scholarly journals Development of an Ultrasensitive Immunoassay for Human Glandular Kallikrein with No Cross-Reactivity from Prostate-specific Antigen

1999 ◽  
Vol 45 (6) ◽  
pp. 790-799 ◽  
Author(s):  
Margot H Black ◽  
Angeliki Magklara ◽  
Christina V Obiezu ◽  
Dimitrios N Melegos ◽  
Eleftherios P Diamandis
Keyword(s):  
Detection Limit ◽  
Prostate Specific Antigen ◽  
Seminal Plasma ◽  
Limit Of Detection ◽  
Biological Fluids ◽  
Specific Antigen ◽  
Cross Reactivity ◽  
Free Form ◽  
Glandular Kallikrein ◽  
Healthy Males

Abstract Background: Studies demonstrating that human glandular kallikrein (hK2) is increased in prostate cancer patients have prompted speculation that this marker may of use in addition to prostate-specific antigen (PSA). Methods: An ultrasensitive hK2 sandwich immunoassay was developed, and its detection limit, cross-reactivity, analytical recovery, precision, and linearity of dilution were evaluated. hK2 was measured in seminal plasma and sera from healthy males, females, and prostatectomized patients. Results: Our assay has an excellent detection limit (6 ng/L) and precision (>90%). Recovery studies indicated that hK2 binds to serum protease inhibitors. All sera from healthy males had measurable hK2 concentrations (median, 402 ng/L). Almost all female sera had undetectable hK2. Serum hK2 and PSA in males correlated positively (r = 0.44), but hK2 was present at concentrations ∼2.5-fold lower than PSA. The PSA/hK2 ratio in male sera was 0.1–34, with a median of 2.6. In seminal plasma, this ratio was 100–500. More than 94% of immunoreactive hK2 in serum was in the free form (∼30 kDa); traces of hK2 complexed to α1-antichymotrypsin were present. Conclusions: The limit of detection of the method for hK2 measurement described here (∼20-fold lower than any other reported assay for hK2) allows the generation of new clinical information. When combined with a previously described method for PSA measurement that has no cross-reactivity from hK2, this methods allows the relative proportions of hK2 and PSA in biological fluids to be measured.

scholarly journals Characterization of Monoclonal Antibodies for Prostate-specific Antigen and Development of Highly Sensitive Free Prostate-specific Antigen Assays

1999 ◽  
Vol 45 (3) ◽  
pp. 347-354 ◽  
Author(s):  
Margot H Black ◽  
C Linda Grass ◽  
Jari Leinonen ◽  
Ulf-Håkan Stenman ◽  
Eleftherios P Diamandis
Keyword(s):  
Monoclonal Antibodies ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Cross Reactivity ◽  
Free Form ◽  
Time Resolved ◽  
Free Psa ◽  
Glandular Kallikrein ◽  
Highly Sensitive

Abstract Background: The recent elucidation of the importance of serological free prostate-specific antigen (PSA) in the diagnosis of prostate cancer has created a demand for immunoassays specific for free PSA. Methods: We developed and characterized 11 monoclonal antibodies with high affinities for PSA (Kavalues from 1.1 × 108 to 1.8 × 1010L/mol), only 3 of which cross-react with human glandular kallikrein (hK2). Using these antibodies and PSA antibodies developed by others, in conjunction with time-resolved fluorometry, we developed ultrasensitive sandwich immunoassays specific for the free form of PSA. Results: The analytical detection limit of these immunoassays is 0.001 μg/L. To our knowledge, this is the most sensitive free PSA assay reported to date. The free PSA immunoassays exhibit <1% cross-reactivity with PSA-α1-antichymotrypsin, show no cross-reactivity with hK2, and correlate well with established free PSA kits. The 11 antibodies developed by our group, in conjunction with 4 commercially available antibodies, were used to generate a putative epitope map of the PSA molecule. Conclusion: The highly sensitive free PSA immunoassays may be used for measuring PSA subfractions in female serum, an application currently impossible with other reported free PSA immunoassays.

scholarly journals Sensitive and Specific Immunodetection of Human Glandular Kallikrein 2 in Serum

2000 ◽  
Vol 46 (2) ◽  
pp. 198-206 ◽  
Author(s):  
Charlotte Becker ◽  
Timo Piironen ◽  
Johanna Kiviniemi ◽  
Hans Lilja ◽  
Kim Pettersson
Keyword(s):  
Detection Limit ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Free Form ◽  
Glandular Kallikrein ◽  
Kallikrein 2 ◽  
Assay Precision ◽  
Analytical Detection

