Time-resolved fluorescence based direct two-site apoA-I immunoassays and their clinical application in patients with suspected obstructive coronary artery disease.

Objective: High-density lipoprotein (HDL) is a heterogeneous group of subpopulations differing in protein/lipid composition and in their anti-atherogenic function. There is a lack of assays that can target the functionality of HDL particles related to atherosclerosis. The objective of this study was to construct two-site apolipoprotein A-I (apoA-I) assays and to evaluate their clinical performance in patients with suspected obstructive coronary artery disease (CAD). Approach and results: Direct two-site apoA-I assays (named 109-121 and 110-525) were developed to identify the presence of apoA-I in the HDL of patients with CAD using apoA-I antibodies as a single-chain variable fragment fused with alkaline phosphatase. ApoA-I109-121 and apoA-I110-525 were measured in 197 patients undergoing coronary computed tomography angiography (CTA) and myocardial positron emission tomography perfusion imaging due to suspected obstructive CAD. Among patients not using lipid-lowering medication (LLM, n = 125), the level of apoA-I110-525 was higher in the presence than in the absence of coronary atherosclerosis [21.88 (15.89-27.44) mg/dl vs. 17.66 (13.38-24.48) mg/dl, P = 0.01)], whereas there was no difference in apoA-I109-121, HDL cholesterol, and apoA-I determined using a polyclonal apoA-I antibody. The levels of apoA-I109-121 and apoA-I110-525 were similar in the presence or absence of obstructive CAD. Among patients not using LLM, apoA-I110-525 adjusted for age and sex identified individuals with coronary atherosclerosis with a similar accuracy to traditional risk factors [area under the curve [AUC] (95% CI): 0.75(0.66-0.84) 0.71 (0.62-0.81)]. However, a combination of apoA-I110-525 with risk factors did not improve the accuracy [AUC (95% CI): 0.73 (0.64-0.82)]. Conclusion: Direct two-site apoA-I assays recognizing heterogeneity in reactivity with apoA-I could provide a potential approach to identify individuals at a risk of coronary atherosclerosis. However, their clinical value remains to be studied in larger cohorts.

Three two-site apoA-I immunoassays using phage expressed detector antibodies - Preliminary clinical evaluation with cardiac patients.

High density lipoproteins (HDL) are a heterogenous group of subpopulations differing in protein/lipid composition and in their anti-atherogenic function. There is a lack of specific and robust assays which can target the functionality of HDL with respect to atherosclerosis. With recently generated CAD HDL targeted, single chain recombinant antibodies (scFvs) we set out to design and optimize apo A-I tests to compare it with conventional HDL-C and apo A-I analyses for diagnosis and risk assessment of coronary artery disease (CAD) and its outcome. Three highly sensitive two-site apo A-I assays: 022-454, 109-121 and 110-525 were optimized. A preliminary clinical evaluation of these assays, after proper sample dilution procedure, was performed using samples derived from 195 chest pain patients (myocardial infarction (MI), n = 86 and non-MI, n = 109), collected at the time of admission and at discharge from hospital (hospital stay ≤ 24 h). The clinical performance of the assays was compared with apo A-I measured with polyclonal anti-apo A-I antibody using conventional ELISA. Apo A-I data was in addition compared with HDL-C concentration of the samples. The concentration of apo A-I was significantly lower in MI patients than in non-MI individuals with assay 022-454 (admission and discharge samples, P < 0.0001 and = 0.004); assay 109-121 (admission and discharge samples, P = 0.04 and 0.0009), and, ELISA based apo A-I test (admission and discharge samples, P = 0.008 and < 0.0001). HDL-C (admission and discharge samples, P = 0.002 and P = 0.01) was also significantly lower in MI patients. In Kaplan- Meier analysis, two-site assay 109-121 assay predicted mortality from admission samples at 1.5 yrs (whole cohort, P = 0.01 and in MI patients, P = 0.05) and at 6 months (whole cohort, P = 0.04). Assay 110-525 predicted mortality at 1.5 yrs from admission samples of non-MI patients (P = 0.01) and at 6 months from whole discharge sample cohort (P = 0.04). Polyclonal anti-apo A-I based conventional assay predicted mortality at 1.5 yrs from admission samples of whole cohort (P = 0.03). Two-site apo A-I assay 022-454 and HDL-C provided no capability of predicting mortality in the whole cohort or any sub-group. In conclusion, two of the tested recombinant apo A-I antibody combinations (sc 109-121 and sc 110-525) display promising outcome to improve diagnosis and prediction of future cardiac events in cardiac patients over polyclonal apo A-I ELISA and HDL-C assays. The noted differences, while interesting, are preliminary and need however to be verified in extensive cohorts of pathological cardiac conditions and healthy controls.

