI values. CPR values obtained by the other two methods were low and similar to each other (Table 1), indicating that the high values obtained by method 1 were due to interference. We calculated ratios of CPR values as indices of the extent of interference since CPR values assayed by the three methods were almost proportional to each other. In case 2, interference appeared first 6 months after surgery and was maximal 22 months after surgery. The concentration of HAMA in case 1 was 45 ng/ml (it was less than 4 ng/ml in normal controls). In case 2, the concentration of HAMA increased above the limit for normal controls 6 months after surgery. Changes in the concentration of HAMA were apparently associated with increases in interference, but the peak level of HAMA (460 ng/ml) was recorded earlier (at 6 months after surgery) than the peak interference.
The concentration of HAMA was determined with Immustrip® (Immunomedics). The assay is based on the ability of HAMA to bridge two mmab (i.e. to bridge a capture antibody and a detection antibody).
3.5. Analysis of the kinetics of binding of HAMA to a bead We analyzed the kinetics of binding of HAMA to a bead in method 1. In this experiment, we assumed that the affinity of HAMA for a bead was the same as that for mouse IgG. Serum from patients was treated with charcoal (final concentration, 1.25%, w/v) to remove C-peptide. Then 75 ml of a solution of mouse IgG at various concentrations (0 – 200 mg/ml) were added to a mixture of 25 ml of charcoal-treated serum, 100 ml of a solution of goat antibodies against C-peptide, 100 ml of a solution of 125I-C-peptide, and a polystyrene bead to which mmab against goat IgG had been bound (reagent in method 1). After incubation at 4°C for 24 h, the radioactivity bound to the bead was determined (F). Total radioactivity (T) was also determined under same conditions except without mouse IgG and serum. When F is the radioactivity of a bead without bound HAMA, the difference (B) between T and F represents radioactivity of a bead with bound HAMA. Thus, the ratio of B to F
4. Results
4.1. CPR 6alues determined with three assay kits and concentrations of HAMA
4.2. Pretreatment of serum with PEG Pretreatment of serum with polyethylene glycol (PEG) eliminated interference in method 1. After PEG treatment, CPR values in the supernatant, as determined by method 1, were similar to those determined by method 2. The relationship between the level of CPR determined by method 1 after PEG treatment and by method 2 was: CPR (method 2)=1.0× CPR (method 1, + PEG)−
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0.1, r= 0.95 (n= 30, data from both cases were used to generate the equation).
4.3. Effects of mouse IgG Addition of mouse IgG to the assay buffer in method 1 decreased interference (Fig. 1). Interference was totally eliminated in serum that showed weak interference (case 1, and case 2 at 43 months after surgery). Interference was increased, paradoxically, in a sample from case 2 at 6 months after surgery. In the serum that showed strong interference (i.e. 6, 22 and 30 months after surgery), interference remained even after addition of mouse IgG at 2000 mg/ml. Complete elimination of interference was achieved by pretreating serum with magnetic particle-bound mouse IgG (Fig. 1). As little as 150 mg/ml IgG was enough to eliminate interference in a sample of serum that showed the strongest interference.
4.4. Modification of method 1 Method 1 was modified to exclude mmab from the assay system, and modified method 1 was free from interference. The relationship between CPR (modified method 1) and CPR (method 2) was: CPR (method 2)=1.1 ×CPR (modified method 1) + 0.1, r= 0.99 (n =30, data from both cases were used to generate the equation).
4.5. Characteristics of HAMA and interference Kinetic analysis revealed that HAMA consisted of populations with at least two different affinities (Fig. 2). Assuming that HAMA consist of two different affinities, we calculated their affinities and titers. As shown in Fig. 3, the extent of interference by HAMA was apparently related to HAMA with high affinity. Either total HAMA or HAMA with low affinity seemed to reflect the concentration of HAMA determined with Immustrip®.
5. Discussion We have had two diabetic patients with high CPR values (as determined by method 1) and low IRI values. They are our first two cases we noticed as having high CPR (method 1)/IRI ratio with normal proinsulin. At first, we suspected that the level of proinsulin might be high but such was not the case. No insulin-specific antibodies were detected either. When three assay kits (methods 1–3) were compared, only method 1 yielded high values, indicating that interference has occurred in method 1. We treated serum with PEG to examine the cause of interference. The supernatant after PEG treatment did not show any interference in
Table 1 Concentrations of CPR and HAMA in cases 1 and 2 Case 2 (months after surgery) Case 1 Fasting CPR (ng/ml) Method 1 Method 2 Method 3 HAMA (ng/ml)
3.0 0.7 0.6 45
Ratio of CPR 6alues a Method 1/method 2 Method 3/method 2
4.19 0.1 (3) 0.9 (1)
1
1.1 1.1 1.0 3 1.090.1 (5) 0.9 (1)
6
22
30
43
2.8 1.0 0.9 460
9.5 0.7 0.7 287
4.9 0.7 0.7 65
2.8 0.9 0.9 12
2.09 0.6 (5) 0.9 (1)
10.49 2.7 (5) 0.99 0.1 (5)
5.09 1.2 (5) 1.0 9 0.0 (5)
2.3 90.4 (5) 1.0 90.0 (5)
Means 9S.D. (number) are shown. The relationship between CPR (method 1) and CPR (method 2) in normal controls was: CPR (method 2) = 1.0×CPR (method 1)−0.1, r = 0.95 (n= 30). The relationship between CPR (method 1) and CPR (method 3) in normal controls was: CPR (method 3) = 0.95 CPR (method 1)+0.12, r =0.95 (n = 30). a
F. Sasakuma et al. / Diabetes Research and Clinical Practice 48 (2000) 105–111
Fig. 1. Effects of mouse IgG. (a) Effects of the addition of mouse IgG to the assay buffer in method 1. (b) Pretreatment of serum with mouse IgG that was bound to magnetic particles. The ratio of the CPR value obtained by method 1 to that obtained by method 2 was calculated as an index of interference. Half-filled squares with a dotted line denote case 1. Closed circles, open circles, open triangles, and open squares denote samples from case 2 collected 6, 22, 30 and 43 months after surgery, respectively.
