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Standardisation of a two-site PTH immunoradiometric assay using various solid phase formats
Reprint requests: Dr Grace Samuel, Radiopharmaceutical Program, Board of Radiation & Isotope Technology, Navi Mumbai 400 703, India e-mail: grace_samuel1955@rediffmail.com
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This article was originally published by Medknow Publications & Media Pvt Ltd and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Background & objectives:
Estimation of parathyroid hormone (PTH) levels is important in the management of metabolic bone disorders. Here we describe a simple, sensitive and specific second generation immunoradiometric assay (IRMA) to detect intact PTH levels using different solid phase matrices. Different methods for immobilization of antibodies have also been evaluated.
Methods:
Experiments were carried out with physical adsorption of antibodies, covalent coupling using 2 per cent glutaraldehyde and N,N’ carbonyldiimidazole. In all cases, antibodies raised against C-terminal were used as solid phase agent. Detector antibodies were N terminal antibodies that were radio-iodinated with 125I followed by gel purification. Several of the antibodies coupled to various solid phase matrices were incubated with PTH standards and the detector antibody as well as the commercially available tracer from DiaSorin kit to identify a suitable match pair.
Results:
The best pair was polyclonal C-terminal PTH antibody along with the kit tracer from DiaSorin with regards to antibody coated to magnetic cellulose particles. Among the various antibodies and the solid phases evaluated, the best assay was obtained with the matched pair of antibodies (70×G67 and 70×G68) from Fitzgerald immobilized on polystyrene tubes. The polyclonal antibody against C-terminal PTH was chosen as the capture antibody and 125I labelled polyclonal antibody against N-terminal PTH as the tracer. The sample values obtained in the antibody coated tubes were comparable to those obtained using a commercial kit.
Interpretation & conclusions:
The results indicated the feasibility of adopting this system for further development into a PTH IRMA for regular production as there is no indigenous kit available for intact PTH.
Keywords
125I-labelled
immobilized antibodies
IRMA
magnetic cellulose
PTH
Parathyroid hormone (PTH), a peptide hormone composed of 84 amino acids, plays a central role in calcium and phosphate homeostasis and bone health. Estimation of parathyroid hormone levels is important in the management of metabolic bone disorders and in certain renal diseases1. Further, PTH inhibition rate is useful in the detection of early-stage primary hyperparathyroidism2. The analysis of PTH is complicated by the heterogeneity of the circulating forms. PTH is rapidly degraded in the peripheral circulation into biologically inactive fragments. The circulating immunoreactive PTH consists of complex mixture of PTH (1-84) [PTH-I], N-terminal fragment (1-34) and various types of C-terminal fragments (36-84, 44-84, 49-84) which are biologically inactive and therefore, interferes with the measurement of biologically active and intact PTH34.
Although the first description of PTH dates back some 40 years5, the heterogeneous nature of parathyroid hormone and technical difficulties encountered in producing specific antiserum led to insufficiently reliable assay methods. The radioimmunoassay of PTH became possible with the availability of highly purified human PTH. This method is based on radiolabelled PTH and polyclonal antibodies directed towards the C- or N-terminal PTH. Such methods allowed the determination of various types of circulating PTH peptide fragments that make up the “family” of heterogeneous parathyroid hormone instead of the intact wholesome PTH (1-84). These were called as “first generation” assays which lacked specificity and sensitivity. In addition, radiolysis of radioactive PTH resulted in limited use of this technique at the clinical front.
In 1987, with the appearance of the first immunoradiometric method6 dosing of intact PTH was made. The first is called capture antibody attached to a solid support while the second is the marker (radioactive, enzymatic or chemiluminescent labelled) for quantifying hormone. These are called second generation assays, which have high sensitivity and specificity owing to the usage of antibodies that recognize the two ends of the molecule. Hence, these were called assays of “intact PTH”. These assays subsequently confirmed their improved clinical importance and diagnostic value, especially in the treatment of renal osteodystrophy7–10.
