Biomedical subjects

N Strick

Publications and source records attributed to N Strick.

At least 55 records · Page 3Linked to original sources

PubMed1986

Detection of antiviral antibodies with predetermined specificity using synthetic peptide--beta-lactamase conjugates: application to antibodies specific for the preS region of the hepatitis B virus envelope proteins.

Amino acid sequences coded for by the preS region of the hepatitis B virus (HBV) envelope gene are present both in HBV and in subviral hepatitis B surface antigen (HBsAg) particles. Consequently, anti-preS-specific antibodies are elicited during the course of HBV infection. Such antibodies are virus-neutralizing. Therefore, it is important to determine whether or not vaccination with HBsAg also induces an anti-preS-specific immune response. We describe here an enzyme-linked immunosorbent assay applicable for the screening of sera from vaccinated individuals for anti-preS antibodies. IgG from serum specimens was adsorbed to staphylococcal Protein A on a superparamagnetic support and subsequently mixed with a synthetic peptide analogue [preS(120-145)] covalently linked to beta-lactamase. The presence of anti-preS in serum specimens resulted in binding of the conjugated beta-lactamase to the magnetic support. The adsorbed enzyme was quantified colorimetrically.

Amino Acid Sequence↗

PubMed1985

Genetic restriction of immune responsiveness to synthetic peptides corresponding to sequences in the pre-S region of the hepatitis B virus (HBV) envelope gene.

Proteins of the HBV envelope (env) are coded for by two adjacent regions of the HBV env gene: the pre-S and S regions. Antigenic determinants corresponding to amino acid sequences of both regions are recognized by human antibodies and are important in virus-neutralizing responses. Protective immune responses to HBV appear to be linked to the major HLA histocompatibility complex. Inbred and congenic strains of mice represent a model system relevant for studies on the genetic control of immune responsiveness of humans to HBV envelope proteins. Such mouse strains were ranked according to their antibody response to the S protein and divided into high [d,q], intermediate [a,k,b], and low [s] responders (letters in brackets indicate H-2 haplotype.) Selected pre-S antigenic determinants can be mimicked with high fidelity by synthetic peptide analogues that are immunogenic without any carriers. Thus it is possible to study directly the genetic control of immune responsiveness to pre-S epitopes mimicked by these peptides without having to consider the influence of carriers or of S protein. The results presented here show that inbred mouse strains can be ranked according to their antibody responses to the synthetic peptide pre-S(120-145) as follows: A/J[a] approximately equal to SWR/J[q] greater than C57BL/6J[b] approximately equal to AKR/J[k] approximately equal to SJL/J[s] much greater than DBA/2J[d] greater than BALB/cJ[d]. Only SJL/J[s] mice responded well to another synthetic peptide pre-S (12-32). Thus, H-2-linked genes regulating the immune response to S protein and to epitopes on pre-S-coded sequences are distinct. Anti-pre-S(120-145) responses in S protein-nonresponders circumvent this nonresponsiveness. This should be considered in the design of hepatitis B vaccines.

Amino Acid Sequence↗

PubMed1985

Radioimmunoassay and enzyme-linked immunoassay of antibodies directed against lymphadenopathy-associated virus (LAV) proteins larger than the core protein (P24).

The acquired immunodeficiency syndrome (AIDS) may be transmitted by blood transfusions and by blood products from donors who have been infected with the lymphadenopathy-associated virus (LAV). Such donors may generally be identified on the basis of a positive test for antibodies-against LAV proteins. We have already described an anti-LAV assay based on the use of crude virus-infected tissue culture medium, which avoids elaborate, expensive and potentially hazardous virus purification steps. This test was operationally specific for antibodies to the approximately 24 kD core protein of the virus (P24; Neurath et al. J. Virol. Methods 11, 75, 1985). Molecular exclusion chromatography of crude LAV antigen preparations allows separation of most of P24 from larger proteins of LAV (PL). PL and 125I- or beta-lactamase-labeled anti-LAV were used as reagents for radioimmunoassay (RIA)--or enzyme-linked immunoassay (ELISA)--inhibition tests to detect antibodies directed predominantly against PL (anti-PL). Among 257 individuals belonging to groups at high risk of developing AIDS, 117 (45.5%) were positive for anti-PL and 108 (42%) for anti-P24, respectively. The 2 individuals among 600 random blood donors found to be anti-P24-positive in the preceding study also had anti-PL in their serum. Sera from 500 additional blood donors were screened for anti-PL and 1 of these was positive. The implication of these findings for screening of blood donors is discussed.