Next generation sequencing of all variable loops of synthetic single framework scFv-Application in anti-HDL antibody selections.

Next generation sequencing (NGS) can be applied to monitoring antibody phage display library selection processes to follow the enrichment of each individual antibody clone. Utilising the recent development of the Illumina sequencing platform enabling sequencing up to 2×300bp, we have developed a method to deep sequence all complementarity determining regions (CDRs) in the clones obtained from a synthetic single framework antibody library. This was complemented by an in-house bioinformatics pipeline for efficient analysis of the sequencing results. The method was utilised to study antibody selections against high density lipoprotein (HDL) particles. Sequencing of the output from each selection round enabled extraction of useful information on both the total copy numbers as well as the relative enrichment rates of the clones. Ten antibody clones showing different ranking in terms of frequency were reproduced from synthetic DNA constructs and their capacity to bind HDL was verified by an immunoassay. The method thus facilitates the isolation of clones of interest, and in particular can assist retrieval of less efficiently enriched, yet interesting clones, which are unlikely to be identified by conventional, random colony picking based, screening.

Identification and analysis of anti-HDL scFv-antibodies obtained from phage display based synthetic antibody library.

OBJECTIVE: In epidemiological studies plasma high density lipoprotein cholesterol (HDL-C) levels are found to correlate inversely with atherosclerotic cardiovascular events. HDL consists of different subpopulations and they vary in their anti-atherogenic properties. The aim of this study is to isolate coronary artery disease (CAD) specific anti-HDL scFv-antibodies. DESIGN AND METHODS: To obtain CAD specific HDL binders, we used phage displayed synthetic antibody libraries to enrich specific antibodies against HDL isolated from CAD patients. The antibodies were affinity purified. Their capability to recognize apolipoproteins A-I and A-II, various HDL forms differing in lipid/protein ratios and plasma HDL, was studied using time-resolved fluorescence based immunoassay. RESULTS: Using different selection strategies and immunoassay based screening we obtained altogether 1200 clones displaying HDL binding activity. By sequencing 337, we identified 264 unique antibodies against HDL. A set of 61 antibodies were selected for further analysis. We found a variety of antibodies with different binding profiles, including apoA-I binding antibodies either in lipid-dependent or lipid-independent manner and binders against apoA-II. Several antibodies were able to discriminate between HDL derived from CAD patients and healthy controls. A majority of the antibodies were immunoreactive with HDL in plasma. CONCLUSION: The novel HDL recognizing antibodies isolated from synthetic antibody phage library have displayed interesting HDL-binding characteristics suggesting that, in addition to use as research tools, a part of them might be useful for the development of diagnostic methods for CAD risk assessment.

Characterization of monoclonal antibodies against prostate specific antigen produced by genetic immunization.