In 1998, even these were denounced due to problems of cross-reactivity with fragments of 7-84 with the detector antibodies10. The carboxy terminal fragments of PTH are known to accumulate in patients with progressive degrees of renal failure as their metabolic clearance depends heavily on glomerular filtration11. This finding could explain the extremely high levels of PTH observed in dialysis patients with the histological features of a dynamic osteopathy. Thus came up the third generation PTH assays, which typically used carboxy terminal antibodies (epitope 39-84) to capture various molecular forms of circulating PTH and a signal amino terminal antibody against epitope 1-4 of PTH. Contrary to 2nd generation intact-PTH assays that reacted with every fragment having the epitope 15-34, this new assay did not react with non-PTH (1-84). However, the use of these 3rd generation assays alone did not appear to improve the distinction of various bone diseases associated with renal failure. The combined use of 3rd and 2nd generation assays has been proposed as a simple way to calculate by subtracting the levels of non-PTH (1-84) and then by taking the ratio of PTH (1-84) to non-PTH (1-84)12.
The present study was undertaken to develop a simple, sensitive and specific second generation solid phase immunoradiometric assay to detect intact PTH levels. Various solid phase matrices viz. polystyrene tubes, beads and magnetisable particles were evaluated1314. In order to have an assay that will measure only intact PTH, one antibody should be against the C terminal while the other against the N terminal PTH. Attempts were made to produce in-house antibodies against C-terminal and N-terminal PTH by conjugating the C-terminal and N-terminal fragments to bovine serum albumin (BSA) and immunizing the rabbits and sheep with these conjugates. We describe here the selection of a matched pair of antibodies and a suitable solid phase system for standardization of an IRMA for PTH.
Material & Methods
Chemicals: Pure PTH, glutaraldehyde, anti-sheep gamma globulin and 0.2 per cent casein were purchased from Sigma Chemical & Co., USA. Antibodies were obtained from two different sources. Polyclonal N-terminal PTH antibody and polyclonal C-terminal PTH antibody were obtained gratis from China Atomic Energy Commission while monoclonal C- terminal PTH antibody, monoclonal mid region PTH antibody , matched pair of polyclonal C- terminal PTH antibody and polyclonal N-terminal PTH antibody, were purchased from Fitzgerald Industries International Inc., USA. Sodium azide, sodium chloride, sephadex G-75, chloramine-T, sodium metabisulphate (Na2S2O5), potassium iodide (KI), BSA were of AR grade obtained from E Merck, India. Na125 I was obtained from IZOTOP, Hungary.
Animal studies: The assay standardization and validation were carried out at the Board of Radiation and Isotope Technology, Navi Mumbai, India. Only the immunizations were carried out at the Sri Venkateswara Veterinary University, Tirupati, India.
Origin and Microsoft Excel Software USA were used for the graphs and statistical analysis.
Ethical clearance was obtained from Animal ethics clearance committee of Sri Venkateswara Institute of Medical Sciences, Tirupati.
Selection of a matched pair of antibodies
Several antibodies were evaluated to arrive at a suitable matched pair for development of an assay for intact PTH.
In-house produced antibodies against C-terminal and N-terminal PTH:
Preparation of N-terminal PTH and C-terminal BSA conjugate - The low molecular weight haptens C-terminal and N-terminal PTH were conjugated to BSA in order to make them immunogenic. Immunization was carried out in both rabbit as well as sheep with 200 μg of N-terminal PTH fragment and 300 μl of 2 mg BSA in 0.05M phosphate buffer (pH 7.4). Two mg of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was added in two lots at an interval of 1.5 h. After 4 h, the mixture was dialyzed overnight against 0.05M phosphate buffer saline and was transferred to a vial, aliquoted and stored at -20°C. The same procedure was followed to prepare the C-terminal PTH- BSA conjugate also. The amount being so low and PTH fragments being expensive, no characterization was carried out before immunization for these conjugates.
Immunisation - Both the conjugates (C- and N-PTH-BSA) were separately emulsified in Freund's adjuvant in 1:1 ratio and injected intradermally into two rabbits each. Two boosters were given at an interval of 4-6 wk each. An initial dose of 100 μg and booster doses of 40 μg were given at monthly intervals. Blood samples were collected 10-15 days after every booster, and tested for the presence of antibodies by incubating with radiolabelled PTH. The same procedure was followed for sheep with the emulsion in proportion to the weight of the animal.
Antibodies from Chinese Atomic Energy Agency - One set of antibodies against N-terminal and C-terminal PTH were obtained as a gift from the Chinese Atomic Energy Agency. These antibodies were also evaluated and used for developing a workable assay.
