Founder's Award, Society for Biomaterials. Sixth World Biomaterials Congress 2000, Kamuela, HI,May 15-20, 2000. Really smart bioconjugates of smart polymers and receptor proteins.

Over the past 18 years we have been deeply involved with the synthesis and applications of stimuli-responsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article.

Activated, N-substituted acrylamide polymers for antibody coupling: application to a novel membrane-based immunoassay.

A room-temperature-precipitable, activated terpolymer consisting of N-isopropylacrylamide (NIPAAm)/N-n-butylacrylamide(nBAAm)/N-acryloxysuccinimide (NASI) (LCST = 7-13 degrees C) at a monomer feed ratio of 60:40:2.5, respectively, was prepared and conjugated to an antibody. The conjugate was evaluated in a novel cellulose acetate (CA) membrane-based immunoassay which utilizes the especially strong physical attachment of the polymer to CA to bind and concentrate the polymer attached protein onto the membrane. When compared in the CA membrane immunoassay to the antibody-poly(NIPAAm) conjugate prepared via anhydrous copolymerization of NIPAAm and NASI at the monomer feed ratio of 40:1, respectively, the performance of the NIPAAm/nBAAm/NASI terpolymer was superior to that of the NIPAAm/NASI copolymer (LCST = 32 degrees C) when the studies were carried out at room temperature. However, the terpolymer and copolymer gave equivalent performance when the assay mixture was heated to 45 degrees C. These results indicate the importance of the LCST of the polymer component of the Ab-polymer conjugate to its adsorption and binding on the CA membrane.

Temperature-dependent absorption/desorption behavior of lower critical solution temperature (LCST) polymers on various substrates.

We have been studying adsorption and retention (resistance to desorption) behavior of temperature sensitive LCST polymers on different substrates as a function of temperature. According to our studies with Poly 64 (a copolymer of 60% (mol) NIPAAm and 40% (mol) NnBAAm, LCST = 8.5 degrees C in water), the copolymer retention depends on the rinse temperature. When the rinse temperature is above the LCST, the polymer adheres well to most surfaces. On the contrary, at rinse temperatures below the LCST, most of the adsorbed polymer is easily rinsed off. These studies are relevant to our work on the thermally reversible adsorption of LCST polymers conjugated to peptides and proteins, such as affinity ligands, for uses in immunoassays and affinity separations. The interaction between the LCST polymer and most hydrophobic polymer surfaces is mainly due to hydrophobic interactions, and the critical surface tension (gamma c) and the solubility parameter (delta) of the solid polymer substrate are the most important factors which influence the LCST polymer adsorption and retention. The critical surface tension appears to correlate best with the LCST polymer adsorption levels on different substrates, while the solubility parameter correlates best with the retention of the adsorbed polymer. According to our preliminary study, n-butyl groups probably interact more strongly with the substrates than isopropyl groups because of the greater hydrophobic surface area of the former groups.

Application of a thermally-reversible polymer-antibody conjugate in a novel membrane-based immunoassay.

We have developed a novel method to immobilize antibodies onto a cellulose acetate membrane using a conjugate of an N-isopropylacrylamide polymer covalently bound to the antibody. When compared with the unconjugated antibody, over 30-fold increase in retention of the antibody on the membrane was observed when it was conjugated to poly (N-isopropylacrylamide). Studies of the polymer-membrane interaction suggest a combination of hydrophobic and ionic forces, especially the former, is responsible for the high retention. We applied this novel immobilization technology in the development of a membrane-based immunoassay.

Phase-separation immunoassays.

Solid-phase-based immunoassays have traditionally been plagued by nonspecific binding to the solid phase and by slow reaction kinetics relative to reactants that are free to diffuse in solution. We have developed two novel immunoassays in which the solid phase is generated in situ after the specific binding reaction has occurred, thereby enhancing reaction kinetics and minimizing the opportunities for non-specific binding. In the first system, the capture antibody is conjugated to an organic monomer, polymerization of which to form insoluble polymer particles is initiated by a reaction involving free radicals. The amount of signal-labeled antibody incorporated into the resulting particles is directly proportional to the concentration of antigen. The principle is illustrated for the simultaneous assay of IgG and IgM in a single sample. In the second system, capture antibody is conjugated to a polymer, the solubility of which is a function of temperature. Specific binding is conducted below the critical solution temperature of the polymer, which is then separated from solution by increasing the temperature above the critical temperature. The incorporation of signal-labeled antibody into the precipitated polymer is directly proportional to the concentration of antigen. This principle is illustrated for the assay of hepatitis B surface antigen and Chlamydia trachomatis.

