Clinicoimmunologic studies on Phoenix sylvestris Roxb. pollen: an aeroallergen from Calcutta, India.

BACKGROUND: This study highlights the allergenicity and allergenic components of the pollen of Phoenix sylvestris Roxb. (PS), or date sugar palm, which is predominantly airborne in the air of Greater Calcutta. METHODS: A 2-year aerobiologic survey was performed by Burkard sampler. PS pollen extract was used in skin tests of allergic patients, fractionated by (NH4)2SO4 and the Sephacryl S-200 column. The allergenicity of each fraction was checked by skin test and IgE ELISA inhibition. The principal allergenic fraction, Fr.lla, was separated in 11% SDS-PAGE, and its allergenicity was confirmed by IgE ELISA inhibition and immunoblotting. RESULTS: PS pollen grains were found to be prevalent in the air of the suburban zone of Calcutta from January to March with a peak in February. The pollen extract showed high (44.07%) positive skin reaction on 540 respiratory allergic patients. Among the (NH4)2SO4 cut fractions, Fr.II was the most active one, and it was resolved into four subfractions in the Sephacryl S-200 column. Fr.lla was the principal allergenic fraction, showing the presence of two components of 33 and 66 kDa in SDS-PAGE. In IgE immunoblotting, both of the components were found to be allergenic. CONCLUSIONS: The PS pollen grain is an important aeroallergen from Calcutta, India. The 33- and 66-kDa components are the major allergens present in the relevant pollen extract.

Expression of abscisic acid-responsive element-binding protein in salt-tolerant indica rice (Oryza sativa L. cv. Pokkali).

As the products of abiotic stress and ABA inducible genes are predicted to play an important role in the mechanism of salt tolerance, the expression of transcription factor that recognizes abscisic acid-responsive element (ABRE) is likely to be regulated when plants are exposed to abiotic stress. Northern analysis of total RNA from control and salt-treated 10-day-old Pokkali (salt tolerant) rice plants was performed to find out the level of transcripts homologous to wheat cDNA (GC19) for EmBP-1 (bZIP class factor), a transcription factor that recognizes ABRE. Salinity stress (72 h)-induced accumulation of two transcripts, of 2.0 kb (r2.0) and 1.5 kb (r1.5), in roots was detected. Both transcripts were detectable even after 6 h of salt or abscisic acid treatment, whereas sheath and lamina showed constitutive levels of r1.5 transcript. When 32P-labeled DNA containing ABRE was used in a gel mobility shift assay, a low level of complex formation by binding factor was detected from the nuclear extract of lamina of control rice plants. Quantitative enhancement of complex formation was found with the nuclear extract prepared from the lamina of plants treated with 200 mM NaCl for 26 h over control nuclear extract, suggesting a step of regulation of expression of ABRE-binding protein in response to salinity stress. South-western blot analysis of equal amounts of nuclear proteins of lamina showed binding of 32P-labeled ABRE-DNA with two polypeptides (22-28 kDa) present at constitutive levels in control or NaCl-treated plants. Preincubation of the laminar nuclear extract of control plants, with spermidine or proline at 5 mM concentration showed quantitative enhancement of ABRE binding activity. Kinetics of spermidine stimulation showed gradual increase of complex formation from 5 mM concentration. Similarly, addition of GTP to the control nuclear extract also showed quantitative enhancement of complex formation and heparin was found to inhibit GTP activated complex formation by about 25%. Results may suggest the presence of ABRE binding protein in presynthesized and inactive form in control plants and GTP mediated activation is probably one of the way to regulate the expression of ABRE-binding factor.

Expression of arginine decarboxylase in seedlings of indica rice (Oryza sativa L.) cultivars as affected by salinity stress.