Precipitated immunoglobulin fraction of N- & C-terminal PTH antiserum - The lyophilized antiserum with N-terminal antibodies as well as C-terminal antibodies was reconstituted in 1 ml of 18 per cent Na2SO4 separately. The precipitate was separated by centrifugation for 30 min at 3000 g. The precipitate was dialyzed against phosphate buffer for 48 h to remove sodium sulphate from the globulins.
Radiodinated N-terminal PTH antibody (N*)
Preparation of 125I-Ab against N-terminal PTH - The radioiodination1516 was carried out in a fume hood. All solutions were made in 0.05M phosphate buffer, pH 7.5, and 20 μg (26 μl) N-terminal PTH antibody, 50 μl of 0.5 M phosphate buffer (pH: 7.5), about 18MBq (10 μl) of Na125I and 5 μg (5 μl) chloramine-T were added in a glass tube, mixed for 1 min at room temperature; 30 μg (15 μl) Na2S2O5 (sodium metabisulphite) was added and mixed to arrest the oxidation reaction. Then 120 μg (100 μl) of KI (potassium iodide) was added as carrier for purification. The reaction mixture (Rx) was transferred to the top of the preconditioned sephadex G-75 gel (15×1 cm) in a glass column. Forty fractions of 1 ml were eluted with 0.05 M phosphate buffer. The fractions were measured for radioactivity in a NaI(Tl), gamma counter, Electronic Corporation of India Ltd. (ECIL), India adjusted for 125I. The radioactivity of the fractions was plotted against the fraction number.
Characterisation of 125I- Ab against N-terminal PTH - An aliquot (~5 μl) of the reaction mixture and peak fractions obtained from the column were spotted on a 30 cm Whatman 3 mm paper strip. The electrophoresis was run for 1 h at 240 V. The electrophoresis pattern of the reaction mixture was used for calculating the percentage radioiodination yield while those of the other fractions were used as a measure of the radiochemical purity of the corresponding fractions. After electrophoresis, the paper was cut into one cm strip and measured for radioactivity.
Immobilization of antibodies
Antibody coupling to magnetic cellulose - The in-house prepared magnetic cellulose particles17 were coupled with purified PTH antiserum using the previously described protocol18. Briefly, these particles were activated with carbonyl diimidazole in acetone. The activated particles were sequentially washed with acetone, double distilled water, acetate buffer and bicarbonate buffer. The antibody in bicarbonate buffer was added to the activated magnetic cellulose particles and incubated for 24 h. The particles were washed with bicarbonate followed by phosphate buffer and suspended in 0.5 per cent BSA. The procedure was repeated for all the different antibodies obtained.
Antibody coating to polystyrene beads and tubes: Direct physical adsorption of primary antibody - Different dilutions (0.3 ml) of the capture antibody in 0.1M bicarbonate buffer (pH 8.5) were added to special polystyrene tubes with star shaped bottom for more surface availability. Separately, polystyrene beads were added in glass vials containing the antibody solution. Both beads and tubes were incubated overnight, washed three times with 0.05M phosphate buffer, pH 7.5. The unoccupied sites on the polystyrene surface were saturated with 1 per cent BSA and finally washed with 0.05 M phosphate buffer.
Coating beads through glutaraldehyde - BSA (2%) in 0.05 M phosphate buffer with 0.1 per cent sodium azide and 0.9 per cent sodium chloride were added to the polystyrene beads taken in a vial and kept for shaking at 100-200 rpm for 3-4 h. The beads were washed with distilled water 6 times to remove traces of BSA, 2 per cent glutaraldehyde was added to the beads and incubated overnight at room temperature for activation. After 24 h, the beads were washed 10-12 times with distilled water to ensure complete removal of unreacted glutaraldehyde. Beads (6-8 in no.) were incubated for 24 h in 2 ml of antibody solution in 0.1M bicarbonate buffer, pH 9. The beads were washed 10 times with distilled water. These coated beads were saturated with 0.5 per cent BSA and 0.2 per cent casein in 0.05 M phosphate buffer for 2 h on a shaker. The beads were washed five times with 0.05 M phosphate buffer.