Acquired Immunodeficiency Syndrome↗

PubMed1985

Radioimmunoassay and enzyme-linked immunoassay of antibodies to the core protein (P24) of human T-lymphotropic virus (HTLV III).

Human T-cell lymphotropic viruses designated HTLV III or LAV are considered to represent the causative agent(s) of the acquired immunodeficiency syndrome (AIDS). Individuals who have been infected with these viruses may generally be identified on the basis of a positive serological test for antibodies against the protein components of these viruses. Purified viruses or viral proteins have been utilized for developing such tests. Since AIDS may be transmitted by blood transfusion and by blood products, screening of donors for antibodies to HTLV III/LAV has become a necessity. Such screening may be facilitated by the application of assays based on the use of crude virus-infected tissue culture media avoiding elaborate, expensive and potentially hazardous virus purification steps. Serum specimens were mixed with an appropriate dilution of an HTLV III-infected tissue culture-derived fraction, obtained by precipitation with polyethylene glycol 6000 and treatment with Tween 80 and tri-n-butylphosphate (to disrupt virus particles), and incubated with polystyrene beads coated with antibodies to HTLV III/LAV (anti-HTLV III). Subsequently, washed beads were incubated with either 125I- or beta-lactamase-labeled anti-HTLV III. The radioactivity or enzymatic activity associated with the beads was proportionate to the quantity of HTLV III antigen originally added to the beads. The presence of anti-HTLV III in serum specimens resulted in decreased antigen binding and thus in decreased radioactivity or diminished beta-lactamase activity associated with the beads. The test was specific for antibodies to the approximately equal to 24 kDa core protein of HTLV III. The prevalence of these antibodies (given in parentheses) in distinct populations was as follows: random blood donors (0.33%); hemophiliacs (36.4%); random homosexual males (25.1%); homosexual males preselected on the basis of positive markers for infection with hepatitis B virus (50%); and those with persistent lymphadenopathy (70%).

Acquired Immunodeficiency Syndrome↗

PubMed1985

Enzyme-linked immunoassay of pre-S gene-coded sequences in hepatitis B vaccines.

Pre-S gene coded domains of the hepatitis B virus (HBV) envelope protein are highly immunogenic in experimental animals and humans. Their presence in HBV and hepatitis B surface antigen (HBsAg) particles leads to production of anti-pre-S-specific antibodies during the course of HBV infection. Since antibodies specific for pre-S domains are capable of preventing the attachment of HBV to hepatocytes and are virus neutralizing, it would seem desirable to produce HBV vaccines with a standardized level of pre-S determinants to ensure their potential for eliciting the same repertoire of protective antibodies as found after recovery from natural infection. However, a test with appropriate sensitivity for detecting pre-S determinants to ensure their potential for eliciting the same repertoire of protective antibodies as found after (ELISA) for detecting pre-S determinants in vaccines containing less than or equal to 20 micrograms of HBsAg. The components of this assay are (1) antibodies to a synthetic peptide pre-S (120-145) adsorbed to polystyrene beads, and (2) beta-lactamase-labelled antibodies purified from anti-HBV serum on the basis of their affinity for a pre-S (120-174) beta-galactosidase fusion protein produced in Escherichia coli. Results of an evaluation of the pre-S content of HBV vaccines from two different commercial sources are discussed.

Amino Acid Sequence↗

PubMed1985

Expression in Escherichia coli of a cloned DNA sequence encoding the pre-S2 region of hepatitis B virus.

A DNA sequence encoding the entire pre-S2 region (amino acids 120-174; serotype ayw) of human hepatitis B virus envelope protein has been inserted into the lacZ gene of the plasmid pSKS105 yielding a recombinant, pWS3. Lac+ colonies of the Escherichia coli M182 delta (lacIOPZYA), isolated after transformation with pWS3, produced a pre-S2 peptide-beta-galactosidase fusion protein. This fusion protein, which comprised as much as 3% of the total bacterial protein, was purified to greater than 90% homogeneity by affinity chromatography on p-aminophenyl-beta-D-thiogalactoside-Sepharose. It is immunoprecipitable with rabbit antibodies to a synthetic peptide corresponding to amino acids 120-145 of the pre-S2 region of serotype adw [pre-S(120-145)] or with antibodies to hepatitis B virus. pre-S(120-145) completely blocked the binding of either antibody to the pre-S2 peptide-beta-galactosidase fusion protein. These results indicate that there are antigenic determinants on the fusion protein that are closely related to, if not identical to, determinants on synthetic pre-S(120-145) and on pre-S2 sequences of native hepatitis B virus particles. Thus, bacteria transformed with pWS3 can provide an abundant source of pre-S2-beta-galactosidase fusion protein, which may prove useful either as a diagnostic reagent possessing marker enzyme activity suitable for ELISA tests or as an immunogen with potential to contribute to active prophylaxis of hepatitis B.

