1. PG-Seph. APCCB 20041 IDENTIFICATION OF MACRO- PROLACTIN WITH PROTEIN-G SEPHAROSE Jones GRD, Giannopoulos P. Departments of Chemical Pathology, St Vincent’s Hospital, Sydney, Australia.

2. PG-Seph. APCCB 20042 Introduction Macroprolactin is a prolactin-immunoglobulin complex which causes false positive results in many prolactin immunoassays. Laboratories require methods to identify this prolactin type. PEG precipitation is not suitable for all analysers (eg Abbott AxSYM), comparing two analysers requires extra equpiment and gel filtration chromatography is impractical in a routine setting. Protein-G is a streptococcus-derived protein with high affinity for immunoglobulin G. We evaluate affinity chromatography with protein-G Sepharose (PG-Sepharose) for use with an Abbott AxSYM analyzer for detection of macroprolactin.

3. PG-Seph. APCCB 20043 Protein-G Sepharose procedure Measure AxSYM prolactin and albumin on serum sample. Aliquot 400uL Protein G Sepharose into microfuge tube. Wash gel twice with phosphate buffered saline (PBS). Add 400uL sample to 400uL gel and mix for 20 minutes. Centrifuge, remeasure prolactin and albumin in supernatant. Use change in albumin concentration to compensate for dilution during chromatography. Protein G Sepharose: Purchased from Sigma Aldrich (Cat# P4691) Recombinant Protein G with albumin-binding site removed immobilised on 4% beaded agarose Regenerate gel, by washing twice with 0.2 M Glycine buffer and neutralise with three PBS washes.

4. PG-Seph. APCCB 20044 Methods and Results (1) A sample with known macroprolactin was treated with PG- Sepharose in quadruplicate. Result: The process was reproducible, with precision better than 3% (table 1). Immunoglobulin G was measured on 7 samples before and after PG-Sepharose treatment using a Beckman-Coulter nephelometer. Result: Immunoglobulin G was almost completely removed with one treatment (table 2) A sample with known macroprolactin was treated twice with PG-Sepharose. Result: First treatment reduced the amount of measured prolactin, repeat testing of the supernatant did not reduce measured prolactin further (table 3). Protein-G Sepharose was shown to be re-usable at least 3 times after regeneration with a low pH buffer.

5. PG-Seph. APCCB 20045 Table 1Table 3Table 2 Table 1. Replicate treatment of a sample containing macro-prolactin. Interpretation: The PG-Sepharose treatment showed good precision. Table 2. Removal of IgG with PG-Seph treatment. The last sample had elevated globulin and elevated IgG. Interpretation: IgG almost completely removed by PG-Seph treatment. Table 3. Repeat PG-Seph treatment of a sample containing macroprolactin. Interpretation: No further fall in prolactin was seen on repeat testing suggesting complete removal with one treatment. * “After” samples corrected for dilution

6. PG-Seph. APCCB 20046 Methods (2) 17 samples with high serum prolactin (>400 mIU/L) were selected and treated with PG-Sepharose. As a comparative method, the ratio of prolactin measurements on a Beckman-Coulter Access and the Abbott AxSYM analyzers was used 1. –Samples without macroprolactin showed an Access/ AxSYM ratio of about 0.63 (due to standardisation differences). –This ratio was previously validated with over 40 samples. –Samples with macroprolactin showed a lower ratio due to higher cross-reactivity of macroprolactin in the AxSYM assay than in the Access assay. –6 samples (from 3 patients) were classified macroprolactin-positive and 11 samples macro-negative.

7. PG-Seph. APCCB 20047 Results (2) After adjustment for dilution, treatment with PG-Sepharose did not change prolactin concentration in most samples without macroprolactin by comparative method. The average prolactin recovery in non-macro samples was 102% (one sample excluded - see below) Six samples with macroprolactin by comparative method showed residual prolactin between 21% and 48%. One “indeterminate” sample showed a fall in prolactin with PG-sepharose treatment (76% residual) but did not show macroprolactin by the comparative method. This data is shown in figure 1. REFERENCE 1. Ismail AAA et al. Ann Clin Biochem. 2003;40:298-300.

8. PG-Seph. APCCB 20048 Figure 1. Residual AxSYM prolactin after PG-Sepharose treatment compared to comparator method results. Dashed lines are the average residual prolactin for each group. The pale blue data point in the No- Macro group (“indeterminate”) was excluded from averages calculation. Interpretation. Most samples show clear separation between the presence and absence of macro-prolactin. No-Macro ( ) Macro ( ) Residual AxSYM Prolactin AxSYM:Access Ratio classification

9. PG-Seph. APCCB 20049 Methods and Results (3) The Access:AxSYM ratio was measured before and after treatment with PG-Sepharose (table 4 & figure 2). Samples without Macroprolactin –No significantly change the Access:AxSYM ratio –Little change in position on graph, ratio remained near 0.63. Samples with Macroprolactin –Returned the Access:AxSYM ratio to near 0.63. –Moved position on graph onto the line consistent with no remaining macroprolactin. –Larger fall in AxSYM result than Access result consistent with known assay cross-reactivity The previously identified “indeterminate sample” showed changes in both measurements, ratio remained near 0.63.

10. PG-Seph. APCCB 200410 Table 4: Effect of PG-Sepharose treatment on Access:AxSYM ratio. Access:AxSYM ratios for samples with and without macroprolactin, before and after PG-Sepharose. “Indeterminate” sample not included. Interpretation: PG-Sepharose treatment returned Access:AxSYM ratio to “normal”, suggesting complete removal of macro-prolactin.

11. PG-Seph. APCCB 200411 Figure 2. Effect of PG-Sepharose treatment on Access:AxSYM ratio. Samples without macroprolactin before ( ) and after ( ) treatment with PG-Sepharose. M acroprolactin sample before ( ) and after ( ). The “indeterminate” sample is shown with red diamonds (, ). Line of best fit for non-macro samples is shown (brown line, y = 0.63 x) AxSYM prolactin Access Prolactin

12. PG-Seph. APCCB 200412 Discussion PG-Sepharose treatment is simple, quick, precise and requires no special equipment. The method allowed clear discrimination between most samples with and without macroprolactin. The “indeterminate” result may be due to misclassification by Access/AxSYM ratio 1. Treatment appears to allow quantitation of free prolactin. –Treatment did not affect normal samples. –Repeat treatment of macroprolactin sample had no further effect. –Treatment normalised the Access:AxSYM ratios of samples with macroprolactin. While the PG-Sepharose is expensive, re-use is possible.

13. PG-Seph. APCCB 200413 Conclusions Sample treatment with protein-G Sepharose is a simple technique for identification of immunoglobulin G-containing macroprolactin for the AxSYM prolactin assay. The method may allow quantitation of uncomplexed prolactin in samples which also contain macroprolactin. Comparison of protein-G Sepharose treatment with a definitive method may assist in further validating this method.