Coating of tubes through second antibody - Another set of tubes was coated with 2 μg/0.3 ml of second antibody (SAb) (rabbit anti-goat IgG) in the star bottom tube and incubated overnight, at room temperature with constant shaking. The tubes were washed thrice with 0.5 ml of 0.05 M phosphate buffers and saturated with 0.5 ml of 1 per cent BSA. The tubes were washed thrice with 0.5 ml/tube of 0.05 M PO4 buffer. Poly C-term PTH antibody 1.5 μg/0.3 ml bicarbonate buffer/tube was incubated for 24 h at room temperature with the immobilized secondary antibody coated tubes for coupling of the first antibody. The tubes were washed with 0.5 ml of 0.05 M phosphate buffer. Then, the beads and coated tubes were incubated with 0 and 2500 pg/ml PTH standards for 24 h. At the end of the incubation, the solution was aspirated from the vial, in case of beads and decanted in case of tubes. Both inclusive as well as exclusive assays were carried out.
Identification of a suitable pair of antibodies: A pair of antibodies, one as the capture and the other as detector is accepted as a suitable pair when it gives the required sensitivity and maximum binding. Several antibodies coupled to magnetic cellulose were incubated with PTH standards and the detector antibody as well as the commercially available kit tracer from DiaSorin, USA.
Standardization of an assay protocol using immobilized antibody: Several standards ranging from 14.6 to 2000 pg/ml in human serum were incubated with about 1 μg of capture antibody (Poly Ab C-terminal PTH) which was coupled to magnetic cellulose, or immobilized on polystyrene tubes or beads, and 100 μl of DiaSorin kit tracer (K*) for 24 h at room temperature. At the end of incubation, the particles were washed twice with wash solution, which was 0.1 per cent tween 20 in 0.05 M phosphate buffer, pH 7.5. The test tubes were placed on a rack with magnetic plate after the addition of wash buffer, for 15 min. The tubes were decanted keeping the tubes in contact with the magnetic rack. The washing was repeated once more and the bound counts were measured in a NaI (Tl) counter. In case of the coated tubes, the tubes were decanted and in case of the beads, the content of the tubes were aspirated. The average bound cpm was plotted against the standard PTH concentration. Exclusive assays were also carried out wherein the tracer was added only after a washing step after the incubation of solid phase antibody and PTH standards.
Results
The rabbit antiserum did not show any appreciable quantity of antibodies against PTH, for any of the boosters when tested for binding with radioiodinated PTH. Similar results were obtained in sheep.
Radiolabeling of N-terminal PTH antibody with 125I and its purification: The reaction mixture after radioiodination showed two radioactive peaks. The first peak corresponded to the radioiodinated antibody and the second was unreacted free iodide (125I), as indicated by the paper electrophoresis pattern. A typical elution pattern is shown in Fig. 1. The average radioiodination yield was about 75 per cent with regards to radioactivity and the radiochemical purity of the pure radioiodinated antibody as estimated by paper electrophoresis was >98 per cent (Fig. 2). The average specific activity ranged from 8-12 μCi/μg. The first fraction was diluted to 400000 counts/min/100μl and was used as detector antibody for evaluation in the assay.
- Elution pattern of iodinated anti PTH against N-terminal PTH.
- Paper electrophoresis of purified fractions of 125I labelled PTH antibody.
Identification of a suitable pair of antibodies and standardization of assay: The bound counts obtained for different combinations of antibodies in the magnetic particle system are given in the Table. The best pair was polyclonal C-terminal PTH antibody from China along with the tracer antibody from DiaSorin kit. The standard curve using this pair is given in Fig. 3. The parameters for an assay using anti-C-PTH-antibody as capture antibody coated on magnetic particle and 125I-anti-N-PTH-antibody (N*) as tracer were also optimized. The developed system was tested with two batches of tracers made in order to observe the behaviour with the life of the tracer. The standard curves using the optimized system are given in Fig. 4. The standard curve for the antibody coated tube assay using the matched pair of antibodies (70×G67 and 70×G68) from Fitzgerald is given in Fig. 5 along with the standard curve obtained by Diasorin kit. In both magnetic particle assay and coated tube assay, the exclusive assays gave better binding. Glutaraldehyde treatment has not shown any added advantage. Several samples were analysed with DiaSorin kit as well as the kit developed in-house. Figs 6 and 7 show the correlation of the sample values obtained by the two methods.
- PTH IRMA standard curve using PTH polyclonal C-terminal antibody coupled to Magnetizable particles. Poly C-Ab: Polyclonal antibodies raised against C-terminal fragment of PTH.