Amino Acid Sequence↗

PubMed1984

Location and chemical synthesis of a pre-S gene coded immunodominant epitope of hepatitis B virus.

Immunodominant, disulfide-bond independent epitopes recognized by human antibodies to hepatitis B virus (HBV) are located within the 55-residue amino terminal portion (coded for by the pre-S region of HBV DNA) of minor HBV envelope components larger than the major protein constituents encoded by the S gene. A peptide having the sequence of the first 26 amino acids from the amino terminal methionine was synthesized and elicited antibodies (at dilutions of greater than or equal to 1 to 10(5) ) to the HBV envelope. These antibodies can be utilized for diagnostic tests. The immunogenicity of the peptide was substantially increased by covalent attachment to liposomes. The disulfide bond-independent determinants on sequences coded for by the pre-S gene may be more easily mimicked by peptide analogs than "conformational" determinants on the S-gene product.

Amino Acid Sequence↗

PubMed1984

Strategies for detection of transfusion-transmitted viruses eluding identification by conventional serologic tests. II. Detection of host DNA in human plasmas with elevated alanine aminotransferase.

As a prelude to the development of nucleic acid probes specific for non-A, non-B hepatitis virus(es) (NANBV), plasmas with alanine aminotransferase levels greater than or equal to 110 IU were assayed for DNA by a radioimmunoassay. Approximately 50% of such plasmas are expected to contain NANBV. One-hundred and seventy-eight of 420 plasma samples tested (42.4%) contained sequestered DNA resistant to DNAse I. The DNA has a molecular weight of approximately equal to 0.8 to 1.4 X 10(6) daltons and hybridizes with a 32P-labeled human DNA probe. The DNA in plasma is mostly bound to IgM. The presence of host DNA will have to be taken into account in planning experiments aiming at the preparation of nucleic acid probes specific for NANBV using infected plasmas as source material. Such experiments will have to utilize recombinant DNA technology and will require the separation of bacterial colonies containing recombinant DNA with viral DNA sequences from colonies with human DNA inserts. The feasibility of this approach is demonstrated using plasma-containing hepatitis B virus.

Alanine Transaminase↗

PubMed1984

Monoclonal antibodies to hepatitis B surface antigen (HBsAg) with anti-alpha specificity recognize a synthetic peptide analogue (S135-155) with unmodified lysine (141).

A synthetic peptide corresponding to residues 135-155 (S135-155) of the major protein component of HBsAg was conjugated to beta-galactosidase. This conjugate reacted with monoclonal anti-HBs antibodies having anti-alpha group specificity. The reaction was inhibited by: HBsAg of either subtype ad or ay; by unconjugated S135-155 or a shorter peptide S140-155, but not by unrelated peptides. Modification of lysine residues of either HBsAg or S135-155 reduced this inhibitory effect. These results indicate that Lys 141 is essential for maintaining the antigenicity of one of the epitopes responsible for the common alpha specificity of HBsAg and that studies involving the use of synthetic peptides and modifications of distinct amino acid residues in the native protein or in the peptide may help in characterizing epitopes of viral antigens in general.

Antibodies, Monoclonal↗

PubMed1984

Antibody response to two synthetic peptides corresponding to residues 45-68 and 69-79 of the major protein of hepatitis B surface antigen.

Peptides corresponding to amino acid residues 48-65 and 69-79 of the major polypeptide component of hepatitis B surface antigen (HBsAg) were synthesized and conjugated to protein (bovine serum albumin and keyhole limpet hemocyanin) and fully synthetic (polyglutaraldehyde and cross-linked liposomes) carriers. The peptide-liposome conjugates appeared the most consistent in eliciting antibodies to HBsAg. Results of competition assays between each of the free synthetic peptides and HBsAg for antibodies suggested that the synthetic analogues and the corresponding segments on intact HBsAg are structurally closely related.