A novel immunoassay system and bioseparation process based on thermal phase separating polymers.

Poly-N-isopropylacrylamide (polyNIPAAm), a water-soluble, thermally precipitating synthetic polymer, has been conjugated together with a monoclonal antibody (MAb) and utilized in a novel separation method for an immunoassay. The PolyNIPAAm precipitates out of water above a critical temperature of 31 degres C, enabling a polymer-bound immune complex to be separated from the solution. The principal advantages of this method are that it utilizes a homogeneous incubation for the antigen-antibody reaction, plus, it has the ability to assay large-molecular-weight antigens with sensitivities equivalent to other nonisotopic heterogeneous immunoassays. In addition, since the polymer-immune complex may be reversibly redissolved by cooling, the method may be used both to concentrate the signal and isolate the analyte. This general technique may also be used for a wide variety of separation processes in addition to immunoassays, in which a specific component in a biological fluid, industrial process stream, or body of water is to be isolated for analysis, recovery, or disposal. Thus, product recovery and/or toxin or pollutant removal processes are possible with this methodology.

Analysis of serum and urinary retinol binding protein in hepato-renal syndrome.

Retinol binding protein (RBP) was analyzed in the sera and urines of 5 patients with hepato-renal syndrome (HRS), 4 with acute tubular necrosis (ATN), 20 liver cirrhosis patients with normal kidney function (NKF), 14 chronic renal failure (CRF) patients, and 19 healthy adults. All renal failure patients had high mean urine RBP (URBP): HRS, 8 mg/L; ATN, 11 mg/L; CRF, 8 mg/L respectively; p less than 0.001 vs the rest. Those with ATN and CRF had high mean serum RPB (SRBP): 146 and 149 mg/L, respectively, p less than 0.001 compared to the other groups. In HRS, in spite of renal failure, SRBP was very low (mean = 12 mg/L). The cirrhotics with NKF averaged less than 50% of the SRBP values of the healthy controls (16 vs 41 mg/L RBP, p less than 0.001); their RBP excretion was normal (mean URBP of 0.1 vs 0.06 mg/L in the control group). RBP analyses before and during HRS in two patients showed a marked increase in urine RBP during HRS (35- and 600-fold respectively) with practically unchanged serum levels. Impaired hepatic production and/or release is proposed to explain the low serum RBP in HRS, and a renal tubular injury or dysfunction to account for its high excretion. The RBP urinary loss could further compromise an already abnormal RBP metabolism and its serum levels. This combination (of low serum and high urine RBP), in the context of renal failure occurring in alcoholic liver cirrhosis, could help in the recognition of HRS.

Retinol binding protein and prealbumin in Reye's syndrome.

Retinol binding protein (RBP) and prealbumin (PA) were analyzed in 29 serum samples from 8 patients with stages II and III Reye's Syndrome (RS), and from 10 healthy fasting children. All RS patients had at least one abnormally low RBP and PA value. A return toward normal was evident within 2-3 days in serial samples. The nadir RBP and PA values in RS (means of 9 and 107 mg/L) were significantly different (p less than 0.001) from those of the controls (RBP mean of 40 and PA of 157 mg/L). A significant correlation was found between RBP and PA serum values in RS (r = 0.7, p less than 0.001); the high admission NH4+ values tended to associate with low RBP levels; however, the correlation was not very high (r = -0.4, p less than 0.01). The presence of an abnormal vitamin A transport system at an early stage of RS raises the possibility of additional abnormalities in vitamin A metabolism in these patients. The findings of reduced serum levels of complement, clotting factors, fibronectin, and now RBP and PA, suggest a marked short-term impairment of hepatic synthesis of proteins and/or their release in RS.

Isolation of the outer membrane components of Bordetella pertussis which enhance the immunogenicity of Haemophilus influenzae type b capsular polysaccharide polyribosyl ribitol phosphate.