- Comparison of standard curves using developed mag PTH assay and DiaSorin kit. MAG Poly C-PTH Ab + N*: Poly C-term PTH antibody coated magnetisable particles + Radioiodinated Poly n- terminal PTH antibody (batch 1)
- MAG Poly C-PTH Ab + N2*: Poly C-term PTH antibody coated magnetisable particles + Radioiodinated Poly n- terminal PTH antibody (batch 2).
- Comparison of standard curves using developed coated tube PTH assay and DiaSorin kit. Poly C-PTH Ab: Polyclonal antibodies raised against C-terminal fragment of PTH.
- Comparison of sample analysis between in-house PTH coated tube assay with DiaSorin kit.
- Correlation studies between in-house PTH coated tube assay and DiaSorin kit.
Discussion
Development of immunoradiometric assay (IRMA) for intact PTH that is devoid of PTH fragment interference is essential for accurate clinical assessment of parathyroid function. For many years, measurement of PTH was performed by single antibody radioimmunoassays that were stymied by PTH fragment intereference. PTH 2-site IRMA based on a match pair of antibodies offers an effective solution to circumvent the problems posed by PTH fragment interference.
Our attempts to produce antibodies against the intact PTH resulted in low avidity antibodies. Further attempts to produce antibodies against conjugates of C- and N-PTH-BSA also resulted in poor titre. Hence, the standardization of the assay was carried out using commercially available antibodies. The preparations of 125I-labelled PTH antibody with relatively uniform specific activities and high purity were realized which helped prevent large variations in assay binding and sensitivity. Based on various experiments that were carried to develop an optimized assay it was seen that the polyclonal antibody against C-terminal PTH coupled to magnetic cellulose could be a good capture antibody for realizing an IRMA for intact PTH, when matched with the tracer from a commercial kit (DiaSorin). However, such a combination will not be feasible for scaling up to regular production. It was also seen that when 125I labelled poly-anti-N-terminal PTH was used as the tracer, a standard curve could be realized, although of much lower sensitivity.
From the results of the above experiments, it was observed that a workable assay can be achieved using a pair of matched antibodies against PTH. As additional validation parameter, the indigenously developed assay was compared with a commercial kit (DiaSorin). Further, it is observed that the maximum binding decreases with the ageing of the tracer and hence it is advisable to use the tracer within a month after its preparation. In order to have a kit that is more user friendly than that employing magnetic cellulose particles as solid phase, it was decided to test the possibility of using the polystyrene assay tubes for coating the capture antibodies. Along with the tubes, polystyrene beads were also tested for their utility in this assay.
The tubes coated with anti-sheep antibody followed by the primary antibody were tested for their performance in kit, by constructing standard curves using these tubes as well as the tubes coated directly with the primary antibody using the poly N-PTH Ab tracer, as optimized earlier. These were also compared with the standard curve obtained using commercial kit assay reagents. It is seen that although the response with secondary antibody coated tubes was positive at high PTH concentrations, at ‘0’ PTH level, the non-specific binding in the assay was high. Several blocking agents were used to decrease this non-specific binding, but were unsuccessful. Finally, an assay for measurement of intact PTH in human serum could be developed using antibody coated by physical adsorption directly on the tubes, and used for further studies. The sample values obtained by the present method correlated well with the values obtained in the commercial kit, indicating the validity of the assay developed. This system is feasible for further development into a PTH-IRMA for regular production and supply.
Among the various antibodies and the solid phases evaluated for development of the PTH-IRMA, the best system was with the matched pair of antibodies (70×G67 and 70×G68) from Fitzgerald and employing antibody coated polystyrene tubes. The polyclonal antibody against C-terminal PTH was chosen as the capture antibody and 125I labelled polyclonal antibody against N-terminal PTH as the tracer. The procedures for radio-iodination, purification and evaluation of the tracer could be optimized for PTH antibodies. The developed system needs to be further validated to take it to the stage of production and supply.
In conclusion, the results indicated the feasibility of adopting this system for further development into a PTH-IRMA for regular production as there is no indigenous kit available for intact PTH.
Acknowledgment
The authors acknowledge CAEC, China, for the gift sample of polyclonal PTH antiserum. The authors thank Dr Vijay Kadwad, Manager, RIA, BRIT, India, and his team for providing the in-house magnetic cellulose, and acknowledge Dr A.K. Kohli, Chief Executive, BRIT for his support during this work.
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