Amino Acid Sequence↗

PubMed1984

Antibodies to hepatitis B surface antigen (HBsAg) elicited by immunization with a synthetic peptide covalently linked to liposomes.

Peptides synthesized for potential application as antiviral vaccines have been mostly tested in the form of conjugates with carrier proteins. The possible use of several distinct synthetic vaccines in prophylaxis would be facilitated by the availability of fully synthetic immunogens. A synthetic peptide corresponding to residues 135 to 155 ( P135 -155) of hepatitis B surface antigen (HBsAg) failed to elicit in free form anti-peptide antibodies or anti-HBs. However, polymers of P135 -155 (prepared by linking to diaminoalkanes ) and synthetic conjugates prepared by binding P135 -155 to liposomes or polylysine were immunogenic. A poor correlation was observed between anti-peptide and anti-HBs responses elicited by these conjugates. Glutaraldehyde-fixed liposomes appeared to be the carriers of choice for inducing anti-HBs.

Epitopes↗

PubMed1983

H-2 linked genetic control of immune responsiveness to hepatitis B surface antigen (HBsAg) in mice.

Recent data suggest that genes involved in the control of (1) immune responses of humans to HBsAg and (2) the susceptibility to the development of chronic hepatitis B are linked to the major HLA histocompatibility complex. Studies on the genetic regulation of anti-HBs responses and on the possible abrogation of nonresponsiveness to HBsAg in humans are difficult. In an attempt to develop a relevant animal model system, the anti-HBs response of inbred and congenic strains of mice was investigated. A great variation in anti-HBs responses among individual mice belonging to the same strains was observed. Nevertheless, it was possible to rank the inbred mouse strains studied according to their decreasing anti-HBs responses as follows: BALB/c[d] congruent to SWR/J[q] greater than C57BL/6J[b] congruent to DBA/2J[a] greater than AKR/J[k] greater than A/J[a] greater than CBA/CaJ[k] greater than SJL/J[s]. (Letters in brackets indicate H-2 haplotype). Only a small proportion of SJL mice had an anti-HBs response. Therefore, this strain may serve as a model for human nonresponders. Studies with the congenic strains B10.D2[d] and B10.S[s] indicated that genes conferring responsiveness to HBsAg are linked to the H-2 histocompatibility complex. However, genes not linked to H-2 also probably play a role in regulating anti-Hbs responses.

Animals↗

PubMed1983

Strategies for detection of transfusion-transmitted viruses eluding identification by conventional serologic tests. I. Radioimmunoassay for picogram quantities of DNA.

The unavailability of serological tests for detection of several not yet characterized infectious agents transmitted by blood transfusion or by blood products prompted the development of alternative tests based on utilization of labeled nucleic acid probes specific for genomes of each of these agents. The prerequisite for the preparation of such probes is the demonstration in human plasma of nucleic acid sequences distinct from those present in host DNA or in genes of already characterized viruses occurring in plasma of infected individuals. To accomplish this, ultrasensitive tests for nucleic acids not dependent on their base sequence are needed. We describe here a radioimmunoassay (RIA) for picogram quantities of DNA. Plasma (serum) specimens are treated with proteinase K in the presence of sodium dodecyl sulfate and extracted with phenol. Nucleic acids are precipitated with ethanol in the presence of dextran (mol. wt. approximately equal to 5 X 10(5)) as carrier. Subsequently, DNA from the redissolved samples is adsorbed onto polylysine-coated wells of microtiter plates and detected by a double-antibody RIA using anti-DNA autoantibodies from NZB/NZW mice and 125I-labeled antibodies to mouse immunoglobulins. DNA which did not hybridize with human DNA was detected by this method in sera containing hepatitis B virus used as a model system.

Blood Transfusion↗

PubMed1983

Specificity of antibodies elicited by a synthetic peptide having a sequence in common with a fragment of a virus protein--the hepatitis B surface antigen.