Abstract Background: Human glandular kallikrein 2 (hK2) is expressed in the prostate and is present in serum from men with prostate cancer. Specific detection in serum is difficult mainly because of low concentrations and immunological cross-reactivity with prostate-specific antigen (PSA). Our objectives were to design an assay with improved analytical detection and functional sensitivity and nonsignificant cross-reactivity with PSA, and to characterize different immunoreactive forms of hK2. Methods: In the assay, critical PSA epitopes were blocked with four monoclonal antibodies (MAbs) specific for PSA. Subsequently, hK2 was captured using a MAb against hK2 (5% cross-reactivity with PSA), and after washing, hK2 was detected by a europium-labeled MAb with identical affinity for hK2 and PSA. Results: The analytical detection limit was <10 ng/L, and functional sensitivity was 30 ng/L. Cross-reaction with PSA was <0.01%. Between-assay imprecision was 3.1% for 1600 ng/L hK2 and 4.8% for 160 ng/L hK2; corresponding values for within-assay precision were 1.9% and 4.5%, respectively. Complexes of hK2-α1-antichymotrypsin (ACT) were detected in vitro with −6% bias compared with the free form of hK2. Gel filtration of patient samples showed that hK2 correlated in size mainly with free hK2; only 4–19% corresponded to hK2 possibly complexed with ACT or protein C inhibitor. Conclusions: Our assay had extremely low cross-reactivity with PSA, provided a very low detection limit, and allowed close to equimolar detection of the free and complexed forms of hK2. Moreover, we found that free hK2 is the predominant immunoreactive form of hK2 in serum.

scholarly journals Benign Prostate-specific Antigen (BPSA) in Serum Is Increased in Benign Prostate Disease

2003 ◽  
Vol 49 (2) ◽  
pp. 253-259 ◽  
Author(s):  
Harry J Linton ◽  
Leonard S Marks ◽  
Lisa S Millar ◽  
Christine L Knott ◽  
Harry G Rittenhouse ◽  
...  
Keyword(s):  
Prostate Specific Antigen ◽  
Limit Of Detection ◽  
Specific Antigen ◽  
Clinical Information ◽  
High Specificity ◽  
Wide Distribution ◽  
Cross Reactivity ◽  
Free Psa ◽  
Control Serum ◽  
Benign Prostate

Abstract Background: BPSA is a “benign” form of free prostate-specific antigen (PSA) that is increased in prostate transition zone tissues of men with pathologic benign prostatic hyperplasia (BPH). We developed an immunoassay to determine the concentration of BPSA in the serum of men with BPH. Methods: The BPSA antigen was purified by HPLC, and murine monoclonal antibodies were prepared by standard methods. A fluorogenic ELISA was developed with high specificity for BPSA and no cross-reactivity with other forms of PSA. Results: The BPSA immunoassay had a lower limit of detection of 6 ng/L and a cross-reactivity of <1% with all other clipped and nonclipped forms of PSA. The BPSA antibody was specific for the internal Lys182 cleavage site that characterizes BPSA. Biopsy-negative men with a median total PSA of 4.8 μg/L had a median of 0.22 μg/L BPSA, representing 25% of the free PSA in serum. BPSA ranged from 0% to 60% of the free PSA in serum. BPSA in a cohort of cancer serum also comprised 25% of the free PSA. Control serum from women or men without increased PSA had nondetectable BPSA. Conclusions: BPSA is a significant percentage of the free PSA in BPH serum but not in control serum. The presence of prostate cancer does not alter the relative proportions of BPSA in sera with <10 μg/L PSA. BPSA has a wide distribution of concentrations in the serum and may provide clinical information for the study of men with BPH.

Serum and Urinary Prostate-specific Antigen and Urinary Human Glandular Kallikrein Concentrations Are Significantly Increased after Testosterone Administration in Female-to-Male Transsexuals

2000 ◽  
Vol 46 (6) ◽  
pp. 859-862 ◽  
Author(s):  
Chrisitna V Obiezu ◽  
Erik J Giltay ◽  
Angeliki Magklara ◽  
Andreas Scorilas ◽  
Louis J G Gooren ◽  
...  
Keyword(s):  
Detection Limit ◽  
Prostate Specific Antigen ◽  
Cultured Cells ◽  
Specific Antigen ◽  
Post Treatment ◽  
Mean Values ◽  
Testosterone Treatment ◽  
Time Points ◽  
Glandular Kallikrein ◽  
Testosterone Administration

Abstract Background: The genes that encode prostate-specific antigen (PSA) and human glandular kallikrein (hK2) are up-regulated by androgens and progestins in cultured cells, but no published studies have described the effect of androgen administration in women on serum and urinary PSA or hK2. Methods: We measured serum and urinary PSA and hK2 before, and 4 and 12 months post testosterone treatment by immunofluorometric methods in 32 female-to-male transsexuals. Results: Mean serum PSA increased from 1.1 ng/L to 11.1 ng/L and then to 22 ng/L by 4 and 12 months post treatment, respectively; the corresponding mean values in urine were 17, 1420, and 18 130 ng/L, respectively. Serum hK2, another kallikrein closely related to PSA, remained undetectable at the three time points. However, urinary hK2 concentration rose from below the detection limit (<6 ng/L) before treatment to 18 and 179 ng/L by the 4th and the 12th month of treatment, respectively. All changes were statistically significant (P <0.001) at 4 months. Conclusions: Testosterone administration increases serum and urinary PSA and urinary hK2 in women. These measurements may be useful as indicators of androgenic stimulation in women.