The outer membrane (OM) component(s) from Bordetella pertussis which potentiated the antigenicity of purified Haemophilus influenzae type b capsular polysaccharide, polyribosyl ribitol phosphate (PRP), has been isolated and partially characterized. The OM was isolated from spheroplasting medium by precipitating at pH 5.0; fractionation was carried out by dissolving the crude OM in Triton X-100 followed by selective precipitation of OM in 50% ethanol. Further purification of OM was accomplished by DEAE-Sepharose 6BCL and Sephacryl S-300 chromatography. The biochemical composition and protein profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of various OM preparations have been examined. Combined vaccines consisting of OM components and PRP were given to young Sprague-Dawley rats, and the serum antibody to PRP was measured by an [3H]PRP binding assay. The purified OM containing mainly a 30,000-dalton protein, but not purified lipopolysaccharide or leukocytosis-promoting toxin, exhibited strong enhancement of PRP immunogenicity.

Nicotine enzyme immunoassay.

In the present study of the use of racemic aminonicotine as a functionalized hapten, nicotine antibodies suitable for use in nicotine determinations have been produced from antigens in which both 'flexible' and 'semi-rigid' chains serve to couple racemic 6-aminonicotine to bovine serum albumin. Nicotine enzyme immunoassay has been developed for the first time using antibodies produced against 6-epsilon-aminocapramido -DL-nicotine and beta-galactosidase nicotine enzyme. The assay is a double antibody method which requires 60 and 15 min incubation respectively. The correlation of nicotine pooled plasma was found to be 0.994 with very good precision and accuracy. The sensitivity, defined as the concentration of nicotine measured at delta F/delta Fo = 90%, was found to be 10 micrograms/1. Samples of human smokers (N = 9) after 1 cigarette at 3 min were 50-100 micrograms/1, and at 15 min were 30-60 micrograms/1.

Quantitation of serum retinol-binding protein by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent technique for human serum retinol-binding protein (RBP) was developed. The assay detects RBP via a double-antibody (rabbit anti-human RBP) sandwich technique. The antibody is immobilized by passive adsorption to a polystyrene tube; the assay is then carried out by successive additions containing known and unknown amounts of RBP (antigen), alkaline phosphatase linked to the same antibody, and p-nitrophenyl phosphate (substrate). Colorimetric analysis of the hydrolysis of the substrate by the enzyme (indirectly) attached to the antigen is used for RBP quantitation. The intra- and interassay coefficients of variation ranged between 4 and 7 and 9 and 12%, respectively. The assay can be performed in less than 7 h and has a sensitivity in the nanogram range (3-48 ng/ml). RBP content was analyzed in serum and urine samples of 20 healthy donors and 17 patients with renal failure and in 20 serum specimens of patients with liver cirrhosis. Renal patients had higher serum (mean 150, range 50-398 micrograms/ml) and urine RBP levels (mean 14, range 1-80 micrograms/ml) than normal donors (mean serum 43, range 30-60 micrograms/ml; mean urine RBP 0.06, range 0.04-0.13 microgram/ml). Liver disease patients had lower than normal serum RBP values (mean 22, range 10-43 micrograms/ml).

A radioactive antigen-binding assay for the measurement of antibody to Haemophilus influenzae type b capsular polysaccharide.

A new polyethylene glycol (PEG) radioimmunoprecipitation assay was developed for the detection of antibody to Haemophilus influenzae b capsular polysaccharide, polyribosylribitol phosphate (PRP). The radioactive antigen, [3H]PRP, with a high specific activity, was produced by growing the organism in the presence of [3H]ribose and was purified by hydroxylapatite and Sepharose 4B column chromatography. In the assay, PEG (12.5%) was used to separate antibody-bound [3H]PRP from free [3H]PRP. The assay covered the range of 0.5 and 20 ng antibody/assay at a maximum sensitivity of 0.5 approximately 1.0 ng antibody/assay. With various dilutions (1-20 ng antibody/assay) of S. Klein reference antiserum, the within-run coefficient of variation (CV) of 10 replicates ranged from 3.5 to 8.5%. Average CVs of 8.9% and 11.0% were obtained in the between-run and day-to-day reproducibility studies. The binding of [3H]PRP to S. Klein reference antiserum was severely inhibited by a minute amount of non-radioactive PRP; however, no significant interference was found in the presence of high concentrations of polysaccharides from Escherichia coli K100 and Streptococcus pneumoniae indicating that the RIA was highly specific for antibody to H. influenzae b PRP. The present RIA is a simple, specific, sensitive and reproducible procedure for the evaluation of antibody responses of young animals and infants to H. influenzae b vaccines and infections.