We predicted the localization of a major hepatitis B surface antigen (HBsAg) determinant within residues 135-155. A peptide corresponding to this sequence (P135-155) was synthesized, linked to macromolecular carriers and used to immunize rabbits. In accordance with published data on shorter peptides within the same amino acid sequence, antibodies to HBsAg were elicited. A detailed analysis of the immune response to P135-155 revealed the following: A heterogeneous population of IgG and IgM antibodies reacting with P135-155 was detected in the antisera by a variety of radioimmunoassays and enzyme linked fluorescence immunoassays. Only a subpopulation of these antibodies reacted with HBsAg. The equilibrium constant (K) for the reaction of the antibodies with HBsAg (K = 4 X 10(5) X M-1) was approximately two orders of magnitude lower than K for the reaction with P135-155, and was below K for the reaction between HBsAg and antibodies elicited by HBsAg (K greater than 10(7) X M-1). Preimmunization with P135-155 did not result in an enhanced response to subsequent immunization with HBsAg. Peptides more accurately mimicking determinants on HBsAg may have to be synthesized for possible application in antiviral prophylaxis.

Amino Acid Sequence↗

PubMed1983

Radioimmunoassays of hidden viral antigens.

Antigens corresponding to infectious agents may be present in biological specimens only in a cryptic form bound to antibodies and thus may elude detection. We describe a solid-phase technique for separation of antigens from antibodies. Immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN and adsorbed onto nitrocellulose or polystyrene supports. Antigens remain topographically separated from antibodies after removal of NaSCN and can be detected with radiolabeled antibodies. Genomes from viruses immobilized on nitrocellulose can be identified by nucleic acid hybridization. Nanogram quantities of sequestered hepatitis B surface and core antigens and picogram amounts of hepatitis B virus DNA are detected.

Antigen-Antibody Complex↗

PubMed1982

Automated enzyme-linked fluorescence immunoassays for antiviral antibodies: a test for anti-HBs.

A solid-phase enzyme-linked fluorescence immunoassay (ELFA) for antiviral antibodies using antigen covalently linked to Escherichia coli beta-galactosidase and a fluorogenic substrate (4-methyl-umbelliferyl-beta-D-galactopyranoside) is described. Antibodies to hepatitis B surface antigen (HBsAg) from test samples were adsorbed to wells of 96-well aminopolystyrene plates precoated with HBsAg. The antibodies were subsequently detected using enzyme-conjugated HBsAg. The measurement of fluorescence was automated. The sensitivity of the test was approximately 50 times higher than that of a commercial radioimmunoassay.

Antibodies, Viral↗

PubMed1982

Radioimmunoassays of hidden viral antigens.

Antigens corresponding to infectious agents may be present in biological specimens only in a cryptic form bound to antibodies and, thus, may elude detection. We describe a solid phase technique for separation of antigens from antibodies. Immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN, and adsorbed onto nitrocellulose or polystyrene supports. Antigens remain topographically separated from antibodies after removal of NaSCN and can be detected with radiolabeled antibodies. Genomes from viruses immobilized on nitrocellulose can be identified by nucleic acid hybridization. Nanogram quantities of sequestered hepatitis B surface and core antigens and picogram amounts of hepatitis B virus DNA were detected. Antibody-bond adenovirus, herpesvirus, and measles virus antigens were discerned by the procedure.

Antigen-Antibody Complex↗

PubMed1982

Specificity of antibodies elicited by a synthetic peptide having a sequence in common with a fragment of a virus protein, the hepatitis B surface antigen.

We predicted the localization of a major hepatitis B surface antigen (HBsAg) determinant within residues 135-155 [Neurath, A.R., Strick, N. & Oleszko, W.R. (1981) J. Virol. Methods 3, 115-125]. A peptide corresponding to this sequence (P135-155) was synthesized, linked to macromolecular carriers, and used to immunize rabbits. In accordance with published data on shorter peptides within the same amino acid sequence, antibodies to HBsAg were elicited. A detailed analysis of the immune response to P135-155 revealed the following: A heterogeneous population of IgG and IgM antibodies reacting with P135-155 was detected in the antisera by a variety of radioimmunoassays and enzyme-linked fluorescence immunoassays. Only a subpopulation of these antibodies reacted with HBsAg. The equilibrium constant (K) for the reaction of the antibodies with HBsAg (K = 4 X 10(5) M-1) was approximately two orders of magnitude lower than K for the reaction with P135-155 and was below K for the reaction between HBsAg and antibodies elicited by HBsAg (K greater than 10(7) M-1). Preimmunization with P135-155 did not result in an enhanced response to subsequent immunization with HBsAg. Peptides more accurately mimicking determinants on HBsAg may have to be synthesized for possible application in antiviral prophylaxis.

Amino Acid Sequence↗