Prostate specific antigen (PSA) is the most important marker for prostate cancer. Antibodies against minor variants of PSA may be useful in the development of novel diagnostic tests for prostate cancer, but it has been difficult to produce such antibodies by protein immunization. In this study, we have compared the characteristics of monoclonal antibodies (MAbs) obtained by genetic immunization with those obtained by protein immunization. The whole coding region of PSA-cDNA was cloned in a mammalian expression vector pCDNA-3. Six mice were immunized four times by intra-muscular (i.m.) injection of the PSA-pCDNA3 plasmid. The MAbs produced were characterized with respect to subclass, epitope specificity, binding to various molecular forms of PSA and affinity. After intra-muscular injection of DNA, anti-PSA antibodies were detected in the serum of all mice, but the antibody titers were markedly lower than after protein immunization. After fusion of the spleen cells from the mice, five hybridomas producing MAbs to PSA were obtained. The MAbs were of IgG1 and IgG2a isotype and they all recognized equally different forms of free PSA, namely enzymatically active, nicked and proPSA. Epitope mapping showed that these MAbs reacted with the same antigenic regions as those obtained by protein immunization. Thus, genetic immunization leads to production of anti PSA MAbs with similar characteristics to those obtained by immunizing with PSA protein. As applied in the present study, it is less efficient than protein immunization, but it is a useful technique when the antigen is not available in the quantities needed for immunization.

Sensitive and specific enzymatic assay for the determination of precursor forms of prostate-specific antigen after an activation step.

BACKGROUND: The proteome of the serine protease prostate-specific antigen (PSA) and its enzymatic properties have been clarified only recently. We have developed a specific and sensitive method for the measurement of active PSA and used it to measure proPSA in blood. METHODS: We used the synthetic peptide KGISSQY, which possesses a PSA-specific cleavage site, as substrate. To ascertain the specificity of the assay, we used an anti-PSA monoclonal antibody that captures known forms of PSA. An activation step enabled us to measure proPSA by converting it to mature, active PSA. RESULTS: The detection limit of the optimized assay was 0.5 microg/L. In blood samples from patients, the activation step substantially increased the concentration of active PSA, thus showing the presence of proPSA in the samples. ProPSA was 0-79% (median, 45%) of the amount of free PSA in 15 samples with total PSA concentrations of 5.3-423 microg/L. In samples obtained from three benign prostatic hyperplasia (BPH) patients after transurethal resection of the prostate, no significant increase in activity was detected after the activation step, thus showing that proPSA was not a portion of free PSA in plasma of BPH patients. CONCLUSIONS: Proforms of PSA are a considerable fraction of free PSA in the blood of patients with increased total PSA. The approach described can be used to study the diagnostic value of proPSA and active PSA in patients with BPH and prostate cancer.

Prostate-specific antigen and other prostate-derived proteases cleave IGFBP-3, but prostate cancer is not associated with proteolytically cleaved circulating IGFBP-3.

BACKGROUND: Proteolysis of insulin-like growth factor-binding proteins (IGFBPs) may increase IGF-mediated growth stimulation and development of cancer in the organs producing large amounts of proteases, such as the prostate. METHODS: We studied proteolysis of IGFBP-3 by three prostate-derived proteases, namely prostate specific antigen (PSA), human kallikrein 2 (hK2), and trypsin, and also by native seminal plasma. Cleavage of 125I-IGFBP-3 was studied by SDS-PAGE and autoradiography. We also used two different sandwich-type IGFBP-3 immunoassays, called "intact" and "total" IGFBP-3 assays. These assays differ in their capacity to recognize proteolytically degraded IGFBP-3. RESULTS: HK2, PSA, and trypsin all cleaved IGFBP-3 at the concentrations normally present in seminal plasma. The IGFBP-3 cleavage by seminal plasma was inhibited by ZnCl2, which strongly inhibits hK2 and PSA, but not by a specific trypsin inhibitor. The IGFBP-3 fragments resulting from proteolytic cleavage by PSA, hK2, or trypsin were undetectable in the "intact IGFBP-3 assay," whereas the "total IGFBP-3 assay" also detected the proteolytic fragments. No increased fragmentation of IGFBP-3 was found in serum of 659 men with elevated PSA concentrations, of whom 178 had a proven prostate cancer. Furthermore, the IGFBP-3 levels were not associated with the PSA concentrations. CONCLUSIONS: These results show that, while seminal plasma and prostate-derived proteases can cleave IGFBP-3, in patients with prostate cancer the circulating IGFBP-3 is not significantly proteolyzed by either PSA, hK2, or trypsin.