Quantification of digoxin by enzyme immunoassay: synthesis of a maleimide derivative of digoxigenin succinate for enzyme coupling.

We synthesized the m-maleimidobenzoyl derivative of digoxigenin-3-0-succinate (through a p-phenylenediamine bridge) as a hapten derivative directed towards coupling to sulfhydryl grouos of beta-galactosidase. Prepared enzyme conjugate had about 97% of the enzyme labeled with the hapten derivative while retaining full enzyme activity. The enzyme immunoassay for digoxin we prepared showed a maximum sensitivity of 30 pg per assay (c.v. = 3%) with minimal cross-reaction with digotoxin (3.8%). Our method for hapten conjugation to beta-galactosidase is highly efficient and is simple and easily replicated.

Phenobarbital specific antibody production: preparation of 5-phenyl-5-(4-aminobutyl) barbituric acid-bovine serum albumin conjugate.

The aminobutyl derivative of phenobarbital, 5-phenyl-5-(4-aminobutyl)barbituric acid hydrochloride, was synthesized through two synthetic pathways for the preparation of immunogen in production of phenobarbital specific antibody. The produced antiserum had high titer, specificity, affinity and sensitivity (0.5ng/ml), when examined by radioimmunoassay.

Nicotine antibodies: comparison of ligand specificities of antibodies produced against two nicotine conjugates.

Conjugates between bovine serum albumin and (R,S)-2-aminonicotine were produced, and these conjugates were employed in rabbits and goats for the production of nicotine antibodies. In the assay of nicotine, an 125I-tyrosine methyl ester derivative of (R,S)-6-aminonicotine was employed as radioligand. The antibody-bound derivative was separated from the free derivative by charcoal adsorption (0.5% charcoal, 0.1% dextran T-70, 0.1% bovine serum albumin pH 7.3). Among the twenty five nicotine derivatives and metabolites examined, (R,S)-6-aminonicotine gave the highest cross-reaction. Cross-reaction with cotinine, a major mammalian metabolite of nicotine, was less than 0.1% for both the rabbit-derived and goat-derived antisera. Cross-reaction by other metabolites, such as (S)-nicotine-N'-oxide, (S)-nornicotine, and N-methylpyrrolidine was less than 1%. The antibodies produced were thus highly specific to nicotine. The radioimmunoassay for nicotine showed a maximum sensitivity of 10 ng/ml in 50-microliter plasma samples for both antisera. After the smoking of a single cigarette (1.2 mg nicotine content in mainstream) the peak of blood plasma level of nicotine in the subjects varied from 20--104 ng/ml, and high levels of nicotine were not necessarily found in heavy smokers.

Synthesis of maleimide derivative of cortisol for enzyme coupling is cortisol enzyme immunoassay.

A new cortisol derivative, cortisol-21-m-maleimidobenzoate (CMB), was synthesized and conjugated with sulfhydryl groups of beta-galactosidase (BG). Both CMB-BG and CHS-BG conjugates have a high immunoreactivity to cortisol antibody, and although CHS-BG does not displace well with the added cortisol, CMB-BG does.

Practical enzyme immunoassay for plasma cortisol using beta-galactosidase as enzyme label.

Enzyme immunoassay for cortisol was developed using beta-galactosidase as an enzyme label and m-maleimidobenzoyl derivatives of cortisol, i.e. cortisol-21-m-maleimidobenzoate (CT-MB) and cortisol-21-hemisuccinate conjugated with m-maleimidobenzoic acid through p-phenylenediamine linkage (CHS-MB), as the haptens coupled to sulfhydryl groups of the enzyme. This enzyme-coupling procedure was highly efficient; over 90% of the enzyme was labeled while full enzyme activity was retained. CHS-MB-beta-galactosidase conjugate showed high immunoreactivity to antibody produced against cortisol-21-hemisuccinate-bovine serum albumin but showed poor displacement with the added cortisol. CT-MB-beta-galactosidase conjugate, however, showed not only a high immunoreactivity to the antibody but also displaced well with cortisol, showing maximum sensitivity of 1 microgram/dl with a 20-microliter sample size. Modification around the linkage of cortisol derivative resulted in high sensitivity to cortisol. Cross-reactions to cortisol-21-acetate, cortisone, and corticosterone were 150%, 10%, and 7%, respectively. The accuracy, precision and correlation with RIA of this method were satisfactory for clinical application.