The effect of salinity stress on the activity of arginine decarboxylase (ADC, EC 4.1.1.19), the first enzyme in biosynthesis of polyamines (PA) from arginine, as well as its transcript level has been compared in salt-sensitive (M-1-48) and salt-tolerant (Pokkali) rice cultivars. Treatment of 72 h grown seedlings either with increasing concentrations of NaCl or with 150 mM NaCl for different time periods, showed a gradual increase of activity in Pokkali. In M-1-48 an immediate increase followed by sharp decrease was observed on prolonged treatment beyond 6 h or above 150 mM NaCl. To generate a DNA probe for ADC, the polymerase chain reaction was used with oat genomic DNA and sequence-specific primers. A region of oat genomic DNA containing a coding sequence for 166 amino acids of the C-terminal part of the ADC enzyme was amplified and called OAD1. Southern analysis of EcoRI- or BamHI-cut genomic DNAs from different cultivars of rice with OAD1 as the probe revealed strong hybridization with one DNA fragment of rice and restriction fragment length polymorphism (RFLP) was noticed. Northern analysis of total RNA of rice with OAD1 as the probe revealed hybridization with a transcript of similar size to the ADC transcript in oat. While in Pokkali, at least a 20-fold accumulation of OAD1 homologous transcript was detected after treatment with 200 mM NaCl, only a seven-fold increase in transcript level was found in M-1-48 after 150 mM NaCl treatment. Results suggest that in the salt-tolerant rice cultivar Pokkali, ADC enzyme activity increases and its transcript also accumulates during the prolonged salinity stress, this mechanism is absent in the salt-sensitive rice cultivar M-1-48 where a prolonged period of salinity stress down-regulates both ADC activity and its transcript level.

A mammalian 2-5A system functions as an antiviral pathway in transgenic plants.

Resistance to virus infections in higher vertebrates is mediated in part through catalysis of RNA decay by the, interferon-regulated 2-5A system. A functional 2-5A system requires two enzymes, a 2-5A synthetase that produces 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A) in response to double-stranded RNA, and the 2-5A-dependent RNase L. We have coexpressed these human enzymes in transgenic tobacco plants by using a single plasmid containing the cDNAs for both human RNase L and a low molecular weight form of human 2-5A synthetase under control of different, constitutive promoters. Expression of the human cDNAs in the transgenic plants was demonstrated from Northern blots, by specific enzyme assays, and by immunodetection (for RNase L). Infection of leaves, detached or in planta, of the coexpressing transgenic plants by tobacco mosaic virus, alfalfa [correction of alfafa] mosaic virus, or tobacco etch virus resulted in necrotic lesions. In contrast, leaves expressing 2-5A synthetase or RNase L alone and leaves containing the plasmid vector alone produced typical systemic infections. While alfalfa mosaic virus produced lesions only in the inoculated leaves regardless of the concentration of virus in the inoculum, high, but not low, levels of tobacco etch virus inoculum resulted in escape of virus to uninoculated leaves. Nevertheless, there was a substantial reduction of tobacco etch virus yield as measured by ELISA assay in the coexpressing transgenic plants. These results indicate that expression of a mammalian 2-5A system in plants provides resistance to virus infections.

Isolation of mammalian pol beta type DNA polymerase in shoot tips of germinated seedlings of IR-8 rice (Oryza sativa L.).

A single polypeptide of approx. 67 kDa mol.wt. with DNA polymerase activity has been chromatographically purified from shoot tips of 72 hr. grown germinated seedlings of rice (Oryza sativa.L.cv IR-8). An approx. 4800 fold enrichment of specific activity was measured by the incorporation of 3H-dTMP into trichloroacetic acid insoluble fraction, using activated calf thymus DNA as template-primer. The enzyme uses different types of DNA but not RNA as a template. The enzyme requires Mg+2, high KCl, and is highly sensitive to dideoxyribonucleoside triphosphate but is unaffected by aphidicolin, suggesting a plant counterpart of mammalian DNA polymerase beta. Replication of M13mp18 single stranded DNA by extension of 5'32P-labeled 17-mer primer(-40) showed distributive type of DNA synthesis by the rice DNA polymerase beta, which is a characteristic feature of mammalian DNA polymerase beta.

Characteristics and contributions of defective, ecotropic, and mink cell focus-inducing viruses involved in a retrovirus-induced immunodeficiency syndrome of mice.

LP-BM5 murine leukemia virus, a derivative of Duplan-Laterjet virus, contains a mixture of replication-competent B-tropic ecotropic and mink cell focus-inducing (MCF) viruses and a defective genome that is the proximal cause of a syndrome, murine AIDS (MAIDS), characterized by lymphoproliferation and immunodeficiency. The defective (BM5d) and ecotropic components of this mixture were molecularly cloned, and complete (BM5d) or partial (ecotropic) nucleotide sequences were determined. BM5d closely resembled the Du5H genome cloned from the Duplan virus, featuring a highly divergent p12 sequence in the gag open reading frame. In MAIDS-sensitive C57BL/6 mice, BM5d was detected in tissues within 2 weeks of infection but was absent from tissues of the MAIDS-resistant strain, A/J, 12 weeks after infection. B-cell-lineage tumors from mice with MAIDS contained and expressed BM5d, and clonal integrations of this genome were variably associated with clonal expansions of B cells in infected mice. Finally, mRNA crosshybridizing with a probe for BM5d was present in spleen but not kidney cells of uninfected B6 mice.