Fig. 2. Analysis of kinetics of binding of MAMA. Closed circles, open circles, and open triangles refer to case 2 at 6, 22 and 30 months after surgery, respectively. HAMA with high affinities were most abundant in serum 22 months after surgery. B/F, bound/free.
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method 1. The material responsible for interference was recovered in the precipitate (data not shown). Antibodies were, therefore, suspected as a cause of interference. Since HAMA are well known as substance that interferes with immunoassays that use mmab [4], we postulated that HAMA were the most probable cause of interference. The incidence of interference in immunoassays due to HAMA was reported to be 74% in patients receiving mmab, and 9–40% in the normal population [5,6]. In 503 healthy Japanese individuals, false positive rates with CEA measurement were reported as high as 29.0% in one HAMA-sensitive assay method, and 5.4 and 5.8% in two commercial kits [7]. Method 3, which also uses mmab, might be resistant to this kind of interference. Therefore, we quantitated HAMA and found that levels of HAMA were above normal limits in both cases. Although the observation that the peak of interference did not coincide with that in the level of HAMA in case 2 appears not to support such proposed inference, two other observations do favor this scenario. Removal of HAMA from serum (using magnetic particle-bound mouse IgG) eliminated interference completely. Furthermore, modified method 1 was no longer susceptible to interference. HAMA were, therefore, considered to be the cause of interference in method 1. This is the first report of HAMA interference of C-peptide assay. Little is known about etiology of HAMA in normal subjects. It was proposed that HAMA may merely reflect a facet of the normal immune system [5]. Case 2 was apparently exposed to antigen in her daily life. We do not consider that interference was related to pancreatectomy since other 80 patients with pancreatectomy did not show such abnormal CPR values. This is the first report of natural course of HAMA in a normal subject. As was apparent in case 2, the extent of interference did not reflect the concentration of HAMA directly. To elucidate the relationship between the concentration of HAMA and interference, we studied the characteristics of HAMA in case 2. Analysis of kinetics showed that HAMA consisted of antibodies with different affinities. Levels of HAMA with high affinities, and not
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with low affinities, changed in parallel with the extent of interference. This observation indicates that HAMA with high affinities were important for interference. The Immustrip® kit detects HAMA that bind two mouse antibodies. What we observed as interference in method 1 was the ability of HAMA to disturb the binding of mmab to its antigen (goat antibodies). All HAMA that were detected by Immustrip® kit do not necessarily disturb this binding of mmab. A difference in affinities probably explain, the different effects of each particular subgroup of HAMA on the assay in case 2. With respect to methods of quantitating HAMA, significant differences in results have been reported [8]. As for epitope, most HAMA are specific for the Fc region, and some HAMA may recognize both the F(ab) and the Fc region [6, 9]. Addition of mouse IgG to the assay mixture did not eliminate interference completely, and a paradoxical increase in interference was observed in the serum at 6 months after surgery in case 2. The limited capacity of co-existing mouse immunoglobulin to eliminate interference has been reported [8, 9]. Pretreatment with magnetic particle-bound mouse IgG proved to be an excellent and straightforward method for elimination of interference. According to the manual for Immustrip® HAMA IgG, serum with HAMA above 40 ng/ml is judged HAMA-positive. We observed interfer-
ence in a sample with as little as 12 ng/ml HAMA. The reference value in the manual is very strict. The concentration of HAMA in normal serum that showed no interference was 2.79 0.6 ng/ml in our hands. When compared three assay kits, each assay kit has its advantage and disadvantage. Method 2 requires manual centrifugation step, and might be vulnerable to the interference by anti-rabbit immunoglobulin antibodies. Method 3 is best for handling, but most expensive. Method 3 may not detect C-peptide fragments in old sample (urine and serum sample preserved without anti-proteolytic reagent). All three methods are similar in respect of sensitivity. We noticed interference in our two cases because CPR values were too high when compared with IRI values. If differences from anticipated values are not striking, interference might be overlooked. This is an important issue in the assessment of insulin-secreting ability in insulinopenic patients who receive insulin therapy. Above consideration also applies to other immunoassays for other hormones, tumor makers, etc. Sera from case 2 interfered the thyroglobulin assay kit (Daiichi Thyroglobulin IRMA Pasteur (Daiichi)) that uses mmab. Thyroglobulin level was 18, 1300, and 51 ng/ml at 1, 6 and 43 months after surgery, respectively (reference value B 40 ng/ml). Medical doctors should always keep in mind the possibilities of interference by heterophilic antibodies in immunoassays. It is also noteworthy that not all immunoassays that use mmab are vulnerable to interference by HAMA.
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Fig. 3. Time course of changes in the HAMA titer and interference in case 2. The ratio of the CPR value obtained by method 1 to that obtained by method 2 is shown by open circles. Closed circles and closed triangles denote titers of HAMA with high affinities and with low affinities, respectively.
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