scholarly journals Epitope Analysis of a Prostate-specific Antigen (PSA) C-Terminal-specific Monoclonal Antibody and New Aspects for the Discrepancy between Equimolar and Skewed PSA Assays

1999 ◽  
Vol 45 (4) ◽  
pp. 486-496 ◽  
Author(s):  
Hiroshi Nagasaki ◽  
Motoyuki Watanabe ◽  
Naoki Komatsu ◽  
Takashi Kaneko ◽  
Jean Y Dubé ◽  
...  
Keyword(s):  
Amino Acid ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Single Amino Acid ◽  
Free Form ◽  
Specific Monoclonal Antibody ◽  
Amino Acid Homology ◽  
Glandular Kallikrein ◽  
Total Psa ◽  
Intact Psa

Abstract Background: Immunoassays to measure prostate-specific antigen (PSA) often give different values for the same patient samples, and the calibrators among commercial immunoassays are not interchangeable. We developed three novel assays to quantify the free and complexed forms of PSA in serum. Methods: We synthesized 46 peptides, which encompassed the entire PSA molecule, and determined the interactions between selected monoclonal antibodies (MAbs) and those peptides or the intact PSA molecule. Results: MAb PA313 did not cross-react with human glandular kallikrein (hK2), which has 78% amino acid homology to PSA. This MAb bound with KD = 40 nmol/L to the C-terminal peptide of PSA and distinguished between a synthetic peptide derived from PSA (PSA46A: NH2-C-R226KWIKDTIVANP237-COOH) that differed from one derived from hK2 (PSA46B: NH2-C-R226KWIKDTAANP237-COOH) by a single amino acid. Only the MAb combination of PA313/PA121 showed equimolar reactivity with PSA and with PSA complexed with α1-antichymotrypsin (PSA-ACT). The free form of PSA (F-PSA) was determined by MAbs PA313/FPA503, and the amount of complexed PSA (C-PSA) in PSA-ACT was determined by αACT/PA313. The total PSA (T-PSA) measured by either of the equimolar assays (PA313/PA121 or Tandem-R) was consistent with the sum of F-PSA and C-PSA. In contrast, T-PSA by a skewed assay (IMx) was higher than F-PSA + C-PSA when the ratio of F-PSA to T-PSA (F/T) was >0.15. T-PSA measured by IMx was nearly equal to F-PSA/0.55 + C-PSA. The coefficient 0.55 reflected different reactivities of the IMx assay with PSA-ACT and PSA. Conclusion: The discrepancy between the values measured by equimolar and skewed assays depends on the ratio of free to total PSA in the sample.

Immunofluorometric assay for sensitive and specific measurement of human prostatic glandular kallikrein (hK2) in serum

1996 ◽  
Vol 42 (7) ◽  
pp. 1034-1041 ◽  
Author(s):  
T Piironen ◽  
J Lövgren ◽  
M Karp ◽  
R Eerola ◽  
A Lundwall ◽  
...  
Keyword(s):  
Detection Limit ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Molecular Size ◽  
Cross Reactivity ◽  
Specific Detection ◽  
Serum Samples ◽  
Free Psa ◽  
Glandular Kallikrein ◽  
Total Psa

Abstract Prostate-specific antigen (PSA) and human prostatic glandular kallikrein (hK2) have 79% identity with the primary structure. When we used recombinant hK2 protein, only 7 of 23 monoclonal anti-PSA IgGs (monoclonal antibodies, MAbs) cross-reacted with hK2, which enabled us to design a novel immunofluorometric MAb-MAb assay for the specific detection of hK2. In the first incubation, an excess of MAb 2H11, which does not cross-react with hK2, is added to prevent both free and complexed PSA from reacting in subsequent immunoreactions. In the second incubation, biotinylated MAb H50, which cross-reacts with hK2 by an epitope overlapping with MAb 2H11, served to bind only hK2 to the microtitration wells coated with streptavidin. In the third step, Eu-labeled MAb H117, which cross-reacts with hK2, detected the immobilized hK2. The hK2 assay was calibrated with recombinant hK2. The detection limit of the assay was 0.1 microgram/L, and the cross-reactivity with recombinant PSA was < or = 0.7%. The concentration of hK2 was measured in serum samples from 334 males with total PSA concentrations ranging from 1 to 3400 microgram/L. Most of the samples (57%) had hK2 concentrations below the detection limit. The proportions of hK2 relative to total PSA were 0-2% in 79%, 2-5% in 14%, 5-10% in 4%, and >10% in 3% of the samples. Gel filtration of 10 serum samples with increased hK2 concentrations showed a single peak of hK2 immunoreactivity with an apparent molecular size of approximately 30 kDa, corresponding to that of recombinant hK2 and free PSA.

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