Direct evidence for translational regulation by leader RNA and Tat protein of human immunodeficiency virus type 1.

Translational effects of the RNA leader and Tat protein of human immunodeficiency virus type 1 (HIV-1) were investigated in rabbit reticulocyte lysate. Hybrid RNA species with natural or mutated HIV-1 leader fused to human interferon- gamma mRNA were produced in vitro from recombinant plasmids. HIV-1 leader RNA was found to inhibit translation through two mechanisms. A 3-fold trans-inhibition of translation was demonstrated by mixing hybrid HIV-1 leader RNA with indicator interferon mRNA. By comparison, HIV-1 leader caused a 50-fold cis-inhibition in lysate in which two trans-inhibitory factors, double-stranded RNA-dependent protein kinase and (2'-5')oligoadenylate synthetase, were suppressed. In contrast, purified HIV-1 Tat protein produced in Escherichia coli enhanced by 4-fold translation from HIV-1 leader-interferon mRNA but not from interferon mRNA lacking HIV sequences or from total poly(A)+ RNA. Translation of mRNA containing either a single base substitution in the loop of the "trans-acting responsive" sequence (TAR) or an alternative stem-loop in TAR was nevertheless stimulated by Tat. The enhancement of translation by Tat was largely due to relief of cis-inhibition, since the effect was found even in lysate in which double-stranded RNA-dependent protein kinase was inhibited with 2-aminopurine. These results suggest that translation is an important level of control in the replication cycle of HIV-1.

Translational regulation by HIV leader RNA, TAT, and interferon-inducible enzymes.

Every step in the replication cycle of HIV provides unique opportunities for controlling the progression of AIDS. In this regard, virus protein synthesis should be an important target for limiting HIV multiplication in cells. Molecular mechanisms in the regulation of HIV protein synthesis were therefore investigated in the context of interferon action. The interferon-inducible enzymes, 2-5A synthetase and dsRNA-dependent protein kinase, which can inhibit translation were activated by HIV-1 leader RNA. In cell-free systems, leader RNA and Tat protein of HIV inhibited and enhanced translation, respectively. An intriguing interplay of these viral and host factors were shown to influence the rate of translation in vitro. A model describing opposing actions of HIV Tat protein and interferon in HIV replication is represented.

Cloning of murine gelsolin and its regulation during differentiation of embryonal carcinoma cells.

The regulation of gelsolin levels during differentiation of the murine embryonal carcinoma cell line, PC-13, was investigated using nucleic acid and immunological probes. A cDNA clone, Mu-319, which contained the entire coding sequence for the cytoplasmic form of murine gelsolin was isolated using a polyclonal antibody. Gelsolin was detected in several cell lines but was not detectable in three undifferentiated embryonal carcinoma cell lines. Levels of gelsolin mRNA increased 10-fold during the differentiation of the murine embryonal carcinoma cell line, PC-13. Differentiation of PC-13 was accompanied by changes in cell shape, from small indistinct cells to large flat cells. The accumulation of gelsolin mRNA in PC-13 cells began 12-24 h after addition of the differentiation-inducing agents. In comparison, 2-5A-dependent RNase activity showed a 40-fold increase beginning after 24 to 36 h and c-fos mRNA were shown to increase about 9-fold beginning 36 to 60 h after induction of differentiation. The levels of gelsolin per se, as determined by immunoreactivity were also shown to increase with differentiation of PC-13 cells. These results suggest that gelsolin may play a role in the restructuring of actin filaments which accompanies the dramatic changes in cell shape during differentiation.

Activation of interferon-regulated, dsRNA-dependent enzymes by human immunodeficiency virus-1 leader RNA.

Human immunodeficiency virus-1 (HIV-1) leader RNA, which contains double-stranded regions due to inverted repeats, was shown to activate the dsRNA-dependent enzymes associated with the interferon system. HIV-1 leader RNA produced in vitro using SP6 RNA polymerase was characterized using probes for antisense and sense-strand RNA. The RNA preparation was free from significant levels of antisense RNA. HIV-1 leader RNA was shown to activate dsRNA-dependent protein kinase in a cell-free system from interferon-treated HeLa cells. Affinity resins, consisting of HIV-1 leader RNA covalently attached to cellulose, immobilized and activated dsRNA-dependent protein kinase and 2-5A-synthetase. HIV-1 leader RNA, therefore, may be a contributing factor in the mechanism by which interferon inhibits HIV replication.

Studies on the mechanism of Escherichia coli DNA polymerase I large fragment. Effect of template sequence and substrate variation on termination of synthesis.

Termination of Escherichia coli DNA polymerase I large fragment after processive synthesis on natural and other well-defined template.primer systems has been examined. We found that after any given deoxynucleoside monophosphate incorporation termination occurs in a nonrandom manner with phi X174 DNA as template: Termination is much more likely at some nucleotide residues along the template than at others. Analysis of these stronger termination sites indicates that the template base:incoming nucleotide combination influences termination. Introduction of a double-stranded region along the phi X174 template induces termination, and reducing dNTP concentrations or substituting 2'-deoxynucleoside 5'-O-(1-thio)triphosphate substrates also increases termination. Observations with the phi X174 DNA template system were extended with a defined template containing 1 inosine residue in an otherwise d(T)n homopolymer. Termination at the I residue is modulated by dCTP and decreases as dCTP concentration increases. A similar relationship is seen with the dCTP (1-thio) derivative, but termination is higher at given concentrations of this derivative than with dCTP. Pyrophosphate decreases general processivity in this system, but does not counteract the effect of increasing dCTP. Hill plot analysis of the dCTP effect in the inosine-containing template system gave a linear plot with Hill coefficient of 0.34, suggesting that dCTP influences termination at several steps in the polymerase reaction scheme. Substituting a methylated template base for I also increased termination, producing very strong blocks to processive synthesis. The results are consistent with a model in which termination occurs with several enzyme forms that are in equilibrium in an ordered catalytic mechanism.

Cell-cycle-specific and serum-dependent expression of gamma-actin mRNA in Swiss mouse 3T3 cells.

We isolated cDNA clones that represent genes whose expression is enhanced when resting Swiss mouse 3T3 cells are stimulated to proliferate with serum. Two clones (designated pME1 and pMR6) were analyzed further. A partial sequence analysis of the pME1 insert DNA indicated that it contained a 104-base-pair stretch with extensive homology to the 3' untranslated region of gamma actin. Similar analysis of the insert DNA from the pMR6 clone indicated that it did not correspond to any previously reported gene sequence. We used the pME1 clone as a probe to determine the level of gamma actin-specific transcript in 3T3 cells under a variety of conditions. The level of gamma actin-specific mRNA began to increase in resting cells upon serum stimulation and reached a peak at 6 h. Thereafter its level declined, and by 24 h it was hardly detectable. In contrast, pMR6-specific transcript was detectable in resting cells but remained elevated even at 24 h poststimulation. The level of gamma-actin mRNA was elevated in resting cells by 12-O-tetradecanoylphorbol-13-acetate, calcium ionophore A23187, and bombesin and to a lesser extent by cholera toxin, fibroblast-derived growth factor, and dibutyryl cyclic AMP. However, insulin, vasopressin, or epidermal growth factor failed to enhance gamma-actin mRNA levels in resting cells. Inhibitors of transcription diminished the induction of gamma-actin mRNA. Gamma-actin gene was superinduced in serum-stimulated cells by cycloheximide, an inhibitor of translation. Analysis of proteins from serum-stimulated cells by two-dimensional gel electrophoresis indicated that enhanced transcription of gamma-actin mRNA resulted in a concomitant increase in the corresponding actin protein. The possible role of gamma actin, a component of the cytoskeleton, in the regulation of cell growth is discussed.

Purification and analysis of murine 2-5A-dependent RNase.

2-5A-dependent RNase (RNase L, RNase F) is an enzyme which mediates effects of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) in cells. 2-5A binding activity present in mouse liver extracts was measured using a 32P-labeled 2-5A derivative. Analysis of Scatchard plots was consistent with a single noninteracting 2-5A binding site with a Ka of 2.5 X 10(10) M-1. Similarly, affinity labeling of proteins with a 32P-labeled 2-5A derivative revealed a single, high-affinity 2-5A-binding protein of Mr 80,000. This 2-5A-binding protein was the only mouse liver protein specifically and consistently eluted by 2-5A from an affinity resin consisting of core(2-5A) covalently attached to cellulose. The 2-5A-eluted protein could degrade polyuridylic acid but not polycytidylic acid. Furthermore, when the 2-5A-eluted protein was electrophoresed into a polyuridylic acid-containing, nondenaturing gel, a band of degraded polyuridylic acid was demonstrated after incubation with 2-5A. There was no band of degraded polyuridylic acid when the elution was performed either in the absence of oligonucleotide or in the presence of low amounts of a closely related analog of 2-5A, p3I2'pA2'pA. Therefore, the Mr 80,000 2-5A-binding protein and the 2-5A-dependent RNase were almost certainly the same protein. Finally, the Mr 80,000 2-5A-binding protein was purified to homogeneity by electroelution from a polyacrylamide gel.

Expression of human DNA polymerase beta in Escherichia coli and characterization of the recombinant enzyme.

The coding region of a human beta-polymerase cDNA, predicting a 335 amino acid protein, was subcloned in the Escherichia coli expression plasmid pRC23. After induction of transformed cells, the crude soluble extract was found to contain a new protein immunoreactive with beta-polymerase antibody and corresponding in size to the protein deduced from the cDNA. This protein was purified in a yield of 1-2 mg/50 g of cells. The recombinant protein had about the same DNA polymerase specific activity as beta-polymerase purified from mammalian tissues, and template-primer specificity and immunological properties of the recombinant polymerase were similar to those of natural beta-polymerases. The purified enzyme was free of nuclease activity. We studied detailed catalytic properties of the recombinant beta-polymerase using defined template-primer systems. The results indicate that this beta-polymerase is essentially identical with natural beta-polymerases. The recombinant enzyme is distributive in mode of synthesis and is capable of detecting changes in the integrity of the single-stranded template, such as methylated bases and double-stranded region. The enzyme recognizes a template region four to seven bases downstream of the primer 3' end and utilizes alternative primers if this downstream template region is double stranded. The enzyme is unable to synthesize past methylated bases N3-methyl-dT or O6-methyl-dG.

Mammalian alpha-polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots.

A new polyclonal antibody against the alpha-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cell alpha-polymerase. The antibody neutralized alpha-polymerase activity and was strong and specific for the alpha-polymerase catalytic polypeptide (Mr 183,000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+) RNA in lambda gt11. A positive phage was identified and plaque purified. This phage, designated lambda pol alpha 1.2, also was found to be positive with an antibody against Drosophila alpha-polymerase. The insert in lambda pol alpha 1.2 (1183 base pairs) contained a poly(A) sequence at the 3' terminus and a short in-phase open reading frame at the 5' terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified alpha-polymerase by enzyme-linked immunosorbent assay and was capable of immunoprecipitating alpha-polymerase. This indicated the lambda pol alpha 1.2 insert encoded an alpha-polymerase epitope and suggested that the cDNA corresponded to an alpha-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(A+) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding alpha-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(A+) RNA revealed that this mRNA is approximately 5.4 kilobases.

Sequence of human DNA polymerase beta mRNA obtained through cDNA cloning.

A cDNA library from polyA+ RNA of a human teratocarcinoma cell line in phage lambda gt11 was screened with a fragment of the rat beta-polymerase cDNA, lambda pol beta-10, as probe. Five positive phage were identified and plaque purified. The cDNA of one positive clone selected for detailed study was 1257 bp. This insert was sequenced and found to contain the coding region for beta-polymerase, as well as 163 bp and 137 bp from the 5' and 3' untranslated regions, respectively. The primary structure of human beta-polymerase (318 amino acids, Mr = 36, 133) deduced from the cDNA was similar to rat beta-polymerase (95% matched residues). The greatest difference between the sequences of the human and rat cDNAs was in the 3' untranslated regions (64% matched base residues). These results provide necessary sequence information for study of the human beta-polymerase gene.

Structure of rodent helix-destabilizing protein revealed by cDNA cloning.

A cDNA library of newborn rat brain poly(A+) RNA in lambda gt 11 was screened with a synthetic oligonucleotide probe corresponding to a five amino acid sequence in the N-terminal region of the calf helix-destabilizing protein, UP1. Six positive phage were isolated after testing 2 X 10(5) recombinants, and each phage was plaque purified. Four of these phage clones were positive with a second oligonucleotide probe corresponding to a 5 amino acid sequence in the C-terminal region of calf UP1; one of the clones positive with both probes was selected for detailed study. This phage, designated lambda HDP-182, contained a 1706-base pair cDNA insert corresponding to an mRNA with a poly(A) sequence at the 3' terminus and a single open reading frame starting 63 bases from the 5' terminus and extending 988 bases. The 3' untranslated region of the mRNA contained 718 bases, including an AAUAAA signal 21 bases from the poly(A) sequence and a 16-residue poly(U) sequence flanked on each side by oligonucleotide repeats. Primer extension analysis of newborn rat brain poly(A+) RNA suggested that the cDNA insert in lambda HDP-182 was full length except for about 35 nucleotide residues missing from the 5' end untranslated region, and Northern blot analysis revealed one relatively abundant mRNA species of approximately the same size as the cDNA insert. The 988-residue open reading frame in the cDNA predicted a 34,215-dalton protein of 320 amino acids. Residues 2 through 196 of this rat protein are identical to the 195-residue sequence of the calf helix-destabilizing protein, UP1. The 124-amino acid sequence in the C-terminal portion of the 34,215-dalton protein is not present in purified calf UP1. This 124-residue sequence has unusual amino acid content in that it is 11% asparagine, 15% serine, and 40% glycine and consists of 16 consecutive oligopeptide repeats. Computer-derived secondary structure predictions for the 34,215-dalton protein revealed two distinct domains consisting of residues 1 through approximately 196 and residues approximately 197 to 320, respectively.

Serology of tuberculosis. III. Crossed immunoelectrophoretic analysis of sera from tuberculosis and leprosy patients with antigens from BCG.

Sera from tuberculous and leprous patients have been examined for antibody reactivities against components of BCG sonicate (BCGS) antigen. A crossed immunoelectrophoresis with intermediate gel reference system was used in which more than 40 components of BCGS could be identified. Forty (74.1%) out of 54 tuberculous sera and 68 (90.7%) out of 75 leprous sera reacted with at least 1 component of BCGS. While tuberculous sera reacted with 9 distinct components of BCGS, leprous sera reacted with at least 12. Components of BCGS precipitated by tuberculous sera were not specific as they were also precipitated by leprous sera. Overall, non-specific antibody responses were found to be dominant among tuberculous sera and by comparison, the reactivity of leprous sera with BCGS components was of a higher magnitude. Among tuberculous sera, precipitating activity was maximal among those taken from chronic treated cases with relapse followed by those obtained from treated and untreated new cases. Some components of BCGS to which both tuberculous and leprous sera showed strong reactivity have been characterized. It is concluded that immunoprecipitation methods with BCG derived antigens are not useful for the detection of a specific antibody response in tuberculosis or for discrimination between tuberculosis and leprosy.

Staphylococcus aureus protein A antibody-mediated haemagglutination assay. A study on the interaction between protein A-bound specific antibodies and red-cell-bound specific antigens.

A method has been described for the enhancement of the sensitivity of the passive haemagglutination (PHA) reaction through intact cells of Staphylococcus aureus (Cowan I) bearing protein A (SAPA) which bind Fc portion of IgG derived from many mammalian species. Interactions between Mycobacterium tuberculosis sonicate antigen-sensitized red cells and antimycobacterial antibodies derived from M. tuberculosis immunized goat, rabbit, guinea pig and mouse sera and sera from human tuberculosis patients have been used as model systems. SAPA cells were allowed to bind antibodies from sera and subsequently cross-react with antigen-sensitized red cells. Co-haemagglutination occurred only when specific antibodies were bound by SAPA cells. The sensitivity of this SAPA antibody-mediated haemagglutination assay (SAPA-AMHA) was found to be higher than that of PHA reaction and it depended upon protein A antibody affinity. The specificity of the interaction between SAPA cell-bound antibodies and red-cell-bound antigens suggests that these two reagents offer a simple and sensitive approach for the study of antigen-antibody reactions.